Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD)

<p>Abstract</p> <p>Background</p> <p>ATP binding cassette (ABC) transporter secretes the protein through inner and outer membranes simultaneously in gram negative bacteria. Thermostable lipase (TliA) of <it>Pseudomonas fluorescens </it>SIK W1 is secreted through the ABC transporter. TliA has four glycine-rich repeats (GGXGXD) in its C-terminus, which appear in many ABC transporter-secreted proteins. From a homology model of TliA derived from the structure of <it>P. aeruginosa </it>alkaline protease (AprA), lipase ABC transporter domains (LARDs) were designed for the secretion of fusion proteins.</p> <p>Results</p> <p>The LARDs included four glycine-rich repeats comprising a β-roll structure, and were added to the C-terminus of test proteins. Either Pro-Gly linker or Factor Xa site was added between fusion proteins and LARDs. We attached different length of LARDs such as LARD0, LARD1 or whole TliA (the longest LARD) to three types of proteins; green fluorescent protein (GFP), epidermal growth factor (EGF) and cytoplasmic transduction peptide (CTP). These fusion proteins were expressed in <it>Escherichia coli </it>together with ABC transporter of either <it>P. fluorescens </it>or <it>Erwinia chrysanthemi</it>. Export of fusion proteins with the whole TliA through the ABC transporter was evident on the basis of lipase enzymatic activity. Upon supplementation of <it>E. coli </it>with ABC transporter, GFP-LARDs and EGF-LARDs were excreted into the culture supernatant.</p> <p>Conclusion</p> <p>The LARDs or whole TliA were attached to C-termini of model proteins and enabled the export of the model proteins such as GFP and EGF in <it>E. coli </it>supplemented with ABC transporter. These results open the possibility for the extracellular production of recombinant proteins in <it>Pseudomonas </it>using LARDs or TliA as a C-terminal signal sequence.</p

Effect of Three-Dimensional Printed Personalized Moisture Chamber Spectacles on the Periocular Humidity

Purpose. To assess the effect of three-dimensional (3D) printed personalized moisture chamber spectacles (PMCS) on the periocular humidity. Methods. Facial computed tomography (CT) scanning was conducted on 10 normal subjects. PMCS was designed based on volume rendered CT images and produced using a 3D printer. Periocular humidity of PMCS and commercially available uniformed moisture chamber spectacles (UMCS) were measured for 30 minutes via microhydrometer. Results. The mean ambient humidity was 15.76±1.18%. The mean periocular humidity was 52.14±3.00% in PMCS and 37.67±8.97% in UMCS. The difference was significant (P<0.001). Additionally, PMCS always demonstrated lower humidity than dew points. Conclusion. PMCS made by 3D printer provides appropriate fitness for the semiclosed humid chamber. PMCS showed higher performance than UMCS. The wearing of PMCS would be an effective method to provide high enough periocular humidity in low humidity environment

Autoimmune Hypoglycemia in a Patient with Characterization of Insulin Receptor Autoantibodies

BackgroundType B insulin resistance syndrome is a manifestation of autoantibodies to the insulin receptor that results in severe hyperglycemia and acanthosis nigricans. However, the mechanisms by which these autoantibodies induce hypoglycemia are largely unknown. In this paper, we report the case of patient with type B insulin resistance syndrome who presented with frequent severe fasting hypoglycemia and acanthosis nigricans.MethodsTo evaluate the mechanism of hypoglycemia, we measured the inhibition of insulin binding to erythrocytes and IM9 lymphocytes in a sample of the patient's dialyzed serum before and after immunosuppressive therapy.ResultsIn the patient's pre-treatment serum IgG, the binding of 125I-insulin to erythrocytes was markedly inhibited in a dose-dependent manner until the cold insulin level reached 10-9 mol/L. We also observed dose-dependent inhibition of insulin binding to IM9 lymphocytes, which reached approximately 82% inhibition and persisted even when diluted 1:20. After treatment with glucocorticoids, insulin-erythrocyte binding activity returned to between 70% and 80% of normal, while the inhibition of insulin-lymphocyte binding was reduced by 17%.ConclusionWe treated a patient with type B insulin resistance syndrome showing recurrent fasting hypoglycemia with steroids and azathioprine. We characterized the patient's insulin receptor antibodies by measuring the inhibition of insulin binding

Spontaneous rupture of the lateral thoracic artery in patients with liver cirrhosis

Bleeding in patients with liver cirrhosis is primarily caused by gastroesophageal varix in association with extensive collateral circulation, portal hypertensive gastropathy, a Mallory-Weiss tear and peptic ulcer disease. The spontaneous rupture of an artery, as a result of coagulopathy, is extremely rare in patients with liver cirrhosis; however, we recently observed a case of a spontaneous rupture of the lateral thoracic artery in a 47 year-old male patient with alcoholic liver cirrhosis. The patient expired despite repeated transcatheter arterial embolization of the lateral thoracic artery and best supportive care. This is, to our knowledge, the first documented case of the spontaneous rupture of the lateral thoracic artery in a patient with liver cirrhosis