Protein over-expression in Escherichia coli triggers adaptation analogous to antimicrobial resistance.

BACKGROUND: The E. coli pET system is the most widely used protein over-expression system worldwide. It relies on the assumption that all cells produce target protein and it is generally believed that integral membrane protein (IMP) over-expression is more toxic than their soluble counterparts. RESULTS: Using GFP-tagged proteins, high level over-expression of either soluble or IMP targets results in > 99.9% cell loss with survival rate of only < 0.03%. Selective pressure generates three phenotypes: large green, large white and small colony variants. As a result, in overnight cultures, ~ 50% of the overall cell mass produces no protein. Genome sequencing of the phenotypes revealed genomic mutations that causes either the loss of T7 RNAP activity or its transcriptional downregulation. The over-expression process is bactericidal and is observed for both soluble and membrane proteins. CONCLUSIONS: We demonstrate that it is the act of high-level over-expression of exogenous proteins in E. coli that sets in motion a chain of events leading to > 99.9% cell death. These results redefine our understanding of protein over-production and link it to the adaptive survival response seen in the development of antimicrobial resistance.

Proinflammatory polarization of stifle synovial macrophages in dogs with cruciate ligament rupture.

OBJECTIVE: To determine polarization of synovial macrophages during development of cruciate ligament rupture (CR) and determine whether differences in synovial macrophage polarization in CR, osteoarthritis (OA), and healthy joints exist. STUDY DESIGN: Prospective case-controlled study. ANIMALS: Client-owned dogs with unstable stifles with CR (n = 22), paired stable contralateral stifles with partial CR (pCR; n = 7), joints with OA not related to CR (n = 6), and clinically normal (Normal; n = 7) joints. METHODS: Synovial fluid samples were collected. Smears were made for differential cytology counts and estimated total nucleated cell counts. Cytospin preparations were made, and immunocytochemical staining was performed with the pan-macrophage marker CD68, M1 macrophage markers inducible nitric oxide synthase (iNOS) and chemokine (C-C motif) receptor 7 (CCR7), and M2 macrophage markers arginase 1 and CD163. Positively stained cells were counted. RESULTS: Numbers of lymphocytes were increased in the CR group compared with the OA and Normal groups (P < .05). Numbers of CD68+ , CCR7+ , and iNOS+ cells in the CR and OA groups were increased compared with the Normal group (P < .05). Globally, the ratio of positively stained M1 polarized CD68+ cells to M2 polarized CD68+ cells was highest for the OA group (2.49), followed by the pCR (2.1), CR (1.63), and Normal (0.7) groups. CONCLUSION: Polarization of synovial macrophages toward an M1 proinflammatory phenotype is an early event in the development of canine CR. CLINICAL SIGNIFICANCE: M1 polarization in pCR stifles provides evidence of a possible role for macrophages in progressive development of cruciate ligament fiber damage. Lymphocytes may play a role in the synovitis found in CR joints. Our findings provide evidence that these cells are therapeutic targets.

Efflux drug transporters at the forefront of antimicrobial resistance.

Bacterial antibiotic resistance is rapidly becoming a major world health consideration. To combat antibiotics, microorganisms employ their pre-existing defence mechanisms that existed long before man's discovery of antibiotics. Bacteria utilise levels of protection that range from gene upregulation, mutations, adaptive resistance, and production of resistant phenotypes (persisters) to communal behaviour, as in swarming and the ultimate defence of a biofilm. A major part of all of these responses involves the use of antibiotic efflux transporters. At the single cell level, it is becoming apparent that the use of efflux pumps is the first line of defence against an antibiotic, as these pumps decrease the intracellular level of antibiotic while the cell activates the various other levels of protection. This frontline of defence involves a coordinated network of efflux transporters. In the future, inhibition of this efflux transporter network, as a target for novel antibiotic therapy, will require the isolation and then biochemical/biophysical characterisation of each pump against all known and new antibiotics. This depth of knowledge is required so that we can fully understand and tackle the mechanisms of developing antimicrobial resistance.

Mitomycin C: a promising agent for the treatment of canine corneal scarring.

OBJECTIVE: To evaluate the safety and efficacy of mitomycin C (MMC) in prevention of canine corneal scarring. METHODS: With an in vitro approach using healthy canine corneas, cultures of primary canine corneal fibroblasts or myofibroblasts were generated. Primary canine corneal fibroblasts were obtained by growing corneal buttons in minimal essential medium supplemented with 10% fetal bovine serum. Canine corneal myofibroblasts were produced by growing cultures in serum-free medium containing transforming growth factor ß1 (1 ng/mL). Trypan blue assay and phase-contrast microscopy were used to evaluate the toxicity of three doses of MMC (0.002%, 0.02% and 0.04%). Real-time PCR, immunoblot, and immunocytochemistry techniques were used to determine MMC efficacy to inhibit markers of canine corneal scarring. RESULTS: A single 2-min treatment of 0.02% or less MMC did not alter canine corneal fibroblast or keratocyte phenotype, viability, or growth. The 0.02% dose substantially reduced myofibroblast formation (up to 67%; P < 0.001), as measured by the change in RNA and protein expression of fibrosis biomarkers (α-smooth muscle actin and F-actin). CONCLUSION: This in vitro study suggests that a single 2-min 0.02% MMC treatment to the canine corneal keratocytes is safe and may be useful in decreasing canine corneal fibrous metaplasia. In vivo studies are warranted.