Search

The predominant mechanism driving hyperuricemia in gout is renal uric acid underexcretion; however, the standard urate-lowering therapy (ULT) recommendation is first-line xanthine oxidase inhibitor (XOI), irrespective of the cause of hyperuricemia. This comparative effectiveness clinical trial was undertaken to compare first-line nontitrated low-dose benzbromarone (LDBen) uricosuric therapy to XOI ULT with low-dose febuxostat (LDFeb) in gout patients with renal uric acid underexcretion.We conducted a prospective, randomized, single-center, open-label trial in men with gout and renal uric acid underexcretion (defined as fractional excretion of urate5.5% and uric acid excretion ≤600 mg/day/1.73 mMore participants in the LDBen group achieved the serum urate target than those in the LDFeb group (61% compared to 32%, P 0.001). Rates of adverse events, including gout flares and urolithiasis, did not differ between groups, with the exception of greater transaminase elevation in the LDFeb group (4% for LDBen compared to 15% for LDFeb, P = 0.008).Compared to LDFeb, LDBen has superior urate-lowering efficacy and similar safety in treating relatively young and healthy patients with renal uric acid underexcretion-type gout.

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.

Objective To investigate the incidence and potential risk factors for development of fenofibrate-associated nephrotoxicity in gout patients. Methods A total of 983 gout patients on fenofibrate treatment who visited the dedicated Gout Clinic at the Affiliated Hospital of Qingdao University between September 2016 and June 2020 were retrospectively enrolled from the electronic records system. Fenofibrate-associated nephrotoxicity was defined as an increase in serum creatinine (SCr) ≥0.3 mg/dl within 6 months of fenofibrate initiation. The change trend of SCr and uric acid levels during the treatment period were assessed by a generalised additive mixed model (GAMM). Multivariate analysis was performed for risk factors affecting elevated SCr. Results A total of 100 (10.2%) patients experienced an increase in SCr ≥0.3 mg/dl within 6 months after fenofibrate initiation. The median change of SCr in the whole cohort was 0.11 mg/dl [interquartile range (IQR) 0.03–0.20], whereas it was 0.36 (0.33–0.45) in the fenofibrate-associated nephrotoxicity group. In a multivariable regression model, chronic kidney disease (CKD) [odds ratio (OR) 2.39 (95% CI 1.48, 3.86)] and tophus [OR 2.29 (95% CI 1.39, 3.78)] were identified to be risk predictors, independent of measured covariates, of fenofibrate-associated nephrotoxicity. During the treatment period, although SCr temporarily increased, serum urate and triglyceride concentrations decreased using the interaction analysis of GAMM. Of those with fenofibrate withdrawal records, the SCr increase in 65% of patients was reversed after an average of 49 days off the drug. Conclusions This observational study implied that fenofibrate-associated nephrotoxicity occurs frequently in gout patients, especially in patients with tophi or CKD. The potential renal risks of fenofibrate usage in gout needs additional research.

Terminal complement inhibition is the standard of care for atypical hemolytic uremic syndrome (aHUS). The optimal duration of complement inhibition is unknown, although indefinite therapy is common. Here, we present the outcomes of a physician-directed eculizumab discontinuation and monitoring protocol in a prospective cohort of 31 patients that started eculizumab for acute aHUS (and without a history of renal transplant). Twenty-five (80.6%) discontinued eculizumab therapy after a median duration on therapy of 2.37 (interquartile range: 1.06, 9.70) months. Eighteen patients discontinued per protocol and 7 because of nonadherence. Of these, 5 (20%) relapsed; however, relapse rate was higher in the case of nonadherence (42.8%) vs clinician-directed discontinuation and monitoring (11.1%). Four of 5 patients who relapsed were successfully retreated without a decline in renal function. One patient died because of recurrent aHUS and hypertensive emergency in the setting of nonadherence. Nonadherence to therapy (odds ratio, 8.25; 95% confidence interval, 1.02-66.19; P = .047) was associated with relapse, whereas the presence of complement gene variants (odds ratio, 1.39; 95% confidence interval, 0.39-4.87; P = .598) was not significantly associated with relapse. Relapse occurred in 40% (2 of 5) with a CFH or MCP variant, 33.3% (2 of 6) with other complement variants, and 0% (0 of 6) with no variants (P = .217). There was no decline in mean glomerular filtration rate from the date of stopping eculizumab until end of follow-up. In summary, eculizumab discontinuation with close monitoring is safe in most patients, with low rates of aHUS relapse and effective salvage with eculizumab retreatment in the event of recurrence.

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.

Objectives Internal fixation with multiple cannulated compression screws is an optional treatment for femoral neck fracture. Recently, fully threaded cannulated compression screws (FTCCS) have been introduced to fix fresh femoral neck fractures (FNF). The purpose of this study was to investigate the effectiveness of FTCCS. Patients and methods Patients with FNF fixed by multiple FTCCS from February 1st, 2014 to August 31st, 2017 were included in this study. They were followed for at least 12 months postoperatively. Nonunion, osteonecrosis of the femoral head (ONFH), fixation failure, reoperation, and femoral neck shortening (FNS) were used to evaluate the outcomes. Risk factors including age, sex, fracture side, fracture displacement, fracture stability, fixation configuration, and screw numbers were analyzed. Results A total of 113 patients including 67 males and 46 females with an average age of 48.4 ± 13.4 years were included. The mean duration of follow-up was 27.1 months (range: 12–51 months). The incidence of nonunion, ONFH, fixation failure, and reoperation was 15.9%, 22.1%, 8.8%, and 24.8%, respectively. The rates of nonunion and reoperation were significantly higher in displaced fractures and unstable fractures. And patients with an unstable fracture had a higher risk of internal fixation failure. The median length of FNS was 2.9 mm (interquartile range: 0.9–6.5 mm, range: 0–17.5 mm). Age was a significant risk factor for FNS. Conclusions The screw fixation method with FTCCS provided encouraging clinical results which may be a rational choice for the treatment of fresh FNF. Displaced fractures and unstable fractures were attributed to the higher incidence of complications. Trial registration: ChiCTR, ChiCTR1800017200. Registered 17 July 2018-Retrospectively registered, http: www.chictr.org.cn/showprojen.aspx?proj=29182.

Background Low doses of febuxostat or benzbromarone are widely used in Asian countries, but lacking studies to compare the efficacy and safety of the two urate-lowering drugs. Methods To compare the efficacy and safety of low-dose febuxostat with low-dose benzbromarone in patients with primary gout, a randomized controlled, open-label trial was performed among male patients with primary gout for urate-lowering therapy (ULT) in a dedicated gout clinic in China. Randomization was carried out by a third-party institution according to random number table. Patients were randomly assigned 1:1 to febuxostat group (Feb group) (20 mg daily) or benzbromarone group (Ben group) (25 mg daily) and treated for 12 weeks. General information and biochemical data were collected at baseline and at every visit monthly. Clinical characteristics before and after the ULT were analyzed in the two groups by SPSS and EmpowerStats software. Results Two hundred forty patients were enrolled and randomized in the two groups, with 214 patients completing 12 weeks’ ULT (105 in the Feb group and 109 in the Ben group). After 12 weeks, substantial percentages of patients in both Feb and Ben group achieved the target serum uric acid (sUA) (

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.

OBJECTIVE: To investigate the risk factors for hypoglycemia after birth in preterm infants with a gestational age of ≤ 32 weeks. METHODS: A retrospective analysis was performed for 86 neonates with hypoglycemia and a gestational age of ≤ 32 weeks who were admitted to the neonatal intensive care unit from January 2017 to June 2020 (hypoglycemia group). A total of 172 preterm infants with normal blood glucose who were hospitalized during the same period were randomly enrolled as the control group. Univariate analysis and multivariate logistic regression analysis were used to screen out the risk factors for hypoglycemia in preterm infants. RESULTS: There were 515 preterm infants during the study, among whom 86 (16.7%) had hypoglycemia. Compared with the control group, the hypoglycemia group had significantly higher percentages of small for gestational age (SGA), cesarean section, maternal hypertension, and antenatal steroid administration (P < 0.05), but significantly lower birth weight and rate of intravenous glucose use before blood glucose test (P < 0.05). SGA (OR=4.311, 95% CI:1.285-14.462, P < 0.05), maternal hypertension (OR=2.469, 95% CI:1.310-4.652, P < 0.05), and antenatal steroid administration (OR=6.337, 95% CI:1.430-28.095, P < 0.05) were risk factors for hypoglycemia in preterm infants, while intravenous glucose use (OR=0.318, 95% CI:0.171-0.591, P < 0.05) was a protective factor against hypoglycemia in preterm infants. CONCLUSIONS: SGA, maternal hypertension, and antenatal steroid administration may increase the risk of early hypoglycemia in preterm infants with a gestational age of ≤ 32 weeks, and intravenous glucose use is recommended as soon as possible after birth for preterm infants with a gestational age of ≤ 32 weeks to reduce the incidence rate of hypoglycemia.

Bisphenols (BPs) are distributed in worldwide as typical environmental hormones, which potentially harm the ecological environment and human health. In this study, four BPs, i.e., bisphenol A, bisphenol F, bisphenol S, and bisphenol AF, were used as prototypes to identify the intrinsic differences in degradation mechanisms correlated with the molecular structures in peroxydisulfate (PDS)-based advanced oxidation processes (AOPs). Electron transfer was the main way of modified biochar to trigger the heterogenous catalysis of PDS, which can cause the degradation of BPs. Phenolic hydroxyl groups on bisphenol pollutants were considered as possible active sites, and the existence of substituents was the main reason for the differentiation in the degradation efficiency of various bisphenols. Results of ecotoxicity prediction showed that most intermediates produced by the degradation of BPs in the β-SB/PDS system, which was dominated by the electron transfer pathway, had a lower toxicity than the parent molecules, while the toxicity of several ring cleavage intermediates was higher. This study presents a simple modification scheme for the conversion of biochar into functional catalysts and provides insights into the mechanism of heterogeneous catalytic degradation mediated by modified biochar as well as the degradation differences of bisphenol pollutants and their potential ecotoxicity.

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.

Data analysis and management of huge volumes of medical data have attracted enormous attention, since discovering knowledge from the data can benefit both caregivers and patients. In this paper, we focus on learning disease labels from medical data of patients in Intensive Care Units (ICU). Specifically, we extract features from two main sources, medical charts and notes. We apply the Bag-of-Words (BoW) model to encode the features. Different from most of the existing multi-label learning algorithms that take correlations among diseases into consideration, our model learns disease specific features to benefit the discrimination of different diseases. To achieve this, we first construct features specific to each disease by conducting clustering analysis on its positive and negative instances, and then perform training and testing by querying the clustering results. Extensive experiments have been conducted on a real-world Intensive Care Units (ICU) database. Evaluation results have shown that our proposed method has better performance against all other compared multi-label learning methods.

Adult-onset Still's disease (AOSD) is a rare autoinflammatory condition with unclear aetiology and highly heterogeneous manifestations, such as spiking fever, skin rash and arthralgia (or arthritis).1 2 AOSD is usually the result of both genetic and environmental factors. However, only a few genetic loci have been associated with AOSD, and none of them have reached the threshold for genome-wide significance (GWS) of p

BACKGROUND Chronic rhinosinusitis with nasal polyps (CRSwNP) is a highly heterogeneous disease and can be categorized into eosinophilic CRSwNP (eCRSwNP) and non-eosinophilic CRSwNP (neCRSwNP). Exploring effective biomarkers to distinguish endotypes is important for personalized therapies. The present study aims to evaluate the predictive value of serum MIF in CRSwNP endotypes. METHODS One hundred and twenty CRSwNP patients, including 51 eCRSwNP and 69 neCRSwNP, 40 chronic rhinosinusitis without nasal polyps (CRSsNP) patients and 40 healthy controls (HC) were enrolled. Serum MIF levels were determined by enzyme-linked immunosorbent assay (ELISA). The patients' clinical variables were analyzed for correlations with serum MIF. Receiver operating characteristic (ROC) curve and multivariate analysis were utilized to assess the predictive value of serum MIF in CRSwNP endotypes. RESULTS The serum MIF levels were significantly higher in CRSwNP group than CRSsNP group and HC group (P

Cadmium (Cd) and lead (Pb) are toxic heavy metals with endocrine-disrupting properties. We investigated the associations of low-level environmental exposure to Cd/Pb and gout status (intercritical gout, gout flare and combined gout) in a cohort study. We measured by ICP-MS the levels of Cd and Pb in blood (Cd-B and Pb-B) and urine (Cd-U and Pb-U) from 408 participants with blood and 346 participants with urine samples recruited from a hospital gout clinic. The median levels of Cd-B and Pb-B (in μg/L) in the gout flare group were 0.87 (range 0.41-2.49) and 31.54 (25.38-41.46), respectively, and the median levels of Cd-U and Pb-U in the gout flare group were 1.05 (0.69-1.91) and 3.86 (3.49-4.44), respectively. These medians were significantly higher than those in the control or intercritical groups (P 0.05). For Cd-B in tertile 2 (T2) and Cd-U in tertile 3, Cd levels were significantly associated with gout flare status compared to the reference tertile 1 (OR = 4.3, P = 0.041 and OR = 25.1, P = 0.002, respectively) after adjustment under Model 3. For Pb-U, the risk of gout flare status was significantly higher in T2 (OR = 51.0, P = 0.002) compared to the T1 under Model 3. Our results show that median levels of Cd-B, Pb-B, Cd-U and Pb-U in the gout flare group were significantly higher than participants without gout or with gout but in the intercritical period. We provide evidence that the risk of gout flare status is associated with increased Cd levels, and that blood and urine levels of Cd are a risk factor for gout flare status.

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.

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.

Thirteen cationic peptidomimetics derived from amino acids bearing an alkyl or ethynylphenyl moiety that mimic the structure of cationic antibacterial peptides were designed and synthesized using a simple coupling reaction of an amino acid with a substituted amine. Antibacterial activities of the resulting peptidomimetics against drug-sensitive bacteria, such as Gram-positive Staphylococcus aureus (S. aureus) and Bacillus subtilis, Gram-negative Escherichia coli (E. coli) and Salmonella enterica, and a drug-resistant bacterium, methicillin-resistant S. aureus (MRSA), were systematically evaluated. Most peptidomimetics show significant broad-spectrum antibacterial activity. A-L-Iso-C12 (isoleucine derivative bearing a dodecyl moiety) show MICs of 2.5 μg/mL against S. aureus and 4 μg/mL against MRSA and A-L-Val-C12 (valine derivative bearing a dodecyl moiety) show MICs of 1.67 μg/mL against E. coli and 8.3 μg/mL against MRSA. A-L-Val-C12 showed low cytotoxicity toward L929 cells in comparison with SGC 7901 cells, indicating tumor-directed killing by peptidomimetics while avoiding toxicity to normal cells. The influences of type of amino acid and substituent, length of substituent, and stereochemistry of amino acids on antibacterial activity and cytotoxicity of peptidomimetics were systematically investigated. The results indicate that this series of cationic peptidomimetics derived from amino acids display antitumor activity and may be useful for treatment of bacterial infections.

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.

BACKGROUND. HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome is a severe variant of hypertensive disorders of pregnancy affecting approximately 1% of all pregnancies, and has significant maternal and fetal morbidity. Previously, we showed that upregulation of the alternative pathway of complement (APC) plays a role in HELLP syndrome. We hypothesize that HELLP syndrome follows a 2-hit disease model similar to atypical hemolytic uremic syndrome (aHUS), requiring both genetic susceptibility and an environmental risk factor. Our objective was to perform a comparative analysis of the frequency of APC activation and germline mutations in affected women and to create a predictive model for identifying HELLP syndrome. METHODS. Pregnant women with HELLP syndrome, and healthy controls after 23 weeks of gestation were recruited, along with aHUS and thrombotic thrombocytopenic purpura participants. We performed a functional assay, the mHam, and targeted genetic sequencing in all groups. RESULTS. Significantly more participants with rare germline mutations in APC genes were present in the HELLP cohort compared with controls (46% versus 8%, P = 0.01). In addition, significantly more HELLP participants were positive for the mHam when compared with controls (62% versus 16%, P = 0.009). Testing positive for both a germline mutation and the mHam was highly predictive for the diagnosis of HELLP syndrome. CONCLUSION. HELLP syndrome is characterized by both activation of the APC and frequent germline mutations in APC genes. Similar to aHUS, treatment via complement inhibition to mitigate maternal and fetal morbidity and mortality may be possible. FUNDING. National Heart Lung and Blood Institute grants T32HL007525 and R01HL133113.

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.

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.

Aims The alteration of ROS level is frequently observed in the course of morphine addiction, and ROS is proverbially involved in this process. This study aims to explore the relationship among morphine addiction, reactive oxygen species (ROS) and expression of μ-opioid receptor (MOR) in differentiated SH-SY5Y cells. Main methods SH-SY5Y cells were induced to differentiation by treatment with retinoic acid (RA); the activity of lactate dehydrogenase (LDH) and the nitro blue tetrazolium (NBT) reduction were assessed by spectrophotometry. Intracellular reactive oxygen species (ROS) was measured with the 2,7-dichlorofluorescin diacetate (DCFH-DA) assay. Cellular cAMP was determined by using a competitive protein binding kit. The mRNA expression of μ-opioid receptor (MOR) was evaluated by qRT-PCR. Key findings Morphine-induced ROS are generated in a concentration- and time-dependent manner and inhibited by naloxone. Exogenous oxidants increase the level of ROS and aggravate morphine addiction, while the exogenous antioxidants efficiently reverse these effects. Morphine decreases the mRNA level of MOR in a concentration-dependent manner. And the mRNA level of MOR is remarkably reduced in the presence of exogenous oxidants and effectively promoted by antioxidants. Significance This study indicates that ROS can affect morphine addiction through involving MOR. Treatment with ROS scavenging can serve as a medical therapy for morphine addiction.

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.

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.

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.

Isoliquiritigenin (ISL), a licorice chalconoid, is a bioactive agent with chemopreventive potential that has been patented for tumor treatment in China. This study investigated the mechanisms of ISL-induced apoptosis in ovarian carcinoma SKOV-3 cells. Cell viability was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. The apoptotic rate was determined via flow cytometry using an annexin V-FITC apoptosis detection kit. The intracellular reactive oxygen species (ROS) levels were assessed using a 2,7-dichlorofluorescein probe assay. Malondialdehyde (MDA) formation was determined via thiobarbituric acid reactive substance test. The expressions of growth arrest and DNA damage-inducible gene (GADD153/CHOP), 78 kDa glucose-regulated protein (GRP 78), α-subunit of eukaryotic initiation factor 2 (eIF2α) phosphorylation, activating transcription factor 6α (ATF6α), and unspliced form of X-box binding protein1 (XBP1U) were analyzed via Western blot. Caspase-3 and caspase-12 activities were assessed using a fluorometric kit. Findings indicate that ISL significantly inhibits SKOV-3 cell proliferation, increases intracellular ROS levels, and causes SKOV-3 cell apoptosis. Moreover, ISL-exposed SKOV-3 cells trigger endoplasmic reticulum (ER) stress, as indicated by the enhancement of ER stress-related molecules p-eIF2α, GADD153/CHOP, GRP78, XBP1 expression, and cleavage of ATF6α. However, caspase-12 inhibitor (Z-ATAD) effectively and partially prevents ROS and MDA formation and inhibits ISL-induced SKOV-3 cell apoptosis. ISL induces apoptosis via ER stress-triggered signaling pathways in SKOV-3 cells. ER stress-induced cancer cell apoptosis has been discussed in some patents.

Introduction: HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets) is a severe variant of preeclampsia that leads to severe morbidity and mortality to both the mother and fetus. Delivery is the treatment of choice at or beyond 34 weeks, but there are no good therapies for pregnant women with HELLP before 34 weeks of pregnancy. Although there are no strict criteria for its diagnosis, the Tennessee and Mississippi classifications have been proposed using platelet count (600 IU/L), bilirubin (>1.2 mg/dL) and aspartate aminotransferase (AST) with or without alanine aminotransferase (ALT) levels (AST >40 or 70 IU/L, ALT>40 IU/L). However, LDH, AST and bilirubin are not specific for liver dysfunction and their elevation may be caused by intravascular hemolysis alone. Recent evidence and clinical similarities suggest a link to atypical hemolytic uremic syndrome (aHUS), a disease of excessive alternative complement pathway (APC) activation. To test this hypothesis we utilized a functional complement assay, the modified Ham test recently described for aHUS diagnosis. Method: Women with classic HELLP, atypical HELLP, preeclampsia with severe features, and women with normal pregnancies were recruited for the study from September 1, 2014 to May 31, 2015. Women with known sickle cell disease, systemic lupus erythematous, antiphospholipid antibody syndrome, or previous diagnosed microangiopathic and hemolytic diseases were excluded. All participants were greater than 23 weeks pregnant. Classic HELLP syndrome was defined as satisfying all Mississippi or Tennessee criteria for HELLP syndrome; while atypical HELLP as having at least one laboratory abnormality in the Mississippi or Tennessee criteria. Sera was collected and sent blinded to the laboratory. APC activation was detected in the modified Ham test and compared to previously described marker of complement activation, serum C5b-9 levels. Furthermore, we tested the in vitro ability of eculizumab to inhibit APC activation. Eculizumab containing serum was collected from a patient with paroxysmal nocturnal hemoglobinuria (PNH) within 60 minutes after the infusion. Results: Serum from 9 women with classic or atypical HELLP, 7 women with severe preeclampsia, and 11 controls (healthy pregnancy) were tested. We found no significant difference in serum C5b-9 levels among patients with HELLP, preeclampsia and controls (p=0.808). However, increased complement activation in the modified Ham test, represented as significantly higher percentage of non-viable cells/cell killing (25.7±19.8% versus 4.1±7.3%, p=0.005), was found in participants with classic or atypical HELLP compared to participants with healthy pregnancy (Figure 1A). Participants with classic HELLP demonstrated significantly more cell killing when compared to severe preeclampsia (38.7+9.8% versus 13.0+11.7, p=0.048). In the ROC (receiver operating curve) analysis, a percentage of killing higher than 20.5% was determined as a cut-off value for the diagnosis of HELLP with 66.7% sensitivity and 88.9% specificity. Importantly, mixing sera from 4 HELLP patients with eculizumab containing serum in different percentages resulted in a significant decrease of cell killing compared to HELLP serum alone (Figure 1B, p=0.007). Conclusions: We have shown that preeclampsia with severe features along with classic and atypical HELLP syndrome may be considered, at least in part, a disease of excessive complement activation. The modified Ham test is a serum-based assay that does not theoretically detect increased complement activation caused by mutations in cell membrane factors. Other pathophysiological mechanisms beyond complement activation may also account for the negative results in 3 HELLP participants. Importantly, the modified Ham test seems a promising tool to identify patients with increased complement activation who may benefit from complement inhibition. If confirmed in a larger cohort, this rapid, inexpensive and highly specific assay may be valuable to select patients for such a clinical trial. Disclosures Brodsky: Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

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.

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.

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy (TMA) characterized by excessive activation of the alternative pathway of complement (APC). Atypical HUS is frequently a diagnosis of exclusion. Differentiating aHUS from other TMAs, especially thrombotic thrombocytopenic purpura (TTP), is difficult due to overlapping clinical manifestations. We sought to develop a novel assay to distinguish aHUS from other TMAs based on the hypothesis that paroxysmal nocturnal hemoglobinuria cells are more sensitive to APC-activated serum due to deficiency of glycosylphosphatidylinositol- anchored complement regulatory proteins (GPI-AP). Here, we demonstrate that phosphatidylinositol-specific phospholipase C-treated EA.hy926 cells and PIGA-mutant TF-1 cells are more susceptible to serum from aHUS patients than parental EA.hy926 and TF-1 cells. We next studied 31 samples from 25 patients with TMAs, including 9 with aHUS and 12 with TTP. Increased C5b-9 deposition was evident by confocal microscopy and flow cytometry on GPI-AP-deficient cells incubated with aHUS serum compared with heat-inactivated control, TTP, and normal serum. Differences in cell viability were observed in biochemically GPI-AP-deficient cells and were further increased in PIGA-deficient cells. Serum from patients with aHUS resulted in a significant increase of nonviable PIGA-deficient TF-1 cells compared with serum from healthy controls (P < .001) and other TMAs (P < .001). The cell viability assay showed high reproducibility, sensitivity, and specificity in detecting aHUS. In conclusion, we developed a simple, rapid, and serum-based assay that helps to differentiate aHUS from other TMAs.

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.

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.

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.

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.

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.

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.

This study is to investigate the mechanism of human promyelocytic leukemia HL-60 cells proliferation induced by alteronol in vitro. Human promyelocytic leukemia HL-60 cells cultured in vitro were treated with different concentrations of alteronol. Inhibition rate was detected by SRB assay. Cellular morphological changes were observed by Hoechst and AO/EB (acridine orange/ethidium bromide dye) staining. The apoptosis rate was determined by Annexin V-FITC/PI assay. Cell cycle distribution was determined by flow cytometry. Western blotting analysis was carried out to determine the cell cycle related proteins. The proliferation of HL-60 cells treated with alteronol was inhibited in a concentration-dependent manner. Based on cell viability assay, observation on cell morphology and apoptosis rate, it confirmed that alteronol played an obvious role in proliferation inhibition of human promyelocytic leukemia HL-60 cells, but it did not induce apoptosis in human promyelocytic leukemia HL-60 cells in different concentrations groups. Alteronol could effectively inhibit the proliferation of human promyelocytic leukemia HL-60 cells inducing cell cycle arrest at G1 phase, as well as, alteration expression of cell cycle proteins level of CyclinD1 and pRb.

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.

Isoliquiritigenin (ISL), a licorice chalconoid, is considered to be a bioactive agent with chemopreventive potential. This study investigates the mechanisms involved in ISL-induced apoptosis in human cervical carcinoma HeLa cells.Cell viability was evaluated using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay. Apoptosis was determined by flow cytometry using an Annexin V-FITC Apoptosis Detection Kit. The intracellular ROS levels were assessed using a 2, 7-dichlorofluorescein probe assay. The mitochondrial membrane potential was measured with the dual-emission potential-sensitive probe 5, 5', 6, 6'-tetra-chloro-1, 1', 3, 3'-tetraethyl-imidacarbocyanine iodide (JC-1). The degradation of poly-ADP-ribose polymerase (PARP) protein, the phosphorylation of PKR-like ER kinase (PERK), the phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α), the expression of the 78 kD glucose-regulated protein (GRP 78), and the activation of caspase-12 were analyzed via western blot analysis.ISL significantly inhibited the proliferation, the increase in ROS levels and apoptotic rates of HeLa cells in a concentration-dependent manner. Moreover, ISL induced mitochondrial dysfunction, caspase activation, and PARP cleavage, which displayed features of mitochondria dependent on apoptotic signals. Besides, exposure of HeLa cells to ISL triggered endoplasmic reticulum (ER) stress, as indicated by the increase in p-eIF2α and GRP78 expression, ER stress-dependent apoptosis is caused by the activation of ER-specific caspase-12.The findings from our study suggest that ISL-induced oxidative stress causes HeLa cell apoptosis via the mitochondrion-dependent and the ER stress-triggered signaling pathways.

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.

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.