Brain-derived neurotrophic factor (BDNF) and its receptor, tyrosine kinase receptor B (TrkB), play a critical role in activity-dependent synaptic plasticity and have been implicated as mediators of hippocampal-dependent learning and memory. The present study is the first to demonstrate a role for BDNF and TrkB in amygdala-dependent learning. Here, the use of Pavlovian fear conditioning as a learning model allows us to examine the concise role of BDNF in the amygdala after a single learning session and within a well understood neural circuit. Using in situ hybridization, mRNA levels of six different trophic factors [BDNF, neurotrophin (NT) 4/5, NGF, NT3, aFGF, and bFGF) were measured at varying time points during the consolidation period after fear conditioning. We found temporally specific changes only in BDNF gene expression in the basolateral amygdala after paired stimuli that supported learning but not after exposure to neutral or aversive stimuli alone. Using Western blotting, we found that the Trk receptor undergoes increased phosphorylation during this consolidation period, suggesting an activation of the receptor subsequent to BDNF release. Furthermore, disruption of neurotrophin signaling with intra-amygdala infusion of the Trk receptor antagonist K252a disrupted acquisition of fear conditioning. To address the specific role of the TrkB receptor, we created a novel lentiviral vector expressing a dominant-negative TrkB isoform (TrkB.T1), which specifically blocked TrkB activation in vitro. In vivo, TrkB.T1 lentivirus blocked fear acquisition without disrupting baseline startle or expression of fear. These data suggest that BDNF signaling through TrkB receptors in the amygdala is required for the acquisition of conditioned fear.

Burkholderia pseudomallei and Burkholderia thailandensis are related pathogens that invade a variety of cell types, replicate in the cytoplasm, and spread to nearby cells. We have investigated temporal and spatial requirements for virulence determinants in the intracellular life cycle, using genetic dissection and photothermal nanoblade delivery, which allows efficient placement of bacterium-sized cargo into the cytoplasm of mammalian cells. The conserved Bsa type III secretion system (T3SS Bsa ) is dispensable for invasion, but is essential for escape from primary endosomes. By nanoblade delivery of B. thailandensis we demonstrate that all subsequent events in intercellular spread occur independently of T3SS Bsa activity. Although intracellular movement was essential for cell-cell spread by B. pseudomallei and B. thailandensis, neither BimA-mediated actin polymerization nor the formation of membrane protrusions containing bacteria was required for B. thailandensis. Surprisingly, the cryptic (fla2) flagellar system encoded on chromosome 2 of B. thailandensis supported rapid intracellular motility and efficient cell-cell spread. Plaque formation by both pathogens was dependent on the activity of a type VI secretion system (T6SS-1) that functions downstream from T3SS Bsa -mediated endosome escape. A remarkable feature of Burkholderia is their ability to induce the formation of multinucleate giant cells (MNGCs) in multiple cell types. By infection and nanoblade delivery, we observed complete correspondence between mutant phenotypes in assays for cell fusion and plaque formation, and time-course studies showed that plaque formation represents MNGC death. Our data suggest that the primary means for intercellular spread involves cell fusion, as opposed to pseudopod engulfment and bacterial escape from double-membrane vacuoles.

While the antibacterial properties of graphene oxide (GO) have been demonstrated across a spectrum of bacteria, the critical role of functional groups is unclear. To address this important issue, we utilized reduction and hydration methods to establish a GO library with different oxidation, hydroxyl, and carbon radical (•C) levels that can be used to study the impact on antibacterial activity. Using antibiotic-resistant (AR) bacteria as a test platform, we found that the •C density is most proximately associated with bacterial killing. Accordingly, hydrated GO (hGO), with the highest •C density, had the strongest antibacterial effects through membrane binding and induction of lipid peroxidation. To explore its potential applications, we demonstrated that coating of catheter and glass surfaces with hGO is capable of killing drug-resistant bacteria. In summary, •C is the principle surface moiety that can be utilized for clinical applications of GO-based antibacterial coatings.

The hsd genes of Mycoplasma pulmonis encode restriction and modification enzymes exhibiting a high degree of sequence similarity to the type I enzymes of enteric bacteria. The S subunits of type I systems dictate the DNA sequence specificity of the holoenzyme and are required for both the restriction and the modification reactions. The M. pulmonis chromosome has two hsd loci, both of which contain two hsdS genes each and are complex, site-specific DNA inversion systems. Embedded within the coding region of each hsdS gene are a minimum of three sites at which DNA inversions occur to generate extensive amino acid sequence variations in the predicted S subunits. We show that the polymorphic hsdS genes produced by gene rearrangement encode a family of functional S subunits with differing DNA sequence specificities. In addition to creating polymorphisms in hsdS sequences, DNA inversions regulate the phase-variable production of restriction activity because the other genes required for restriction activity (hsdR and hsdM) are expressed only from loci that are oriented appropriately in the chromosome relative to the hsd promoter. These data cast doubt on the prevailing paradigms that restriction systems are either selfish or function to confer protection from invasion by foreign DNA.Bacterial restriction and modification (R-M) systems function as a defense mechanism conferring protection from phage infection and other types of DNA invasion. Incoming DNA lacking the appropriate base modifications is cleaved by the restriction endonuclease while host DNA is protected by the presence of sequence-specific base modifications introduced by the activity of the DNA methyltransferase (MTase). These systems usually confer incomplete protection from phage infection because some phage DNA molecules are modified by the MTase before the endonucleolytic cleavage can occur, giving rise to progeny phage containing DNA modifications identical to that of the host and, therefore, resistant to restriction activity.The type I restriction enzymes are considerably more complex than the more prevalent type II enzymes. Type II restriction activity and MTase activity are performed by two distinct enzymes encoded by gene pairs. The endonuclease reaction is sequence-specific and occurs at or very near to the site modified by the MTase. In the type I systems, the nuclease and MTase activities are performed by the same holoenzyme, which consists of three types of subunits. The S subunit dictates the sequence specificity of the MTase activity. Both the S and M subunits are necessary and sufficient for MTase activity, which occurs at specific adenine residues. All three types of subunits (S, R, and M) are required for the nuclease reaction, which occurs at essentially random sites up to 7 kilobases from the recognition sequence (1). Translocation of the holoenzyme from the recognition site to the cleavage site occurs through the ATP-dependent helicase activity of the R subunit.It has been proposed that some restriction systems are selfish beca...

Pseudomallei group Burkholderia species are facultative intracellular parasites that spread efficiently from cell to cell by a mechanism involving the fusion of adjacent cell membranes. Intercellular fusion requires the function of the cluster 5 type VI secretion system (T6SS-5) and its associated valine-glycine repeat protein, VgrG5. Here we show that VgrG5 alleles are conserved and functionally interchangeable between Burkholderia pseudomallei and its relatives B. mallei, B. oklahomensis, and B. thailandensis. We also demonstrate that the integrity of the VgrG5 C-terminal domain is required for fusogenic activity, and we identify sequence motifs, including two hydrophobic segments, that are important for fusion. Mutagenesis and secretion experiments using B. pseudomallei strains engineered to express T6SS-5 in vitro show that the VgrG5 C-terminal domain is dispensable for T6SS-mediated secretion of Hcp5, demonstrating that the ability of VgrG5 to mediate membrane fusion can be uncoupled from its essential role in type VI secretion. We propose a model in which a unique fusogenic activity at the C terminus of VgrG5 facilitates intercellular spread by B. pseudomallei and related species following injection across the plasma membranes of infected cells.