Phosphofructokinase-1 (PFK1) catalyzes the rate-limiting step of glycolysis, committing glucose to conversion into cellular energy. PFK1 is highly regulated to respond to the changing energy needs of the cell. In bacteria, the structural basis of PFK1 regulation is a textbook example of allostery; molecular signals of low and high cellular energy promote transition between an active R-state and inactive T-state conformation, respectively. Little is known, however, about the structural basis for regulation of eukaryotic PFK1. Here, we determine structures of the human liver isoform of PFK1 (PFKL) in the R- and T-state by cryoEM, providing insight into eukaryotic PFK1 allosteric regulatory mechanisms. The T-state structure reveals conformational differences between the bacterial and eukaryotic enzyme, the mechanisms of allosteric inhibition by ATP binding at multiple sites, and an autoinhibitory role of the C-terminus in stabilizing the T-state. We also determine structures of PFKL filaments that define the mechanism of higher-order assembly and demonstrate that these structures are necessary for higher-order assembly of PFKL in cells.
- MeSH
- Adenosine Triphosphate * metabolism MeSH
- Allosteric Regulation MeSH
- Cryoelectron Microscopy MeSH
- Phosphofructokinase-1 * metabolism chemistry genetics MeSH
- Glycolysis MeSH
- Liver enzymology metabolism MeSH
- Protein Conformation MeSH
- Humans MeSH
- Models, Molecular MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
As is the case with batch-based tableting processes, continuous tablet manufacturing can be conducted by direct compression or with a granulation step such as dry or wet granulation included in the production procedure. In this work, continuous manufacturing tests were performed with a commercial tablet formulation, while maintaining its original material composition. Challenges were encountered with the feeding performance of the API during initial tests which required designing different powder pre-blend compositions. After the pre-blend optimization phase, granules were prepared with a roller compactor. Tableting was conducted with the granules and an additional brief continuous direct compression run was completed with some ungranulated mixture. The tablets were assessed with off-line tests, applying the quality requirements demanded for the batch-manufactured product. Chemical maps were obtained by Raman mapping and elemental maps by scanning electron microscopy with energy-dispersive X-ray spectroscopy. Large variations in both tablet weights and breaking forces were observed in all tested samples, resulting in significant quality complications. It was suspected that the API tended to adhere to the process equipment, accounting for the low API content in the powder mixture and tablets. These results suggest that this API or the tablet composition was unsuitable for manufacturing in a continuous line; further testing could be continued with different materials and changes in the process.
With the use of proton-NMR and powder XRD (XRPD) studies, the suitability of specific Au-focused electron beam induced deposition (FEBID) precursors has been investigated with low electron energy, structure, excited states and resonances, structural crystal modifications, flexibility, and vaporization level. 4,5-Dichloro-1,3-diethyl-imidazolylidene trifluoromethyl gold(I) is a compound that is a uniquely designed precursor to meet the needs of focused electron beam-induced deposition at the nanostructure level, which proves its capability in creating high purity structures, and its growing importance in other AuImx and AuClnB (where x and n are the number of radicals, B = CH, CH3, or Br) compounds in the radiation cancer therapy increases the efforts to design more suitable bonds in processes of SEM (scanning electron microscopy) deposition and in gas-phase studies. The investigation performed of its powder shape using the XRPD XPERT3 panalytical diffractometer based on CoKα lines shows changes to its structure with change in temperature, level of vacuum, and light; the sensitivity of this compound makes it highly interesting in particular to the radiation research. Used in FEBID, though its smaller number of C, H, and O atoms has lower levels of C contamination in the structures and on the surface, it replaces these bonds with C-Cl and C-N bonds that have lower bond-breaking energy. However, it still needs an extra purification step in the deposition process, either H2O, O2, or H jets.
- Publication type
- Journal Article MeSH
Two dimensional (2D) nanomaterials display properties with significant biological utility (e.g., antimicrobial activity). In this study, MXene-functionalized graphene (FG) nanocomposites with Ti3C2T x in varying ratios (FG : Ti3C2T x , 25 : 75%, 50 : 50%, and 75 : 25%) were prepared and characterized via scanning electron microscopy, scanning electron microscopy-energy dispersive X-ray (SEM-EDX), high-resolution transmission electron microscopy (HRTEM), and zeta potential analysis. Their cytotoxicity was assessed using immortalized human keratinocytes (HaCaT) cells at three different timepoints, and antibacterial activity was assessed using Gram-positive Methicillin resistant Staphylococcus aureus, MRSA, and Gram-negative neuro-pathogenic Escherichia coli K1 (E. coli K1) in vitro. The nanomaterials and composites displayed potent antibacterial effects against both types of bacteria and low cytotoxicity against HaCaT cells at 200 μg mL-1, which is promising for their utilization for biomedical applications.
- Publication type
- Journal Article MeSH
The work was aimed at evaluating the efficiency of multifunctional magnesium aluminosilicate materials (MAS) as a novel glidant in solid dosage forms. MAS are known for their very low cohesive interactions and their utilization could avoid the disadvantages associated with conventional glidant usage. Flow properties of several mixtures comprising a model excipient (microcrystalline cellulose) and a glidant were characterized using a powder rheometer FT4. The mixtures were formulated to represent effects of glidant types, various levels of glidant loading, particle size and mixing time on flow properties of the model excipient. Pre-conditioning, shear testing, compressibility, flow energy measurements and an additional tapping test were carried out to monitor flow properties. Mixtures were analyzed employing scanning electron microscopy, using a detector of back-scattered electrons to identify a mechanism of MAS towards improving the mixture flow properties. All studied parameters were found to have substantial effects on mixture flow properties, but the effect of mixing time was much less important compared to mixtures based on traditional glidant. The mechanism of MAS glidant action was found to be different compared to that of traditional one, having less process sensitivity, so that MAS utilization as glidant could be advantageous for the formulation performance.
- MeSH
- Excipients * MeSH
- Powders MeSH
- Rheology MeSH
- Silicates * MeSH
- Aluminum Compounds MeSH
- Magnesium Compounds MeSH
- Particle Size MeSH
- Publication type
- Journal Article MeSH
The aim of this study was to fabricate novel microparticles (MPs) for efficient and long-term delivery of amikacin (AMI). The emulsification method proposed for encapsulating AMI employed low-molecular-weight poly(lactic acid) (PLA) and poly(lactic acid-co-polyethylene glycol) (PLA-PEG), both supplemented with poly(vinyl alcohol) (PVA). The diameters of the particles obtained were determined as less than 30 μm. Based on an in-vitro release study, it was proven that the MPs (both PLA/PVA- and PLA-PEG/PVA-based) demonstrated long-term AMI release (2 months), the kinetics of which adhered to the Korsmeyer-Peppas model. The loading efficiencies of AMI in the study were determined at the followings levels: 36.5 ± 1.5 μg/mg for the PLA-based MPs and 106 ± 32 μg/mg for the PLA-PEG-based MPs. These values were relatively high and draw parallels with studies published on the encapsulation of aminoglycosides. The MPs provided antimicrobial action against the Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae bacterial strains. The materials were also comprehensively characterized by the following methods: differential scanning calorimetry; gel permeation chromatography; scanning electron microscopy; Fourier transform infrared spectroscopy-attenuated total reflectance; energy-dispersive X-ray fluorescence; and Brunauer-Emmett-Teller surface area analysis. The findings of this study contribute toward discerning new means for conducting targeted therapy with polar, broad spectrum antibiotics.
- MeSH
- Amikacin administration & dosage chemistry MeSH
- Anti-Bacterial Agents administration & dosage chemistry MeSH
- Escherichia coli drug effects MeSH
- Klebsiella pneumoniae drug effects MeSH
- Lactates chemistry MeSH
- Microbial Sensitivity Tests MeSH
- Molecular Weight MeSH
- Drug Carriers chemistry MeSH
- Polyesters chemistry MeSH
- Polyethylene Glycols chemistry MeSH
- Polyvinyl Alcohol chemistry MeSH
- Drug Compounding methods MeSH
- Pseudomonas aeruginosa drug effects MeSH
- Solubility MeSH
- Staphylococcus aureus drug effects MeSH
- Capsules MeSH
- Drug Liberation MeSH
- Particle Size MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Inspired by the standard computed tomography, a new method of 3D X-ray imaging embedded in FIB-SEM microscope is proposed. The unique combination of TEM-like specimen stage enabling in lens STEM detection (referred to as CompuStage), nanomanipulator (referred to as EasyLift) facilitating in-situ sample transfer from bulk sample to TEM-like stage and pixelated in-situ Timepix X-ray detector in Helios G4 FX FIB-SEM system offers an unprecedented workflow. Motivated by common circular CT scan known from microCT world, the object under study is placed on CompuStage rod which enables two possible rotation (in TEM/SEM terminology called tilt) movements - α-tilt - rotation of the CompuStage rod around its axis, and β-tilt - rotation around axis perpendicular to CompuStage rod. β-tilt rotation enables a circular movement of the sample while α-tilt sets the correct position of sample with respect to target and detector. Thin metal lamella of suitable material welded to EasyLift manipulator needle is used as an X-ray target. The final target-sample geometry - position, distance - can be fine-tuned using position control of CompuStage and EasyLift and in-situ monitored by SEM. Both sample and target can also be easily prepared in-situ. Radiographs are recorded by Timepix detector with inherent noise-free operation and energy filtration. For the 3D reconstruction standard microCT reconstruction algorithm is used with the procedure adjusted for the format and quality of nanoCT images. The experiments were carried out on Helios G4 FX DualBeam using titanium and tungsten targets and various semiconductor samples. The ultimate resolution of the proposed method in orders of tens of nanometers was achieved both by the possibility of close target to sample positioning and of adjustment of primary beam energy down to low energies reducing the interaction volume in the target. Since the lower energy radiation is well suited for life-science, the method was also tested on several bio-samples using silver target. The silver target, thanks to its massive low energy Lα line, allowed to distinguish subtle structures in the resin embedded stained mouse brain and also to observe and reconstruct canaliculi in the mouse bone (earlier reported by Dierolf et al. 2010, Nature 467 436).
- MeSH
- Algorithms MeSH
- Phantoms, Imaging MeSH
- Femur ultrastructure MeSH
- Microscopy, Electron, Scanning * instrumentation methods MeSH
- Mice MeSH
- Image Processing, Computer-Assisted methods MeSH
- X-Ray Microtomography * instrumentation methods MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
The limited availability of biological samples hinders phylogenetic efforts to define structural differences among various biological groups. A novel workflow enabling the analysis of protists in low cell numbers by electron microscopy (EM) is described with cysts of Giardia intestinalis, a single-celled eukaryotic parasite. Correlative light and electron microscopy (CLEM) allows for the selection of individual cells and is economical in terms of time and cost. We describe a cyst purification protocol in combination with an adhesive coating for fixation and ultrathin embedding that results in excellent preservation of cell morphology. The application of advanced structural and analytical EM methods, such as high-resolution field emission scanning electron microscopy (FESEM), focused ion beam tomography (FIB/SEM), and energy-dispersive X-ray spectroscopy (EDX) analysis, is demonstrated. The workflow represents a new approach for studying the cellular and organelle architecture of rare and "difficult to culture" microorganisms.
Arsenic compounds are carcinogenic to humans and are typically removed from contaminated water using various sorbents. The ionic composition plays a significant role in arsenate removal efficiency during the process of water remediation. Here, we quantify the effects of natural ions (chlorides, nitrates, carbonates, sulfates, and phosphates) and humic acid on the removal of arsenates by ferrate(VI) at pH = 6.6. In the experiments, the initial concentration of arsenates was 10 mg L-1 (as As) and the concentrations of ions varied in the range from 5 to 100 mg L-1 of element in ionic form and humic acid. The achieved results show that only phosphate ions had principle influence on the efficiency of arsenate removal by ferrate(VI). The effect of phosphates was elucidated by applying transmission electron microscopy, energy-dispersive X-ray spectroscopy, and low temperature in-field 57Fe Mössbauer spectroscopy to solid samples, prepared under different weight ratios of ferrate(VI), arsenates, and phosphates. These results show three crucial effects of phosphates on the arsenate removal mechanisms. At low P:As weight ratio (up to 1:1), the incorporation of arsenate ions into the crystalline structure of γ-Fe2O3/γ-FeOOH nanoparticles was found to be suppressed by the presence of phosphates. Thus, arsenates were mainly adsorbed onto the surface of γ-Fe2O3/γ-FeOOH nanoparticles. Further increase in the P:As weight ratio (more than 1:1) resulted in the competition between arsenates and phosphates sorption. With the increased concentration of phosphate ions, the number of arsenates on the surface of γ-Fe2O3/γ-FeOOH nanoparticles was reduced. Finally, the complexation of iron(III) ions with phosphate ions occurred, leading to a decrease in the arsenates removal efficiency, which resulted from a lower content of precipitated γ-Fe2O3/γ-FeOOH nanoparticles. All these aspects need to be considered prior to application of ferrate(VI) for arsenates removal in real natural waters.
- MeSH
- Adsorption MeSH
- Arsenates chemistry MeSH
- Water Pollutants, Chemical chemistry MeSH
- Chlorides chemistry MeSH
- Water Purification methods MeSH
- Nitrates chemistry MeSH
- Phosphates chemistry MeSH
- Humic Substances * MeSH
- Hydrogen-Ion Concentration MeSH
- Sulfates chemistry MeSH
- Spectroscopy, Mossbauer MeSH
- Carbonates chemistry MeSH
- Ferric Compounds chemistry MeSH
- Iron chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Wear particles from automotive friction brake pads of various sizes, morphology, and chemical composition are significant contributors towards particulate matter. Knowledge concerning the potential adverse effects following inhalation exposure to brake wear debris is limited. Our aim was, therefore, to generate brake wear particles released from commercial low-metallic and non-asbestos organic automotive brake pads used in mid-size passenger cars by a full-scale brake dynamometer with an environmental chamber simulating urban driving and to deduce their potential hazard in vitro. The collected fractions were analysed using scanning electron microscopy via energy-dispersive X-ray spectroscopy (SEM-EDS) and Raman microspectroscopy. The biological impact of the samples was investigated using a human 3D multicellular model consisting of human epithelial cells (A549) and human primary immune cells (macrophages and dendritic cells) mimicking the human epithelial tissue barrier. The viability, morphology, oxidative stress, and (pro-)inflammatory response of the cells were assessed following 24 h exposure to ~ 12, ~ 24, and ~ 48 µg/cm2 of non-airborne samples and to ~ 3.7 µg/cm2 of different brake wear size fractions (2-4, 1-2, and 0.25-1 µm) applying a pseudo-air-liquid interface approach. Brake wear debris with low-metallic formula does not induce any adverse biological effects to the in vitro lung multicellular model. Brake wear particles from non-asbestos organic formulated pads, however, induced increased (pro-)inflammatory mediator release from the same in vitro system. The latter finding can be attributed to the different particle compositions, specifically the presence of anatase.
- MeSH
- Models, Biological * MeSH
- A549 Cells MeSH
- Cytokines metabolism MeSH
- Dendritic Cells drug effects metabolism ultrastructure MeSH
- Coculture Techniques MeSH
- Air Pollutants toxicity MeSH
- Humans MeSH
- Macrophages drug effects metabolism ultrastructure MeSH
- Motor Vehicles MeSH
- Oxidative Stress drug effects MeSH
- Particulate Matter toxicity MeSH
- Lung drug effects metabolism pathology MeSH
- Surface Properties MeSH
- Particle Size MeSH
- Cell Survival drug effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
