degenerative and regenerative roles of tumor necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine with pleiotropic functions, were investigated by using TNF receptor 1 and 2 double knockout (TNFR-DKO) and TNF-alpha antibody neutralized mice following traumatic freeze injury to the tibialis anterior muscle. In wild-type control mice, TNF-alpha mRNA transcripts and protein increased following injury and gradually returned to control (uninjured) levels by 13 days. A reduction in MyoD mRNA expression occurred in TNF-alpha-deficient mice, although there were no visible differences in MyoD immunostaining or histological characteristics in regenerating muscles. At 5 days post-injury, the reductions in isometric strength in TNFR-DKO and TNF-alpha-depleted mice did not differ from that of wild-type mice but by 13 days after injury, the TNFR-DKO and TNF-alpha-depleted mice exhibited strength deficits twice that of wild-type mice (i.e., 27-31% vs 13%). Muscle injury was also accompanied by increased expression of interleukin-6 (IL-6), but IL-6-deficient mice demonstrated MyoD expression and recovery of isometric strength similar to that of wild-type mice. These data indicate that TNF-alpha is involved in the recovery of muscle function after traumatic muscle injury, and this effect might be associated with modulation of muscle regulatory genes, including MyoD.

With current concerns of antibiotic-resistant bacteria and biodefense, it has become important to rapidly identify infectious bacteria. Traditional technologies involving isolation and amplification of the pathogenic bacteria are time-consuming. We report a rapid and simple method that combines in vivo biotinylation of engineered host-specific bacteriophage and conjugation of the phage to streptavidin-coated quantum dots. The method provides specific detection of as few as 10 bacterial cells per milliliter in experimental samples, with an Ϸ100-fold amplification of the signal over background in 1 h. We believe that the method can be applied to any bacteria susceptible to specific phages and would be particularly useful for detection of bacterial strains that are slow growing, e.g., Mycobacterium, or are highly infectious, e.g., Bacillus anthracis. The potential for simultaneous detection of different bacterial species in a single sample and applications in the study of phage biology are discussed.bacteriophage T7 ͉ BirA ͉ Escherichia coli ͉ water sample T he number and diversity of bacteriophages in the environment provide a promising natural pool of specific detection tools for pathogenic bacteria. Currently there are several phagebased methods for detection of pathogenic bacteria (1): a plaque assay for detection of Mycobacterium tuberculosis (2); fluorescence-labeled phage and immunomagnetic separation assay for detection of Escherichia coli O157:H7 (3, 4); phage-based electrochemical assays (5); a luciferase reporter mycobacteriophage and Listeria phage assays (6, 7); and detection of the phagemediated bacterial lysis and release of host enzymes (e.g., adenylate kinase) (8).Two limiting features when detecting pathogenic bacteria are sensitivity and rapidity. Common fluorophores (e.g., GFP and luciferase) used as reporters have two major disadvantages: low signal-to-noise ratio due to autofluorescence of clinical samples and of bacterial cells and low photostability, such as fast photobleaching. To overcome these disadvantages, we used new fluorescent semiconductor nanocrystals, quantum dots (QDs) (9). QDs are colloidal semiconductor (e.g., CdSe) crystals of a few nanometers in diameter. They exhibit broadband absorption spectra, and their emissions are of narrow bandwidth with size-dependent local maxima. The presence of an outer shell of a few atomic layers (e.g., ZnS) increases the quantum yield and further enhances the photostability, resulting in photostable fluorescent probes superior to conventional organic dyes. Recently, development in surface chemistry protocols allows conjugation of biomolecules onto these QDs to target specific biological molecules and probe nanoenvironments (10-12). The power to observe and trace single QDs or a group of bioconjugated QDs, enabling more precise quantitative biology, has been claimed to be one of the most exciting new capabilities offered to biologists today (13,14).Typically, the detection of small numbers of bacteria in environmental or clinical samples require...

The genome sequence of a Bacillus anthracis-specific clear plaque mutant phage, AP50c, contains 31 open reading frames spanning 14,398 bp, has two mutations compared to wild-type AP50t, and has a colinear genome architecture highly similar to that of gram-positive Tectiviridae phages. Spontaneous AP50c-resistant B. anthracis mutants exhibit a mucoid colony phenotype.

No abstract

Traumatic skeletal muscle injury causes a specific sequence of cellular events consisting of degeneration, inflammation, regeneration, and fibrosis. The role of early posttraumatic mechanisms, including acute inflammatory response, in muscle repair is not well understood. In the present study, oligonucleotide microarray analyses were used to examine the candidate genes that are involved in these early events of the muscle injury/repair process. cDNA was prepared from the injured and control tibialis anterior (TA) muscle of mice 24 h postinjury and labeled with the fluorescent dye Cy5 or Cy3 prior to hybridization to a DNA microarray. The microarray analysis, including 732 genes, was conducted in triplicate, and we describe only genes modulated by the injury showing a differential expression (both increased and decreased) 1.7-fold or greater (p < 0.05) from control uninjured TA muscle. Selected expression patterns were confirmed by other gene expression detection methods, including real-time reverse transcription-polymerase chain reaction (RT-PCR) and RNase protection assay (RPA) or immunohistochemistry detection methods. The upregulated genes (2.8%) were mainly associated with inflammation, oxidative stress, and cell proliferation, whereas the downregulated genes (3.2%) were related to metabolic and cell signaling pathways. In addition, the study suggested that chemokines, such as monocyte chemoattractant protein-1 (MCP-1), associated with monocyte/macrophage influx and activation, are abundantly expressed in postinjured muscle, and they might play a role in traumatic muscle injury/recovery processes.