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Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) may manifest as thrombosis, stroke, renal failure, myocardial infarction, and thrombocytopenia, reminiscent of other complement- mediated diseases. Multiple clinical and preclinical studies have implicated complement in the pathogenesis of COVID-19 illness. We previously found that the SARS-CoV-2 spike protein activates the alternative pathway of complement (APC) in vitro through interfering with the function of complement factor H, a key negative regulator of APC. Here, we demonstrated that serum from 58 COVID-19 patients (32 patients with minimal oxygen requirement, 7 on high flow oxygen, 17 requiring mechanical ventilation and 2 deaths) can induce complementmediated cell death in a functional assay (the modified Ham test) and increase membrane attack complex (C5b-9) deposition on the cell surface. A positive modified Ham assay (>20% cell-killing) was present in 41.2% COVID-19 patients requiring intubation (n=7/17) and only 6.3% in COVID-19 patients requiring minimal oxygen support (n=2/32). C5 and factor D inhibition effectively mitigated the complement amplification induced by COVID-19 patient serum. Increased serum factor Bb level was associated with disease severity in COVID-19 patients, suggesting that APC dysregulation plays an important role. Moreover, SARS-CoV-2 spike proteins directly block complement factor H from binding to heparin, which may lead to complement dysregulation on the cell surface. Taken together, our data suggest that complement dysregulation contributes to the pathogenesis of COVID-19 and may be a marker of disease severity.

There is a Blood Commentary on this article in this issue., Key Points SARS-CoV-2 spike proteins bind heparan sulfate and activate the alternative complement pathway on cell surfaces. Factor D inhibitor (ACH145951) blocks the complement activation induced by SARS-CoV-2 spike proteins., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly contagious respiratory virus that can lead to venous/arterial thrombosis, stroke, renal failure, myocardial infarction, thrombocytopenia, and other end-organ damage. Animal models demonstrating end-organ protection in C3-deficient mice and evidence of complement activation in humans have led to the hypothesis that SARS-CoV-2 triggers complement-mediated endothelial damage, but the mechanism is unclear. Here, we demonstrate that the SARS-CoV-2 spike protein (subunit 1 and 2), but not the N protein, directly activates the alternative pathway of complement (APC). Complement-dependent killing using the modified Ham test is blocked by either C5 or factor D inhibition. C3 fragments and C5b-9 are deposited on TF1PIGAnull target cells, and complement factor Bb is increased in the supernatant from spike protein–treated cells. C5 inhibition prevents the accumulation of C5b-9 on cells, but not C3c; however, factor D inhibition prevents both C3c and C5b-9 accumulation. Addition of factor H mitigates the complement attack. In conclusion, SARS-CoV-2 spike proteins convert nonactivator surfaces to activator surfaces by preventing the inactivation of the cell-surface APC convertase. APC activation may explain many of the clinical manifestations (microangiopathy, thrombocytopenia, renal injury, and thrombophilia) of COVID-19 that are also observed in other complement-driven diseases such as atypical hemolytic uremic syndrome and catastrophic antiphospholipid antibody syndrome. C5 inhibition prevents accumulation of C5b-9 in vitro but does not prevent upstream complement activation in response to SARS-CoV-2 spike proteins., Visual Abstract

The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity in the presence of antiphospholipid antibodies, including anti-β2-glycoprotein-I (anti-β2GPI), that are considered central to APS pathogenesis. Based on animal studies showing a role of complement in APS-related clinical events, we used the modified Ham (mHam) assay (complement-dependent cell killing) and cell-surface deposition of C5b-9 to test the hypothesis that complement activation is associated with thrombotic events in APS. A positive mHam (and corresponding C5b-9 deposition) were present in 85.7% of catastrophic APS (CAPS), 35.6% of APS (and 68.5% of samples collected within 1 year of thrombosis), and only 6.8% of systemic lupus erythematosus (SLE) sera. A positive mHam assay was associated with triple positivity (for lupus anticoagulant, anticardiolipin, and anti-β2GPI antibodies) and recurrent thrombosis. Patient-derived anti-β2GPI antibodies also induced C5b-9 deposition, which was blocked completely by an anti-C5 monoclonal antibody, but not by a factor D inhibitor, indicating that complement activation by anti-β2GPI antibodies occurs primarily through the classical complement pathway. Finally, patients with CAPS have high rates of rare germline variants in complement regulatory genes (60%), compared with patients with APS (21.8%) or SLE (28.6%) or normal controls (23.3%), and have mutations at a rate similar to that of patients with atypical hemolytic uremic syndrome (51.5%). Taken together, our data suggest that anti-β2GPI antibodies activate complement and contribute to thrombosis in APS, whereas patients with CAPS have underlying mutations in complement regulatory genes that serve as a “second hit,” leading to uncontrolled complement activation and a more severe thrombotic phenotype.

Hyperuricemia (HU) is a cause of gout. Clinical studies show a link between HU and cardiovascular disease. However, the role of soluble serum urate (SU) on atherosclerosis development remains elusive. We aimed to use a new HU mouse model [Uricase/Uox knockout (KO)] to further investigate the relationship between HU and atherosclerosis. A mouse model by perivascular collar placement of induced carotid atherosclerosis was established in male Uox‐ KO mice. The Uox‐ KO mice had elevated SU levels and enhanced levels of atherosclerosis inflammatory response proteins. In contrast, Uox‐ KO mice with carotid atherosclerosis showed severe neointimal changes in histology staining consistent with increases in intimal area and increases in proliferating cell nuclear antigen (PCNA)‐ and F4/80‐positive cells. Allopurinol reduced neointimal areas induced by the perivascular collar in hyperuricemic mice, accompanied by decreased expression of PCNA‐ and F4/80‐positive cells. Urate‐lowering treatment alleviated atherosclerosis inflammatory response factors and reactive oxygen species (ROS) intensities in both collar placement Uox‐ KO mice and urate‐stimulated human umbilical vein endothelial cells (HUVECs). In vitro results using HUVECs showed ROS was induced by urate and ROS induction was abrogated using antioxidants. These data demonstrate that urate per se does not trigger atherosclerosis intima lesions in male mice. Urate worsens carotid neointimal lesions induced by the perivascular collar and urate‐lowering therapy partially abrogates the effects. The current study warrants clinical studies on the possible benefits of urate‐lowering therapy in atherosclerosis patients with HU., Clinical studies show a link between hyperuricemia (HU) and cardiovascular disease. However, the role of soluble urate on atherosclerosis development remains unclear. A mouse model of induced carotid atherosclerosis was established in a spontaneous HU mouse with the uricase gene inactivated. This study demonstrates that urate worsens carotid neointimal lesions induced by the perivascular collar and that urate‐lowering therapy partially abrogates the effects.

In this study, a bioflocculation method assisted by fungal pellets was developed for highly efficient microalgae harvesting. Effects of critical parameters, including flocculation type, temperature, rotation speed and initial pH, on the bioflocculation of fungal Aspergillus niger for microalgae Scenedesmus sp. were investigated. Results showed that the maximum flocculation efficiency of 99.4% was obtained when the fungal pellets were inoculated in the algal solution at the initial pH of 8.0, temperature of 30 °C and rotation speed of 160 rpm for 48 h in BG-11 medium. Furthermore, microscopy examination, scanning electron microscopy, Fourier transform infrared spectroscopy, Zeta potential measurement and three-dimensional excitation emission matrix fluorescence spectroscopy were used to explore the mechanism of bioflocculation process. It was found that the interaction of fungi and microalgae was related to the surface functional groups of fungal pellets. This study provides a interpretation of conceivable mechanism for microalgal bioflocculation by fungal pellets.

The increasingly growing epidemics of multidrug-resistant bacteria are becoming severe public health threat. There is in an urgent need to develop new antibacterial agents with broad-spectrum antibacterial activity and high selectivity. Here, a series of N-terminal dipeptide mimetics with an aromatic amide moiety were synthesized from amino acids. The effects of amino acid type and aromatic moiety on the biological activities of the mimetics were evaluated. The dipeptide mimetics not only showed significant broad-spectrum antibacterial activity against Gram-negative (Escherichia coli and Klebsiella pneumoniae), Gram-positive (Staphylococcus aureus) and drug-resistant bacterium MRSA (methicillin-resistant S. aureus) but also demonstrated high selectivity for S. aureus versus mammalian erythrocytes. The coupling product of L-valine with p-alkynylaniline (dipeptide mimetic 7) exhibited the best antibacterial activities with minimum inhibitory concentration (MIC) ranging from 2.5 to 5 μg/mL. Moreover, the bactericidal kinetics and multi-passage resistance tests indicated that the mimetic 7 both rapidly killed bacteria and had a low probability of emergence of antimalarial resistance. Meanwhile, the mimetic 7 possessed the ability to both inhibit bacterial biofilm formation and eradicate mature biofilm. The depolarization and destruction of the bacterial cell membrane is the main sterilization mechanism, which hinders the propensity to develop bacterial resistance. Furthermore, the mimetic 7 also showed good antineoplastic activity against gastric cancer cell (SGC 7901, IC50 = 70.8 μg/mL), while it had very low toxicity to mammalian cell (L929). The mimetics bear considerable potential to be used as antibacterial and anticancer agents to combat antibiotic resistance.

Publisher's Note: There is an Inside Blood Commentary on this article in this issue.

Gout is a chronic disease resulting from elevated serum urate (SU). Previous genome-wide association studies (GWAS) have identified dozens of susceptibility loci for SU/gout, but few have been conducted for Chinese descent. Here, we try to extensively investigate whether these loci contribute to gout risk in Han Chinese. A total of 2255 variants in linkage disequilibrium (LD) with GWAS identified SU/gout associated variants were analyzed in a Han Chinese cohort of 1255 gout patients and 1848 controls. Cumulative genetic risk score analysis was performed to assess the cumulative effect of multiple “risk” variants on gout incidence. 23 variants (41%) of LD pruned variants set (n = 56) showed nominal association with gout in our sample (p ALDH16A1), including ABCG2, SLC2A9, GCKR, ALDH2 and CNIH2, were replicated. Cumulative genetic risk score analyses showed that the risk of gout increased for individuals with the growing number (≥8) of the risk alleles on gout associated loci. Most of the gout associated loci identified in previous GWAS were confirmed in an independent Chinese cohort, and the SU associated loci also confer susceptibility to gout. These findings provide important information of the genetic association of gout.

Background Simultaneous wastewater treatment and lipid production by oleaginous microalgae show great potential to alleviate energy shortage and environmental pollution, because they exhibit tremendous advantages over traditional activated sludge. Currently, most research on wastewater treatment by microalgal are carried out at optimized temperature conditions (25–35 °C), but no information about simultaneous wastewater treatment and lipid production by microalgae at low temperatures has been reported. Microalgal growth and metabolism will be inhibited at low temperature conditions, and satisfactory wastewater treatment performance will be not obtained. Therefore, it is critical to domesticate and screen superior microalgal strains with low temperature adaptability, which is of great importance for wastewater treatment and biodiesel production. Results In this work, simultaneous wastewater treatment and lipid production were achieved by a microalgal mutant Scenedesmus sp. Z-4 at the low temperature conditions (4, 10, and 15 °C). The results showed that algal growth was inhibited at 4, 10, and 15 °C compared to that at the optimal temperature of 25 °C. However, decreased temperature had no significant effect on the total cellular lipid content of algae. Importantly, lipid productivity at 10 °C was compromised by more net energy output relevant to biodiesel production, which demonstrated that the low temperature of 10 °C was favorable to wastewater treatment and energy recovery by Scenedesmus sp. Z-4. When molasses wastewater with optimal COD concentration of 8000 mg L−1, initial inoculation ratio of 15%, and C/N ratio of 15 was used to cultivate microalgae, the maximum removal rate of COD, TN, and TP at 10 °C reached 87.2, 90.5, and 88.6%, respectively. In addition, lipid content of 28.9% and lipid productivity of 94.4 mg L−1 day−1 were obtained. Conclusions Scenedesmus sp. Z-4 had good adaptability to low temperature conditions, and showed great potential to realize simultaneous wastewater treatment and lipid production at low temperatures. The proposed approach in the study was simple compared to other wastewater treatment methods, and this potential novel process was still efficient to remove COD, N, and P at low temperatures. Thus, it had a vital significance for the wastewater treatment in low temperature regions.

Paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome are diseases of excess activation of the alternative pathway of complement that are treated with eculizumab, a humanized monoclonal antibody against the terminal complement component C5. Eculizumab must be administered intravenously, and moreover some patients with paroxysmal nocturnal hemoglobinuria on eculizumab have symptomatic extravascular hemolysis, indicating an unmet need for additional therapeutic approaches. We report the activity of two novel small-molecule inhibitors of the alternative pathway component Factor D using in vitro correlates of both paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Both compounds bind human Factor D with high affinity and effectively inhibit its proteolytic activity against purified Factor B in complex with C3b. When tested using the traditional Ham test with cells from paroxysmal nocturnal hemoglobinuria patients, the Factor D inhibitors significantly reduced complement-mediated hemolysis at concentrations as low as 0.01 μM. Additionally the compound ACH-4471 significantly decreased C3 fragment deposition on paroxysmal nocturnal hemoglobinuria erythrocytes, indicating a reduced potential relative to eculizumab for extravascular hemolysis. Using the recently described modified Ham test with serum from patients with atypical hemolytic uremic syndrome, the compounds reduced the alternative pathway-mediated killing of PIGA-null reagent cells, thus establishing their potential utility for this disease of alternative pathway of complement dysregulation and validating the modified Ham test as a system for pre-clinical drug development for atypical hemolytic uremic syndrome. Finally, ACH-4471 blocked alternative pathway activity when administered orally to cynomolgus monkeys. In conclusion, the small-molecule Factor D inhibitors show potential as oral therapeutics for human diseases driven by the alternative pathway of complement, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome.

Adult-onset Still's disease (AOSD) is a rare autoinflammatory disease with systemic involvement, and its pathophysiology remains unclear. Genome-wide association studies (GWAS) in the Chinese population have revealed an association between AOSD and the major histocompatibility complex (MHC) locus; however, causal variants in the MHC remain undetermined. In the present study, we identified independent amino-acid polymorphisms in human leukocyte antigen (HLA) molecules that are associated with Han Chinese patients with AOSD by fine-mapping the MHC locus. Through conditional analyses, we identified position 34 in HLA-DQα1 (p = 1.44 × 10−14) and Asn in HLA-DRβ1 position 37 (p = 5.12 × 10−11) as the major determinants for AOSD. Moreover, we identified the associations for three main HLA class II alleles: HLA-DQB1*06:02 (OR = 2.70, p = 3.02 × 10−14), HLA-DRB1*15:01 (OR = 2.44, p = 3.66 × 10−13), and HLA-DQA1*01:02 (OR = 1.97, p = 1.09 × 10−9). This study reveals the relationship between functional variations in the class II HLA region and AOSD, implicating the MHC locus in the pathogenesis of AOSD.

In complement-driven thrombotic microangiopathies, failure to regulate complement activation leads to end-organ damage. The modified Ham (mHam) test measures complement-mediated killing of a nucleated cell in vitro but lacks a confirmatory assay and reliable positive controls. We demonstrate that C5b-9 accumulation on the surface of TF1 PIGAnull cells correlates with cell killing in the mHam. We also show that Sialidase treatment of cells or addition of Shiga toxin 1 to human serum serve as a more reliable positive control for the mHam than cobra venom factor or lipopolysaccharide. Simultaneously performing the mHam and measuring C5b-9 accumulation either in GVB(++) or GVB(0) MgEGTA buffer with the addition of complement pathway specific inhibitors (anti-C5 antibody or a factor D inhibitor, ACH-145951) can be used to localize defects in complement regulation. As more targeted complement inhibitors become available, these assays may aid in the selection of personalized treatments for patients with complement-mediated diseases.

Primordial germ cells (PGCs) share many properties with embryonic stem cells (ESCs) and innately express several key pluripotency-controlling factors, including OCT4, NANOG, and LIN28. Therefore, PGCs may provide a simple and efficient model for studying somatic cell reprogramming to induced pluripotent stem cells (iPSCs), especially in determining the regulatory mechanisms that fundamentally define pluripotency. Here, we report a novel model of PGC reprogramming to generate iPSCs via transfection with SOX2 and OCT4 using integrative lentiviral. We also show the feasibility of using nonintegrative approaches for generating iPSC from PGCs using only these two factors. We show that human PGCs express endogenous levels of KLF4 and C-MYC protein at levels similar to embryonic germ cells (EGCs) but lower levels of SOX2 and OCT4. Transfection with both SOX2 and OCT4 together was required to induce PGCs to a pluripotent state at an efficiency of 1.71%, and the further addition of C-MYC increased the efficiency to 2.33%. Immunohistochemical analyses of the SO-derived PGC-iPSCs revealed that these cells were more similar to ESCs than EGCs regarding both colony morphology and molecular characterization. Although leukemia inhibitory factor (LIF) was not required for the generation of PGC-iPSCs like EGCs, the presence of LIF combined with ectopic exposure to C-MYC yielded higher efficiencies. Additionally, the SO-derived PGC-iPSCs exhibited differentiation into representative cell types from all three germ layers in vitro and successfully formed teratomas in vivo. Several lines were generated that were karyotypically stable for up to 24 subcultures. Their derivation efficiency and survival in culture significantly supersedes that of EGCs, demonstrating their utility as a powerful model for studying factors regulating pluripotency in future studies.

Introduction: The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity along with persistent antiphospholipid antibodies (aPL). Despite adequate anticoagulation, 10-30% of patients have recurrent thrombosis. Catastrophic APS (CAPS) is associated with approximately 40% mortality despite treatment. The pathogenesis of APS complications is incompletely understood. Recent animal studies indicate that complement is required for aPL-associated thrombosis, and complement has emerged as an attractive therapeutic target for refractory thrombotic APS and CAPS. Methods: We first evaluated complement activation in sera of patients with thrombotic APS by ISTH criteria (N=53), catastrophic APS (CAPS; N=8, sera available for 6), and systemic lupus erythematosus (SLE; N=74) who presented to our institution from June 2015 to June 2019 (and four patients with CAPS from other institutions). We used the modified Ham (mHam) test, a functional assay for complement activation as described previously (Gavriilaki et al. Blood 2015). The mHam assay is based on the principle that a PNH cell line (PIGA-null TF-1 cells) lacking the cell surface complement regulators CD55 and CD59 undergoes lysis in serum containing activated complement. Cell death (measured by a cell viability assay) is a measure of complement activation. Cell surface deposition of complement products (C3c, C5b-9) is also detected by flow cytometry. We then evaluated whether adding purified patient-derived aPL (anti-β2 glycoprotein IgG) to normal serum induced complement activation. Finally, we performed targeted sequencing of 15 complement genes in the study subjects, as well as 22 patients with aHUS and 36 healthy individuals as positive and negative controls, respectively. Results: (A) Complement activation is associated with thrombotic APS. A positive mHam assay (>20% cell killing) was detected in 32.1% (17 of 53) patients with thrombotic APS and 100% (6 of 6 with available sera) of CAPS compared with 6.8% (5 of 74) with SLE, (P (B) aPL from patients activate complement in vitro. Patient-derived anti- β2 glycoprotein IgG from all four patients induced complement activation in the mHam assay (Fig 2A). Flow cytometry confirmed cell surface deposition of complement activation products (C4d, C5b-9), which was inhibited by adding anti-C5 monoclonal Ab or a factor D inhibitor (representative sample in fig. 1B). (C) Catastrophic APS is associated with complement mutations. Rare (minor allele frequency Conclusions: APS serum activates complement in vitro shown by a functional assay (mHam) and increased C5b-9 deposition on the cell surface. A positive mHam test strongly associates with both recent thrombosis and triple positive APS. Purified human anti-B2GPI antibody from APS patients activates complement when added to normal human serum, suggesting that complement activation plays a pathophysiologic role in APS associated thrombosis. Finally, CAPS patients have a high rate of mutations in complement genes, which likely serves as a 'second-hit' (in addition to aPL) leading to uncontrolled complement activation and a more severe phenotype (Figure 3). Taken together, our results provide a rationale for complement inhibition as a therapeutic strategy in patients with CAPS and refractory thrombotic APS. Disclosures Chaturvedi: Shire/Takeda: Research Funding; Sanofi: Consultancy; Alexion: Consultancy. Streiff:Pfizer: Consultancy, Honoraria; Bayer: Consultancy, Honoraria; Portola: Consultancy, Honoraria; Roche: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria. Petri:Astellas: Consultancy; Novartis: Consultancy; Exagen: Consultancy, Research Funding; Glenmark Pharmaceuticals: Consultancy; EMD Serono: Consultancy; Bristol-Myers Squibb: Consultancy; IQVIA: Consultancy; Janssen Pharmaceuticals: Consultancy; Aleon Pharmaceuticals: Consultancy; Momenta Pharmaceuticals: Consultancy; Blackrock Pharmaceuticals: Consultancy; Astrazeneca: Consultancy, Research Funding; UCB Pharmaceuticals: Consultancy; GSK: Consultancy; Qiagen: Consultancy; Abbive: Consultancy; Amgen: Consultancy; Decision Resources: Consultancy; Principia Biopharma: Consultancy; Eli Lilly: Consultancy; Kezaar Life Sciences: Consultancy. McCrae:Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Rigel Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Sanofi Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Brodsky:Alexion: Membership on an entity's Board of Directors or advisory committees, Other: Grant funding; Achillion: Research Funding.

Background Defects in the human glycosylphosphatidylinositol anchor biosynthetic pathway are associated with inherited glycosylphosphatidylinositol (GPI)-deficiencies characterized by a broad range of clinical phenotypes including multiple congenital anomalies, dysmorphic faces, developmental delay, hypotonia, and epilepsy. Biallelic variants in PIGN, encoding phosphatidylinositol-glycan biosynthesis class N have been recently associated with multiple congenital anomalies hypotonia seizure syndrome. Case presentation Our patient is a 2 year old male with hypotonia, global developmental delay, and focal epilepsy. Trio whole-exome sequencing revealed heterozygous variants in PIGN, c.181G > T (p.Glu61*) and c.284G > A (p.Arg95Gln). Analysis of FLAER and anti-CD59 by flow-cytometry demonstrated a shift in this patient’s granulocytes, confirming a glycosylphosphatidylinositol-biosynthesis defect, consistent with PIGN-related disease. Conclusions To date, a total of 18 patients have been reported, all but 2 of whom have congenital anomalies and/or obvious dysmorphic features. Our patient has no significant dysmorphic features or multiple congenital anomalies, which is consistent with recent reports linking non-truncating variants with a milder phenotype, highlighting the importance of functional studies in interpreting sequence variants. Electronic supplementary material The online version of this article (10.1186/s12881-017-0481-9) contains supplementary material, which is available to authorized users.

The current study investigated the mechanisms by which licochalcone C induces apoptosis of T24 human malignant bladder cancer cells. Cell viability was evaluated using an MTT assay. Apoptosis was investigated using a morphological assay, flow cytometry and a caspase‑3 activity assay. Alterations in the gene expression levels of Bcl‑2 family members were measured by semi‑quantitative reverse transcription‑polymerase chain reaction assays. The protein levels of pro‑caspase‑3 and cleaved poly(ADP ribose) polymerase were measured using western blotting. The results indicated that licochalcone C induced T24 cell apoptosis in a concentration‑dependent manner. Licochalcone C treatment reduced the levels of the anti‑apoptotic mRNAs (Bcl‑2, Bcl‑w and Bcl‑XL) and increased expression of the pro‑apoptotic mRNAs (Bax and Bim). The Bcl‑2 family inhibitor (ABT‑737) reduced apoptosis induced by licochalcone C in T24 cells. The current study demonstrated that licochalcone C may be a potential adjuvant therapeutic agent for bladder cancer.

Mutations in genes involved in glycosylphosphatidylinositol (GPI) anchor biosynthesis underlie a group of congenital syndromes characterized by severe neurodevelopmental defects. GPI anchored proteins have diverse roles in cell adhesion, signaling, metabolism and complement regulation. Over 30 enzymes are required for GPI anchor biosynthesis and PIGA is involved in the first step of this process. A hypomorphic mutation in the X-linked PIGA gene (c.1234C>T) causes multiple congenital anomalies hypotonia seizure syndrome 2 (MCAHS2), indicating that even partial reduction of GPI anchored proteins dramatically impairs central nervous system development, but the mechanism is unclear. Here, we established a human induced pluripotent stem cell (hiPSC) model containing the PIGAc.1234C>T mutation to study the effects of a hypomorphic allele of PIGA on neuronal development. Neuronal differentiation from neural progenitor cells generated by EB formation in PIGAc.1234C>T is significantly impaired with decreased proliferation, aberrant synapse formation and abnormal membrane depolarization. The results provide direct evidence for a critical role of GPI anchor proteins in early neurodevelopment. Furthermore, neural progenitors derived from PIGAc.1234C>T hiPSCs demonstrate increased susceptibility to complement-mediated cytotoxicity, suggesting that defective complement regulation may contribute to neurodevelopmental disorders.

To examine the mechanisms by which licochalcone B (LCB) inhibits the proliferation of human malignant bladder cancer cell lines (T24 and EJ) in vitro and antitumor activity in vivo in MB49 (murine bladder cancer cell line) tumor model. Exposure of T24 or EJ cells to LCB significantly inhibited cell lines proliferation in a concentration-dependent and time-dependent manner, and resulted in S phase arrest in T24 or EJ cells, respectively. LCB treatment decreased the expression of cyclin A, cyclin-dependent kinase (CDK1 and CDK2) mRNA, cell division cycle 25 (Cdc25A and Cdc25B) protein. In addition, LCB treatment down-regulated Bcl-2 and survivin expression, enhanced Bax expression, activated caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) protein. Consistently, the tumorigenicity of LCB-treated MB49 cells was limited significantly by using the colony formation assay in vitro and the MB49 tumor model performed in C57BL/6 mice in vivo. These findings provide support for the use of LCB in chemoprevention and bladder cancer therapy.

Alternol is a novel compound purified from the fermentation products of a microorganism in the yew tree bark. This study looks at the effects of alternol on the proliferation and cell cycle distribution of mouse melanoma cells. The inhibition of cell proliferation and changes in cell cycle distribution were analysed by sulforhodamine B and flow cytometry assays, respectively. mRNA expression of cyclin A, cyclin-dependent kinase 2 (CDK2), proliferating cell nuclear antigen (PCNA) and CDK inhibitor1A (p21) were measured by real-time reverse transcription PCR (RT-PCR). The protein levels of cyclin A, CDK2 and PCNA were analysed by Western blot analysis. p21 was measured by ELISA. Alternol treatment caused a significant decrease in the proliferation rate of B16F0 and B16F10 cells, which were significantly arrested in S phase, but this treatment had less effect on normal human embryonic kidney 293T cells. The mechanism by which alternol inhibits B16F0 proliferation in vitro may be associated with the inhibition of CDK2 and PCNA, and the activation of p21.

This study evaluates antioxidative and cardioprotective effects of total flavonoids extracted from Dracocephalum moldavica L. (DML). The total flavonoids showed remarkable scavenging effects against 1,1-diphenyl-2-picrylhydrazyl, hydroxyl and superoxide anion radicals in vitro. Compared with the ischemia/reperfusion (I/R) group as demonstrated by the use of improved Langendorff retrograde perfusion technology, the total flavonoids (5 μg/mL) pretreatment improved the heart rate and coronary flow, rised left ventricular developed pressure and decreased creatine kinase, lactate dehydrogenase levels in coronary flow. The infarct size/ischemic area at risk of DML-treated hearts was smaller than that of I/R group; the superoxide dismutase activity and glutathione/glutathione disulfide ratio increased and malondialdehyde content reduced obviously (P < 0.01) in total flavonoids treatment groups. In conclusion, the total flavonoids possess obvious protective effects on myocardial I/R injury, which may be related to the improvement of myocardial oxidative stress states.

The aim of this study was to determine the relationship between proliferation inhibition and the production of reactive oxygen species (ROS) induced by Licochalcone A (LCA). Cell viability was evaluated using sulforhodamine B (SRB) assay. Intracellular ROS level was assessed using the 2, 7-dichlorofluorescein diacetate (H2DCFDA) probe and dihydroethidium (DHE) probe assay. The results indicate that LCA inhibits human bladder cancer T24 proliferation in a concentration-dependent manner, with an IC50 value of approximately 55 μM. The LCA-induced ROS production is inhibited by the co-treatment of LCA and free radical scavenger N-acetyl-cysteine (NAC), on the contrary, the proliferation rate and ROS production increase when treated by the combination of LCA and L-buthionine-(S,R)-sulfoximine (BSO). The ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) decreases in a concentration-dependent manner. The results suggest that LCA inhibits proliferation by increasing intracellular ROS levels resulted in an oxidative stress status in T24 cells.

The urate oxidase ( Uox ) gene encodes uricase that in the rodent liver degrades uric acid into allantoin, forming an obstacle for establishing stable mouse models of hyperuricemia. The loss of uricase in humans during primate evolution causes their vulnerability to hyperuricemia. Thus, we generated a Uox- knockout mouse model on a pure C57BL/6J background using the transcription activator-like effector nuclease (TALEN) technique. These Uox -knockout mice spontaneously developed hyperuricemia (over 420 μmol/l) with about 40% survival up to 62 weeks. Renal dysfunction (elevated serum creatinine and blood urea nitrogen) and glomerular/tubular lesions were observed in these Uox -knockout mice. Male Uox -knockout mice developed glycol-metabolic disorders associated with compromised insulin secretion and elevated vulnerability to streptozotocin-induced diabetes, whereas female mice developed hypertension accompanied by aberrant lipo-metabolism. Urate-lowering drugs reduced serum uric acid and improved hyperuricemia-induced disorders. Thus, uricase knockout provides a suitable mouse model to investigate hyperuricemia and associated disorders mimicking the human condition, suggesting that hyperuricemia has a causal role in the development of metabolic disorders and hypertension.

Introduction aHUS is thrombotic microangiopathy associated with excess activation of alternative complement pathway (AP). Virtually all germline mutations that predispose to aHUS [e.g., factor H (fH)] occur in genes that regulate the amplification loop in the AP. The modified Ham test (mHam) measures the ability of a nucleated cell to protect itself from complement-mediated cell death (in vitro) in the absence of downstream cell-surface complement regulators, CD55 and CD59. Thus, the mHam exposes defects in serum-based complement regulators/activators that involve the amplification loop. The mHam is useful in distinguishing aHUS from thrombotic thrombocytopenic purpura (TTP); limitations are the lack of a reliable positive control and the lack of a confirmatory assay. In order to develop a positive control for the mHam, three complement activators, cobra venom factor (CVF), lipopolysaccharide (LPS) and sialidase (neuraminidase) were studied. CVF and LPS can activate and deplete serum of complement. Sialidase (Sia) makes cells more susceptible to complement mediated killing by removing sialic acid residues that are critical for binding the complement regulator, fH. Here, we show that Sia is a reliable positive control for the mHam and that cell surface C5b-9 accumulation correlates with a positive mHam. Methods/Results: The mHam assay was performed utilizing the PIGAnull TF1 cell line as previously described. CVF and LPS in dosages of 1.0 to 10ug/mL were added to 20% normal human serum (NHS) individually for 15 minutes at 37°C, and then added to cells in GVB++ buffer for 30 minutes. Sia (6.25unit/mL to 200unit/mL) was added to cells in GVB++ buffer for 15 minutes at 37°C first, and then incubated with 20% normal human serum (NHS) for 30 minutes. Complement-mediated cell killing in the mHam was evaluated using WST-1 absorbance as described previously. We found that sia treatment was more effective and reliable than LPS and CVF at inducing complement-mediated killing. A sia dose of 50U/mL or greater increased complement mediated killing by more than 3-fold in the mHam. Cell killing correlated with increased C5b-9 staining and was abrogated by the addition of an anti-C5 antibody and a factor D inhibitor. Next, we loaded PIGAnull TF1 cells with increasing amounts of C3b, added 20% NHS, and simultaneously performed the mHam and stained cells with an anti-C5b-9 monoclonal antibody for FACS staining to see if C5b-9 accumulation correlates with mHam killing. Briefly, PIGAnull TF1 cells in GVB+ buffer were incubated with 17.5ug C3, 4ug factor B and 0.1ug factor D for 20 minutes at room temperature (RT). Cells were washed and then incubated with 2ug factor B and 0.1ug factor D at RT for 3 minutes first, and treated with 0.25M EDTA in GVB0 buffer. Then 17.5ug C3 was added to cells for 15 minutes at 37°C. After three consecutive cycles of C3b loading, 20% NHS was added and complement activity was measured in the mHam and by flow cytometry. Cells loaded with one or two cycles of C3b showed minimal C5b-9 staining and minimal cell death ( Conclusion We describe an AP pathway specific flow cytometric assay that correlates with the mHam and may further aid in the diagnosis of aHUS. Sialidase treatment of TF1PIGAnull cells serves as a reliable positive control for the mHam by making cells more susceptible to AP killing. Disclosures No relevant conflicts of interest to declare.

Low non-freezing temperature is one of the major environmental factors affecting growth, development and geographical distribution of chilling-sensitive plants, Jatropha curcas is considered as a sustainable energy plants with great potential for biodiesel production. In this study, chilling shock at 5 °C followed by recovery at 26 °C for 4 h significantly improved survival percentage of J. curcas seedlings under chilling stress at 1 °C. In addition, chilling shock could obviously enhance the activities of antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR), and the levels of antioxidants ascorbic acid (AsA) and glutathione (GSH), as well as the contents of osmolytes proline and betaine in leaves of seedlings of J. curcas compared with the control without chilling shock. During the process of recovery, GR activity, AsA, GSH, proline and betaine contents sequentially increased, whereas SOD, APX and CAT activities gradually decreased, but they markedly maintained higher activities than those of control. Under chilling stress, activities of SOD, APX, CAT, GR and GPX, and contents of AsA, GSH, proline and betaine, as well as the ratio of the reduced antioxidants to total antioxidants [AsA/(AsA + DHA) and GSH/(GSH + GSSG)] in the shocked and non-shock seedlings all dropped, but shocked seedlings sustained significantly higher antioxidant enzyme activity, antioxidant and osmolyte contents, as well as ratio of reduced antioxidants to total antioxidants from beginning to end compared with control. These results indicated that the chilling shock followed by recovery could improve chilling tolerance of seedlings in J. curcas, and antioxidant enzymes and osmolytes play important role in the acquisition of chilling tolerance.

Phosphatidylinositol glycan class A (PIGA) is involved in the first step of glycosylphosphatidylinositol (GPI) biosynthesis. Many proteins, including CD55 and CD59, are anchored to the cell by GPI. Loss of CD55 and CD59 on erythrocytes causes complement-mediated lysis in paroxysmal nocturnal hemoglobinuria (PNH), a disease that manifests after clonal expansion of hematopoietic cells with somatic PIGA mutations. Although somatic PIGA mutations have been identified in many PNH patients, it has been proposed that germline mutations are lethal. We report a family with an X-linked lethal disorder involving cleft palate, neonatal seizures, contractures, central nervous system (CNS) structural malformations, and other anomalies. An X chromosome exome next-generation sequencing screen identified a single nonsense PIGA mutation, c.1234CT, which predicts p.Arg412(∗). This variant segregated with disease and carrier status in the family, is similar to mutations known to cause PNH as a result of PIGA dysfunction, and was absent in 409 controls. PIGA-null mutations are thought to be embryonic lethal, suggesting that p.Arg412(∗) PIGA has residual function. Transfection of a mutant p.Arg412(∗) PIGA construct into PIGA-null cells showed partial restoration of GPI-anchored proteins. The genetic data show that the c.1234CT (p.Arg412(∗)) mutation is present in an affected child, is linked to the affected chromosome in this family, is rare in the population, and results in reduced, but not absent, biosynthesis of GPI anchors. We conclude that c.1234CT in PIGA results in the lethal X-linked phenotype recognized in the reported family.

The aim of our study was to investigate the neuroprotective properties of shikonin, a naphthoquinone pigment isolated from the roots of the traditional Chinese herb Lithospermum erythrorhizon. In the present study, mice were divided randomly into sham, model, shikonin and edaravone-treated groups. Shikonin (50, 25, and 12.5 mg/kg, i.g.) or maize oil was administered three times before ischemia and once at 2 h after the onset of ischemia. Mice were anesthetized with chloral hydrate and subjected to middle cerebral artery 2 h of occlusion and then 22 h of reperfusion. Different antioxidant assays were employed in order to evaluate the antioxidant activities of shikonin in vitro. Neurological deficit, infarct size, histopathology changes and oxidative stress markers were evaluated after 22 h of reperfusion. In comparison with the model group, treatment with shikonin significantly decreased neurological deficit scores, infarct size, the levels of malondialdehyde(MDA), carbonyl and reactive oxygen species, and attenuated neuronal damage, up-regulated superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) activities and reduced glutathione (GSH)/glutathione disulfide (GSSG) ratio. Taken together, these results suggested that the neuroprotective effects of shikonin against cerebral ischemia/reperfusion injury may be attributed to its antioxidant effects.

Recent characterization of hemangioblasts differentiated from human embryonic stem cells (hESC) has further confirmed evidence from murine, zebrafish and avian experimental systems that hematopoietic and endothelial lineages arise from a common progenitor. Such progenitors may provide a valuable resource for delineating the initial developmental steps of human hemato-endotheliogenesis, which is a process normally difficult to study due to the very limited accessibility of early human embryonic/fetal tissues. Moreover, efficient hemangioblast and hematopoietic stem cell (HSC) generation from patient-specific pluripotent stem cells has enormous potential for regenerative medicine, since it could lead to strategies for treating a multitude of hematologic and vascular disorders. However, significant scientific challenges remain in achieving these goals, and the generation of transplantable hemangioblasts and HSC derived from hESC currently remains elusive. Our previous work has suggested that the failure to derive engraftable HSC from hESC is due to the fact that current methodologies for differentiating hESC produce hematopoietic progenitors developmentally similar to those found in the human yolk sac, and are therefore too immature to provide adult-type hematopoietic reconstitution. Herein, we outline the nature of this challenge and propose targeted strategies for generating engraftable human pluripotent stem cell-derived HSC from primitive hemangioblasts using a developmental approach. We also focus on methods by which reprogrammed somatic cells could be used to derive autologous pluripotent stem cells, which in turn could provide unlimited sources of patient-specific hemangioblasts and HSC.

Both phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen activated protein kinase (MAPK) signaling cascades play an important role in cell proliferation, survival, angiogenesis and metastasis of tumor cells. In the present report, we investigated the effects of licochalcone A (LA), a flavonoid extracted from licorice root, on the PI3K/AKT/mTOR and MAPK activation pathways in human gastric cancer BGC-823 cells. LA increased reactive oxygen species (ROS) levels, which is associated with the induction of apoptosis as characterized by positive Annexin V binding and activation of caspase-3 and cleavage of poly-ADP-ribose polymerase (PARP). Inhibition of ROS generation by N-acetylcysteine (NAC) significantly prevented LA-induced apoptosis. Interestingly, we also observed that LA caused the activation of ERK, JNK and p38 MAPK in BGC-823 cells. The antitumour activity of LA-treated BGC-823 cells was significantly distinct in KM mice in vivo. All the findings from our study suggest that LA can interfere with MAPK signaling cascades, initiate ROS generation, induce oxidative stress and consequently cause BGC cell apoptosis.

Flavonoids are important components of ‘functional foods’, with beneficial effects on cardiovascular function. The present study was designed to investigate whether licochalcone D (LD) could be a cardioprotective agent in ischemia/reperfusion (I/R) injury and to shed light on its possible mechanism. Compared with the I/R group, LD treatment enhanced myocardial function (increased LVDP, dp/dt max, dp/dt min, HR and CR) and suppressed cardiac injury (decreased LDH, CK and myocardial infarct size). Moreover, LD treatment reversed the I/R-induced cleavage of caspase-3 and PARP, resulting in a significant decrease in proinflammatory factors and an increase in antioxidant capacity in I/R myocardial tissue. The mechanisms underlying the antiapoptosis, antiinflammation and antioxidant effects were related to the activation of the AKT pathway and to the blockage of the NF-κB/p65 and p38 MAPK pathways in the I/R-injured heart. Additionally, LD treatment markedly activated endothelial nitric oxide synthase (eNOS) and reduced nitric oxide (NO) production. The findings indicated that LD had real cardioprotective potential and provided support for the use of LD in myocardial I/R injury.

Neurodevelopmental disorders (NDDs) affect more than 3% of children and are attributable to single-gene mutations at more than 1000 loci. Traditional methods yield molecular diagnoses in less than one-half of children with NDD. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) can enable diagnosis of NDD, but their clinical and cost-effectiveness are unknown. One hundred families with 119 children affected by NDD received diagnostic WGS and/or WES of parent-child trios, wherein the sequencing approach was guided by acuity of illness. Forty-five percent received molecular diagnoses. An accelerated sequencing modality, rapid WGS, yielded diagnoses in 73% of families with acutely ill children (11 of 15). Forty percent of families with children with nonacute NDD, followed in ambulatory care clinics (34 of 85), received diagnoses: 33 by WES and 1 by staged WES then WGS. The cost of prior negative tests in the nonacute patients was $19,100 per family, suggesting sequencing to be cost-effective at up to $7640 per family. A change in clinical care or impression of the pathophysiology was reported in 49% of newly diagnosed families. If WES or WGS had been performed at symptom onset, genomic diagnoses may have been made 77 months earlier than occurred in this study. It is suggested that initial diagnostic evaluation of children with NDD should include trio WGS or WES, with extension of accelerated sequencing modalities to high-acuity patients.

This study investigated the mechanisms by which licochalcone B (LCB) inhibits the adhesion,invasion and metastasis of human malignant bladder cancer T24 cells. Cell viability was evaluated using a sulforhodamine B (SRB) assay. Cell migration and invasion ability were conducted using wound-healing assay and matrigel transwell invasion assay. The activities of matrix metalloproteinases (MMP)-2 and MMP-9 were measured by gelatin zymography protease assays. The expression in protein level of NF-κBP65 and AP-1 was determined using the ELISA method; the protein levels of MMP-9, NF-κBP65, IκBα and P-IκBα were detected by Western blot. The expression in mRNA level of MMP-9 was assessed using quantitative real-time polymerase chain reaction (PCR) and reverse transcription PCR. The results indicated that LCB attenuated T24 cell migration, adhesion and invasion in a concentration-dependent manner. LCB treatment down-regulated the mRNA expression, protein expression and activity of MMP-9 but had no effect on MMP-2. In addition, LCB treatment decreased the protein level of NF-кBP65 and nuclear translocation of NF-кB. These findings suggested that LCB attenuated migration of bladder cancer T24 cells and adhesion and invasion accompanied with down-regulated protein expression of MMP-9 and the nuclear translocation of NF-кB. Our results provide support that LCB may be a potent adjuvant therapeutic agent in the prevention and therapy of bladder cancer.

Introduction: Antiphospholipid antibody syndrome (APS) is characterized by recurrent thrombosis and pregnancy loss, in the presence of antibodies directed against phospholipid-binding proteins. Although persistence of antiphospholipid antibodies (aPL), including lupus anticoagulant (LA), anti-cardiolipin (aCL) or anti-β2-glycoprotein (aβ2-GPI) antibodies, is a key diagnostic feature of APS and its severe variant, catastrophic APS (CAPS), the mechanism by which aPL contribute to the clinical manifestations of these disorders remains unclear. There is evidence that excessive activation of complement plays a role in the pathophysiology of APS and CAPS, and limited case reports suggest a therapeutic role for complement inhibition; however, a clear mechanistic understanding of this process has not been elucidated. The modified Ham (mHam) is a functional assay for detection of systemic activation of the alternative pathway of complement (APC). Here, we show that the APC is active in serum from patients with APS and CAPs using the mHam assay, and provide evidence that inhibition of complement factor H (CFH), a homolog of β2-GPI, by aβ2-GPI antibodies is a plausible mechanistic link between APS/CAPS and excessive APC activation. Methods: The patient population studied included adults with APS who presented to our institution between January 2015 and January 2016. A diagnosis of APS or CAPS was based on diagnostic criteria from 2002 and 2006. Patients with a CAPS-like phenotype, who did not meet all criteria for definite or probable CAPS (Asherson et al. 2003), were also included. Patient serum was evaluated in the mHam using the PIGA-null TF1 cell line and percentage of cell killing was measured using a cell proliferation assay. Additional studies testing the addition of purified phospholipids or commercially available β2-GPI antibodies (and isotype controls) also utilized the mHam. Serum from 5 patients with atypical hemolytic uremic syndrome (aHUS) was used as a control in the phospholipid study. It was hypothesized that CFH, the principal negative regulator of the APC, may be the target of pathogenic aPL given its homology to β2-GPI. To test this hypothesis, competitive binding assays were performed with commercially available recombinant β2-GPI and purified CFH. Results: Serum from 18 patients with APS, 1 patient with definite CAPS, 3 patients with CAPS-like phenotype, 5 healthy volunteers and 14 patients with other hypercoagulable states (9 patients with lupus who did not meet criteria for APS, 4 patients with TTP and 1 patient with DIC) was analyzed in the mHam. APC-mediated cell killing in the mHam was higher in the serum from patients with APS and CAPS, compared to normal controls and patients with other hypercoagulable states (mean of 27.9% and 36.2% versus 5.2% and 12.2%, respectively; see Figure). Addition of purified phospholipids to serum from patients with APS reduced APC-mediated cell killing, but had no effect on serum from patients with aHUS, consistent with divergent mechanisms for APC activation in these disorders. Addition of a β2-GPI IgG antibody to normal human serum resulted in increased APC-mediated cell killing in the mHam, relative to addition of a β2-GPI IgM antibody or isotype controls, suggesting that antibodies directed against β2-GPI may directly activate the APC. Finally, the addition of CFH blocked binding of a commercially available β2-GPI IgG antibody to purified β2-GPI, supporting the hypothesis that aβ2-GPI antibodies may promote excessive activation of the APC in APS and CAPS by binding to and inhibiting CFH. Conclusions: Systemic activation of the APC can be demonstrated by the mHam test in serum from patients with APS and CAPS. This suggests that, in at least a subset of patients with APS and CAPS, excessive activation of the APC may be an important driver of the disease manifestations. Further study is required to determine whether there is a relationship between seropositivity for LA, aCL and aβ2-GPI and activation of the APC. Autoantibodies directed against β2-GPI may directly activate the APC by inhibiting CFH, providing further rationale for the potential use of therapies aimed at complement inhibition in these devastating disorders. Figure Figure. Disclosures Streiff: Janssen: Consultancy, Research Funding; CSL Behring: Consultancy, Research Funding; Portola: Research Funding; Roche: Research Funding. Brodsky:Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Achillion Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees.

PIG-A is an X-linked gene required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; thus, PIG-A mutant cells have a deficiency or absence of all GPI-anchored proteins (GPI-APs). Acquired mutations in hematopoietic stem cells result in the disease paroxysmal nocturnal hemoglobinuria, and hypomorphic germline PIG-A mutations lead to severe developmental abnormalities, seizures, and early death. Human induced pluripotent stem cells (iPSCs) can differentiate into cell types derived from all three germ layers, providing a novel developmental system for modeling human diseases. Using PIG-A gene targeting and an inducible PIG-A expression system, we have established, for the first time, a conditional PIG-A knockout model in human iPSCs that allows for the production of GPI-AP-deficient blood cells. PIG-A-null iPSCs were unable to generate hematopoietic cells or any cells expressing the CD34 marker and were defective in generating mesodermal cells expressing KDR/VEGFR2 (kinase insert domain receptor) and CD56 markers. In addition, PIG-A-null iPSCs had a block in embryonic development prior to mesoderm differentiation that appears to be due to defective signaling through bone morphogenetic protein 4. However, early inducible PIG-A transgene expression allowed for the generation of GPI-AP-deficient blood cells. This conditional PIG-A knockout model should be a valuable tool for studying the importance of GPI-APs in hematopoiesis and human development.

The phosphatidylinositol glycan class A (PIGA) protein is a member of the glycosylphosphatidylinositol anchor pathway. Germline mutations in PIGA located at Xp22.2 are thought to be lethal in males. However, a nonsense mutation in the last coding exon was recently described in two brothers with multiple congenital anomalies-hypotonia-seizures syndrome 2 (MCAHS2) who survived through birth likely because of the hypomorphic nature of the truncated protein, but died in their first weeks of life. Here, we report on a frameshift mutation early in the PIGA cDNA (c.76dupT; p.Y26Lfs*3) that cosegregates with the disease in a large family diagnosed with a severe syndromic form of X-linked intellectual disability. Unexpectedly, CD59 surface expression suggested the production of a shorter PIGA protein with residual functionality. We provide evidence that the second methionine at position 37 may be used for the translation of a 36 amino acids shorter PIGA. Complementation assays confirmed that this shorter PIGA cDNA was able to partially rescue the surface expression of CD59 in a PIGA-null cell line. Taken together, our data strongly suggest that the early frameshift mutation in PIGA produces a truncated hypomorph, which is sufficient to rescue the lethality in males but not the MCAHS2-like phenotype.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, clonal, hematopoietic stem cell disorder that manifests with a complement-mediated hemolytic anemia, bone marrow failure and a propensity for thrombosis. These patients experience both intra- and extravascular hemolysis in the context of underlying complement activation. Currently eculizumab effectively blocks the intravascular hemolysis PNH. There remains an unmet clinical need for a complement inhibitor with activity early in the complement cascade to block complement at the classical and alternative pathways. C1 esterase inhibitor (C1INH) is an endogenous human plasma protein that has broad inhibitory activity in the complement pathway through inhibition of the classical pathway by binding C1r and C1s and inhibits the mannose-binding lectin-associated serine proteases in the lectin pathway. In this study, we show that commercially available plasma derived C1INH prevents lysis induced by the alternative complement pathway, of PNH erythrocytes in human serum. Importantly, C1INH was able to block the accumulation of C3 degradation products on CD55 deficient erythrocytes from PNH patient on eculizumab therapy. This could suggest a role for inhibition of earlier phases of the complement cascade than that currently inhibited by eculizumab for incomplete or non-responders to that therapy.

To evaluate the role of redox homeostasis in differentiation in human promyelocytic leukemia cells (HL-60) induced by isoliquiritigenin (ISL) through modulation of the nuclear erythroid-related factor 2/antioxidant responsive element (Nrf2/ARE) pathway. Morphological changes, cell surface markers CD11b/CD14, and nitroblue tetrazolium (NBT)-reducing ability were used to determine the differentiation of HL-60, and 2,7-dichlorofluorescein was used to detect the level of intracellular reactive oxygen species (ROS). Thiobarbituric acid test was utilised to determine the levels of malondialdehyde production in ISL-treated HL-60. The study determines and presents the redox state of the ratio of reduced/oxidised glutathione as a consequence of progression from differentiation in HL-60. Expression levels of the Nrf2/ARE downstream target genes were determined by quantitative polymerase chain reaction. Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) inhibitors, apocynin (APO), and diphenyleneiodonium (DPI) were used for the preliminary study to determine the potential downstream targets regulated by NADPH oxidase in ISL-induced HL-60 differentiation. The data showed a strong dose-response relationship between ISL exposure and the characteristics of HL-60 differentiation, namely, morphology changes, NBT reductive activities, and expression levels of surface antigens CD11b/CD14. Intercellular redox homeostasis changes toward oxidation during drug exposure are necessary to support ISL-induced differentiation. The unique expression levels of the Nrf2/ARE downstream target genes in the differentiation of HL-60 recorded a statistically significant and dose-dependent increase (P < 0.05), which were suppressed by NADPH oxidase inhibitor, APO, and DPI. ISL as a differentiation-inducing agent with mechanisms involved in the Nrf2/ARE pathway to modulate intercellular redox homeostasis, and thus, facilitate differentiation.

The aim of this study was to evaluate the antitumour activities and genotoxic effects of isoliquiritigenin (ISL) combined with cyclophosphamide (CP) in vitro and in vivo. U14 cells were treated with either of ISL (5-25 μg/mL) or CP (0.25-1.25 mg/mL) alone or with combination of ISL (5-25 μg/mL) and CP (1.0 mg/mL) for 48 h. The proliferation inhibitory effect in vitro was evaluated by MTT and colony formation assays. KM mice bearing U14 mouse cervical cancer cells were used to estimate the antitumour activity in vivo. The genotoxic activity in bone marrow polychromatic erythrocytes was assayed by frequency of micronuclei. The DNA damage in peripheral white blood cells was assayed by single cell gel electrophoresis. The results showed that ISL enhanced antitumour activity of CP in vitro and in vivo, and decreased the micronucleus formation in polychromatic erythrocytes and DNA strand breaks in white blood cells in a dose-dependent way.

Licochalcone A (LCA), a licorice chalconoid, is considered to be a bioactive agent with chemopreventive potential. This study investigated the mechanisms involved in LCA-induced apoptosis in human bladder cancer T24 cells. LCA significantly inhibited cells proliferation, increased reactive oxygen species (ROS) levels, and caused T24 cells apoptosis. Moreover, LCA induced mitochondrial dysfunction, caspase-3 activation, and poly-ADP-ribose polymerase (PARP) cleavage, which displayed features of mitochondria-dependent apoptotic signals. Besides, exposure of T24 cells to LCA triggered endoplasmic reticulum (ER) stress; as indicated by the enhancement in 78 kDa glucose-regulated protein (GRP 78), growth arrest and DNA damage-inducible gene 153/C/EBP homology protein (GADD153/CHOP) expression, ER stress-dependent apoptosis is caused by the activation of ER-specific caspase-12. All the findings from our study suggest that LCA initiates mitochondrial ROS generation and induces oxidative stress that consequently causes T24 cell apoptosis via the mitochondria-dependent and the ER stress-triggered signaling pathways.

The air-dried aerial parts of Lavandula angustifolia Mill, a traditional Uygur herbal drug, is used as resuscitation-inducing therapy to treat neurodisfunctions, such as stroke. This study was designed to assess the neuroprotective effects of lavender oil against ischemia/reperfusion (IR) injury in mice. Focal cerebral ischemia was induced by the intraluminal occlusion method with a nylon string. The neurodysfuntion was evaluated by neurological deficit and the infarct area was showed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The histopathological changes were observed by hematoxylin and eosin staining. The levels of mitochondria-generated reactive oxygen species (ROS), malondialdehyde (MDA) and carbonyl, the ratio of reduced glutathione (GSH)/glutathione disulfide (GSSG), the activities of superoxide dismutase (SOD), catalase (CAT) and glutathion peroxidase (GSH-Px) in brain tissue were measured to estimate the oxidative stress state. Neurological deficit, infarct size, histopathology changes and oxidative stress markers were evaluated after 22 h of reperfusion. In comparison with the model group, treatment with lavender oil significantly decreased neurological deficit scores, infarct size, the levels of MDA, carbonyl and ROS, and attenuated neuronal damage, upregulated SOD, CAT, GSH-Px activities and GSH/GSSG ratio. These results suggested that the neuroprotective effects of lavender oil against cerebral ischemia/reperfusion injury may be attributed to its antioxidant effects.

This study focuses on the relationship between the apoptosis induced by isoliquiritigenin (ISL) and the production of reactive oxygen species (ROS). Cell viability was evaluated using sulforhodamine B assay. The apoptotic rate was determined via flow cytometry. Intracellular ROS level was assessed using the 2,7-dichlorofluorescein probe assay. Poly-ADP-ribose polymerase (PARP) protein expression was examined using Western blot analysis. The results showed that ISL treatment inhibited cell proliferation by inducing apoptosis. The increased apoptotic rate and ROS production induced by ISL were inhibited by the co-treatment of ISL and free radical scavenger N-acetyl-cysteine (NAC), catalase (CAT), and 4,5-dihydroxyl-1,3-benzededisulfonic acid (Tiron). On the contrary, the increased apoptotic rate and the ROS production were compensated by the co-treatment of ISL and l-buthionine-(S,R)-sulfoximine (BSO). ISL treatment increased the degradation of PARP, which was counteracted by antioxidants (NAC or CAT), whereas the combination treatment of ISL and pro-oxidant (BSO) enhanced the PARP degradation induced by ISL. Our findings suggested that ISL treatment induced apoptosis by increasing intracellular ROS levels in HeLa cells.

The chemotherapeutical treatment is very limited for malignant melanoma, a highly lethal disease occurs globally. Natural products derived from traditional Chinese medicine licorice are attractive in quest new treatments due to their anti-tumor activities. A new dietary flavonoid isoliquiritigenin (ISL) were thus investigated to indentify its anti-melanoma activities on mouse melanoma B16F0 cells in present study. Using biochemical and free radical biological experiments in vitro, we identified the pro-differentiated profiles of ISL and evaluated the role of reactive oxygen species (ROS) during B16F0 cell differentiation. The data showed a strong dose-response relationship between ISL exposure and the characteristics of B16F0 differentiation in terms of morphology changes and melanogenesis. The accumulated intercellular ROS during exposure are necessary to support ISL-induced differentiation, which was proven by additional redox modulators. It was confirmed further by the relative activities of enzymes and genes modulated melanogenesis in ISL-treatments with or without ROS modulators. The tumorigenicity of ISL-treated cells was limited significantly by using the colony formation assay in vitro and an animal model assay in vivo respectively. Our research demonstrated that isoliquiritigenin is a differentiation-inducing agent, and its mechanisms involve ROS accumulation facilitating melanogenesis.

Introduction: Factor D is a highly specific serine protease that cleaves factor B as its only substrate. It is the rate-limiting step of the alternative pathway of complement (APC). Therefore, factor D is a promising therapeutic target in diseases of excess activation of the APC, such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). Terminal complement inhibition by eculizumab is currently the treatment of choice for PNH and aHUS. Eculizumab must be administered intravenously and indefinitely in PNH; moreover, up to 20% of PNH patients treated with eculizumab have symptomatic hemolysis due to extravascular hemolysis. Glycosylphosphatidylinositol (GPI) anchor protein deficient erythrocytes from PNH patients or erythrocytes from animals can be used to test novel complement inhibitors for PNH; however, there are no in vitro assays available to model complement-mediated attack in aHUS. Here, we demonstrate that the modified Ham test (PIGA-null reagent cell line exposed to serum from aHUS patients) is the first reliable in vitro model to test complement inhibition for aHUS. In addition, we demonstrate that small molecule factor D inhibitors specifically block the APC in PNH and aHUS. Method: Three factor D inhibitors (ACH-3856, ACH-4100, ACH-4471) that reversibly bind factor D and are being developed for oral administration were studied. We obtained blood samples from 5 PNH and 4 aHUS patients after written informed consent. Serum and erythrocytes from PNH patients on eculizumab were collected within 60 minutes of eculizumab administration. All 3 compounds were tested in half-log dilutions ranging from 0.001 ìM to 10 ìÌ. The binding affinity to factor D was determined by surface plasmon resonance (Biacore) analysis. The inhibitory effect on factor D protease was evaluated biochemically using a natural substrate consisting of C3b and the complement factor B. The inhibition of APC-mediated hemolysis was first evaluated with rabbit erythrocytes incubated with 8% normal human serum (NHS). The inhibition of APC-mediated C3 fragment deposition was also initially evaluated with rabbit erythrocytes incubated with 20% C5-depleted NHS (to mimic the effect of eculizumab) by flow cytometry. To confirm the observation with rabbit erythrocytes, erythrocytes from PNH patients were also tested in the hemolytic assay (20% acidified NHS). Regarding aHUS, the inhibitory effect of the compounds was evaluated in the recently described modified Ham test. Briefly, PIGA-null TF-1 cells were incubated with 20% of aHUS serum for 30 minutes at 37 o C and complement-mediated cell killing was evaluated using the cell proliferation reagent (WST-1). Absorbance was measured and expressed as a ratio to the heat-inactivated control (% non-viable cells) Results: All 3 small molecules showed high binding affinity to human factor D and effectively inhibited factor D proteolytic activity in a dose-dependent manner (mean IC50 9.8-20 nM). Similarly, we observed a dose-dependent inhibition of hemolysis on rabbit erythrocytes (EC50 9-17 nM). C3 fragment deposition on rabbit erythrocytes after incubation with C5-depleted serum was reduced to undetectable levels with all 3 compounds from concentrations of 0.1 ìÌ and above. Next, we studied samples from 5 PNH patients (2 treatment naïve and 3 on eculizumab) and 4 aHUS patients (1 on eculizumab, 1 in acute phase and on eculizumab and 2 in remission). When tested on the hemolytic assay with erythrocytes from PNH patients and acidified NHS, factor D inhibitors significantly reduced hemolysis in a dose-dependent manner from concentrations as low as 0.01 ìM. Lastly, cell killing was observed as previously reported in the presence of the serum from aHUS patients in the modified Ham test and addition of factor D inhibitors caused significant dose-dependent reduction in the cell killing at concentration as low as 0.01 ìÌ. Mean percentage of non-viable cells before and after addition of compound ACH-4471 is shown in Figure 1. Conclusions: We demonstrate that factor D inhibitors, potentially the first-ever oral complement inhibitors, efficiently block hemolysis of PNH cells in vitro and mitigate the accumulation of C3 fragments. Importantly, our findings indicate that the recently described modified Ham test can be used as a preclinical model to test complement inhibitors in aHUS. Figure 1. Figure 1. Disclosures Thanassi: Achillion Pharmaceuticals: Employment. Yang:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment. Brodsky:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Background The production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34+ cord blood using non-viral, non-integrating methods. Methodology/Principal Findings We systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34+ cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64–89%) of cardiac troponin I+ cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs. Conclusion/Significance This efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine.

Previous reports reveal that Isoliquiritigen(ISL), a licorice flavonoid, possesses various antitumour properties, cyclophosphamide, an alkylating agent, has various genotoxic and carcinogenic effects, and to be involved in some secondary neoplasmas. However, it is still used extensively as an antitumour agent and immunosuppressant in the clinic. In order to find out whether isoliquiritigen could enhance antitumour activity, as well as decrease genotoxic effects of cyclophosphamide or not, the antitumour activity and genotoxic effect of oral intake of ISL combined with intraperitoneal injection of cyclophosphamide was investigated. Mice bearing mouse sarcoma S180 cells and cervical cancer U14 cells were respectively used to estimate the antitumour activity in vivo. The clastogenic activity in bone marrow polychromatic erythrocytes was assayed by frequency of micronucleus. The DNA damage in peripheral white blood cells was assayed by single cell gel electrophoresis. The results indicated that oral administration of ISL (5, 10 and 20 mg/kg body weight) alone has no obvious antitumour activity and genotoxic effect in mice, while ISL synergistically enhanced the antitumour activity of cyclophosphamide (40 mg/kg body weight) in a dose-dependent manner. in addition, ISL can decrease the micronucleus formation in polychromatic erythrocytes and DNA strand breaks in white blood cells in a dose-dependent way. Our results suggested that ISL is able to enhance the antitumour activity and decrease the genotoxic effect of cyclophosphamide.

We report that angiotensin-converting enzyme (ACE), a critical physiologic regulator of blood pressure, angiogenesis, and inflammation, is a novel marker for identifying hemangioblasts differentiating from human embryonic stem cells (hESC). We demonstrate that ACE+CD45−CD34+/− hemangioblasts are common yolk sac (YS)–like progenitors for not only endothelium but also both primitive and definitive human lymphohematopoietic cells. Thrombopoietin and basic fibroblast growth factor are identified as critical factors for the proliferation of human hemangioblasts. The developmental sequence of human embryoid body hematopoiesis is remarkably congruent to the timeline of normal human YS development, which occurs during weeks 2 to 6 of human gestation. Furthermore, ACE and the renin-angiotensin system (RAS) directly regulate hemangioblast expansion and differentiation via signaling through the angiotensin II receptors AGTR1 and AGTR2. ACE enzymatic activity is required for hemangioblast expansion, and differentiation toward either endothelium or multipotent hematopoietic progenitors is dramatically augmented after manipulation of angiotensin II signaling with either AGTR1- or AGTR2-specific inhibitors. The RAS can therefore be exploited to direct the hematopoietic or endothelial fate of hESC-derived hemangioblasts, thus providing novel opportunities for human tissue engineering. Moreover, the initial events of human hematoendotheliogenesis can be delineated in a manner previously impossible because of inaccessibility to early human embryonic tissues.

Patients with malignant gliomas have a very poor prognosis. To explore a novel and more effective approach for the treatment of malignant gliomas, a strategy that combined tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy and radiation treatment (RT) was designed in this study. Plasmid pE4-GFP was constructed by including the radioinducible early growth response gene 1 (Egr-1) promoter, and it yielded the best response with fractionated RT. Plasmid pE4-TRAIL was constructed by including the Egr-1 promoter and evaluated using U251 and U87 glioma cells. In the assay of apoptosis and killing activities, pE4-TRAIL exhibited radioresponse. pE4-TRAIL combined with RT is capable of inducing cell death synergistically. The expression of TRAIL death receptors was evaluated; which may be influenced by RT. Glioma cells with wild-type p53 showed upregulated expression of death receptors, and more synergistic effects on killing activities are expected. pE4-TRAIL was transfected into the subcutaneous U251 glioma cells in nude mice by the in vivo electroporation method. In the mice treated with pE4-TRAIL and RT, apoptotic cells were detected in pathological sections, and a significant difference of tumor volumes was observed when compared with the other groups (P

Objective Our laboratory has established two unique methods to isolate murine hematopoietic stem cells on the basis of functional characteristics such as the ability of stem cells to home to bone marrow and aldehyde dehydrogenase (ALDH) activity. An essential component of both protocols is the separation of whole bone marrow into small-sized cells by counter-flow elutriation. We sought to provide the scientific community with an alternate approach to acquire our stem cells by replacing elutriation with the use of density-gradient centrifugation. Methods The elutriated fraction 25 population was characterized based on density using a discontinuous gradient. The long-term reconstituting potential of whole bone marrow cells collected at each density interface was determined by subjecting the fractions to the two-day homing protocol, transplanting them into lethally irradiated recipient mice, and assessing peripheral blood chimerism. We also investigated the ability of high-density bone marrow cells isolated in conjunction with the ALDH protocol to repopulate the hematopoietic system of myeloablated recipients. Results Bone marrow cells collected at the high-density interface of 1.081/1.087 g/mL (fraction 3) had the capacity for homing to marrow and the ability to provide long-term hematopoietic reconstitution. Fraction three lineage-depleted ALDH-bright cells could also engraft and provide long-term hematopoiesis at limiting dilutions. Conclusions Density-gradient centrifugation can be used in conjunction with either of our stem cell isolation protocols to obtain cells with long-term reconstitution ability. We anticipate that this strategy will encourage and enable investigators to study the biology of HSCs isolated using functional characteristics.

Caspase-3 plays a critical role as an executioner of apoptosis. The aim of this study is to evaluate the potential of the combination of caspase-3 gene therapy and radiation treatment. We prepared a plasmid (pCI-CSP3) that contained the human caspase-3 gene and the cytomegalovirus promoter. We introduced this plasmid into U251 and U87 human glioma cells and subjected the cells to radiation treatment. The degree of cell death and apoptosis were evaluated. None of the cell lines underwent apoptosis by the overexpression of caspase-3 alone, but the degree of cell death and apoptosis were markedly enhanced by the addition of radiation treatment. Next, we prepared another plasmid (EGR-CSP3) that contained the caspase-3 gene and a radiation-sensitive promoter. Each treatment system using either pCI-CSP3 or EGR-CSP3 showed radio response. The treatment system using pCI-CSP3 more effectively induced apoptosis than that using EGR-CSP3. Caspase-3 gene therapy in combination with radiation treatment has the potential to serve as a radio-responsive gene therapy without any radiation-sensitive promoter.

Human embryonic stem cells (hESCs) self-renew indefinitely while maintaining pluripotency. The molecular mechanism underlying hESCs self-renewal and pluripotency is poorly understood. To identify the signaling pathway molecules that maintain the proliferation of hESCs, we performed a microarray analysis comparing an aneuploid H1 hESC line (named H1T) versus euploid H1 hESC line because the H1T hESC line demonstrates a self-renewal advantage while maintaining pluripotency. We find differential gene expression for the Nodal/Activin, fibroblast growth factor (FGF), Wnt, and Hedgehog (Hh) signaling pathways in the H1T line, which implicates each of these molecules in maintaining the undifferentiated state, whereas the bone morphogenic protein (BMP) and Notch pathways could promote hESCs differentiation. Experimentally, we find that Activin A is necessary and sufficient for the maintenance of self-renewal and pluripotency of hESCs and supports long-term feeder and serum-free growth of hESCs. We show that Activin A induces the expression of Oct4, Nanog, Nodal, Wnt3, basic FGF, and FGF8 and suppresses the BMP signal. Our data indicates Activin A as a key regulator in maintenance of the stemness in hESCs. This finding will help elucidate the complex signaling network that maintains the hESC phenotype and function.