The underlying causes of severe malarial anaemia are multifactorial. In previously reports, Plasmodium vivax was found to be able to directly inhibited erythroid cell proliferation and differentiation. The molecular mechanisms underlying the suppression of erythropoiesis by P. vivax are remarkably complex and remain unclear. In this study, a phosphoproteomic approach was performed to dissect the molecular mechanism of phosphoprotein regulation, which is involved in the inhibitory effect of parasites on erythroid cell development.

Mesenchymal stem cell (MSC) is a type of stem cell that is capable of differentiating into osteoblasts and adipocytes. The pathological perturbation of MSC fate determination is well demonstrated by the replacement of bone tissues with fat in those with osteoporosis and osteopenia. Cell fate determination can be regulated by epigenetic and post-transcriptional mechanisms. MicroRNAs (miRNAs) are small endogenous non-coding RNA molecules that mediates the post-transcriptional regulation of genes expression. We hypothesized that miRNA specified to PPARγ, a major transcription factor of adipogenesis, is responsible for the differentiation of MSCs into osteoblasts. Candidate miRNA that is responsible for target gene inhibition was identified from the miRNA database via bioinformatic analyses. In this study, miR-130a and miR-27b were selected for investigation on their role in specifically binding to peroxisome proliferator-activated receptor γ (PPARγ) via in vitro osteogenesis of human MSCs. During osteogenic differentiation of human MSCs, the expression level of miR-130a and miR-27b were found to be upregulated. In the meanwhile, adipogenic marker genes (PPARγ and C/EBPβ) were found to decrease, which is in contrary to the increased expression of osteogenic marker genes (RUNX2 and Osterix). MSCs were transfected with mimics and inhibitors of miR-130a and miR-27b during in vitro osteogenesis followed by evaluation for the presence of osteogenic markers via quantitative gene expression, Western blot analysis and alkaline phosphatase activity assay. The overexpression of miR-130a and miR-27b is shown to enhance osteogenesis by increasing the gene expression of RUNX2 and Osterix, the protein expression of RUNX2, COL1A1, and Osterix as well as the alkaline phosphatase activity. Taken altogether, these results suggested that miR-130a and miR-27b could promote osteogenesis in human MSCs by targeting the PPARγ.

Retinoblastoma (RB) is a retinal tumor that most commonly occurs in children. Approximately 40% of RB patients carry germline mutations in the RB1 gene. RB survivors with germline mutations are at increased risk of passing on the disease to future offspring and of secondary cancer in adulthood. This highlights the importance of genetic testing in disease management and counseling. This study aimed to identify germline RB1 mutations and to correlate the mutations with clinical phenotypes of RB patients.

β-thalassemia/Hb E is a global health issue, which is characterized by a range of clinical symptoms from a mild and asymptomatic anemia to severe disorders that require transfusions from infancy. Pathological mechanisms of the disease involve the excess of unmatched alpha globin and iron overload, leading to ineffective erythropoiesis and ultimately to the premature death of erythroid precursors in bone marrow (BM) and peripheral organs. However, it is unclear as to how BM microenvironment factors contribute to the defective erythropoiesis in β-thalassemia/Hb E patients. Here, we employed mass spectrometry-based comparative proteomics to analyze BM plasma that was collected from six β-thalassemia/Hb E patients and four healthy donors. We identified that the differentially expressed proteins are enriched in secretory or exosome-associated proteins, many of which have putative functions in the oxidative stress response. Using Western blot assay, we confirmed that atypical lipoprotein, Apolipoprotein D (APOD), belonging to the Lipocalin transporter superfamily, was significantly decreased in BM plasma of the tested pediatric β-thalassemia/Hb E patients. Our results highlight that the disease condition of ineffective erythropoiesis and oxidative stress found in BM microenvironment of β-thalassemia/Hb E patients is associated with the impaired expression of APOD protein.

Since December 2019, the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused severe pneumonia, a disease named COVID-19, that became pandemic and created an acute threat to public health. The effective therapeutics are in urgent need. Here, we developed a high-content screening for the antiviral candidates using fluorescence-based SARS-CoV-2 nucleoprotein detection in Vero E6 cells coupled with plaque reduction assay. Among 122 Thai natural products, we found that Boesenbergia rotunda extract and its phytochemical compound, panduratin A, exhibited the potent anti-SARS-CoV-2 activity. Treatment with B. rotunda extract and panduratin A after viral infection drastically suppressed SARS-CoV-2 infectivity in Vero E6 cells with IC50 of 3.62 μg/mL (CC50 = 28.06 µg/mL) and 0.81 μΜ (CC50 = 14.71 µM), respectively. Also, the treatment of panduratin A at the pre-entry phase inhibited SARS-CoV-2 infection with IC50 of 5.30 µM (CC50 = 43.47 µM). Our study demonstrated, for the first time, that panduratin A exerts the inhibitory effect against SARS-CoV-2 infection at both pre-entry and post-infection phases. Apart from Vero E6 cells, treatment with this compound was able to suppress viral infectivity in human airway epithelial cells. This result confirmed the potential of panduratin A as the anti-SARS-CoV-2 agent in the major target cells in human. Since B. rotunda is a culinary herb generally grown in China and Southeast Asia, its extract and the purified panduratin A may serve as the promising candidates for therapeutic purposes with economic advantage during COVID-19 situation.

Induction of fetal hemoglobin (HbF) ameliorates the clinical severity of β-thalassemias. Histone methyltransferase LSD1 enzyme removes methyl groups from the activating chromatin mark histone 3 lysine 4 at silenced genes, including the γ-globin genes. LSD1 inhibitor RN-1 induces HbF levels in cultured human erythroid cells. Here, the HbF-inducing activity of RN-1 was investigated in erythroid progenitor cells derived from β0-thalassemia/ hemoglobin E (HbE) patients. The significant and reproducible increases in γ-globin transcript and HbF expression upon RN-1 treatment were demonstrated in erythroid cells with divergent HbF baseline levels, the average of HbF induction was 17.7±0.8%. RN-1 at low concentration did not affect viability and proliferation of erythroid cells, but decreases in cell number were observed in cells treated with RN-1 at high concentration. Delayed terminal erythroid differentiation was revealed in β0-thalassemia/HbE erythroid cells treated with RN-1 as similar to other compounds that target LSD1 activity. Downregulation of repressors of γ- globin expression; NCOR1 and SOX6, was observed in RN-1 treatment. These findings provide proof of the concept that LSD1 epigenetic enzyme is a potential therapeutic target for β0-thalassemia/HbE patients.

The interaction of SARS-CoV-2 infection with extracellular vesicles (EVs) is of particular interest at the moment. Studying SARS-CoV-2 contaminated-EV isolates in instruments located outside of the biosafety level-3 (BSL-3) environment requires knowing how viral inactivation methods affect the structure and function of extracellular vesicles (EVs). Therefore, three common viral inactivation methods, ultraviolet-C (UVC; 1350 mJ/cm2 ), β-propiolactone (BPL; 0.005%), heat (56°C, 45 min) were performed on defined EV particles and their proteins, RNAs, and function. Small EVs were isolated from the supernatant of SARS-CoV-2-infected human lung epithelial Calu-3 cells by stepwise centrifugation, ultrafiltration and qEV size-exclusion chromatography. The EV isolates contained SARS-CoV-2. UVC, BPL and heat completely abolished SARS-CoV-2 infectivity of the contaminated EVs. Particle detection by electron microscopy and nanoparticle tracking was less affected by UVC and BPL than heat treatment. Western blot analysis of EV markers was not affected by any of these three methods. UVC reduced SARS-CoV-2 spike detectability by quantitative RT-PCR and slightly altered EV-derived β-actin detection. Fibroblast migration-wound healing activity of the SARS-CoV-2 contaminated-EV isolate was only retained after UVC treatment. In conclusion, specific viral inactivation methods are compatible with specific measures in SARS-CoV-2 contaminated-EV isolates. UVC treatment seems preferable for studying functions of EVs released from SARS-CoV-2 infected cells.

Hemoglobin Constant Spring (HbCS) is unstable hemoglobin resulting from a nucleotide substitution at the termination codon of the HBA2 gene (c.427 T > C). The homozygous state for HbCS is non-transfusion dependent in adults. Nevertheless, severe anemia is often observed in fetuses. Here, human induced pluripotent stem cell line MUi034-A was generated from peripheral blood CD34+ hematopoietic stem/progenitor cells (HSPCs) derived from a 14-year-old female with homozygous HbCS who had a history of severe anemia and hydrops during fetal period. The MUi034-A cell line represented embryonic-like characteristics as they expressed specific pluripotency markers, differentiated into the three germ layers, and retained normal karyotyping.

The study aimed to generate a stepwise method to reduce the workload of full-scale RB1 sequencing for germline mutation screening in retinoblastoma (RB) patients. The implication of germline mutation in tumor focality was also determined in this study.

Human mesenchymal stem cells (hMSCs) have the potential to differentiate into hepatocyte-like cells, indicating that these cells may be the new target cell of interest to produce biopharmaceuticals. Our group recently established a hMSC-derived immortalized hepatocyte-like cell line (imHC) that demonstrates several liver-specific phenotypes. However, the ability of imHC to produce coagulation factors has not been characterized. Here, we examined the potential for imHC as a source of coagulation protein production by investigating the ability of imHC to produce human factor VII (FVII) using a lentiviral transduction system. Our results showed that imHC secreted a low amount of FVII (~22 ng/mL) into culture supernatant. Moreover, FVII from the transduced imHC (0.11 ± 0.005 IU/mL) demonstrated a similar coagulant activity compared with FVII from transduced HEK293T cells (0.12 ± 0.004 IU/mL) as determined by chromogenic assay. We demonstrate for the first time, to the best of our knowledge, that imHC produced FVII, albeit at a low level, indicating the unique characteristic of hepatocytes. Our study suggests the possibility of using imHC for the production of coagulation proteins.

Autophagy is a conserved lysosomal-dependent cellular degradation process and its dysregulation has been linked to numerous diseases including neurodegeneration, infectious diseases, and cancer. Modulation of autophagy is therefore considered as an attractive target for disease intervention. We carried out a high-content image analysis screen of natural product-derived compounds to discover novel autophagy modulating molecules. Our screen identified ECDD-S27 as the most effective compound for increasing the number of autophagic vacuoles inside cells. The structure of ECDD-S27 revealed that it is a derivative of cleistanthin A, a natural arylnaphthalene lignan glycoside found in plants. ECDD-S27 increases the number of autophagic vacuoles by inhibiting the autophagic flux and is able to restrict the survival of different cancer cells at low nanomolar concentrations. Molecular docking and SERS analysis showed that ECDD-S27 may potentially target the V-ATPase. Upon treatment of various cancer cells with ECDD-S27, the V-ATPase activity is potently inhibited thereby resulting in the loss of lysosomal acidification. Taken together, these data indicated that ECDD-S27 retards the autophagy pathway by targeting the V-ATPase and inhibits cancer cell survival. The observed antitumor activity without cytotoxicity to normal cells suggests the therapeutic potential warranting further studies on lead optimization of the compound for cancer treatment.

Cancer is currently ineffectively treated using therapeutic drugs, and is also able to resist drug action, resulting in increased side effects following drug treatment. A novel therapeutic strategy against cancer cells is the use of anticancer peptides (ACPs). The physicochemical properties, amino acid composition and the addition of chemical groups on the ACP sequence influences their conformation, net charge and orientation of the secondary structure, leading to an effect on targeting specificity and ACP‑cell interaction, as well as peptide penetrating capability, stability and efficacy. ACPs have been developed from both naturally occurring and modified peptides by substituting neutral or anionic amino acid residues with cationic amino acid residues, or by adding a chemical group. The modified peptides lead to an increase in the effectiveness of cancer therapy. Due to this effectiveness, ACPs have recently been improved to form drugs and vaccines, which have sequentially been evaluated in various phases of clinical trials. The development of the ACPs remains focused on generating newly modified ACPs for clinical application in order to decrease the incidence of new cancer cases and decrease the mortality rate. The present review could further facilitate the design of ACPs and increase efficacious ACP therapy in the near future.

Erythropoietin (Epo) is widely used for the treatment of anemia; however, non-hematopoietic effects and cancer risk limit its clinical applications. Therefore, alternative molecules to improve erythropoiesis in anemia patients are urgently needed. Here, we investigated the potential effects of a phytoestrogen diarylheptanoid (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol, (ASPP 049) isolated from Curcuma comosa on promoting erythropoiesis. Treatment with C. comosa extract improved anemia symptoms demonstrated by increasing red blood cell numbers, hematocrit, and hemoglobin content in anemic mice. In addition, ASPP 049, the major compound isolated from C. comosa, enhanced the suboptimal Epo dosages to improve erythroid cell differentiation from hematopoietic stem cells, which was inhibited by the estrogen receptor (ER) antagonist, ICI 182,780. Moreover, the ASPP 049-activated Epo-Epo receptor (EpoR) complex subsequently induced phosphorylation of EpoR-mediated erythropoiesis pathways: STAT5, MAPK/ERK, and PI3K/AKT in Epo-sensitive UT-7 cells. Taken together, these results suggest that C. comosa extract and ASPP 049 increased erythropoiesis through ER- and EpoR-mediated signaling cascades. Our findings provide insight into the specific interaction between a phytoestrogen diarylheptanoid and Epo-EpoR in a hematopoietic system for the potential treatment of anemia.

Tissue factor (TF) pathway inhibitor (TFPI) is a Kunitz-type anticoagulation protein that inhibits activated factor VII (FVIIa)/TF complex. Incidentally, many different F7 gene variants, including TFPI-binding exosite mutations, have been reported in patients with congenital FVII deficiency and clinical bleeding variabilities. Here, TFPI-binding exosites (R147 and K192) on FVII zymogen were selectively disrupted to understand their roles in the pathogenesis of bleeding phenotypes. Expression of recombinant FVII variants (R147A, K192A, and R147A/K192A) demonstrated markedly reduced secretion of FVII owing to intracellular retention in the endoplasmic reticulum, as demonstrated by upregulation of the unfolded protein response genes in all FVII variants. FVII variants showed a similar FVII activation pattern and FVIIa amidolytic activity than FVII wild-type (WT). In contrast to FVII activation, R147A and K192A showed a 90% reduction in FX activation relative to WT, whereas the R147A/K192A variant demonstrated a 99% decrease in FX activation. The clotting time was markedly prolonged with R147A and K192A than WT, and no FVII coagulant activity was detected in R147A/K192A. In addition, the thrombin generation assay revealed a significant prolongation of lag time in all FVII variants. Our study explains how mutations of TFPI-binding exosites of FVII can lead to bleeding phenotypes in individuals carrying these aberrancies.

Allergic contact dermatitis (ACD) is a type IV hypersensitivity mainly mediated by Th1/Th17 immune response. Topical corticosteroid is currently the first-line treatment for allergic contact dermatitis (ACD) and systemic administration of immunosuppressive drugs are used in patients with severe disseminated cases. However, increased risk of adverse effects has limited their use. Thus, the development of a novel immunosuppressant for ACD with low toxicity is a challenging issue. In this study, we began our study by using a murine contact hypersensitivity (CHS) model of ACD to examine the immunosuppressive effects of DYRK1B inhibition. We found that mice treated with a selective DYRK1B inhibitor show reduced ear inflammation. In addition, a significant reduction of Th1 and Th17 cells in the regional lymph node upon DYRK1B inhibition was observed by FACS analysis. Studies in vitro further revealed that DYRK1B inhibitor does not only suppressed Th1 and Th17 differentiation, but also promotes regulatory T cells (Treg) differentiation. Mechanistically, FOXO1 signaling was enhanced due to the suppression of FOXO1Ser329 phosphorylation in the presence of DYRK1B inhibitor. Therefore, these findings suggest that DYRK1B regulates CD4 T cell differentiation through FOXO1 phosphorylation and DYRK1B inhibitor has a potential as a novel agent for treatment of ACD.

Recombinant factor (F)VIIa (rFVIIa) has been approved by the US Food and Drug Administration for the treatment of hemophilia A and B with inhibitors and congenital FVII deficiency. Moreover, the investigational uses of rFVIIa are becoming of interest since it can be used to treat various clinical bleeding conditions. However, there is evidence showing that rFVIIa is a potent procoagulant agent that potentially leads to an increased risk of thrombotic complications.

The diarylheptanoid ASPP 049 has improved the quality of adult hematopoietic stem cell (HSC) expansion ex vivo through long-term reconstitution in animal models. However, its effect on hematopoietic regeneration from human induced pluripotent stem cells (hiPSCs) is unknown.

Mutations in MYH9 gene is one of the major causes of inherited thrombocytopenia resulted from nonfunctional myosin-9 protein. We have generated a human induced pluripotent stem cell line MUi010-A from skin fibroblasts of a patient who had a point mutation c.2104C>T (p.R702C) in the exon 16 of MYH9 gene using a non-integrative reprogramming method. The MUi010-A exhibited embryonic stem cell-like characteristics with consistent pluripotent markers expression, was capable of all three embryonic germ layers differentiation, and had a normal karyotype.

Hepatitis C virus (HCV) could induce chronic liver diseases and hepatocellular carcinoma in human. The use of primary human hepatocyte as a viral host is restrained with the scarcity of tissue supply. A culture model restricted to HCV genotype 2a (JFH-1) has been established using Huh7-derived hepatocyte. Other genotypes including the wild-type virus could not propagate in Huh7, Huh7.5 and Huh7.5.1 cells.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic stem cells. It is characterized at cytogenetic level by the Philadelphia (Ph) chromosome and at the molecular level by the BCR/ABL gene rearrangement. Bone marrow derived mesenchymal stem cells (MSCs) are also pluripotent stem cells that can differentiate into several mesenchymal tissues. To date, no study has been performed to characterize whether MSCs from CML harbor the abnormal Ph chromosome similar to CML bone marrow cells. We isolated and characterized MSCs from diagnostic marrow samples (n=11) and showed that MSCs can be readily isolated from CML marrow and exhibit major expansion potential as well as intact osteogenic differentiation ability. Moreover, they do not harbor the Ph chromosome confirmed by fluorescence in situ hybridization (FISH) and reverse transcriptase polymerase chain reaction (RT-PCR). Thus, we demonstrated that CML marrow is an abundant source of MSCs appearing through both FISH and RT-PCR not to be involved by the malignant process of CML. Furthermore, these MSCs from a CML patient could support in vitro cord blood expansion as those MSCs from a normal donor. Since MSCs are able to support engraftment of hematopoietic stem cells in stem cell transplantation (SCT) as well as suppress alloreactive T cells causing graft-versus-host disease, this current report thus provides evidence that in a SCT setting of CML patients, autologous MSCs could be a source of stem cell support in future cell therapy applications.