A streamlined, automated workflow to screen and triage large numbers of baculoviruses for protein expression.

The baculovirus expression system is a powerful and widely used method to generate large quantities of recombinant protein. However, challenges exist in workflows utilizing either liquid baculovirus stocks or the Titerless Infected-Cells Preservation and Scale-Up (TIPS) method, including the time and effort to generate baculoviruses, screen for protein expression and store large numbers of baculovirus stocks. To mitigate these challenges, we have developed a streamlined, hybrid workflow which utilizes high titer liquid virus stocks for rapid plate-based protein expression screening, followed by a TIPS-based scale-up for larger protein production efforts. Additionally, we have automated each step in this screening workflow using a custom robotic system. With these process improvements, we have significantly reduced the time, effort and resources required to manage large baculovirus generation and expression screening campaigns.

Tendinosis develops from age- and oxygen tension-dependent modulation of Rac1 activity.

Age-related tendon degeneration (tendinosis) is characterized by a phenotypic change in which tenocytes display characteristics of fibrochondrocytes and mineralized fibrochondrocytes. As tendon degeneration has been noted in vivo in areas of decreased tendon vascularity, we hypothesized that hypoxia is responsible for the development of the tendinosis phenotype, and that these effects are more pronounced in aged tenocytes. Hypoxic (1% O2 ) culture of aged, tendinotic, and young human tenocytes resulted in a mineralized fibrochondrocyte phenotype in aged tenocytes, and a fibrochondrocyte phenotype in young and tendinotic tenocytes. Investigation of the molecular mechanism responsible for this phenotype change revealed that the fibrochondrocyte phenotype in aged tenocytes occurs with decreased Rac1 activity in response to hypoxia. In young hypoxic tenocytes, however, the fibrochondrocyte phenotype occurs with concomitant decreased Rac1 activity coupled with increased RhoA activity. Using pharmacologic and adenoviral manipulation, we confirmed that these hypoxic effects on the tenocyte phenotype are linked directly to the activity of RhoA/Rac1 GTPase in in vitro human cell culture and tendon explants. These results demonstrate that hypoxia drives tenocyte phenotypic changes, and provide a molecular insight into the development of human tendinosis that occurs with aging.

Assessing subsistence and its relationship to cultural complexity in the late prehistoric upper Midwest: A new perspective provided by dental health.

OBJECTIVE: Previous researchers have assumed that the Late Prehistoric Oneota were less reliant on maize agriculture than their Middle Mississippian neighbors to the south. This assumption is based on the idea that intensive maize agriculture is related to sociopolitical complexity, and that the climate of the Great Lakes region was less conducive to full-scale agriculture than that of the American Bottom. Here, we assess the diet of the Oneota using dental pathology to test the hypothesis that the Oneota in Eastern Wisconsin were highly reliant on maize agriculture. MATERIALS AND METHODS: To test our hypothesis, skeletal remains representing 187 individuals and 1,102 teeth were examined from nine Oneota sites from the Middle Fox and Koshkonong Localities, as well as the Late Woodland/Middle Mississippian site of Aztalan. Dental caries, antemortem tooth loss (AMTL), and dental abscesses were assessed for each individual in the sample. Dental pathologies in the Oneota groups were compared to each other based on Locality and to the Aztalan population using chi-squared tests. RESULTS: Dental caries rates for the Oneota, based on the tooth count approach, were observed at 16.8% for the Middle Fox Locality, and 49% for the Koshkonong Locality. Comparatively, the Late Woodland/Middle Mississippian population from Aztalan had a tooth count rate of 19.5%. AMTL rates were similar across samples. Dental abscessing was universally low. DISCUSSION: The relatively high rate of dental caries among the Oneota is comparable to Middle Mississippian populations from throughout the Midwest, suggesting similar reliance on maize between the groups.

Favorable outcomes in patients with high donor-derived T cell count after in vivo T cell-depleted reduced-intensity allogeneic stem cell transplantation.

Patients with hematologic malignancies were conditioned using a rabbit antithymocyte globulin-based reduced-intensity conditioning regimen for allogeneic stem cell transplantation. Donor-derived CD3(+) cell count (ddCD3), a product of CD3(+) cell chimerism and absolute CD3(+) cell count, when <110/µL at 8 weeks post-stem cell transplantation predicted a high risk of sustained mixed chimerism and relapse. Alternatively, patients with a higher ddCD3 developed graft-versus-host disease more frequently, and when partially chimeric, had higher rates of conversion to full donor chimerism after withdrawal of immunosuppression. Early data from our small cohort of patients indicate that ddCD3 at 8 weeks may be used to guide decisions regarding withdrawal of immunosuppression and administration of donor lymphocyte infusion in partially T cell-depleted reduced-intensity regimens.