Split flow humidity generator equilibration and stability study.

Generation and control of humidity in a testing environment is crucial when evaluating a chemical vapor sensor as water vapor in the air can not only interfere with the sensor itself, but also react with a chemical analyte changing its composition. Upon constructing a split-flow humidity generator for chemical vapor sensor development, numerous issues were observed due to instability of the generated relative humidity level and drift of the humidity over time. By first fixing the initial relative humidity output of the system at 50%, we studied the effects of flowrate on stabilization time along with long term stability for extended testing events. It was found that the stabilization time can be upwards of 7 h, but can be maintained for greater than 90 h allowing for extended experiments. Once the stabilization time was known for 50% relative humidity output, additional studies at differing humidity levels and flowrates were performed to better characterize the system. At a relative humidity of 20% there was no time required to stabilize, but when increased to 80% this time increased to over 4 h. With this information we were better able to understand the generation process and characterize the humidity generation system, output stabilization and possible modifications to limit future testing issues.

Branched ubiquitin chain binding and deubiquitination by UCH37 facilitate proteasome clearance of stress-induced inclusions.

UCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently, it was reported that UCH37 activity is stimulated by branched ubiquitin (Ub) chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and Nuclear Magnetic Resonance (NMR) structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal Ubs that emanate from a branched Ub. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear Ub chains from having access to the UCH37 active site. In cultured human cells under conditions of proteolytic stress, we show that substrate clearance by the proteasome is promoted by both binding and deubiquitination of branched polyubiquitin by UCH37. Proteasomes containing UCH37(C88A), which is catalytically inactive, aberrantly retain polyubiquitinated species as well as the RAD23B substrate shuttle factor, suggesting a defect in recycling of the proteasome for the next round of substrate processing. These findings provide a foundation to understand how proteasome degradation of substrates modified by a unique Ub chain architecture is aided by a DUB.