Structural basis of RGD-hirudin binding to thrombin: Tyr3 and five C-terminal residues are crucial for inhibiting thrombin activity

Background

p300/CBP associating factor (PCAF, also known as KAT2B for lysine acetyltransferase 2B) is a catalytic subunit of megadalton metazoan complex ATAC (Ada-Two-A containing complex) for acetylation of histones. However, relatively little is known about the regulation of the enzymatic activity of PCAF.

Results

Here we present two dimeric structures of the PCAF acetyltransferase (HAT) domain. These dimerizations are mediated by either four-helical hydrophobic interactions or a ß-sheet extension. Our chemical cross-linking experiments in combined with site-directed mutagenesis demonstrated that the PCAF HAT domain mainly forms a dimer in solution through one of the observed interfaces. The results of maltose binding protein (MBP)-pulldown, co-immunoprecipitation and multiangle static light scattering experiments further indicated that PCAF dimeric state is detectable and may possibly exist in vivo.

Conclusions

Taken together, our structural and biochemical studies indicate that PCAF appears to be a dimer in its functional ATAC complex.     

Dose-escalation of human anti-interferon-α receptor monoclonal antibody MEDI-546 in subjects with systemic sclerosis: a phase 1, multicenter,open label study

Introduction

Type I interferons (IFNs) are implicated in the pathogenesis of systemic sclerosis (SSc). MEDI-546 is an investigational human monoclonal antibody directed against the type I IFN receptor. This Phase 1 study evaluated the safety/tolerability, pharmacokinetics (PK), immunogenicity, and pharmacodynamics (PD) of single and multiple intravenous doses of MEDI-546 in adults with SSc.

Methods

Subjects (≥18 years) with SSc were enrolled in an open-label, dose-escalation study to receive single (0.1, 0.3, 1.0, 3.0, 10.0, or 20.0 mg/kg), or 4 weekly intravenous doses (0.3, 1.0, or 5.0 mg/kg/week) of MEDI-546. Subjects were followed for 12 weeks. Safety assessments included adverse events (AEs), laboratory results, and viral monitoring. Blood samples were collected from all subjects for determination of PK, presence of anti-drug antibodies (ADAs), and expression of type I IFN-inducible genes.

Results

Of 34 subjects (mean age 47.4 years), 32 completed treatment and 33 completed the study. Overall, 148 treatment-emergent AEs (TEAEs) were reported (68.9% mild, 27.7% moderate). TEAEs included one grade 1 infusion reaction (5.0 mg/kg/week multiple dose). Of 4 treatment-emergent serious AEs (skin ulcer, osteomyelitis, vertigo, and chronic myelogenous leukemia (CML)), only CML (1.0 mg/kg/week multiple dose) was considered possibly treatment-related. MEDI-546 exhibited non-linear PK at lower doses. ADAs were detected in 5 subjects; no apparent impact on PK was observed. Peak inhibition of the type I IFN signature in whole blood was achieved within 1 day and in skin after 7 days.

Conclusion

The safety/tolerability, PK, and PD profiles observed in this study support further clinical development of MEDI-546.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00930683","term_id":"NCT00930683"}}NCT00930683     

Improvement and Optimization of Standards for a Preclinical Animal Test Model of Laser Induced Choroidal Neovascularization

Background

As the murine model of laser-induced choroidal neovascularization (CNV) is becoming the most established and commonly utilized model worldwide for studying the pathogenesis of CNV and its response to treatment, specific operating standards are yet to be clarified. The purpose of this study is to compare the lesion size of CNV in mice with different ages, sex, durations of CNV process, and treated positions of laser spots, to make recommendations that may improve and optimize the quality of the model.

Methods and Results

C57/BL6 mice of different ages were treated with diode laser photocoagulation per eye and perfused with PBS containing fluorescein-labeled dextran at different time of observation. Choroid flat mounts, were then examined by fluorescence microscopy for the measurement of CNV area. Messenger-RNA expression levels of several angiogenic cytokines in eye cups of male and female C57BL/6 mice at 5–8 and 16–20 week-old were analyzed by real-time RT-PCR assay. The results showed significantly more CNV area in eyes of female mice compared to male mice with the expression level of several angiogenic cytokines elevated. 16–20-week-old female mice developed the biggest area of CNV. The mean area of CNV increased significantly at the 14^th day after photocoagulation. Laser spots delivered 1PD away from the optic disc induced the biggest area of CNV compared to those 2PD or 3PD away. Interaction of NV was observed in laser spots delivered less than 1PD away from each other.

Conclusion

The current results suggest that 16–20-week-old female C57BL/6 mice developed the most distinct CNV lesion size with laser spots delivered 1PD away from the optic disc. The best time to observe and analyze is the 14^th day after photocoagulation.     

Diagnostic Accuracy of the PURE-LAMP Test for Pulmonary Tuberculosis at the County-Level Laboratory in China

Background

Early and effective detection of Mycobacterium tuberculosis (MTB), particularly in smear-negative tuberculosis (TB), is a priority for global TB control. Loop-mediated isothermal amplification with a procedure for ultra rapid DNA extraction (PURE-LAMP) can detect TB in sputum samples rapidly and with high sensitivity and specificity. However, the PURE-LAMP test has not been effectively evaluated, especially in resource-limited laboratories. In this study, we evaluated the performance of the PURE-LAMP test for TB detection in TB suspects from two county-level TB dispensaries in China.

Methodology/Principal Findings

From April 2011 to February 2012, patients with suspected TB were continuously enrolled from two county-level TB laboratories in China. Three sputum samples (spot, night, and morning sputum) were collected from each recruited patient. Detection of MTB by PURE-LAMP was compared to a reference standard L-J culture. The results showed that the sensitivity of the PURE-LAMP test based on spot sputum for MTB detection was 70.67%, while the sensitivity of the PURE-LAMP test based on spot sputum for MTB detection in smear positive and culture positive patients and smear negative and culture positive patients was 92.12% and 53.81%, respectively. The specificity of PURE-LAMP based on spot sputum for MTB detection was 98.32%. The sensitivity and specificity of the PURE-LAMP test based on three sputa combination for MTB detection was 88.80% and 96.86%, respectively. The results also showed that the PURE-LAMP test had a significantly lower contamination rate than did solid culture.

Conclusions/Significance

The study suggested that, in peripheral-level TB laboratories in China, the PURE-LAMP test showed high sensitivity and specificity for TB detection in TB suspects, making it a more effective, rapid, and safe method worthy of broader use in the future.     

Insight of the Cytotoxicity of the Aggregates of Peptides or Aberrant Proteins: A Meta-Analysis

Aberrant proteins or peptide aggregates form soluble oligomers or nanofibrils that can cause a wide range of amyloidosis diseases, including Alzheimer''s disease (AD). The mechanisms of their cytotoxicity, however, remain controversial and poorly understood, greatly hindering the development of AD drugs. Here we report a comprehensive evaluation of the cytotoxicity of the aggregates by meta-analysis. The analysis indicates that the cytotoxicity of the aggregates converges in a narrower range in the mass concentrations than in the molar concentrations, suggesting that it is the weight of the aggregates rather than the number of the molecules that dictates the cytotoxicity. This new perspective implies that these aggregates are likely to have non-specific interactions with cells to cause cell death. The comparison of several existing theories regarding cellular volumes supports that the aggregates may result in crowding effect and increase the free energy, thus resulting in instability of the cells.