Expression, purification and crystallization of the (3R)-hydroxyacyl-ACP dehydratase HadAB complex from Mycobacterium tuberculosis

AbstractThe (3R)-hydroxyacyl-ACP dehydratase HadAB, involved in the biosynthetic pathway for mycolic acid (MA) of Mycobacterium tuberculosis, catalyzes the third step in the fatty acid (FA) elongation cycle, which is an ideal and actual target for anti-tubercular agent. Though HadAB is predicted to be a member of the hotdog superfamily, it shares no sequence identity with typical hotdog fold isoenzyme FabZ. To characterize the significance of HadAB from the perspective of structural biology, large amount of pure HadAB complex is required for biochemical characterization and crystallization. Here, we used a unique expression and purification method. HadA and HadB were cloned separately and co-expressed in Escherichia coli. After GST affinity chromatography, two steps of anion exchange chromatography and gel filtration, the purity of the protein as estimated by SDS–PAGE was >95%. Using hanging-drop vapor-diffusion method, crystals were obtained and diffracted X-rays to 1.75Å resolution. The crystal belongs to space group P41212, with unit-cell parameters a=b=82.0Å, c=139.8Å, α=β=γ=90.0°

Precursors and Pathways Leading to Enhanced Secondary Organic Aerosol Formation during Severe Haze Episodes

Publisher Copyright: © 2021 American Chemical SocietyMolecular analyses help to investigate the key precursors and chemical processes of secondary organic aerosol (SOA) formation. We obtained the sources and molecular compositions of organic aerosol in PM2.5in winter in Beijing by online and offline mass spectrometer measurements. Photochemical and aqueous processing were both involved in producing SOA during the haze events. Aromatics, isoprene, long-chain alkanes or alkenes, and carbonyls such as glyoxal and methylglyoxal were all important precursors. The enhanced SOA formation during the severe haze event was predominantly contributed by aqueous processing that was promoted by elevated amounts of aerosol water for which multifunctional organic nitrates contributed the most followed by organic compounds having four oxygen atoms in their formulae. The latter included dicarboxylic acids and various oxidation products from isoprene and aromatics as well as products or oligomers from methylglyoxal aqueous uptake. Nitrated phenols, organosulfates, and methanesulfonic acid were also important SOA products but their contributions to the elevated SOA mass during the severe haze event were minor. Our results highlight the importance of reducing nitrogen oxides and nitrate for future SOA control. Additionally, the formation of highly oxygenated long-chain molecules with a low degree of unsaturation in polluted urban environments requires further research.Peer reviewe

Capsaicin Protects Mice from Community-Associated Methicillin-Resistant Staphylococcus aureus Pneumonia

BACKGROUND: α-toxin is one of the major virulence factors secreted by most Staphylococcus aureus strains, which played a central role in the pathogenesis of S. aureus pneumonia. The aim of this study was to investigate the impact of capsaicin on the production of α-toxin by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain USA 300 and to further assess its performance in the treatment of CA-MRSA pneumonia in a mouse model. METHODOLOGY/PRINCIPAL FINDINGS: The in vitro effects of capsaicin on α-toxin production by S. aureus USA 300 were determined using hemolysis, western blot, and real-time RT-PCR assays. The influence of capsaicin on the α-toxin-mediated injury of human alveolar epithelial cells was determined using viability and cytotoxicity assays. Mice were infected intranasally with S. aureus USA300; the in vivo protective effects of capsaicin against S. aureus pneumonia were assessed by monitoring the mortality, histopathological changes and cytokine levels. Low concentrations of capsaicin substantially decreased the production of α-toxin by S. aureus USA 300 without affecting the bacterial viability. The addition of capsaicin prevented α-toxin-mediated human alveolar cell (A549) injury in co-culture with S. aureus. Furthermore, the in vivo experiments indicated that capsaicin protected mice from CA-MRSA pneumonia caused by strain USA 300. CONCLUSIONS/SIGNIFICANCE: Capsaicin inhibits the production of α-toxin by CA-MRSA strain USA 300 in vitro and protects mice from CA-MRSA pneumonia in vivo. However, the results need further confirmation with other CA-MRSA lineages. This study supports the views of anti-virulence as a new antibacterial approach for chemotherapy

Evaluation of the antioxidant effects of different histone deacetylase inhibitors (HDACis) on human lens epithelial cells (HLECs) after UVB exposure

Abstract Background To compare the protective effects of the histone deacetylase inhibitors (HDACis) β-hydroxybutyrate (βOHB), trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA) on human lens epithelial cells(HLECs) following ultraviolet-B (UVB) exposure. Methods HLECs were divided into subgroups: four HDACi groups, a control group, a UVB-treated group and a DMSO group (cells treated with DMSO and UVB irradiation). In the HDACi groups, HLECs were cultured with different concentrations of HDACis 12 h prior to UVB irradiation. The protective effects of the HDACis were evaluated by assessing apoptosis rates, cell activity and expression levels of genes associated with apotosis (caspase-3, Bcl-2, BAX, SOD1, FOXO3A and MT2). The levels of superoxide dismutase (SOD), reactive oxygen species (ROS), malondialdehyde (MDA) and total antioxidant capacity (T-AOC) were detected in order to evaluate oxidative stress. Results The results showed that SAHA (1 μmol/L, 2 μmol/L) and TSA (0.2 μmol/L) had mild protective effects on cell viability. βOHB (4 mmol/L) and TSA (0.2 mol/L) demonstrated protective effects on BCL-2 expression. TSA (0.2 mol/L) showed protective effects on SOD1 expression. TSA (0.2 mol/L) and SAHA (1 μmol/L) suppressed BAX and caspase-3 expression. TSA (0.2 mol/L, 0.8 mol/L) and SAHA (1 μmol/L, 2 μmol/L) suppressed the expression of FOXO3A and MT2. SOD levels were increased after treatment with βOHB (4 mmol/L), SAHA (8 μmol/L) and TSA (0.1 mol/L, 0.2 mol/L). T-AOC levels were increased in UVB-treated HLECs after treatment with SAHA (2 μmol/L). MDA levels decreased in UVB-treated HLECs following treatment with TSA (0.2 mol/L, 0.8 mol/L). ROS levels decreased in UVB-treated HLECs following treatment with βOHB (4 mmol/L), SAHA (1 μmol/L, 2 μmol/L) and TSA (0.2 mol/L). Western blotting results demonstrated that SOD1 levels significantly increased in the βOHB (4 mmol/L), SAHA (1 μmol/L, 2 μmol/L), TSA (0.1 mol/L, 0.2 mol/L) and VPA (5 mmol/L) groups. Only SAHA (1 μmol/L) had an anti-apoptotic effect on UVB-treated HLECs. Conclusions Our findings indicate that low concentrations of HDACis (1 μmol/L of SAHA) mildly inhibit oxidative stress, thus protecting HLECs from oxidation. These results may suggest that there is a possibility to explore the clinical applications of HDACis for treatment and prevention of cataracts

Efficient Generation of Lens Progenitor Cells from Cataract Patient–Specific Induced Pluripotent Stem Cells

The development of a technique to induce the transformation of somatic cells to a pluripotent state via the ectopic expression of defined transcription factors was a transformational event in the field of regenerative medicine. The development of this technique also impacted ophthalmology, as patient-specific induced pluripotent stemcells (iPSCs) may be useful resources for some ophthalmological diseases. The lens is a key refractive element in the eye that focuses images of the visual world onto the retina. To establish a new model for drug screening to treat lens diseases and investigating lens aging and development, we examined whether human lens epithelial cells (HLECs) could be induced into iPSCs and if lensspecific differentiation of these cells could be achieved under defined chemical conditions. We first efficiently reprogrammed HLECs from age-related cataract patients to iPSCs with OCT-4, SOX-2, and KLF-4. The resulting HLECderived iPS (HLE-iPS) colonies were indistinguishable from human ES cells with respect to morphology, gene expression, pluripotent marker expression and their ability to generate all embryonic germ-cell layers. Next, we performed a 3-step induction procedure: HLE-iPS cells were differentiated into large numbers of lens progenitor-like cells with defined factors (Noggin, BMP and FGF2), and we determined that these cells expressed lens-specific markers (PAX6, SOX2, SIX3, CRYAB, CRYAA, BFSP1, and MIP). In addition, HLE-iPS-derived lens cells exhibited reduced expression of epithelial mesenchymal transition (EMT) markers compared with human embryonic stem cells (hESCs) and fibroblast-derived iPSCs. Our stud

Toric Intraocular Lens Implantation in the Correction of Moderate-To-High Corneal Astigmatism in Cataract Patients: Clinical Efficacy and Safety

Background/Aims. To investigate the long-term efficacy and rotational stability of toric intraocular lenses (IOLs) implanted for the correction of moderate-to-high corneal astigmatism. Methods. A total of 57 cataract patients (57 eyes) with regular corneal astigmatism (≥2.57 D) were enrolled in this retrospective cohort study. Phacoemulsification with toric IOL implantation was performed for all patients. The uncorrected visual acuity (UCVA) and best corrected visual acuity (BCVA) were recorded before and one year after surgery, and statistical analysis of preoperative corneal astigmatism, postoperative residual astigmatism, aberrations, IOL rotation, and related factors was performed to evaluate the efficacy, safety, and stability of toric IOLs in correcting moderate-to-high corneal astigmatism. Results. One year after surgery, visual acuity was significantly improved compared with that before surgery (preoperative log MAR 0.87 ± 0.34 vs. postoperative log MAR 0.31 ± 0.26, p<0.001), and the self-reported spectacle independence rate was 68.42%. The total residual astigmatism was 1.18 ± 0.85 D, which was significantly less than the preoperative value (3.41 ± 0.99 D) (p<0.001). The degree of toric IOL rotation was 4.93 ± 3.02°, and 54.39% of patients had a lens rotation of less than 5°. The IOLs of 5.26% (3 eyes) of patients rotated more than 10°, and these patients received glasses instead of undergoing IOL repositioning. Conclusions. Toric IOL implantation provided optimal vision outcomes and low spectacle dependence during a one-year follow-up period. The results from our study show that toric IOL implantation is a safe and effective option for cataract patients with moderate-to-high corneal astigmatism

Simulation analysis of the diode laser spectral beam combining

For high power diode laser, the multi-wavelength combining is an effective technology of improving the beam brightness. Based on the combing technology, the diode laser bar is described, with the different sample grating in every emitter ridge, which can have stability of the wavelength and the single longitudinal mode. Mathematics computing model based on the spilt-step fourier (SSF) method combining is built using MatLab. It simulated the multi-wavelength combining process of emitters. In simulation, the diode laser beam successively go through a focus lens and a diffraction grating, respectively. In result, the model accords with the law of the combining which is verified by existing report. The simulation solution satisfied the reported experiment

Simulation analysis of the diode laser spectral beam combining

For high power diode laser, the multi-wavelength combining is an effective technology of improving the beam brightness. Based on the combing technology, the diode laser bar is described, with the different sample grating in every emitter ridge, which can have stability of the wavelength and the single longitudinal mode. Mathematics computing model based on the spilt-step fourier (SSF) method combining is built using MatLab. It simulated the multi-wavelength combining process of emitters. In simulation, the diode laser beam successively go through a focus lens and a diffraction grating, respectively. In result, the model accords with the law of the combining which is verified by existing report. The simulation solution satisfied the reported experiment

Clinical Characteristics of Intraocular Lens Dislocation in Chinese Han Populations

Purpose. To investigate the clinical characteristics of patients with intraocular lens (IOL) dislocation after IOL implantation in Chinese Han populations. Methods. The medical records of patients with IOL dislocation were retrospectively reviewed from January 2007 to December 2017, and a total of 312 patients (male: 231, female: 97) (328 eyes) were included in this study. The axial length (AL), IOL power, and the time interval between cataract surgery and IOL dislocation as well as the ocular conditions associated with IOL dislocation were recorded. The IOL dislocation was classified and graded based on its relationship with the capsule and the position of the dislocated IOL. Results. The mean time between original cataract surgery and IOL dislocation was 5.63 ± 5.13 years; IOL dislocation occurred in up to 56.1% (184 eyes) of the eyes within 5 years. Trauma was found in 136 eyes (41.5%); pars plana vitrectomies were performed in 61 eyes (18.6%), and high myopia was detected in 108 eyes (32.9%). A total of 243 eyes (74.1%) had out-of-the-bag IOL dislocations, while 85 eyes (25.9%) had in-the-bag IOL dislocations. There was a statistically significant difference in the constituent ratio of trauma between in-the-bag dislocation and out-of-the-bag dislocation (Pearson’s chi2 = 33.3992, P<0.001); ocular blunt traumas were significantly higher in in-the-bag dislocations, while open-globe injuries were significantly higher in out-of-the-bag dislocations. A statistically significant difference was found for the ratio of patients with AL longer than 30 mm between in-the-bag dislocation and out-of-the-bag dislocation (Pearson’s chi2 = 9.7355, P<0.002). Conclusions. In Chinese Han populations, the most common IOL dislocation is out-of-the-bag dislocation; the most common risk factors were trauma, long axial length, and eyes undergoing pars plana vitrectomy; a minimum follow-up of 5 years is suggested for IOL dislocation-predisposed eyes undergoing cataract surgery

Consensus for non-linear multi-agent systems modelled by PDEs based on spatial boundary communication

There is spatio-temporal nature for many multi-agent systems such as infight hose-and-drogue aerial refuelling systems. To deal with consensus control of such cases, this study establishes a non-linear leader-following spatio-temporal multi-agent system modelled by partial differential equations. Initially, a boundary controller based on boundary coupling is studied to ensure consensus of the multi-agent system. A sufficient condition on the existence of the controller for consensus is presented in terms of linear matrix inequalities. To simplify the obtained result, a second boundary controller is studied and a simple sufficient condition of its existence is investigated. Finally, two numerical examples demonstrate the effectiveness of the proposed methods. The merits of the proposed controllers lie in making use of only spatial boundary communication and requiring actuators and sensors only at spatial boundary positions