Basement membrane extract potentiates the endochondral ossification phenotype of bone marrow-derived mesenchymal stem cell-based cartilage organoids.

Endochondral ossification is a developmental process in the skeletal system and bone marrow of vertebrates. During endochondral ossification, primitive cartilaginous anlages derived from mesenchymal stem cells (MSCs) undergo vascular invasion and ossification. In vitro regeneration of endochondral ossification is beneficial for research on the skeletal system and bone marrow development as well as their clinical aspects. However, to achieve the regeneration of endochondral ossification, a stem cell-based artificial cartilage (cartilage organoid, Cart-Org) that possesses an endochondral ossification phenotype is required. Here, we modified a conventional 3D culture method to create stem cell-based Cart-Org by mixing it with a basement membrane extract (BME) and further characterized its chondrogenic and ossification properties. BME enlarged and matured the bone marrow MSC-based Cart-Orgs without any shape abnormalities. Histological analysis using Alcian blue staining showed that the production of cartilaginous extracellular matrices was enhanced in Cart-Org treated with BME. Transcriptome analysis using RNA sequencing revealed that BME altered the gene expression pattern of Cart-Org to a dominant chondrogenic state. BME triggered the activation of the SMAD pathway and inhibition of the NK-κB pathway, which resulted in the upregulation of SOX9, COL2A1, and ACAN in Cart-Org. BME also facilitated the upregulation of genes associated with hypertrophic chondrocytes (IHH, PTH1R, and COL10A1) and ossification (SP7, ALPL, and MMP13). Our findings indicate that BME promotes cartilaginous maturation and further ossification of bone marrow MSC-based Cart-Org, suggesting that Cart-Org treated with BME possesses the phenotype of endochondral ossification.

Decrease in electrolyte after vitrectomy surgery may affect the results of forensic investigations using vitreous humor.

PURPOSE: Vitreous humor (VH) is used for postmortem biochemical studies because it is well protected in an uncontaminated state even after death. The goal of this research was to investigate electrolyte concentrations in the VH from human eyes with and without a history of vitrectomy surgery. METHODS: We analyzed the sodium (Na), potassium (K), chloride (Cl) and magnesium (Mg) concentrations from 34 VH samples from 34 patients. Eleven samples were from eyes with a history of vitrectomy, and the remaining 23 eyes had no history of vitrectomy. The correlations of Na, K, Cl and Mg concentrations with patient age, interval between first and second vitrectomy, and lens status (history of cataract surgery) were also evaluated. RESULTS: The Na, K, Cl and Mg concentrations in VH from vitrectomized eyes were 134.1 ± 7.9 mmol/L, 3.7 ± 0.2 mmol/L, 99.7 ± 6.7 mmol/L and 0.59 ± 0.09 mmol/L, respectively; all were significantly lower than the corresponding concentrations in VH from control eyes (lower by 5.0%, 11.0%, 11.7%, and 22.6%, respectively). Na, K, Cl and Mg concentrations in VH from vitrectomized eyes did not show significant correlations with patient ages or the interval between their first and second vitrectomies. There were no significant differences in Na, K, Cl and Mg concentrations in VH between phakic eyes and intraocular lens-implanted eyes. CONCLUSIONS: With the increasing number of vitrectomies being performed, it is necessary to consider the history of vitrectomy when using a subject's VH in forensic examination.

Periosteum-derived podoplanin-expressing stromal cells regulate nascent vascularization during epiphyseal marrow development.

Bone marrow development and endochondral bone formation occur simultaneously. During endochondral ossification, periosteal vasculatures and stromal progenitors invade the primary avascular cartilaginous anlage, which induces primitive marrow development. We previously determined that bone marrow podoplanin (PDPN)-expressing stromal cells exist in the perivascular microenvironment and promote megakaryopoiesis and erythropoiesis. In this study, we aimed to examine the involvement of PDPN-expressing stromal cells in postnatal bone marrow generation. Using histological analysis, we observed that periosteum-derived PDPN-expressing stromal cells infiltrated the cartilaginous anlage of the postnatal epiphysis and populated on the primitive vasculature of secondary ossification center. Furthermore, immunophenotyping and cellular characteristic analyses indicated that the PDPN-expressing stromal cells constituted a subpopulation of the skeletal stem cell lineage. In vitro xenovascular model cocultured with human umbilical vein endothelial cells and PDPN-expressing skeletal stem cell progenies showed that PDPN-expressing stromal cells maintained vascular integrity via the release of angiogenic factors and vascular basement membrane-related extracellular matrices. We show that in this process, Notch signal activation committed the PDPN-expressing stromal cells into a dominant state with basement membrane-related extracellular matrices, especially type IV collagens. Our findings suggest that the PDPN-expressing stromal cells regulate the integrity of the primitive vasculatures in the epiphyseal nascent marrow. To the best of our knowledge, this is the first study to comprehensively examine how PDPN-expressing stromal cells contribute to marrow development and homeostasis.

The usefulness of tranexamic acid for bleeding symptoms of chronic consumptive coagulopathy complicated by aortic disease: a single-institute, retrospective study of 14 patients.

BACKGROUND: Tranexamic acid (TXA) is an antifibrinolytic drug that blocks lysine-binding sites on the profibrinolytic enzyme plasminogen. Aortic diseases with chronic consumption coagulopathy may lead to disseminated intravascular coagulation (DIC) and cause fatal bleeding. Although the use of antifibrinolytic agents in DIC is generally not recommended due to enhanced fibrin deposition risking thrombotic symptoms, the efficacy of TXA has been reported in several cases of DIC with aortic diseases. However, the efficacy and safety of TXA for bleeding symptoms of chronic consumption coagulopathy with aortic diseases have not been studied in detail. METHODS: We evaluated the efficacy of TXA in 14 patients with chronic consumptive coagulopathy due to aortic disease complicated by bleeding symptoms. Changes in coagulation and fibrinolysis parameters from baseline were analyzed with Wilcoxon matched-pairs signed-rank tests, excluding missing values. Kaplan-Meier curves were used to analyze overall survival. RESULTS: Median age was 78.5 years (range, 66-89 years) and median observation period was 448 days (range, 0-2282 days). Twelve patients had chronic renal failure and 1 patient had chronic liver failure. Before starting treatment, median Japanese Ministry of Health and Welfare DIC diagnostic criteria score was 8 (range, 4-11) and median platelet count was 64 × 109/L (range, 25-97 × 109/L). Twelve patients underwent evaluation of bleeding symptoms after introduction of TXA, and 10 of those 12 patients showed improved bleeding tendencies within 30 days (median, 5.0 days). One patient with chronic liver failure showed worsening of bleeding symptoms. Although only one patient was initiated TXA in combination with anticoagulants, no significant worsening of thrombotic events was observed within 30 days. CONCLUSIONS: TXA therapy appears effective against chronic consumptive coagulopathy with bleeding due to aortic disease, with few side effects.

Thrombosis from a prothrombin mutation conveying antithrombin resistance.

We identified a novel mechanism of hereditary thrombosis associated with antithrombin resistance, with a substitution of arginine for leucine at position 596 (p.Arg596Leu) in the gene encoding prothrombin (called prothrombin Yukuhashi). The mutant prothrombin had moderately lower activity than wild-type prothrombin in clotting assays, but the formation of thrombin-antithrombin complex was substantially impaired. A thrombin-generation assay revealed that the peak activity of the mutant prothrombin was fairly low, but its inactivation was extremely slow in reconstituted plasma. The Leu596 substitution caused a gain-of-function mutation in the prothrombin gene, resulting in resistance to antithrombin and susceptibility to thrombosis.

Ribavirin-induced intracellular GTP depletion activates transcription elongation in coagulation factor VII gene expression.

Coagulation FVII (Factor VII) is a vitamin K-dependent glycoprotein synthesized in hepatocytes. It was reported previously that FVII gene (F7) expression was up-regulated by ribavirin treatment in hepatitis C virus-infected haemophilia patients; however, its precise mechanism is still unknown. In the present study, we investigated the molecular mechanism of ribavirin-induced up-regulation of F7 expression in HepG2 (human hepatoma cell line). We found that intracellular GTP depletion by ribavirin as well as other IMPDH (inosine-5'-monophosphate dehydrogenase) inhibitors, such as mycophenolic acid and 6-mercaptopurine, up-regulated F7 expression. FVII mRNA transcription was mainly enhanced by accelerated transcription elongation, which was mediated by the P-TEFb (positive-transcription elongation factor b) complex, rather than by promoter activation. Ribavirin unregulated ELL (eleven-nineteen lysine-rich leukaemia) 3 mRNA expression before F7 up-regulation. We observed that ribavirin enhanced ELL3 recruitment to F7, whereas knockdown of ELL3 diminished ribavirin-induced FVII mRNA up-regulation. Ribavirin also enhanced recruitment of CDK9 (cyclin-dependent kinase 9) and AFF4 to F7. These data suggest that ribavirin-induced intracellular GTP depletion recruits a super elongation complex containing P-TEFb, AFF4 and ELL3, to F7, and modulates FVII mRNA transcription elongation. Collectively, we have elucidated a basal mechanism for ribavirin-induced FVII mRNA up-regulation by acceleration of transcription elongation, which may be crucial in understanding its pleiotropic functions in vivo.

Size-dependent ability of AtaA to immobilize cells in Acinetobacter sp. Tol 5.

Microbial cells serve as efficient and environmentally friendly biocatalysts, but their stability and reusability in practical applications must often be improved through immobilization. Acinetobacter sp. Tol 5 shows high adhesiveness to materials due to its large cell surface protein AtaA, which consists of 3630 amino acids (aa). Previously, we developed a method for immobilizing bacteria using AtaA. Herein, we investigated the cell immobilization ability of in-frame deletion (IFD) mutants of AtaA with different sizes in Tol 5. Mini-AtaA, which consists of 775 aa and is functional in Escherichia coli, was produced and present on the cell surface; however, mini-AtaA showed no immobilization ability in Tol 5. A cell immobilization assay was performed with cells expressing 16 IFD mutants of AtaA with different sizes, revealing that a length of at least 1417 aa was required for the sufficient immobilization of Tol 5 cells; thus, the minimum length needed to achieve the adhesive function of AtaA varies among bacterial species. The constructed mutant library of AtaA ranging from 3630 to 775 aa will allow researchers to quickly and easily explore the optimal size of AtaA, even for bacteria newly introduced to AtaA.

International council for standardisation in haematology recommendations on fibrinogen assays, thrombin clotting time and related tests in the investigation of bleeding disorders.

This guidance was prepared on behalf of the International Council for Standardisation in Haematology (ICSH) by an international working group of clinicians and scientists. The document focuses on tests and assays used for the assessment of fibrinogen function, particularly in the scenario of bleeding disorders. Thrombin clotting time (TT) is used as a screening test in some laboratories and also has some utility when direct anticoagulants are in use. The Clauss fibrinogen assay remains the method of choice for the assessment of fibrinogen function, but there are some situations where the results may be misleading. Prothrombin time derived fibrinogen assays are frequently used, but should be interpreted with caution; the results are not interchangeable between different methods and fibrinogen can be overestimated in certain clinical scenarios. Viscoelastic point of care methods may be helpful in emergency situations, while Reptilase time (and similar tests) are useful combined with TT in distinguishing heparin contamination of samples (i.e., if an incorrect blood draw is suspected) and the presence of direct thrombin inhibitors. Fibrinogen antigen assays should be used in the investigation of functional fibrinogen abnormalities; fibrinogen antigen and genetic testing are recommended in the confirmation of congenital fibrinogen disorders. The following recommendations for fibrinogen function assessment are based on published literature and expert opinion and should supplement local regulations and standards.

Variability in combinations of APTT reagent and substrate plasma for a one-stage clotting assay to measure factor VIII products.

INTRODUCTION: An investigation of the suitability of reagents for measuring FVIII products in a one-stage clotting assay (OSA) showed variations in their FVIII activity (FVIII:C). Most studies have focused on the activated partial thromboplastin time (APTT) reagent rather than FVIII-deficient plasma (F8DP), even though the APTT-based OSA is comprised of APTT reagents and factor-deficient plasma. AIM: A single-centre study was conducted to clarify variations in measurements of FVIII products in an OSA using a total of 12 reagent combinations, including four APTT reagents and three types of F8DP. METHODS: FVIII:C in nine types of FVIII product-spiked plasma was measured using an OSA with four different APTT reagents and three types of F8DP. RESULTS: F8DP-dependent variations were found in addition to differences derived from APTT reagents. Variations in target recovery (TR) were observed for NovoEight®, Eloctate®, and Jivi®. Reduced TR for Jivi was found only for Pathromtin SL in combination with congenital F8DP (F8DP-3). This lower TR was not observed with alternative manufacturing lots of F8DP-3. The reduced TR for Jivi might be related to impaired contact activation due to lower factor XI activity in F8DP-3. CONCLUSION: In addition to APTT reagents, variations in F8DPs used for OSAs can also affect FVIII:C results. F8DPs as well as the APTT reagent used for OSA should be chosen with caution, and laboratories should evaluate reagents for F8DPs as they currently do for APTT reagents, especially when lot changes occur.

Newly diagnosed multiple myeloma with bleeding and coagulation abnormalities caused by a thrombin-inhibiting substance.

Approximately 7% of patients with newly diagnosed multiple myeloma (MM) experience bleeding complications with varying causes, but few reports have described these complications. Here we report the case of a patient with newly diagnosed MM who presented with a bleeding tendency and various coagulation abnormalities. Chromogenic assays, thrombin time, and reptilase time revealed the presence of a thrombin-inhibiting substance that inhibited release of fibrinopeptide A from fibrinogen. The coagulation abnormalities improved after treatment with daratumumab, lenalidomide, and dexamethasone. As the thrombin inhibition mechanism remains unclear, no previous studies have reported recent treatment outcomes in older patients producing thrombin-inhibiting substances, which can hinder clinical treatment. Therefore, we believe that the diagnosis and the treatment course of this case provide valuable information. Moreover, such case reports provide significant insights into the pathophysiology of bleeding complications associated with MM.

Does Rhizobial Inoculation Change the Microbial Community in Field Soils? A| |Comparison with Agricultural Land-use Changes.

Although microbial inoculation may be effective for sustainable crop production, detrimental aspects have been argued because of the potential of inoculated microorganisms to behave as invaders and negatively affect the microbial ecosystem. We herein compared the impact of rhizobial inoculation on the soil bacterial community with that of agricultural land-use changes using a 16S rRNA amplicon ana-lysis. Soybean plants were cultivated with and without five types of bradyrhizobial inoculants (Bradyrhizobium diazoefficiens or Bradyrhizobium ottawaense) in experimental fields of Andosol, and the high nodule occupancy (35-72%) of bradyrhizobial inoculants was confirmed by nosZ PCR. However, bradyrhizobial inoculants did not significantly affect Shannon's diversity index (α-diversity) or shifts (ß-diversity) in the bacterial community in the soils. Moreover, the soil bacterial community was significantly affected by land-use types (conventional cropping, organic cropping, and original forest), where ß-diversity correlated with soil chemical properties (pH, carbon, and nitrogen contents). Therefore, the effects of bradyrhizobial inoculation on bacterial communities in bulk soil were minor, regardless of high nodule occupancy. We also observed a correlation between the relative abundance of bacterial classes (Alphaproteobacteria, Gammaproteobacteria, and Gemmatimonadetes) and land-use types or soil chemical properties. The impact of microbial inoculation on soil microbial ecosystems has been exami-ned to a limited extent, such as rhizosphere communities and viability. In the present study, we found that bacterial community shifts in soil were more strongly affected by land usage than by rhizobial inoculation. Therefore, the results obtained herein highlight the importance of assessing microbial inoculants in consideration of the entire land management system.

Identification of the adhesive domain of AtaA from Acinetobacter sp. Tol 5 and its application in immobilizing Escherichia coli.

Cell immobilization is an important technique for efficiently utilizing whole-cell biocatalysts. We previously invented a method for bacterial cell immobilization using AtaA, a trimeric autotransporter adhesin from the highly sticky bacterium Acinetobacter sp. Tol 5. However, except for Acinetobacter species, only one bacterium has been successfully immobilized using AtaA. This is probably because the heterologous expression of large AtaA (1 MDa), that is a homotrimer of polypeptide chains composed of 3,630 amino acids, is difficult. In this study, we identified the adhesive domain of AtaA and constructed a miniaturized AtaA (mini-AtaA) to improve the heterologous expression of ataA. In-frame deletion mutants were used to perform functional mapping, revealing that the N-terminal head domain is essential for the adhesive feature of AtaA. The mini-AtaA, which contains a homotrimer of polypeptide chains from 775 amino acids and lacks the unnecessary part for its adhesion, was properly expressed in E. coli, and a larger amount of molecules was displayed on the cell surface than that of full-length AtaA (FL-AtaA). The immobilization ratio of E. coli cells expressing mini-AtaA on a polyurethane foam support was significantly higher compared to the cells with or without FL-AtaA expression, respectively. The expression of mini-AtaA in E. coli had little effect on the cell growth and the activity of another enzyme reflecting the production level, and the immobilized E. coli cells could be used for repetitive enzymatic reactions as a whole-cell catalyst.

VWF-Gly2752Ser, a novel non-cysteine substitution variant in the CK domain, exhibits severe secretory impairment by hampering C-terminal dimer formation.

BACKGROUND: Von Willebrand factor (VWF) is a multimeric glycoprotein that plays important roles in hemostasis and thrombosis. C-terminal interchain-disulfide bonds in the cystine knot (CK) domain are essential for VWF dimerization. Previous studies have reported that missense variants of cysteine in the CK domain disrupt the intrachain-disulfide bond and cause type 3 von Willebrand disease (VWD). However, type 3 VWD-associated noncysteine substitution variants in the CK domain have not been reported. OBJECTIVE: To investigate the molecular mechanism of a novel non-cysteine variant in the CK domain, VWF c.8254 G>A (p.Gly2752Ser), which was identified in a patient with type 3 VWD as homozygous. METHODS: Genetic analysis was performed by whole exome sequencing and Sanger sequencing. VWF multimer analysis was performed using SDS-agarose electrophoresis. VWF production and subcellular localization were analyzed using ex vivo endothelial colony forming cells (ECFCs) and an in vitro recombinant VWF (rVWF) expression system. RESULTS: The patient was homozygous for VWF-Gly2752Ser. Plasma VWF enzyme-linked immunosorbent assay showed that the VWF antigen level of the patient was 1.2% compared with healthy subjects. A tiny amount of VWF was identified in the patient's ECFC. Multimer analysis revealed that the circulating VWF-Gly2752Ser presented only low molecular weight multimers. Subcellular localization analysis of VWF-Gly2752Ser-transfected cell lines showed that rVWF-Gly2752Ser was severely impaired in its ER-to-Golgi trafficking. CONCLUSION: VWF-Gly2752Ser causes severe secretory impairment because of its dimerization failure. This is the first report of a VWF variant with a noncysteine substitution in the CK domain that causes type 3 VWD.

F9 mRNA splicing aberration due to a deep Intronic structural variation in a patient with moderate hemophilia B.

INTRODUCTION: Hemophilia B (HB) is a hereditary bleeding disorder caused by the genetic variation of the coagulation factor IX (FIX) gene (F9). Several F9 structural abnormalities, including large deletion and/or insertion, have been observed to cause HB development. However, there is limited information available on F9 deep intronic variations. In this study, we report about a novel large deletion/insertion observed in a deep region of F9 intron 1 that causes mRNA splicing abnormalities. PATIENT AND METHODS: The patient was a Japanese male diagnosed with moderate HB (FIX:C = 3.0 IU/dL). The genomic DNA of the patient was isolated from peripheral blood leukocytes. DNA sequences of F9 exons and splice donor/acceptor sites were analyzed via polymerase chain reaction and Sanger sequencing. Variant-affected F9 mRNA aberration and FIX protein production, secretion, and coagulant activity were analyzed by cell-based exon trap and splicing-competent FIX expression vector systems. RESULTS: A 28-bp deletion/476-bp insertion was identified in the F9 intron 1 of a patient with moderate HB. A DNA sequence identical to a part of the inverted HNRNPA1 exon 12 was inserted. Cell-based transcript analysis revealed that this large intronic deletion/insertion disrupted F9 mRNA splicing pattern, resulting in reduction of protein-coding F9 mRNA. CONCLUSION: A novel deep intronic F9 rearrangement was identified in a Japanese patient with moderate HB. Abnormal F9 mRNA splicing pattern due to this deep intronic structural variation resulted in a reduction of protein-coding F9 mRNA, which probably caused the moderate HB phenotype.

Protein S-Leu17Pro disrupts the hydrophobicity of its signal peptide causing a proteasome-dependent degradation.

INTRODUCTION: Protein S is a vitamin K-dependent glycoprotein with important anticoagulant, fibrinolytic, anti-inflammatory, anti-apoptotic, and cytoprotective functions. Congenital protein S deficiency is an autosomal dominant thrombophilia due to protein S gene (PROS1) variations. Our group identified a variation in PROS1 that translates into protein S deficiency: c.50 T > C (p.Leu17Pro). Here, we investigated the mechanisms by which this variation results in protein S deficiency. MATERIALS AND METHODS: The effect of L17P substitution on protein S signal peptide was predicted by in silico (a computational prediction technique) analysis of hydrophobicity and signal peptide cleavage. Recombinant protein S was overexpressed in HEK293 and COS-7 cells. Intracellular kinetics and extracellular secretion of recombinant protein S-L17P were analyzed by western blotting and immunocytochemistry. RESULTS: In silico hydrophobicity analysis showed that protein S-L17P had disrupted hydrophobic status in the h-region of its signal peptide. Under normal culture conditions, recombinant protein S -L17P was not detected in either transfectant cell lysates or medium. Upon treatment with a proteasome inhibitor, recombinant protein S-L17P was clearly detected in the cell lysate, but not in the culture medium. Recombinant protein S-L17P did not undergo post-translational modification with N-glycosylation, suggesting that the nascent polypeptide of recombinant protein S-L17P is not transported to the endoplasmic reticulum lumen, but is mislocalized to the cytosol. CONCLUSION: PROS1-L17P variation translates into protein S deficiency. Protein S-L17P causes its cytosolic mislocalization resulting in its proteasome-dependent degradation.

Development and validation of a novel qualitative test for plasma fibrinogen utilizing clot waveform analysis.

Plasma fibrinogen is commonly examined by Clauss fibrinogen assay, which cannot distinguish between quantitative and qualitative fibrinogen anomalies. However, our previously reported Clauss fibrinogen assay utilizing clot waveform analysis (Clauss-CWA) provides additional information that contributes to the classification of fibrinogen anomalies. In this study, we adopted the Clauss-CWA method for an autoanalyzer to automatically measure the antigenic estimate (eAg) of fibrinogen in addition to the functional amount (Ac), and to thus provide the Ac/eAg ratio as a qualitative indicator. Performance was validated by receiver operating characteristics (ROC) and precision recall (PR) curve analyses using a patient cohort, consisting of a training cohort (n = 519) and a validation cohort (n = 523), both of which contained cases of congenital (hypo)dysfibrinogenemia as qualitative defects. We obtained an optimal cutoff of 0.65 for Ac/eAg by ROC curve analysis of the training cohort, offering superior sensitivity (> 0.9661) and specificity (1.000). This cutoff was validated in the validation cohort, providing positive predictive value > 0.933 and negative predictive value > 0.998. PR curve analysis also showed that Clauss-CWA provided excellent performance for detecting qualitative fibrinogen anomalies. The Clauss-CWA method may represent a useful approach for detecting qualitative fibrinogen abnormalities in routine laboratory testing.

Crystal structure of yeast rpn14, a chaperone of the 19 S regulatory particle of the proteasome.

The ubiquitin-proteasome pathway is a major proteolytic system in eukaryotic cells and regulates various cellular processes. The 26 S proteasome, the central enzyme of this pathway, consists of a proteolytic core particle and two 19 S regulatory particles (RPs) composed of ATPase (Rpt) and non-ATPase (Rpn) subunits. Growing evidence indicates that proteasome assembly is assisted by a variety of chaperones. In particular, it has been reported recently that Nas2, Nas6, Rpn14, and Hsm3 bind specific Rpt subunits, thereby contributing to the formation of 19 S RP. Rpn14 transiently binds to the C-terminal domain of the Rpt6 subunit (Rpt6-C) during maturation of the ATPase ring of 19 S RP. In this study, we determined the crystal structure of yeast Rpn14 at 2.0 A resolution, which revealed that this chaperone consists of a unique N-terminal domain with unknown function and a C-terminal domain assuming a canonical seven-bladed beta-propeller fold. The Rpt6-binding site on Rpn14 was predicted based on structural comparison with the complex formed between Nas6 and Rpt3-C. The top face of Rpn14 exhibits a highly acidic surface area, whereas the putative interacting surface of Rpt6-C is basic. By inspection of structural data along with genetic and biochemical data, we determined the specific residues of Rpn14 and Rpt6 for complementary charge interactions that are required for 19 S RP assembly.

Down-regulation of PROS1 gene expression by 17beta-estradiol via estrogen receptor alpha (ERalpha)-Sp1 interaction recruiting receptor-interacting protein 140 and the corepressor-HDAC3 complex.

Pregnant women show a low level of protein S (PS) in plasma, which is known to be a risk for deep venous thrombosis. 17Beta-estradiol (E(2)), an estrogen that increases in concentration in the late stages of pregnancy, regulates the expression of various genes via the estrogen receptor (ER). Here, we investigated the molecular mechanisms behind the reduction in PS levels caused by E(2) in HepG2-ERalpha cells, which stably express ERalpha, and also the genomic ER signaling pathway, which modulates the ligand-dependent repression of the PSalpha gene (PROS1). We observed that E(2) repressed the production of mRNA and antigen of PS. A luciferase reporter assay revealed that E(2) down-regulated PROS1 promoter activity and that this E(2)-dependent repression disappeared upon the deletion or mutation of two adjacent GC-rich motifs in the promoter. An electrophoretic mobility shift assay and DNA pulldown assay revealed that the GC-rich motifs were associated with Sp1, Sp3, and ERalpha. In a chromatin immunoprecipitation assay, we found ERalpha-Sp protein-promoter interaction involved in the E(2)-dependent repression of PROS1 transcription. Furthermore, we demonstrated that E(2) treatment recruited RIP140 and the NCoR-SMRT-HDAC3 complex to the PROS1 promoter, which hypoacetylated chromatin. Taken together, this suggested that E(2) might repress PROS1 transcription depending upon ERalpha-Sp1 recruiting transcriptional repressors in HepG2-ERalpha cells and, consequently, that high levels of E(2) leading to reduced levels of plasma PS would be a risk for deep venous thrombosis in pregnant women.

Myh9 R702C is associated with erythroid abnormality with splenomegaly in mice.

MYH9 disorders are characterized by giant platelets, thrombocytopenia, and Döhle body-like cytoplasmic inclusion bodies in granulocytes. However, whether these disorders cause any changes in erythroid cells has yet to be determined. This study analyzed the influence of Myh9 R702C, as one of the most commonly detected MYH9 disorders, on erythroid cells in a mouse model. Knock-in mice expressing Myh9 R702C mutation either systemically or specific to hematological cells (R702C and R702C vav1 mice, respectively) were used in this study. Both displayed lower hemoglobin and higher erythropoietin levels than wild-type (WT) mice, along with significant splenomegaly. Flow cytometric analysis revealed erythroblasts present at a higher rate than WT mice in the spleen. However, no obvious abnormalities were seen in erythroid differentiation from megakaryocyte/erythroid progenitor to erythrocyte. Cell culture assay by fetal liver and colony assay also showed normal progression of erythroid differentiation from erythroid burst-forming unit to red blood cell. In conclusion, R702C and R702C vav1 mice displayed erythroid abnormality with splenomegaly. However, erythroid differentiation showed no obvious abnormality. Further research is required to elucidate the underlying mechanisms.

Endostatin and Vascular Endothelial Growth Factor-A165b May Contribute to Classification of Pulmonary Hypertension.

Background: Pulmonary hypertension (PH) is characterized by dysregulation of small pulmonary arteries. In addition to endostatin (ES), placenta growth factor (PlGF), vascular endothelial growth factor-A (VEGF-A), and the anti-angiogenesis isoform of VEGF-A (VEGF-A165b) are associated with PH. However, the usefulness of these biomarkers in PH in unknown. We investigated whether these 4 biomarkers are related to PH classification. Methods and Results: Between July 2015 and August 2017, 33 control patients and 107 PH patients were enrolled in the study. Among the PH patients, 48 had pulmonary arterial hypertension (PAH), 5 had left heart disease-associated PH (LHD-PH), 4 had lung disease-associated PH (LD-PH), and 50 had chronic thromboembolic PH (CTEPH). Among the PAH patients, 16 had idiopathic PAH (IPAH) and 17 had connective tissue disease-associated PAH (CTD-PAH). PlGF, total VEGF-A, and VEGF-A165b levels were measured in the control and PH groups. ES was only measured in the PH group. VEGF-A165b levels were significantly higher in the LD-PH group than in the PAH, LHD-PH, and CTEPH groups (all P<0.001). PlGF levels were significantly higher in the CTD-PAH group than in the IPAH and control groups. ES levels were significantly correlated with the 6-min walk distance (P<0.001), B-type natriuretic peptide (P<0.001), and pulmonary vascular resistance (P=0.008). Conclusions: ES could detect CTD-PAH in PAH and may be an indicator of PH severity. VEGF-A165b was useful in detecting LD-PH.