An Automated Model Test System for Systematic Development and Improvement of Gene Expression Models.

Gene expression models greatly accelerate the engineering of synthetic metabolic pathways and genetic circuits by predicting sequence-function relationships and reducing trial-and-error experimentation. However, developing models with more accurate predictions remains a significant challenge. Here we present a model test system that combines advanced statistics, machine learning, and a database of 9862 characterized genetic systems to automatically quantify model accuracies, accept or reject mechanistic hypotheses, and identify areas for model improvement. We also introduce model capacity, a new information theoretic metric for correct cross-data-set comparisons. We demonstrate the model test system by comparing six models of translation initiation rate, evaluating 100 mechanistic hypotheses, and uncovering new sequence determinants that control protein expression levels. We then applied these results to develop a biophysical model of translation initiation rate with significant improvements in accuracy. Automated model test systems will dramatically accelerate the development of gene expression models, and thereby transition synthetic biology into a mature engineering discipline.

Automated design of thousands of nonrepetitive parts for engineering stable genetic systems.

Engineered genetic systems are prone to failure when their genetic parts contain repetitive sequences. Designing many nonrepetitive genetic parts with desired functionalities remains a difficult challenge with high computational complexity. To overcome this challenge, we developed the Nonrepetitive Parts Calculator to rapidly generate thousands of highly nonrepetitive genetic parts from specified design constraints, including promoters, ribosome-binding sites and terminators. As a demonstration, we designed and experimentally characterized 4,350 nonrepetitive bacterial promoters with transcription rates that varied across a 820,000-fold range, and 1,722 highly nonrepetitive yeast promoters with transcription rates that varied across a 25,000-fold range. We applied machine learning to explain how specific interactions controlled the promoters' transcription rates. We also show that using nonrepetitive genetic parts substantially reduces homologous recombination, resulting in greater genetic stability.

Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays.

Engineering cellular phenotypes often requires the regulation of many genes. When using CRISPR interference, coexpressing many single-guide RNAs (sgRNAs) triggers genetic instability and phenotype loss, due to the presence of repetitive DNA sequences. We stably coexpressed 22 sgRNAs within nonrepetitive extra-long sgRNA arrays (ELSAs) to simultaneously repress up to 13 genes by up to 3,500-fold. We applied biophysical modeling, biochemical characterization and machine learning to develop toolboxes of nonrepetitive genetic parts, including 28 sgRNA handles that bind Cas9. We designed ELSAs by combining nonrepetitive genetic parts according to algorithmic rules quantifying DNA synthesis complexity, sgRNA expression, sgRNA targeting and genetic stability. Using ELSAs, we created three highly selective phenotypes in Escherichia coli, including redirecting metabolism to increase succinic acid production by 150-fold, knocking down amino acid biosynthesis to create a multi-auxotrophic strain and repressing stress responses to reduce persister cell formation by 21-fold. ELSAs enable simultaneous and stable regulation of many genes for metabolic engineering and synthetic biology applications.

Successful treatment of cystoid macular edema with valdecoxib.

PURPOSE: To evaluate the safety and efficacy of the COX-2 inhibitor valdecoxib in treating macular edema after cataract surgery. SETTING: University Eye Clinic, Freiburg, Germany and Reis Medical Institution, Liechtenstein. METHODS: The COX-2 inhibitor valdecoxib (Bextra) was administered systemically to patients with significant visual loss resulting from macular edema in a prospective clinical trial. RESULTS: Ten patients were enrolled. Valdecoxib was tolerated well and led to a significant visual improvement within 10 days of therapy in all patients. CONCLUSION: The fast and persistent control of macular edema with valdecoxib warrants further investigation.

An open prospective pilot study on the use of rapamycin after penetrating high-risk keratoplasty.

BACKGROUND: The purpose of this study was to prove efficacy and safety of systemic immunosuppression with rapamycin following penetrating high-risk keratoplasty. Rapamycin has shown its immunosuppressive potential in the rat keratoplasty model and is a component of several immunosuppressive protocols after solid organ transplantation. In this pilot study, we compared the efficacy and safety of rapamycin and mycophenolate mofetil (MMF). METHODS: Ten patients (group 1) undergoing high-risk keratoplasty were included in this study, receiving rapamycin as postoperative immunoprophylaxis. Rapamycin was administered orally once daily (blood trough level 4-10 ng/ml) for 6 months. Thereafter, it was tapered over 2 weeks. The control group (group 2) consisted of 24 patients who received 1000 mg MMF twice daily for 6 months. All of the patients received postoperative medication with fluocortolone 1 mg/kg/day (tapered over 3 weeks) and prednisolone acetate eyedrops 5 times per day (tapered over 5 months). RESULTS: Mean follow-up of all patients (n=34) was 739 days. No immune reaction was observed in groups 1 and 2 during the first 6 months under immunosuppression. Two immune reactions occurred in group 1, and five in group 2 within a 2-year follow-up. All of the immune reactions were reversible. The side effects observed in both groups were mostly reversible. CONCLUSIONS: Rapamycin and mycophenolate mofetil seem to be similarly efficacious in preventing immune reactions after high-risk keratoplasty, as long as they are administered. However, we observed a broad spectrum of side effects from rapamycin.

Cyclooxygenase-2 inhibitors: a new therapeutic option in the treatment of macular edema after cataract surgery.

Two patients had uneventful phacoemulsification. After initial improvement, vision deteriorated because of cystoid macular edema (CME). In 1 patient, treatment with systemic nonsteroidal antiinflammatory drugs showed significant improvement in visual acuity but had to be discontinued because of side effects and a relapse of the disease. In the other patient, this therapy was not sufficient. Both patients were given valdecoxib, a cyclooxygenase-2 inhibitor. The new therapy was tolerated well and led to significant and stable improvement in visual acuity in both patients. To our knowledge, this is the first report of using a cyclooxygenase-2 inhibitor in the treatment of clinically significant CME.

Topical FK506 as immunoprophylaxis after allogeneic penetrating normal-risk keratoplasty: a randomized clinical pilot study.

The purpose of this study was to evaluate for the first time the efficacy and safety of topical FK506 in patients undergoing penetrating normal-risk keratoplasty in a prospectively randomized clinical trial. Twenty patients were treated with FK506 0.06% three times per day for 6 months postoperatively. An additional 20 patients received five drops of prednisolone acetate 1% tapered within 6 months. All patients received 1 mg/kg bodyweight/day of systemic fluocortolon tapered within 3 weeks postoperatively. Clear graft survival, ratio of immune reactions and side effects were the main outcome measures. One year postoperatively all patients of the FK 506 group were free from immune reactions, in contrast to 84% in the steroid group (Kaplan-Meier values; P = 0.9 in the log rank test). None of the patients developed irreversible graft failure so far. In eight patients of the FK506 group premature withdrawal of the drug was deemed appropriate because of local side effects. FK506 might turn out to become an effective immunoprophylaxis in subjects undergoing penetrating normal-risk keratoplasty. Local discomfort should be further reduced.

Synergistic antiherpetic effect of acyclovir and mycophenolate mofetil following keratoplasty in patients with herpetic eye disease: first results of a randomised pilot study.

PURPOSE: The main reasons for graft failure following penetrating keratoplasty in patients with herpetic eye disease are recurrence of herpetic disease and allograft rejection. In a randomised trial the effect of systemic acyclovir and mycophenolate mofetil (MMF) on these post-keratoplasty complications was evaluated. PATIENTS AND METHODS: Patients with typical clinical findings of recurrent herpetic keratitis were enrolled in this single-centre study after contraindications to systemic immunosuppression were ruled out. In a prospective randomised trial 30 patients were treated in three groups. In group A patients received acyclovir 200 mg five times/day for 3 weeks. In group B patients were treated with acyclovir 200 mg five times/day for 1 year, and patients in group C received acyclovir 200 mg five times/day in combination with MMF 1 g twice daily for 1 year. RESULTS: In group A 3 patients experienced seven herpes recurrences. One patient had a moderate and one further patient a severe allograft rejection. In group B three severe allograft rejections were observed. Herpes recurrences did not occur while receiving acyclovir prophylaxis, but only once after the prophylaxis had been stopped. In group C no herpes recurrence was observed, and only two mild allograft rejections occurred while being under combined acyclovir-MMF therapy. Another mild and one moderate allograft rejection were observed after cessation of MMF. CONCLUSIONS: These results demonstrate that systemic acyclovir protects the grafts from recurrences of herpetic disease as long as it is administered at efficient doses. Simultaneously administered mycophenolate mofetil does not trigger herpes recurrences and protects the graft from severe allograft rejections, but mild, less dangerous immune reactions may still occur while receiving MMF. The combination of systemic acyclovir and mycophenolate mofetil therefore is recommended for patients at high risk for herpes recurrence and allograft rejections.

Differential contribution of natural killer cells to corneal graft rejection in 3-week-old versus mature rats.

BACKGROUND: The aim of this study was to compare immunologic graft rejection in adult and 3-week-old immature recipients in the rat keratoplasty model. METHODS: Forty orthotopic penetrating keratoplasties were performed in four different donor-recipient combinations. Group 1 consisted of adult Fisher donors and adult Lewis recipients, group 2 consisted of adult Fisher donors and immature Lewis recipients, group 3 consisted of adult Lewis donors and recipients, and group 4 consisted of adult Lewis donors and immature Lewis recipients. An immunohistologic evaluation of the grafts was performed on day 14. RESULTS: Grafts in both allogeneic groups (groups 1 and 2) showed infiltration with CD4+ cells, CD8+ cells, natural killer (NK) cells, interleukin-2-receptor+ cells, macrophages, and intercellular adhesion molecule-1+ cells. The density of infiltrating CD4+, CD8+, interleukin-2-receptor+, and intercellular adhesion molecule-1+ cells in the graft stroma, however, was statistically significantly lower in the immature group (group 2) than in the adult group (group 1). The density of CD161+ NK cells, in contrast, was statistically significantly higher in the immature group than in the adult group. There were no or only a few infiltrating inflammatory cells in grafts of both syngeneic groups (groups 3 and 4). CONCLUSIONS: We were able to establish for the first time an animal model for keratoplasty in infants that showed that the mechanism of graft rejection in young recipients seems to be different from that in mature rats. In adult recipients, alloreactive T cells are the main mediators of rejection, whereas NK cells seem to play a more dominant role in immature recipients.

[Topical Fk506 in inflammatory corneal and conjunctival diseases. A pilot study]. / Lokales Fk506 bei entzündlichen Erkrankungen von Hornhaut und Bindehaut--Eine Pilotstudie.

BACKGROUND: Topical corticosteroids are the only effective measure in serious inflammatory corneal and conjunctival diseases. Although results obtained with topical cyclosporin A are encuraging it is not effective in all patients. The mode of action of Fk506 is similar to that of cyclosporin A, i.e. it exerts an inhibitory effect on transcription of interleukin 2 in T lymphocytes. The immunosuppressive potential of Fk506, however, is much larger. Furthermore, it penetrates more easily into cornea and conjunctiva. To find out whether the theoretical advantages of topical Fk506 can be translated into clinical practice, a selected group of patients refractory to conventional therapy was treated in this pilot study. PATIENTS AND METHODS: Fk506 0.06 % was administered initially three times daily in 15 patients with atopic blepharokeratoconjunctivitis, Mooren's ulcer, ocular pemphigoid, Thygeson's superficial punctate keratitis, nummular adenoviral keratitis, graft-versus-host reaction of the conjunctiva and steroid response glaucoma after penetrating keratoplasty. RESULTS: Within a follow-up of 26 +/- 15 weeks improvement was recorded in 5/15 patients and stabilization in 5/15 patients. In two patients progression of the disease was noted (one patient with progression of ocular pemphigoid, another patient with suspected automutilation). Premature withdrawal the drug was judged to be necessary in two patients with ocular surface disorders and in one patient with non-compliance. CONCLUSIONS: Topical Fk506 seems to be a promising new immunosuppressive drug for patients with atopic blepharokeratoconjunctivitis, Thygeson's superficial punctate keratitis and nummular adenoviral keratitis. Exact efficacy in these and other corneal and conjunctival inflammatory diseases has to be determined in randomised clinical studies. Before these studies may start the risk of side-effects must be reduced via an improvement of the drops.

Synergism of RAD and cyclosporin A in prevention of acute rat corneal allograft rejection.

PURPOSE: The novel immunosuppressant RAD, 40-0-(2-hydroxy-ethyl)-rapamycin, has synergistic effects with cyclosporin A. The aim of this study was to evaluate the combined effect of RAD and cyclosporin A in the prevention of acute allograft rejection after murine corneal transplantation. METHODS: Fisher donor corneas were implanted into Lewis recipients. Postoperative evaluation included slit-lamp biomicroscopy and immunohistology. Treatment groups were comprised of rats treated orally with RAD 2.5 mg/kg/day, cyclosporin A 10 mg/kg/day, RAD 1.5 mg/kg/day plus cyclosporin A 5 mg/kg/day. RESULTS: Therapy with RAD 2.5 mg/kg and cyclosporin A 10 mg/kg led to a statistically significant and comparable prolongation of transplant survival. However. combination therapy was significantly superior. There was a significant reduction in the number of infiltrating cells in the animals treated with RAD and cyclosporin A. CONCLUSIONS: This is the first study on the efficacy of a double drug regimen with RAD and cyclosporin A for the control of acute corneal allograft rejection. Combination therapy resulted in superior graft survival.