Energy complexes are apparently associated with the switch-motor complex of bacterial flagella.

Recently, the switch-motor complex of bacterial flagella was found to be associated with a number of non-flagellar proteins, which, in spite of not being known as belonging to the chemotaxis system, affect the function of the flagella. The observation that one of these proteins, fumarate reductase, is essentially involved in electron transport under anaerobic conditions raised the question of whether other energy-linked enzymes are associated with the switch-motor complex as well. Here, we identified two additional such enzymes in Escherichia coli. Employing fluorescence resonance energy transfer in vivo and pull-down assays invitro, we provided evidence for the interaction of F(0)F(1) ATP synthase via its ß subunit with the flagellar switch protein FliG and for the interaction of NADH-ubiquinone oxidoreductase with FliG, FliM, and possibly FliN. Furthermore, we measured higher rates of ATP synthesis, ATP hydrolysis, and electron transport from NADH to oxygen in membrane areas adjacent to the flagellar motor than in other membrane areas. All these observations suggest the association of energy complexes with the flagellar switch-motor complex. Finding that deletion of the ß subunit in vivo affected the direction of flagellar rotation and switching frequency further implied that the interaction of F(0)F(1) ATP synthase with FliG is important for the function of the switch of bacterial flagella.

Porcine small diameter arterial extracellular matrix supports endothelium formation and media remodeling forming a promising vascular engineered biograft.

Patients with small caliber artery disorders, often lack the suitable autologous tissue needed for bypassing diseased vessels or for other vascular reconstructive procedures. We propose to decellularize small caliber porcine carotid artery, then recellularize it with vascular cells and function as scaffold for tissue engineering vascular graft replacements. Based on a modified decellularization method developed in our laboratory, the cellular contents of small caliber (<4 mm) arteries were carefully removed using an enzymatic and detergent decellularization procedure. Decellularization efficiency was evaluated using histology and scanning electron microscopy, which demonstrated the absence of cellular remains in the artery wall. Proteomic analysis of the scaffold revealed that the decellularized vessels retained their major extracellular matrix protein composition. Mechanical analyses revealed no significant change in the extracellular matrix (ECM) properties versus the native artery. The decellularized artery was reseeded with human umbilical vein endothelial cells (HUVECs) and smooth muscle cells (SMCs) and cultured under static or dynamic conditions in a perfusion bioreactor designed and developed in our laboratory for these studies. Dynamic co-culturing of SMC and HUVEC, in this custom-made perfusion bioreactor, led to a higher infiltration, migration and proliferation of SMC toward the media and to a more confluent endothelium formation on the luminal surface when compared with static culturing. In addition, vascular media remodeling by SMC correlated to the expression of remodeling related genes assessed by real-time reverse transcription-polymerase chain reaction and HUVEC cultivation contributed to the remodeling of several basement membrane proteins stained using immunohistochemistry. All together, these findings indicate the potential of such decellularized arterial ECM for future small caliber vascular graft reconstruction therapies.

Engineering blood vessels by gene and cell therapy.

Cardiovascular-related syndromes are the leading cause of morbidity and mortality worldwide. Arterial narrowing and blockage due to atherosclerosis cause reduced blood flow to the brain, heart and legs. Bypass surgery to improve blood flow to the heart and legs in these patients is performed in hundreds of thousands of patients every year. Autologous grafts, such as the internal thoracic artery and saphenous vein, are used in most patients, but in a significant number of patients such grafts are not available and synthetic grafts are used. Synthetic grafts have higher failure rates than autologous grafts due to thrombosis and scar formation within graft lumen. Cell and gene therapy combined with tissue engineering hold a great promise to provide grafts that will be biocompatible and durable. This review describes the field of vascular grafts in the context of tissue engineering using cell and gene therapies.