Dynamic control of gene expression can have far-reaching implications for biotechnological applications and biological discovery. Thanks to the advantages of light, optogenetics has emerged as an ...ideal technology for this task. Current state-of-the-art methods for optical expression control fail to combine precision with repeatability and cannot withstand changing operating culture conditions. Here, we present a novel fully automatic experimental platform for the robust and precise long-term optogenetic regulation of protein production in liquid Escherichia coli cultures. Using a computer-controlled light-responsive two-component system, we accurately track prescribed dynamic green fluorescent protein expression profiles through the application of feedback control, and show that the system adapts to global perturbations such as nutrient and temperature changes. We demonstrate the efficacy and potential utility of our approach by placing a key metabolic enzyme under optogenetic control, thus enabling dynamic regulation of the culture growth rate with potential applications in bacterial physiology studies and biotechnology.
Botulinum toxin type A (BOTOX
®) has been used to treat pathological pain conditions although the mechanism is not entirely understood. Subcutaneous (s.c.) BOTOX
® also inhibits inflammatory pain in ...the rat formalin model, and the present study examined whether this could be due to a direct action on sensory neurons. BOTOX
® (3.5–30
U/kg) was injected s.c. into the subplantar surface of the rat hind paw followed 1–5 days later by 50
mL of 5% formalin. Using microdialysis, we found that BOTOX
® significantly inhibited formalin-induced glutamate release (peak inhibitions: 35%, 41%, and 45% with 3.5, 7, and 15
U/kg, respectively). BOTOX
® also dose dependently reduced the number of formalin-induced Fos-like immunoreactive cells in the dorsal horn of the spinal cord and significantly (15 and 30
U/kg) inhibited the excitation of wide dynamic range neurons of the dorsal horn in Phase II but not Phase I of the formalin response. These results indicate that s.c. BOTOX
® inhibits neurotransmitter release from primary sensory neurons in the rat formalin model. Through this mechanism, BOTOX
® inhibits peripheral sensitization in these models, which leads to an indirect reduction in central sensitization.
The neurotoxin, botulinum toxin type A, has been used successfully, in some patients, as an analgesic for myofascial pain syndromes, migraine, and other headache types. The toxin inhibits the release ...of the neurotransmitter, acetylcholine, at the neuromuscular junction thereby inhibiting striated muscle contractions. In the majority of pain syndromes where botulinum toxin type A is effective, inhibiting muscle spasms is an important component of its activity. Even so, the reduction of pain often occurs before the decrease in muscle contractions suggesting that botulinum toxin type A has a more complex mechanism of action than initially hypothesized. Current data points to an antinociceptive effect of botulinum toxin type A that is separate from its neuromuscular activity. The common biochemical mechanism, however, remains the same between botulinum toxin type A's effect on the motor nerve or the sensory nerve: enzymatic blockade of neurotransmitter release. The antinociceptive effect of the toxin was reported to block substance P release using in vitro culture systems.1
The current investigation evaluated the in vivo mechanism of action for the antinociceptive action of botulinum toxin type A. In these studies, botulinum toxin type A was found to block the release of glutamate. Furthermore, Fos, a product of the immediate early gene, c‐fos, expressed with neuronal stimuli was prevented upon peripheral exposure to the toxin.
These findings suggest that botulinum toxin type A blocks peripheral sensitization and, indirectly, reduces central sensitization. The recent hypothesis that migraine involves both peripheral and central sensitization may help explain how botulinum toxin type A inhibits migraine pain by acting on these two pathways. Further research is needed to determine whether the antinociceptive mechanism mediated by botulinum toxin type A affects the neuronal signaling pathways that are activated during migraine.
Light has emerged as a control input for biological systems due to its precise spatiotemporal resolution. The limited toolset for light control in bacteria motivated us to develop a light-inducible ...transcription system that is independent from cellular regulation through the use of an orthogonal RNA polymerase. Here, we present our engineered blue light-responsive T7 RNA polymerases (Opto-T7RNAPs) that show properties such as low leakiness of gene expression in the dark state, high expression strength when induced with blue light, and an inducible range of more than 300-fold. Following optimization of the system to reduce expression variability, we created a variant that returns to the inactive dark state within minutes once the blue light is turned off. This allows for precise dynamic control of gene expression, which is a key aspect for most applications using optogenetic regulation. The regulators, which only require blue light from ordinary light-emitting diodes for induction, were developed and tested in the bacterium Escherichia coli, which is a crucial cell factory for biotechnology due to its fast and inexpensive cultivation and well understood physiology and genetics. Opto-T7RNAP, with minor alterations, should be extendable to other bacterial species as well as eukaryotes such as mammalian cells and yeast in which the T7 RNA polymerase and the light-inducible Vivid regulator have been shown to be functional. We anticipate that our approach will expand the applicability of using light as an inducer for gene expression independent from cellular regulation and allow for a more reliable dynamic control of synthetic and natural gene networks.
Background
Meningeal and other trigeminal nociceptors are thought to play important roles in the initiation of migraine headache. Currently, the only approved peripherally administered chronic ...migraine prophylactic drug is onabotulinumtoxinA. The purpose of this study was to determine how botulinum neurotoxin type A (BoNT-A) affects naïve and sensitized meningeal nociceptors.
Material and methods
Using electrophysiological techniques, we identified 43 C- and 36 Aδ-meningeal nociceptors, and measured their spontaneous and evoked firing before and after BoNT-A administration to intracranial dura and extracranial suture-receptive fields.
Results
As a rule, BoNT-A inhibited C- but not Aδ-meningeal nociceptors. When applied to nonsensitized C-units, BoNT-A inhibited responses to mechanical stimulation of the dura with suprathreshold forces. When applied to sensitized units, BoNT-A reversed mechanical hypersensitivity. When applied before sensitization, BoNT-A prevented development of mechanical hypersensitivity. When applied extracranially to suture branches of intracranial meningeal nociceptors, BoNT-A inhibited the mechanical responsiveness of the suture branch but not dural axon. In contrast, BoNT-A did not inhibit C-unit responses to mechanical stimulation of the dura with threshold forces, or their spontaneous activity.
Discussion
The study provides evidence for the ability of BoNT-A to inhibit mechanical nociception in peripheral trigeminovascular neurons. These findings suggest that BoNT-A interferes with neuronal surface expression of high-threshold mechanosensitive ion channels linked preferentially to mechanical pain by preventing their fusion into the nerve terminal membrane.
Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency ...assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.
Interfacial instability and atomization behavior on acoustically levitated droplets for further stable liquid manipulation were investigated. We visualized the atomization behavior of water and ...ethanol droplets. Atomization was clearly affected by the difference in surface tension. The pressure difference between the inside and the outside of the droplet was estimated from rapid droplet deformation immediately before its atomization. Finally, the capillary wave on the droplet surface that can trigger atomization was quantified and elucidated with the theory. The size distribution of atomized daughter droplets was compared with the length scale of the capillary wave on the droplet surface.
Molecular dynamics (MD) methods suitable for treating anisotropic fluctuations in condensed matter are discussed. Barostats with two anisotropic factors to control the simulation cell dynamics are ...introduced in three MD simulation methods; constant pressure, constant surface tension, and canonical hydrostatic. To demonstrate the possibilities of these methods, calculations of two systems, one consisting of anisotropic particles and other consisting of isotropic particles, are presented.
Abstract Botulinum toxin A has been traditionally viewed as a motor nerve specific treatment. However, clinical uses for botulinum toxin A have continued to expand, with increased use in conditions ...implicating sensory pain nerve dysfunction. Chronic pain is associated with excess pain fiber activity. When the site of this excess activity resides in the peripheral portion of the pain pathway, a condition of peripheral sensitization can establish. During this state, excess pain signaling reaches the central nervous system, which can then lead to a condition of central sensitization, manifesting as the symptoms associated with chronic pain (i.e. burning, electric pain, lowered pain threshold to normal stimuli, etc). Experimentally , botulinum toxin type A has been shown to reduce neuropeptides and neurotransmitter release from treated cells or nerve endings and to attenuate nociception in both neuropathic and non-neuropathic pain models. This review summarizes the literature to update the hypothesis for the mechanism by which botulinum toxin type A can modulate chronic pain.
Bacteria have developed remarkable systems that sense neighboring target cells upon contact and initiate a series of events that enhance their survival and growth at the expense of the target cells. ...Four main classes of bacterial cell surface structures have been identified that interact with prokaryotic or eukaryotic target cells to deliver DNA or protein effectors. Type III secretion systems (T3SS) use a flagellum-like tube to deliver protein effectors into eukaryotic host cells, whereas Type IV systems use a pilus-based system to mediate DNA or protein transfer into recipient cells. The contact-dependent growth inhibition system (CDI) is a Type V system, using a long β-helical cell surface protein to contact receptors in target cells and deliver a growth inhibitory signal. Type VI systems utilize a phage-like tube and cell puncturing device to secrete effector proteins into both eukaryotic and prokaryotic target cells.
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