CD40 Negatively Regulates ATP-TLR4-Activated Inflammasome in Microglia

During acute brain injury and/or sterile inflammation, release of danger-associated molecular patterns (DAMPs) activates pattern recognition receptors (PRRs). Microglial toll-like receptor (TLR)-4 activated by DAMPs potentiates neuroinflammation through inflammasome-induced IL-1β and pathogenic Th17 polarization which critically influences brain injury. TLR4 activation accompanies increased CD40, a cognate costimulatory molecule, involved in microglia-mediated immune responses in the brain. During brain injury, excessive release of extracellular ATP (DAMPs) is involved in promoting the damage. However, the regulatory role of CD40 in microglia during ATP-TLR4-mediated inflammasome activation has never been explored. We report that CD40, in the absence of ATP, synergizes TLR4-induced proinflammatory cytokines but not IL-1β, suggesting that the response is independent of inflammasome. The presence of ATP during TLR4 activation leads to NLRP3 inflammasome activation and caspase-1-mediated IL-1β secretion which was inhibited during CD40 activation, accompanied with inhibition of ERK1/2 and reactive oxygen species (ROS), and elevation in p38 MAPK phosphorylation. Experiments using selective inhibitors prove indispensability of ERK 1/2 and ROS for inflammasome activation. The ATP-TLR4-primed macrophages polarize the immune response toward pathogenic Th17 cells, whereas CD40 activation mediates Th1 response. Exogenous supplementation of IFN-γ (a Th1 cytokine and CD40 inducer) results in decreased IL-1β, suggesting possible feedback loop mechanism of inflammasome inhibition, whereby IFN-γ-mediated increase in CD40 expression and activation suppress neurotoxic inflammasome activation required for Th17 response. Collectively, the findings indicate that CD40 is a novel negative regulator of ATP-TLR4-mediated inflammasome activation in microglia, thus providing a checkpoint to regulate excessive inflammasome activation and Th17 response during DAMP-mediated brain injury.     

Potential of 2, 2′-dipyridyl diselane as an adjunct to antibiotics to manage cadmium-induced antibiotic resistance in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhi Ty2 strain

One of the reasons for increased antibiotic resistance in Salmonella enterica serovar Typhi Ty2 is the influx of heavy metal ions in the sewage, from where the infection is transmitted. Therefore, curbing these selective agents could be one of the strategies to manage the emergence of multidrug resistance in the pathogen. As observed in our earlier study, the present study also confirmed the links between cadmium accumulation and antibiotic resistance in Salmonella. Therefore, the potential of a chemically-synthesised compound 2, 2′-dipyridyl diselane (DPDS) was explored to combat the metal-induced antibiotic resistance. Its metal chelating and antimicrobial properties were evidenced by fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FE-SEM), and microbroth dilution method. Owing to these properties of DPDS, further, this compound was evaluated for its potential to be used in combination with conventional antibiotics. The data revealed effective synergism at much lower concentrations of both the agents. Thus, it is indicated from the study that the combination of these two agents at their lower effective doses might reduce the chances of emergence of antibiotic resistance, which can be ascribed to the multi-pronged action of the agents.     

Detailed role of microRNA-mediated regulation of PI3K/AKT axis in human tumors

The regulation of signal transmission and biological processes, such as cell proliferation, apoptosis, metabolism, migration, and angiogenesis are greatly influenced by the PI3K/AKT signaling pathway. Highly conserved endogenous non-protein-coding RNAs known as microRNAs (miRNAs) have the ability to regulate gene expression by inhibiting mRNA translation or mRNA degradation. MiRNAs serve key role in PI3K/AKT pathway as upstream or downstream target, and aberrant activation of this pathway contributes to the development of cancers. A growing body of research shows that miRNAs can control the PI3K/AKT pathway to control the biological processes within cells. The expression of genes linked to cancers can be controlled by the miRNA/PI3K/AKT axis, which in turn controls the development of cancer. There is also a strong correlation between the expression of miRNAs linked to the PI3K/AKT pathway and numerous clinical traits. Moreover, PI3K/AKT pathway-associated miRNAs are potential biomarkers for cancer diagnosis, therapy, and prognostic evaluation. The role and clinical applications of the PI3K/AKT pathway and miRNA/PI3K/AKT axis in the emergence of cancers are reviewed in this article.     

Scanning Mutagenesis of ��-Conotoxin Vc1.1 Reveals Residues Crucial for Activity at the ��9��10 Nicotinic Acetylcholine Receptor

Vc1.1 is a disulfide-rich peptide inhibitor of nicotinic acetylcholine receptors that has stimulated considerable interest in these receptors as potential therapeutic targets for the treatment of neuropathic pain. Here we present an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp, or Lys. The effect on acetylcholine (ACh)-evoked membrane currents at the α9α10 nicotinic acetylcholine receptor (nAChR), which has been implicated as a target in the alleviation of neuropathic pain, was then observed. The analogs were characterized by NMR spectroscopy to determine the effects of mutations on structure. The structural fold was found to be preserved in all peptides except where Pro was substituted. Electrophysiological studies showed that the key residues for functional activity are Asp⁵–Arg⁷ and Asp¹¹–Ile¹⁵, because changes at these positions resulted in the loss of activity at the α9α10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. A second generation of mutants was synthesized, namely N9G, N9I, N9L, S4R, and S4K+N9A, all of which were more potent than Vc1.1 at both the rat α9α10 and the human α9/rat α10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the α3β2, α3β4, and α7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the α9α10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the α9α10 nAChR subtype and will help in the further development of analogs of Vc1.1 as analgesic drugs.Marine snails belonging to the Conus genus produce a variety of neurotoxic peptides in their venom glands that they use for the capture of prey (1–3). Within this repertoire of conopeptides, those that are disulfide-rich are referred to as conotoxins. Conotoxins typically range in size from 12 to 30 amino acids, contain 4 or more Cys residues, and exhibit high potency and selectivity toward a variety of membrane receptors and ion channels (4, 5). The α-conotoxin subfamily members typically range in size from 12 to 19 amino acids, contain 2 disulfide bonds in a Cys^I–Cys^III and Cys^II–Cys^IV connectivity, and have an amidated C terminus, as depicted in Fig. 1. They interact with nicotinic acetylcholine receptors (nAChRs),⁴ of both the muscle and the neuronal type, which have been implicated in a range of neurological disorders varying from Alzheimer disease to addiction (6–8).Open in a separate windowFIGURE 1.α-Conotoxin sequences and structure of Vc1.1. a, the sequences of selected α-conotoxins relevant to this study are shown by one-letter amino acid codes. The asterisk indicates an amidated C terminus, which is a common post-translational modification found in α-conotoxins. The conserved cysteine residues are highlighted in yellow, and the Cys^I–Cys^III and Cys^II–Cys^IV disulfide connectivity is indicated by the connecting lines under the sequence. The number of residues between the cysteines define two backbone “loops,” which are used to classify α-conotoxins into subclasses. For example, RgIA has four residues in loop 1 and three residues in loop 2, making this a 4/3 loop subclass α-conotoxin. b, structural representation of Vc1.1 (PDB 2H8S), with disulfide bonds depicted in yellow. The cysteines, the loops, and the termini are labeled.The nAChRs are ligand-gated ion channels that respond to ACh, nicotine, and other competitive agonists/antagonists. They are composed of five subunits, with differing nAChR subunit composition according to the site of expression. The muscle-type nAChRs are composed of two α subunits, a β and δ subunit, and either an ϵ or a γ subunit (9–12). The neuronal forms exist either as homomeric channels composed of α subunits alone or αβ heteromeric channels. The wide variety of possible subunit combinations has led to unique subtypes with distinct pharmacological properties. This makes α-conotoxins valuable neuropharmacological tools and drug leads, because they have the ability to distinguish between different nAChR subtypes. Effectively, they are small rigid scaffolds that display amino acids on their surface to selectively target their receptors (13).Of particular interest in this study is the α-conotoxin Vc1.1, a synthetic derivative of a naturally occurring peptide from the venom of the marine cone snail, Conus victoriae. It was discovered using PCR screening of cDNA extracted from the snail venom duct (14). Fig. 1 depicts the sequences of selected α-conotoxins, including Vc1.1, which is 16 amino acids in length and displays the classic disulfide bond connectivity observed for α-conotoxins, together with a short helical segment as depicted in Fig. 1b. The conserved Cys framework of α-conotoxins defines two backbone loops, which vary in size and residue composition, and are classified by an n/m nomenclature to define subclasses of α-conotoxins. For example, Vc1.1 is a 4/7 subclass α-conotoxin, because it contains four residues in loop 1 and seven in loop 2. RgIA (15–17) is another conotoxin of interest in this study, because it is also selective for the α9α10 nAChR subtype, and has a 4/3 framework. Vc1.1 contains an amidated C terminus, a post-translational modification common to most α-conotoxins, but it is not present in RgIA. Vc1.1 lacks the post-translationally modified hydroxyproline and γ-carboxyglutamate residues present in the native peptide, vc1a, isolated from the venom duct of C. victoriae (18).Vc1.1 has been under development as a drug lead for neuropathic pain (19). When tested in rat models of neuropathic pain, Vc1.1 induced analgesia when injected intramuscularly near the site of injury (20). Initially, it was thought that α3-containing subtypes of nAChRs may be the target for Vc1.1 (21); however, it was then reported that Vc1.1 has a 100-fold higher affinity at the α9α10 nAChR subtype (22, 23). The α9α10 nAChR mediates synaptic transmission between efferent olivocochlear fibers and cochlear hair cells (24–26). The mRNA of these receptor subtypes is expressed in many different tissue types from the inner ear, dorsal root ganglion (27), skin keratinocytes (28), and lymphocytes (29) to the pituitary (26). The α10 subunit has to be expressed with the α9 subunit to form a functional receptor. In the auditory system, the α9α10 nAChR plays an important role in hair cell development, but its role in other tissues is yet to be characterized (22, 26, 30, 31).Owing to the promising antinociceptive effects of Vc1.1 in animals, its analogs are of interest as leads for the treatment of neuropathic pain (14, 20). To date, studies have predominantly focused on the α9α10 nAChR, but the very recent finding that Vc1.1 also targets the γ-aminobutyric acid, type B receptor (32) has raised interest in the molecular mode of action of Vc1.1 in analgesia. Hence there is a need to define structure-activity relationships of this peptide at several targets, including human and rat forms of the α9α10 nAChR. In particular, we were interested in analogs that maintain potency at the rat α9α10 nAChR but also show significant improvement in potency at human forms of the receptor, while maintaining selectivity over other nAChR subtypes.In this study we determined such structure-activity relationships for Vc1.1 at the α9α10 nAChR by successively mutating each non-Cys residue of Vc1.1 to either an “inert” residue (Ala), a negatively charged residue (Asp), or a positively charged residue (Lys) and observing the impact on the structure and functional activity of Vc1.1. Once the key residues had been identified, a second generation of analogs with new substitutions was synthesized and tested at the rat α9α10 nAChR. The analogs were also analyzed at the human α9/rat α10 (hα9rα10) hybrid clone, because a recent report⁵ suggested differences in the activity of Vc1.1 at the human and rat clones of the α9α10 nAChR. We also examined the effect of pH change on the structure of Vc1.1 using NMR αH chemical shift analysis. The results from this study provide valuable insight into the key residues involved in the interaction of Vc1.1 with the α9α10 nAChR subtype and have the potential to assist in the development of conotoxin analogs as drug leads for the treatment of neuropathic pain (4, 33).