In vitro models of the blood-brain barrier (BBB) are critical tools for the study of BBB transport and the development of drugs that can reach the CNS. Brain endothelial cells grown in culture are ...often used to model the BBB; however, it is challenging to maintain reproducible BBB properties and function. 'BBB organoids' are obtained following coculture of endothelial cells, pericytes and astrocytes under low-adhesion conditions. These organoids reproduce many features of the BBB, including the expression of tight junctions, molecular transporters and drug efflux pumps, and hence can be used to model drug transport across the BBB. This protocol provides a comprehensive description of the techniques required to culture and maintain BBB organoids. We also describe two separate detection approaches that can be used to analyze drug penetration into the organoids: confocal fluorescence microscopy and mass spectrometry imaging. Using our protocol, BBB organoids can be established within 2-3 d. An additional day is required to analyze drug permeability. The BBB organoid platform represents an accurate, versatile and cost-effective in vitro tool. It can easily be scaled to a high-throughput format, offering a tool for BBB modeling that could accelerate therapeutic discovery for the treatment of various neuropathologies.
A cyber-physical system (CPS) is composed of tightly-integrated computation, communication and physical elements. Medical devices, buildings, mobile devices, robots, transportation and energy systems ...can benefit from CPS co-design and optimization techniques. Cyber-physical vehicle systems (CPVSs) are rapidly advancing due to progress in real-time computing, control and artificial intelligence. Multidisciplinary or multi-objective design optimization maximizes CPS efficiency, capability and safety, while online regulation enables the vehicle to be responsive to disturbances, modeling errors and uncertainties. CPVS optimization occurs at design-time and at run-time. This paper surveys the run-time cooperative optimization or co-optimization of cyber and physical systems, which have historically been considered separately. A run-time CPVS is also cooperatively regulated or co-regulated when cyber and physical resources are utilized in a manner that is responsive to both cyber and physical system requirements. This paper surveys research that considers both cyber and physical resources in co-optimization and co-regulation schemes with applications to mobile robotic and vehicle systems. Time-varying sampling patterns, sensor scheduling, anytime control, feedback scheduling, task and motion planning and resource sharing are examined.
Culture-based blood-brain barrier (BBB) models are crucial tools to enable rapid screening of brain-penetrating drugs. However, reproducibility of in vitro barrier properties and permeability remain ...as major challenges. Here, we report that self-assembling multicellular BBB spheroids display reproducible BBB features and functions. The spheroid core is comprised mainly of astrocytes, while brain endothelial cells and pericytes encase the surface, acting as a barrier that regulates transport of molecules. The spheroid surface exhibits high expression of tight junction proteins, VEGF-dependent permeability, efflux pump activity and receptor-mediated transcytosis of angiopep-2. In contrast, the transwell co-culture system displays comparatively low levels of BBB regulatory proteins, and is unable to discriminate between the transport of angiopep-2 and a control peptide. Finally, we have utilized the BBB spheroids to screen and identify BBB-penetrant cell-penetrating peptides (CPPs). This robust in vitro BBB model could serve as a valuable next-generation platform for expediting the development of CNS therapeutics.
In vitro bioassays are sensitive, effect-based tools used to quantitatively screen for chemicals with nuclear receptor activity in environmental samples. We measured in vitro estrogen (ER), androgen ...(AR), and glucocorticoid receptor (GR) activity, along with a broad suite of chemical analytes, in streamwater from 35 well-characterized sites (3 reference and 32 impacted) across 24 states and Puerto Rico. ER agonism was the most frequently detected with nearly all sites (34/35) displaying activity (range, 0.054–116 ng E2Eq L–1). There was a strong linear relationship (r 2 = 0.917) between in vitro ER activity and concentrations of steroidal estrogens after correcting for the in vitro potency of each compound. AR agonism was detected in 5/35 samples (range, 1.6–4.8 ng DHTEq L–1) but concentrations of androgenic compounds were largely unable to account for the in vitro activity. Similarly, GR agonism was detected in 9/35 samples (range, 6.0–43 ng DexEq L–1); however, none of the recognized GR-active compounds on the target-chemical analyte list were detected. The utility of in vitro assays in water quality monitoring was evident from both the quantitative agreement between ER activity and estrogen concentrations, as well as the detection of AR and GR activity for which there were limited or no corresponding target-chemical detections to explain the bioactivity. Incorporation of in vitro bioassays as complements to chemical analyses in standard water quality monitoring efforts would allow for more complete assessment of the chemical mixtures present in many surface waters.
Exon‐skipping antisense oligonucleotides are effective treatments for genetic diseases, yet exon‐skipping activity requires that these macromolecules reach the nucleus. While cell‐penetrating ...peptides can improve delivery, proteolytic instability often limits efficacy. It is hypothesized that the bicyclization of arginine‐rich peptides would improve their stability and their ability to deliver oligonucleotides into the nucleus. Two methods were introduced for the synthesis of arginine‐rich bicyclic peptides using cysteine perfluoroarylation chemistry. Then, the bicyclic peptides were covalently linked to a phosphorodiamidate morpholino oligonucleotide (PMO) and assayed for exon skipping activity. The perfluoroaryl cyclic and bicyclic peptides improved PMO activity roughly 14‐fold over the unconjugated PMO. The bicyclic peptides exhibited increased proteolytic stability relative to the monocycle, demonstrating that perfluoroaryl bicyclic peptides are potent and stable delivery agents.
Assembling a new bicycle: Peptide bicycles are synthesized using cysteine perfluoroarylation chemistry. The bicyclic peptides demonstrate enhanced proteolytic stability relative to the monocyclic peptide. Additionally, after conjugation to antisense oligonucleotides, the bicyclic peptide conjugates exhibit a 14‐fold increase in cellular activity when compared to the unconjugated oligonucleotide.
Bacterial iron detoxification at the molecular level Bradley, Justin M.; Svistunenko, Dimitry A.; Wilson, Michael T. ...
The Journal of biological chemistry,
12/2020, Letnik:
295, Številka:
51
Journal Article
Recenzirano
Odprti dostop
Iron is an essential micronutrient, and, in the case of bacteria, its availability is commonly a growth-limiting factor. However, correct functioning of cells requires that the labile pool of ...chelatable “free” iron be tightly regulated. Correct metalation of proteins requiring iron as a cofactor demands that such a readily accessible source of iron exist, but overaccumulation results in an oxidative burden that, if unchecked, would lead to cell death. The toxicity of iron stems from its potential to catalyze formation of reactive oxygen species that, in addition to causing damage to biological molecules, can also lead to the formation of reactive nitrogen species. To avoid iron-mediated oxidative stress, bacteria utilize iron-dependent global regulators to sense the iron status of the cell and regulate the expression of proteins involved in the acquisition, storage, and efflux of iron accordingly. Here, we survey the current understanding of the structure and mechanism of the important members of each of these classes of protein. Diversity in the details of iron homeostasis mechanisms reflect the differing nutritional stresses resulting from the wide variety of ecological niches that bacteria inhabit. However, in this review, we seek to highlight the similarities of iron homeostasis between different bacteria, while acknowledging important variations. In this way, we hope to illustrate how bacteria have evolved common approaches to overcome the dual problems of the insolubility and potential toxicity of iron.
Iron is an essential micronutrient, and, in the case of bacteria, its availability is commonly a growth-limiting factor. However, correct functioning of cells requires that the labile pool of ...chelatable "free" iron be tightly regulated. Correct metalation of proteins requiring iron as a cofactor demands that such a readily accessible source of iron exist, but overaccumulation results in an oxidative burden that, if unchecked, would lead to cell death. The toxicity of iron stems from its potential to catalyze formation of reactive oxygen species that, in addition to causing damage to biological molecules, can also lead to the formation of reactive nitrogen species. To avoid iron-mediated oxidative stress, bacteria utilize iron-dependent global regulators to sense the iron status of the cell and regulate the expression of proteins involved in the acquisition, storage, and efflux of iron accordingly. Here, we survey the current understanding of the structure and mechanism of the important members of each of these classes of protein. Diversity in the details of iron homeostasis mechanisms reflect the differing nutritional stresses resulting from the wide variety of ecological niches that bacteria inhabit. However, in this review, we seek to highlight the similarities of iron homeostasis between different bacteria, while acknowledging important variations. In this way, we hope to illustrate how bacteria have evolved common approaches to overcome the dual problems of the insolubility and potential toxicity of iron.
Compartmentalization of metabolic enzymes through protein–protein interactions is an emerging mechanism for localizing and regulating metabolic activity. Self-assembly into linear filaments is a ...common strategy for cellular compartmentalization of enzymes. Polymerization is often driven by changes in the metabolic state of the cell, suggesting that it is a strategy for shifting metabolic flux in response to cellular demand. Although polymerization of metabolic enzymes is widespread, observed from bacteria to humans, we are just beginning to appreciate their role in regulating cellular metabolism. In most cases, one functional role of metabolic enzyme filaments is allosteric control of enzyme activity. Here, we highlight recent findings, providing insight into the structural and functional significance of filamentation of metabolic enzymes in cells.
In neutrophils, nicotinamide adenine dinucleotide phosphate (NADPH) generated via the pentose phosphate pathway fuels NADPH oxidase NOX2 to produce reactive oxygen species for killing invading ...pathogens. However, excessive NOX2 activity can exacerbate inflammation, as in acute respiratory distress syndrome (ARDS). Here, we use two unbiased chemical proteomic strategies to show that small-molecule LDC7559, or a more potent designed analog NA-11, inhibits the NOX2-dependent oxidative burst in neutrophils by activating the glycolytic enzyme phosphofructokinase-1 liver type (PFKL) and dampening flux through the pentose phosphate pathway. Accordingly, neutrophils treated with NA-11 had reduced NOX2-dependent outputs, including neutrophil cell death (NETosis) and tissue damage. A high-resolution structure of PFKL confirmed binding of NA-11 to the AMP/ADP allosteric activation site and explained why NA-11 failed to agonize phosphofructokinase-1 platelet type (PFKP) or muscle type (PFKM). Thus, NA-11 represents a tool for selective activation of PFKL, the main phosphofructokinase-1 isoform expressed in immune cells.
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•Small molecules LDC7559 and NA-11 inhibit the phagocytic oxidative burst•NA-11 dampens flux through the pentose phosphate pathway to limit cellular NADPH•Cryo-EM confirms agonism of PFKL through binding at the AMP/ADP allosteric pocket•PFKL is a regulatory node for NOX2-dependent NETosis and tissue damage
The small molecule LDC7559 and its more potent analog, NA-11, suppress excessive NOX2-dependent oxidative burst and NETosis, as well as subsequent tissue damage and inflammation, without compromising basal ROS production. They selectively activate the glycolytic enzyme phosphofructokinase-1 liver type (PFKL) to suppress glycolytic flux through the pentose phosphate pathway that leads to NOX2-dependent outputs.
Despite abundant knowledge of the regulation and biochemistry of glycolytic enzymes, we have limited understanding on how they are spatially organized in the cell. Emerging evidence indicates that ...nonglycolytic metabolic enzymes regulating diverse pathways can assemble into polymers. We now show tetramer- and substrate-dependent filament assembly by phosphofructokinase-1 (PFK1), which is considered the "gatekeeper" of glycolysis because it catalyzes the step committing glucose to breakdown. Recombinant liver PFK1 (PFKL) isoform, but not platelet PFK1 (PFKP) or muscle PFK1 (PFKM) isoforms, assembles into filaments. Negative-stain electron micrographs reveal that filaments are apolar and made of stacked tetramers oriented with exposed catalytic sites positioned along the edge of the polymer. Electron micrographs and biochemical data with a PFKL/PFKP chimera indicate that the PFKL regulatory domain mediates filament assembly. Quantified live-cell imaging shows dynamic properties of localized PFKL puncta that are enriched at the plasma membrane. These findings reveal a new behavior of a key glycolytic enzyme with insights on spatial organization and isoform-specific glucose metabolism in cells.
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