We have synthesized a 582,970-base pair Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted "watermarks" at intergenic sites known to tolerate transposon insertions. Overlapping "cassettes" of 5 to 7 kilobases (kb), assembled from chemically synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approximately 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4 genome"), which were all cloned as bacterial artificial chromosomes in Escherichia coli. Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments.

Selective inhibition of T cell costimulation using the B7-specific fusion protein CTLA4-Ig has been shown to induce long-term allograft survival in rodents. Antibodies preventing the interaction between CD40 and its T cell-based ligand CD154 (CD40L) have been shown in rodents to act synergistically with CTLA4-Ig. It has thus been hypothesized that these agents might be capable of inducing long-term acceptance of allografted tissues in primates. To test this hypothesis in a relevant preclinical model, CTLA4-Ig and the CD40L-specific monoclonal antibody 5C8 were tested in rhesus monkeys. Both agents effectively inhibited rhesus mixed lymphocyte reactions, but the combination was 100 times more effective than either drug alone. Renal allografts were transplanted into nephectomized rhesus monkeys shown to be disparate at major histocompatibility complex class I and class II loci. Control animals rejected in 5-8 days. Brief induction doses of CTLA4-Ig or 5C8 alone significantly prolonged rejection-free survival (20-98 days). Two of four animals treated with both agents experienced extended (>150 days) rejection-free allograft survival. Two animals treated with 5C8 alone and one animal treated with both 5C8 and CTLA4-Ig experienced late, biopsy-proven rejection, but a repeat course of their induction regimen successfully restored normal graft function. Neither drug affected peripheral T cell or B cell counts. There were no clinically evident side effects or rejections during treatment. We conclude that CTLA4-Ig and 5C8 can both prevent and reverse acute allograft rejection, significantly prolonging the survival of major histocompatibility complex-mismatched renal allografts in primates without the need for chronic immunosuppression.Unmodified organ transplantation between genetically nonidentical individuals invariably results in immunological rejection of the organ through T cell-dependent mechanisms. Successful transplantation of allogeneic organs has therefore required the administration of drugs directed at suppressing recipient T cell function. Both calcineurin phosphatase inhibitors and glucocorticosteroids are used clinically, and both prevent the T cell-mediated release of activating cytokines, particularly IL-2. Therapy with these agents is imperfect, however. Both act by impairing T cell antigen receptor (TCR) signal transduction, the sole mediator of T cell antigen recognition, thus minimizing the potential for specific immune interaction between the recipient and donor. They also act on all T cells indiscriminately. In addition, the effect of these drugs is not lasting, such that cessation of immunosuppression has generally resulted in graft loss even after prolonged rejection-free survival. Thus, transplant patients are required to suffer the consequences of nonspecific immunosuppression to avoid rejection. These consequences include an increased risk to the patient of infection and malignancy as well as significant drug related expense and toxicity.Data establishing that T cell activation require...

We recently reported the chemical synthesis, assembly, and cloning of a bacterial genome in yeast. To produce a synthetic cell, the genome must be transferred from yeast to a receptive cytoplasm. Here we describe methods to accomplish this. We cloned a Mycoplasma mycoides genome as a yeast centromeric plasmid and then transplanted it into Mycoplasma capricolum to produce a viable M. mycoides cell. While in yeast, the genome was altered by using yeast genetic systems and then transplanted to produce a new strain of M. mycoides. These methods allow the construction of strains that could not be produced with genetic tools available for this bacterium.

Both chronic and acute dermal wounds are susceptible to infection due to sterile loss of the innate barrier function of the skin and dermal appendages, facilitating the development of microbial communities, referred to as biofilms, within the wound environment. Microbial biofilms are implicated in both the infection of wounds and failure of those wounds to heal. The aim of this review is to provide a summary of published papers detailing biofilms in wounds, the effect they have on infection and wound healing, and detailing methods employed for their detection. The studies highlighted within this paper provide evidence that biofilms reside within the chronic wound and represent an important mechanism underlying the observed, delayed healing and infection. The reasons for this include both protease activity and immunological suppression. Furthermore, a lack of responsiveness to an array of antimicrobial agents has been due to the biofilms' ability to inherently resist antimicrobial agents. It is imperative that effective strategies are developed, tested prospectively, and employed in chronic wounds to support the healing process and to reduce infection rates. It is increasingly apparent that adoption of a biofilm-based management approach to wound care, utilizing the "antibiofilm tool box" of therapies, to kill and prevent reattachment of microorganisms in the biofilm is producing the most positive clinical outcomes and prevention of infection.

Chronic leg and foot wounds represent an increasing burden to healthcare systems as the age of the population increases. The deep dermal tissues of all chronic wounds harbour microorganisms, however, the precise interaction between microbes in the wounds and impaired healing is unknown. With regard to antibiotic therapy, there is a lack of evidence concerning its effectiveness, optimal regimens or clinical indications for treatment. Despite this lack of evidence, antibiotics are frequently a feature of the management of chronic wounds and these patients receive significantly more antibiotic prescriptions (both systemic and topical) than age and sex-matched patients. Current guidelines for antibiotic prescribing for such wounds are often based on expert opinion rather than scientific fact and may present difficulties in interpretation and implementation to the clinician. Although the increasing prevalence of antibiotic resistance is widely recognized, the relationships between antibiotic resistance, chronic wound microbiology and rationales for antibiotic therapy have yet to be determined. This review discusses the role of microbes in chronic wounds from a clinical perspective with particular focus on the occurrence of bacteria and their impact on such wounds. The evidence and role of antibiotics in the treatment of such wounds are outlined and current practice of antibiotic usage for chronic wounds in the primary care setting described. The implications of antibiotic usage with regard to antibiotic resistance are also considered.