Optimal cultivation of Chlamydia requires testing of serum on individual species.

OBJECTIVE: This report is a side product of experiments aimed at identifying serum for culturing obligate intracellular bacteria Chlamydia trachomatis and C. muridarum in mouse fibroblast L929 cells. RESULTS: Of five commercial serum samples tested, two showed optimal efficiencies at supporting growth of the human pathogen Chlamydia trachomatis as control fetal bovine serum, whereas two showed modest ~ 40% inhibitions in progeny production, and the remaining one showed a 20% inhibition. Three of the six sera poorly supported growth of the murine pathogen Chlamydia muridarum, resulting in 73-90% reduction in progeny formation. Most significantly, the one with the strongest (90%) C. muridarum inhibition activity showed optimal C. trachomatis-supporting efficiency. These findings indicate that in laboratories that study multiple Chlamydia species, serum samples should be prescreened on a species basis. Considering Chlamydial biology and epidemiology, it may even be necessary to perform serum tests on a serovar- or strain-basis for studying some animal chlamydiae.

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription.

A transcription factor (TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two σ factors, namely σ66 and σ28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Role for GrgA in Regulation of σ28-Dependent Transcription in the Obligate Intracellular Bacterial Pathogen Chlamydia trachomatis.

The obligate intracellular bacterial pathogen Chlamydia trachomatis has a unique developmental cycle consisting of two contrasting cellular forms. Whereas the primary Chlamydia sigma factor, σ66, is involved in the expression of the majority of chlamydial genes throughout the developmental cycle, expression of several late genes requires the alternative sigma factor, σ28 In prior work, we identified GrgA as a Chlamydia-specific transcription factor that activates σ66-dependent transcription by binding DNA and interacting with a nonconserved region (NCR) of σ66 Here, we extend these findings by showing GrgA can also activate σ28-dependent transcription through direct interaction with σ28 We measure the binding affinity of GrgA for both σ66 and σ28, and we identify regions of GrgA important for σ28-dependent transcription. Similar to results obtained with σ66, we find that GrgA's interaction with σ28 involves an NCR located upstream of conserved region 2 of σ28 Our findings suggest that GrgA is an important regulator of both σ66- and σ28-dependent transcription in C. trachomatis and further highlight NCRs of bacterial RNA polymerase as targets for regulatory factors unique to particular organisms.IMPORTANCEChlamydia trachomatis is the number one sexually transmitted bacterial pathogen worldwide. A substantial proportion of C. trachomatis-infected women develop infertility, pelvic inflammatory syndrome, and other serious complications. C. trachomatis is also a leading infectious cause of blindness in underdeveloped countries. The pathogen has a unique developmental cycle that is transcriptionally regulated. The discovery of an expanded role for the Chlamydia-specific transcription factor GrgA helps us understand the progression of the chlamydial developmental cycle.

Engineering goals for future thoracic endografts-how can we make them more effective?

Endovascular treatments for catastrophic aortic conditions have gained increasing popularity over the past 20 years. Originally developed for abdominal aortic aneurysms (EVAR), treatment has been modified for use in thoracic aortic repair (TEVAR). As expanding numbers of patients with increasingly intractable conditions and more hostile anatomies are treated, endovascular stent designs are maturing to be suitable for these more demanding situations. This article discusses the engineering considerations that apply to changing stent graft designs for current and evolving thoracic applications. The biological parameters that differentiate thoracic from abdominal aortic environments are outlined. Factors concerning materials, sealing mechanisms, deployment, stent frame architecture, and migration resistance are described, and eagerly awaited potential future developments are summarized.

4-methylpyrazole decreases 1,4-butanediol toxicity by blocking its in vivo biotransformation to gamma-hydroxybutyric acid.

1,4-Butanediol (1,4-BD), a prodrug converted in vivo to gamma-hydroxybutyric acid by alcohol dehydrogenase, has resulted in life-threatening overdoses and deaths. We investigated whether 4-methylpyrazole (4-MP), an alcohol dehydrogenase antagonist, can be used as an antidote in a murine model of 1,4-BD overdose. CD-1 mice were overdosed with 1,4-BD, 600 mg/kg i.p. Mice then received 4-MP, 25 mg/kg i.p., or control injections after 1 min, 5 min, and symptom appearance. Mice were then evaluated for toxicity by the righting reflex and rotarod test every 10 min after intervention. When 4-MP was administered 1 and 5 min after 1,4-BD overdose, mice completely maintained their righting reflex. Conversely, control mice lost their righting reflex for 110 and 130 min, respectively (P < 0.05). When 4-MP was administered after symptomatic 1,4-BD overdose, mice lost their righting reflex but recovered it by 60 min. Conversely, control mice lost their righting reflex and recovered it by 140 min (P < 0.05). When 4-MP was administered at 1 min after 1,4-BD overdose, mice never failed the rotarod test. Conversely, control mice failed the rotarod test for 210 min (P < 0.05). When 4-MP was administered 5 min after 1,4-BD and after symptomatic 1,4-BD overdose, mice failed the rotarod test for 100 and 110 min, respectively. Conversely, control mice failed the rotarod test for 210 and 180 min, respectively (P < 0.05). In addition, treatment of mice with 4-MP significantly attenuated increases in blood gamma-hydroxybutyric acid concentrations and prevented loss of the righting reflex and failure of the rotarod test. In this murine model of 1,4-BD overdose, 4-MP conferred antidotal effects by inhibiting alcohol dehydrogenase-mediated biotransformation of 1,4-BD to gamma-hydroxybutyric acid.

Enzyme and receptor antagonists for preventing toxicity from the gamma-hydroxybutyric acid precursor 1,4-butanediol in CD-1 mice.

1,4-Butanediol (1,4-BD), the diol alcohol precursor of gamma-hydroxybutyric acid (GHB), undergoes in vivo enzymatic biotransformation to GHB by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase. The subsequent metabolite, GHB, is pharmacologically active at GABA(B) and GHB receptors. GHB can be metabolized in vivo to gamma-aminobutyric acid (GABA) and trans-4-hydroxycrotonic acid (T-HCA), which are also pharmacologically active at GABA(B) receptors and GHB receptors, respectively. Therefore, we speculate that 1,4-BD overdose toxicity can be prevented or attenuated with the ADH enzyme inhibitor 4-methylpyrazole (4-MP) as well as with CGP-35348 and NCS-382, novel high-affinity receptor antagonists of GABA(B) receptors and GHB receptors, respectively. In our murine model of acute 1,4-BD overdose, pretreatment of CD-1 mice with 4-MP significantly attenuated increases in blood GHB concentrations and prevented loss of the righting reflex and failure of the rotarod test. Also, pretreatment with CGP-35348 and its combination with NCS-382 significantly decreased the duration of failure for the rotarod test and the percentage of animals failing the rotarod test, respectively. However, pretreatment of CD-1 mice with NCS-382 alone produced prolonged failure of the rotarod test, an unexpected synergistic effect with 1,4-BD and presumably GHB, which has not previously been demonstrated.