Articular cartilage is a load-bearing tissue covering the ends of long bones that normally allows for painless motion of joints. Adult articular cartilage has limited capacity for self-repair when injured. Current surgical treatments for cartilage defects include the transplantation of fresh osteochondral tissue from autologous or allogeneic sources to restore the architecture and characteristics of native tissue at the defect site. Such treatments have a successful clinical history but are limited by tissue availability. Osteochondral tissue engineering aims to fabricate osteochondral grafts in vitro but faces the challenge of integrating the cartilaginous and osseous layers to form a mechanically functional unit. The native osteochondral interface consists of a zone of calcified cartilage (ZCC) that anchors articular cartilage to subchondral bone through an interdigitated zone of intermediate stiffness. Thus, the overall goal of this dissertation was to form a functional osteochondral tissue graft in vitro by integrating articular cartilage to bone through a biomimetic transitional zone of calcified cartilage. The ZCC at the human osteochondral interface was found to have increased hydraulic conductance, or ease of fluid transport, and undergo changes in thickness and vascularity with increasing stages of osteoarthritis, suggesting that the ZCC is a zone of active tissue remodeling during cartilage repair. Cartilage explants were used to investigate conditions for in vitro calcification of cartilage and the resulting functional biomechanical consequences of matrix calcification. The in vitro calcification of immature articular cartilage in medium supplemented with beta-glycerophosphate was dependent on cell viability and occurred only in the deep zone, resulting in a local increase in mechanical stiffness. Calcification of mature cartilage occurred within 3 days in medium with supersaturated calcium and phosphate and was dependent on supplementation with fetuin, an abundant serum protein. Cartilage explants cultured atop devitalized bone underwent fetuin-mediated calcification in the zone adjacent to bone, thereby attaching cartilage to bone through a calcified cartilage interface and achieving integration strengths similar to that of native immature osteochondral tissue. These studies demonstrate that calcification can be used to integrate cartilage and bone to form a mechanically functional osteochondral graft. Such methods can be useful to provide biological fixation of cartilaginous grafts to bone for the formation of osteochondral grafts suitable for the treatment of large defects or ultimately, for resurfacing damaged or osteoarthritic joints

The radical S-adenosylmethionine (SAM) protein PqqE is predicted to function in the pyrroloquinoline quinone (PQQ) biosynthetic pathway via catalysis of carbon-carbon bond formation between a glutamate and tyrosine side chain within the small peptide substrate PqqA. We report here that PqqE activity is dependent on the accessory protein PqqD, which was recently shown to bind PqqA tightly. In addition, PqqE activity in vitro requires the presence of a flavodoxin- and flavodoxin reductase-based reduction system, with other reductants leading to an uncoupled cleavage of the co-substrate SAM. These results indicate that PqqE, in conjunction with PqqD, carries out the first step in PQQ biosynthesis: a radical-mediated formation of a new carbon-carbon bond between two amino acid side chains on PqqA.

Clinically evident sarcoidosis involving the heart has been noted in at least 2 to 7% of patients with sarcoidosis, but occult involvement is much higher (> 20%). Cardiac sarcoidosis is often not recognized antemortem, as sudden death may be the presenting feature. Cardiac involvement may occur at any point during the course of sarcoidosis and may occur in the absence of pulmonary or systemic involvement. Sarcoidosis can involve any part of the heart, with protean manifestations. Prognosis of cardiac sarcoidosis is related to extent and site(s) of involvement. Most deaths due to cardiac sarcoidosis are due to arrhythmias or conduction defects, but granulomatous infiltration of the myocardium may be lethal. The definitive diagnosis of isolated cardiac sarcoidosis is difficult. The yield of endomyocardial biopsies is low; treatment of cardiac sarcoidosis is often warranted even in the absence of histologic proof. Radionuclide scans are integral to the diagnosis. Currently, 18F-fluorodeoxyglucose positron emission tomography/computed tomography and gadolinium-enhanced magnetic resonance imaging scans are the key imaging modalities to diagnose cardiac sarcoidosis. The prognosis of cardiac sarcoidosis is variable, but mortality rates of untreated cardiac sarcoidosis are high. Although randomized therapeutic trials have not been done, corticosteroids (alone or combined with additional immunosuppressive medications) remain the mainstay of treatment. Because of the potential for sudden cardiac death, implantable cardioverter-defibrillators should be placed in any patient with cardiac sarcoidosis and serious ventricular arrhythmias or heart block, and should be considered for cardiomyopathy. Cardiac transplantation is a viable option for patients with end-stage cardiac sarcoidosis refractory to medical therapy.

Intense interest centers on the role of the human gut microbiome in health and disease, but optimal methods for analysis are still under development. Here we present a study of methods for surveying bacterial communities in human feces using 454/Roche pyrosequencing of 16S rRNA gene tags. We analyzed fecal samples from 10 individuals and compared methods for storage, DNA purification and sequence acquisition. To assess reproducibility, we compared samples one cm apart on a single stool specimen for each individual. To analyze storage methods, we compared 1) immediate freezing at -80 degrees C, 2) storage on ice for 24 or 3) 48 hours. For DNA purification methods, we tested three commercial kits and bead beating in hot phenol. Variations due to the different methodologies were compared to variation among individuals using two approaches--one based on presence-absence information for bacterial taxa (unweighted UniFrac) and the other taking into account their relative abundance (weighted UniFrac). In the unweighted analysis relatively little variation was associated with the different analytical procedures, and variation between individuals predominated. In the weighted analysis considerable variation was associated with the purification methods. Particularly notable was improved recovery of Firmicutes sequences using the hot phenol method. We also carried out surveys of the effects of different 454 sequencing methods (FLX versus Titanium) and amplification of different 16S rRNA variable gene segments. Based on our findings we present recommendations for protocols to collect, process and sequence bacterial 16S rDNA from fecal samples--some major points are 1) if feasible, bead-beating in hot phenol or use of the PSP kit improves recovery; 2) storage methods can be adjusted based on experimental convenience; 3) unweighted (presence-absence) comparisons are less affected by lysis method.