Neuronal nicotinic acetylcholine receptors (nAChR), recognized targets for drug development in cognitive and neuro-degenerative disorders, are allosteric proteins with dynamic interconversions ...between multiple functional states. Activation of the nAChR ion channel is primarily controlled by the binding of ligands (agonists, partial agonists, competitive antagonists) at conventional agonist binding sites, but is also regulated in either negative or positive ways by the binding of ligands to other modulatory sites. In this review, we discuss models for the activation and desensitization of nAChR, and the discovery of multiple types of ligands that influence those processes in both heteromeric nAChR, such as the high-affinity nicotine receptors of the brain, and homomeric α7-type receptors. In recent years, α7 nAChRs have been identified as a potential target for therapeutic indications leading to the development of α7-selective agonists and partial agonists. However, unique properties of α7 nAChR, including low probability of channel opening and rapid desensitization, may limit the therapeutic usefulness of ligands binding exclusively to conventional agonist binding sites. New enthusiasm for the therapeutic targeting of α7 has come from the identification of α7-selective positive allosteric modulators (PAMs) that work effectively on the intrinsic factors that limit α7 ion channel activation. While these new drugs appear promising for therapeutic development, we also consider potential caveats and possible limitations for their use, including PAM-insensitive forms of desensitization and cytotoxicity issues.
The CDC biofilm reactor is a robust culture system with high reproducibility in which biofilms can be grown for a wide variety of analyses. Multiple material types are available as growth substrates, ...yet data from biofilms grown on biologically relevant materials is scarce, particularly for antibiotic efficacy against differentially supported biofilms. In this study, CDC reactor holders were modified to allow growth of biofilms on collagen, a biologically relevant substrate. Susceptibility to multiple antibiotics was compared between biofilms of varying species grown on collagen versus standard polycarbonate coupons. Data indicated that in 13/18 instances, biofilms on polycarbonate were more susceptible to antibiotics than those on collagen, suggesting that when grown on a complex substrate, biofilms may be more tolerant to antibiotics. These outcomes may influence the translatability of antibiotic susceptibility profiles that have been collected for biofilms on hard plastic materials. Data may also help to advance information on antibiotic susceptibility testing of biofilms grown on biologically relevant materials for future in vitro and in vivo applications.
The rise in antibiotic resistance has stimulated research into adjuvants that can improve the efficacy of broad-spectrum antibiotics. Lactoferrin is a candidate adjuvant; it is a multifunctional ...iron-binding protein with antimicrobial properties. It is known to show dose-dependent antimicrobial activity against Staphylococcus aureus through iron sequestration and repression of beta-lactamase expression. However, S. aureus can extract iron from lactoferrin through siderophores for their growth, which confounds the resolution of lactoferrin's method of action. We measured the minimum inhibitory concentration (MIC) for a range of lactoferrin/ beta-lactam antibiotic dose combinations and observed that at low doses ( 6.25 muM), iron-depleted native lactoferrin reduced bacterial growth and reduced the MIC of the beta-lactam-antibiotic cefazolin. This differential behaviour points to a bacterial population response to the lactoferrin/ beta-lactam dose combination. Here, with the aid of a mathematical model, we show that lactoferrin stratifies the bacterial population, and the resulting population heterogeneity is at the basis of the dose dependent response seen. Further, lactoferrin disables a sub-population from beta-lactam-induced production of beta-lactamase, which when sufficiently large reduces the population's ability to recover after being treated by an antibiotic. Our analysis shows that an optimal dose of lactoferrin acts as a suitable adjuvant to eliminate S. aureus colonies using beta-lactams, but sub-inhibitory doses of lactoferrin reduces the efficacy of beta-lactams.
MyoD is a key regulator of skeletal myogenesis that directs contractile protein synthesis, but whether this transcription factor also regulates skeletal muscle metabolism has not been explored. In a ...genome-wide ChIP-seq analysis of skeletal muscle cells, we unexpectedly observed that MyoD directly binds to numerous metabolic genes, including those associated with mitochondrial biogenesis, fatty acid oxidation, and the electron transport chain. Results in cultured cells and adult skeletal muscle confirmed that MyoD regulates oxidative metabolism through multiple transcriptional targets, including PGC-1β, a master regulator of mitochondrial biogenesis. We find that PGC-1β expression is cooperatively regulated by MyoD and the alternative NF-κB signaling pathway. Bioinformatics evidence suggests that this cooperativity between MyoD and NF-κB extends to other metabolic genes as well. Together, these data identify MyoD as a regulator of the metabolic capacity of mature skeletal muscle to ensure that sufficient energy is available to support muscle contraction.
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•MyoD regulates the oxidative metabolic capacity of adult skeletal muscle•ChIP-seq analysis identified MyoD binding on the PGC-1β, but not PGC-1α, gene locus•MyoD cooperates with alternative NF-κB to regulate PGC-1β transcription•MyoD and RelB co-occupy many other genes involved in aerobic respiration
Shintaku et al. discovered that MyoD is a major regulator of skeletal muscle oxidative metabolism. MyoD and the alternative NF-κB transcription factor RelB cooperatively bind enhancers along the PGC-1β gene to regulate its transcription. In addition to PGC-1β, MyoD and RelB preferentially co-occupy numerous other oxidative metabolic genes.
Although α7 nicotinic acetylcholine receptors are considered potentially important therapeutic targets, the development of selective agonists has been stymied by the α7 receptor's intrinsically low ...probability of opening (P(open)) and the concern that an agonist-based therapeutic approach would disrupt endogenous cholinergic function. Development of α7 positive allosteric modulators (PAMs) holds promise of avoiding both issues. N-(5-Chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)-urea (PNU-120596) is one of the most effective α7 PAMs, with a mechanism associated, at least in part, with the destabilization of desensitized states. We studied the mechanism of PNU-120596 potentiation of α7 receptors expressed in Xenopus laevis oocytes and outside-out patches from BOSC 23 cells. We identify two forms of α7 desensitization: one is destabilized by PNU-120596 (D(s)), and the other is induced by strong episodes of activation and is stable in the presence of the PAM (D(i)). Our characterization of prolonged bursts of single-channel currents that occur with PNU-120596 provide a remarkable contrast to the behavior of the channels in the absence of the PAM. Individual channels that avoid the D(i) state show a 100,000-fold increase in P(open) compared with receptors in the nonpotentiated state. In the presence of PNU-120596, balance between D(s) and D(i) is dynamically regulated by both agonist and PAM binding, with maximal ion channel activity at intermediate levels of binding to both classes of sites. In the presence of high agonist concentrations, competitive antagonists may have the effect of shifting the balance in favor of D(s) and increasing ion channel currents.
Biofilm-impaired tissue is a significant factor in chronic wounds such as diabetic foot ulcers. Most, if not all, anti-biotics in clinical use have been optimized against planktonic phenotypes. In ...this study, an in vitro assessment was performed to determine the potential efficacy of a first-in-class series of antibiofilm antibiotics and compare outcomes to current clinical standards of care. The agent, CZ-01179, was formulated into a hydrogel and tested against mature biofilms of a clinical isolate of methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa ATCC 27853 using two separate methods. In the first method, biofilms were grown on cellulose discs on an agar surface. Topical agents were spread on gauze and placed over the biofilms for 24 h. Biofilms were quantified and imaged with confocal and scanning electron microscopy. In the second method, biofilms were grown on bioabsorbable collagen coupons in a modified CDC biofilm reactor. Coupons were immersed in treatment for 24 h. The first method was limited in its ability to assess efficacy. Efficacy profiles against biofilms grown on collagen were more definitive, with CZ-01179 gel eradicating well-established biofilms to a greater degree compared to clinical standards. In conclusion, CZ-01179 may be a promising topical agent that targets the biofilm phenotype. Pre-clinical work is currently being performed to determine the translatable potential of CZ-01179 gel.
Periprosthetic joint infection (PJI) is a devastating complication of orthopedic implant surgeries, such as total knee and hip arthroplasties. Treatment requires additional surgeries because ...antibiotics have limited efficacy due to biofilm formation and resistant bacterial strains such as methicillin‐resistant Staphylococcus aureus (MRSA). A non‐pharmaceutical approach is needed, and examples of this are found in nature; dragonfly and cicada wings are antibacterial because of their nanopillar surface structure rather than their chemistry. Carbon‐infiltrated carbon nanotube (CICNT) surfaces exhibit a similar nanopillar structure, and have been shown to facilitate osseointegration, and it is postulated that they might provide a structurally‐derived resistance to bacterial proliferation and biofilm formation. The objective of this study was to test the biofilm resistance of CICNT coatings. Two types of CICNT were produced: a vertically aligned CNT forest on a silicon substrate using a layer of iron as the catalyst (CICNT‐Si) and a random‐oriented CNT forest on stainless steel (SS) substrate using the substrate as the catalyst (CICNT‐SS). These were tested against SS and carbon controls. After 48 h in an MRSA biofilm reactor, samples demonstrated that both types of CICNT coatings significantly (p < 0.0001) reduced MRSA biofilm formation by 60%–80%. Morphologically, biofilm presence on both types of CICNT was also significantly reduced. Clinical Significance: Results suggest that a CICNT surface modification could be suitable and advantageous for medical devices susceptible to MRSA cell attachment and biofilm proliferation, particularly orthopedic implants.
Bacterial biofilms pose a challenge in treating implant-associated infections. Biofilms provide bacteria with protection against antimicrobial agents and the immune response and often are invisible ...to the naked eye. As a biofilm-disclosing agent, methylene blue (MB) has shown promise, but lacks rigorous in vitro evaluation. The purposes of the present study were to assess MB as a biofilm-disclosing agent in vitro for common biofilm-forming organisms and to determine performance characteristics across implant materials and healthy tissue types.
Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 27853) biofilms were grown on culture for 2 days in CDC biofilm reactors on titanium, cobalt chromium, polyethylene, and polyether ether ketone (PEEK) coupons. Biofilms were stained with MB solutions of either 0.005% or 0.01% and then were washed with normal saline solution. Digital photographs were obtained to compare the visual sensitivity of the blue dye at these dilutions. Scanning electron microscopy (SEM) was performed to confirm the absence or presence of biofilm on MB-stained areas. Uninoculated controls were also assessed. Healthy adult sheep tissues were also stained to determine the staining characteristics of the host tissue. ImageJ was used to determine the relative blue intensity of stained implants and tissues compared with standard curves.
S. aureus and P. aeruginosa biofilms stained avidly on titanium, cobalt chromium, polyethylene, and PEEK coupons. There was visible dose-dependent staining based on dye concentration. MB was visible only where biofilms were present as confirmed by SEM. MB did not stain uninoculated controls. Articular cartilage and meniscus demonstrated appreciable staining; bone, tendon, muscle, nerve, and fat did not. Bacterial biofilms demonstrated both dose-dependent and species-specific staining.
MB is an effective disclosing agent for S. aureus and P. aeruginosa biofilms in vitro. MB did not stain implant materials, nor did it stain most healthy tissues in vitro. MB may allow surgeons to see biofilms and may allow for enhanced debridement once visualized.
The literature demonstrates that obtaining a biopsy of the physis may be beneficial for diagnostic purposes. A small biopsy of the epiphyseal plate may allow for earlier detection of certain ...conditions and be used to monitor the healing of diseased and/or damaged physes. However, due to the fear of a growth arrest in a growing child, biopsies are not currently performed. In this study, we investigated the effects of a biopsy of the epiphyseal plate in 3‐month‐old lambs. A total of 4.2 mm biopsy samples were captured in the proximal tibiae and distal femora physes. The lambs were monitored 12‐ and 24‐week post‐biopsy. Computed tomography (CT) and micro‐CT scans were obtained to determine if any angular deformities occurred, while scanning electron microscope (SEM) and histological analysis were utilized to assess the bone response due to the biopsy. The contralateral limbs served as unaltered controls for direct comparison within each lamb. The data demonstrated no signs of angular deformities following a 4.2 mm biopsy of the physis. Bone growth/elongation was confirmed by CT, SEM, and fluorochrome analyses and indicated that the lambs were in fact immature and still growing at the time of the biopsy. Clinical Significance: This investigation demonstrated that a small biopsy of the epiphyseal plate can be obtained safely without the cause of growth arrest and angular deformities. The ability to precisely diagnose, treat, and/or monitor at‐risk children at an earlier timepoint by way of a biopsy sample could be an important advancement in regard to researching diseased and/or damaged physes.
We investigated the effects of a biopsy of the epiphyseal plate in 3‐month old lambs. A 4.2 mm biopsy sample was captured in the proximal tibiae and distal femora physes. The lambs were monitored for 12‐ and 24‐week. The data demonstrated no signs of angular deformities following a 4.2 mm biopsy of the physis. Bone growth/elongation was confirmed by computed tomography, scanning electron microscope (SEM), and fluorochrome analyses. This investigation demonstrated that a small biopsy of the epiphyseal plate can be obtained safely for diagnostic purposes.
Antimicrobial blue light (aBL) is an attractive option for managing biofilm burden at the skin-implant interface of percutaneous osseointegrated (OI) implants. However, marketed aBL devices have both ...structural and optical limitations that prevent them from being used in an OI implant environment. They must be handheld, preventing even irradiation of the entire skin-implant interface, and the devices do not offer sufficient optical power outputs required to kill biofilms. We present the developmental process of a unique aBL device that overcomes these limitations. Four prototypes are detailed, each being a progressive improvement from the previous iteration as we move from proof-of-concept to in vivo application. Design features focused on a cooling system, LED orientation, modularity, and "sheep-proofing". The final prototype was tested in an in vivo OI implant sheep model, demonstrating that it was structurally and optically adequate to address biofilm burdens at the skin-implant of percutaneous OI implants. The device made it possible to test aBL in the unique OI implant environment and compare its efficacy to clinical antibiotics-data which had not before been achievable. It has provided insight into whether or not continued pursual of light therapy research for OI implants, and other percutaneous devices, is worthwhile. However, the device has drawbacks concerning the cooling system, complexity, and size if it is to be translated to human clinical trials. Overall, we successfully developed a device to test aBL therapy for patients with OI implants and helped progress understanding in the field of infection management strategies.
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