A dps promoter based expression system for improved solubility of expressed proteins in Escherichia coli

Escherichia coli is widely used for recombinant protein production due to its well established genetic manipulation techniques and cost effectiveness of the associated production processes. Soluble expression of heterologous recombinant proteins constitutes a major problem in the deployment of bacterial expression systems. We have developed a dps promoter based expression system in E. coli for improved solubility of expressed proteins. The resulting expression system was found to be superior to the IPTG inducible T7 promoter based pET expression system for production of soluble beta-galactosidase, tdTomato, and mCherry. The dps promoter based expression system was shown to be functional in most commonly used strains of E. coli without need for prior genetic manipulation of the host genome

Case study: Correction of angular deformity post-trans-tibial amputation to improve prosthesis fit and comfort

Background: This case report describes the revision of a trans-tibial amputation complicated by a proximal malunited fracture. It demonstrates the complexity of decisions involved in revisions of this nature. Case description and methods: The patient presented has a painful stump due to breakdown of soft tissues. She had been suffering for over a year with pressure sores and inability to use a prosthesis resulting from this, decreasing her quality of life. Malunion of a tibial fracture proximal to the amputation complicated her condition. Findings and outcomes: Initially it was feared that the amputation level would be converted to a through-knee amputation, but wedge osteotomy of the tibia allowed coverage of the stump, increasing functionality for the patient, while still maintaining a below-knee stump. Conclusion: This report highlights the importance of addressing the cause of soft tissue problems in an amputation revision, which must be addressed on a case-by-case basis. Clinical relevance: This case offers an insight into the decision-making process in limb reconstruction and suggests a solution for similar cases. It is important that all surgical options are considered before amputation length is sacrificed

Functional and molecular identification of pH-sensitive K+ channels in murine urinary bladder smooth muscle

The definitive version may be found at www.wiley.comObjectiveTo examine the role of pH-sensitive K(+) channels in setting the resting membrane potential in murine bladder smooth muscle, as bladder contractility is influenced by the resting membrane potential, which is mainly regulated by background K(+) conductances.Materials and methodsUsing conventional microelectrode recordings, isometric tension measurements, patch-clamp recordings, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry, we assessed bladder smooth muscle cells and tissues.ResultsAcidic pH (pH 6.5) depolarized the resting membrane potential of murine bladder smooth muscles and increased muscle tone and contractility. The pH-induced changes were not abolished by neuronal blockers or classical K(+)-channel antagonists. Lidocaine (1 mM) and bupivacaine (100 microm) mimicked the effects of acidifying the external solution, and in the presence of lidocaine no further increase in contractility was induced by reducing the pH to 6.5. Voltage-clamp experiments on freshly dispersed bladder myocytes showed that pH 6.5 decreased the outward current. Pre-treatment of bladder myocytes with the classical K(+) antagonists tetraethylammonium (10 mm), 4-aminopyridine (5 mM), glibenclamide (10 microm) or apamin (300 nM) did not inhibit the effects of low pH on outward current. However, treatment with lidocaine (1 mM) abolished the effects of acidic pH on outward current. RT-PCR showed the expression of the acid-sensitive K(+) channel (TASK)-1 and TASK-2 gene transcripts in murine bladder, and immunohistochemistry and Western blot analysis showed TASK-1 and TASK-2 channel expression and distribution in smooth muscle tissues and cells.ConclusionTASK channels are expressed in bladder smooth muscle and contribute to the basal K(+) conductances responsible for resting membrane potential.Elizabeth A.H. Beckett, Insoo Han, Salah A. Baker, Junguk Han, Fiona C. Britton and Sang Don Ko