Deletion of a previously uncharacterized lipoprotein lirL confers resistance to an inhibitor of type II signal peptidase in Acinetobacter baumannii.

Acinetobacter baumannii is a clinically important, predominantly health care-associated gram-negative bacterium with high rates of emerging resistance worldwide. Given the urgent need for novel antibacterial therapies against A. baumannii, we focused on inhibiting lipoprotein biosynthesis, a pathway that is essential for envelope biogenesis in gram-negative bacteria. The natural product globomycin, which inhibits the essential type II signal peptidase prolipoprotein signal peptidase (LspA), is ineffective against wild-type A. baumannii clinical isolates due to its poor penetration through the outer membrane. Here, we describe a globomycin analog, G5132, that is more potent against wild-type and clinical A. baumannii isolates. Mutations leading to G5132 resistance in A. baumannii map to the signal peptide of a single hypothetical gene, which we confirm encodes an alanine-rich lipoprotein and have renamed lirL (prolipoprotein signal peptidase inhibitor resistance lipoprotein). LirL is a highly abundant lipoprotein primarily localized to the inner membrane. Deletion of lirL leads to G5132 resistance, inefficient cell division, increased sensitivity to serum, and attenuated virulence. Signal peptide mutations that confer resistance to G5132 lead to the accumulation of diacylglyceryl-modified LirL prolipoprotein in untreated cells without significant loss in cell viability, suggesting that these mutations overcome a block in lipoprotein biosynthetic flux by decreasing LirL prolipoprotein substrate sensitivity to processing by LspA. This study characterizes a lipoprotein that plays a critical role in resistance to LspA inhibitors and validates lipoprotein biosynthesis as a antibacterial target in A. baumannii.

Peripheral blood-derived mesenchymal stem cells: candidate cells responsible for healing critical-sized calvarial bone defects.

Postnatal tissue-specific stem/progenitor cells hold great promise to enhance repair of damaged tissues. Many of these cells are retrieved from bone marrow or adipose tissue via invasive procedures. Peripheral blood is an ideal alternative source for the stem/progenitor cells because of its ease of retrieval. We present a coculture system that routinely produces a group of cells from adult peripheral blood. Treatment with these cells enhanced healing of critical-size bone defects in the mouse calvarium, a proof of principle that peripheral blood-derived cells can be used to heal bone defects. From these cells, we isolated a subset of CD45(-) cells with a fibroblastic morphology. The CD45(-) cells were responsible for most of the differentiation-induced calcification activity and were most likely responsible for the enhanced healing process. These CD45(-) fibroblastic cells are plastic-adherent and exhibit a surface marker profile negative for CD34, CD19, CD11b, lineage, and c-kit and positive for stem cell antigen 1, CD73, CD44, CD90.1, CD29, CD105, CD106, and CD140α. Furthermore, these cells exhibited osteogenesis, chondrogenesis, and adipogenesis capabilities. The CD45(-) fibroblastic cells are the first peripheral blood-derived cells that fulfill the criteria of mesenchymal stem cells as defined by the International Society for Cellular Therapy. We have named these cells "blood-derived mesenchymal stem cells."

A simple running model with rolling contact and its role as a template for dynamic locomotion on a hexapod robot.

We report on the development of a robot's dynamic locomotion based on a template which fits the robot's natural dynamics. The developed template is a low degree-of-freedom planar model for running with rolling contact, which we call rolling spring loaded inverted pendulum (R-SLIP). Originating from a reduced-order model of the RHex-style robot with compliant circular legs, the R-SLIP model also acts as the template for general dynamic running. The model has a torsional spring and a large circular arc as the distributed foot, so during locomotion it rolls on the ground with varied equivalent linear stiffness. This differs from the well-known spring loaded inverted pendulum (SLIP) model with fixed stiffness and ground contact points. Through dimensionless steps-to-fall and return map analysis, within a wide range of parameter spaces, the R-SLIP model is revealed to have self-stable gaits and a larger stability region than that of the SLIP model. The R-SLIP model is then embedded as the reduced-order 'template' in a more complex 'anchor', the RHex-style robot, via various mapping definitions between the template and the anchor. Experimental validation confirms that by merely deploying the stable running gaits of the R-SLIP model on the empirical robot with simple open-loop control strategy, the robot can easily initiate its dynamic running behaviors with a flight phase and can move with similar body state profiles to those of the model, in all five testing speeds. The robot, embedded with the SLIP model but performing walking locomotion, further confirms the importance of finding an adequate template of the robot for dynamic locomotion.

Bio-inspired step-climbing in a hexapod robot.

Inspired by the observation that the cockroach changes from a tripod gait to a different gait for climbing high steps, we report on the design and implementation of a novel, fully autonomous step-climbing maneuver, which enables a RHex-style hexapod robot to reliably climb a step up to 230% higher than the length of its leg. Similar to the climbing strategy most used by cockroaches, the proposed maneuver is composed of two stages. The first stage is the 'rearing stage,' inclining the body so the front side of the body is raised and it is easier for the front legs to catch the top of the step, followed by the 'rising stage,' maneuvering the body's center of mass to the top of the step. Two infrared range sensors are installed on the front of the robot to detect the presence of the step and its orientation relative to the robot's heading, so that the robot can perform automatic gait transition, from walking to step-climbing, as well as correct its initial tilt approaching posture. An inclinometer is utilized to measure body inclination and to compute step height, thus enabling the robot to adjust its gait automatically, in real time, and to climb steps of different heights and depths successfully. The algorithm is applicable for the robot to climb various rectangular obstacles, including a narrow bar, a bar and a step (i.e. a bar of infinite width). The performance of the algorithm is evaluated experimentally, and the comparison of climbing strategies and climbing behaviors in biological and robotic systems is discussed.

Herpes simplex virus-1 induces expression of a novel MxA isoform that enhances viral replication.

MxA is an antiviral protein induced by interferon (IFN)-α/ß that is known to inhibit the replication of many RNA viruses. In these experiments, the 76-kDa MxA protein expressed in IFN-α-treated cells was shown to have antiviral activity against herpes simplex virus-1 (HSV-1), a human DNA virus. However, MxA was expressed as a 56-kDa protein in HSV-1-infected cells in the absence of IFN-α. This previously unrecognized MxA isoform was produced from an alternatively spliced MxA transcript that had a deletion of Exons 14-16 and a frame shift altering the C-terminus. The variant MxA (varMxA) isoform was associated with HSV-1 regulatory proteins and virions in nuclear replication compartments. varMxA expression enhanced HSV-1 infection as shown by a reduction in infectious virus titers from cells in which MxA had been inhibited by RNA interference and by an increase in HSV-1 titers when the 56-kDa varMxA was expressed constitutively. Thus, the human MxA gene encodes two MxA isoforms, which are expressed differentially depending on whether the stimulus is IFN-α or HSV-1. These findings show that alternative splicing of cellular mRNA can result in expression of a novel isoform of a host defense gene that supports instead of restricting viral infection.

Endonuclease G: a role for the enzyme in recombination and cellular proliferation.

Our earlier studies had suggested that endonuclease G (EndoG), a member of the evolutionarily conserved DNA/RNA nonspecific betabetaalpha-Me-finger nuclease family, functioned in the a sequence-mediated segment inversion observed during herpes simplex virus 1 replication. To test this hypothesis, we used RNA interference to reduce the level of EndoG in mammalian cells in culture. Reduction of EndoG produced a small but statistically significant decrease in a sequence-mediated recombination, suggesting that EndoG does play a role in this process. We also observed that reduction in the level of EndoG resulted in a deficiency in cell proliferation. Cells with a reduced level of EndoG also showed changes in cell distribution in the cell cycle, producing a pattern characteristic of cells that have been arrested in the G(2) phase. These findings suggest that EndoG is required for normal cellular proliferation.

Endonuclease G, a candidate human enzyme for the initiation of genomic inversion in herpes simplex type 1 virus.

The herpes simplex virus type 1 (HSV-1) a sequence is present as a direct repeat at the two termini of the 152-kilobase viral genome and as an inverted repeat at the junction of the two unique components L and S. During replication, the HSV-1 genome undergoes inversion of L and S, producing an equimolar mixture of the four possible isomers. Isomerization is believed to result from recombination triggered by breakage at the a sequence, a recombinational hot spot. We have identified an enzyme in HeLa cell extracts that preferentially cleaves the a sequence and have purified it to near homogeneity. Microsequencing showed it to be human endonuclease G, an enzyme with a strong preference for G+C-rich sequences. Endonuclease G appears to be the only cellular enzyme that can specifically cleave the a sequence. Endonuclease G also showed the predicted recombination properties in an in vitro recombination assay. Based on these findings, we propose that endonuclease G initiates the a sequence-mediated inversion of the L and S components during HSV-1 DNA replication.