Although Bcl-XL and Bax are structurally similar, activated Bax forms large oligomers that permeabilize the outer mitochondrial membrane, thereby committing cells to apoptosis, whereas Bcl-XL inhibits this process. Two different models of Bcl-XL function have been proposed. In one, Bcl-XL binds to an activator, thereby preventing Bax activation. In the other, Bcl-XL binds directly to activated Bax. It has been difficult to sort out which interaction is important in cells, as all three proteins are present simultaneously. We examined the mechanism of Bax activation by tBid and its inhibition by Bcl-XL using full-length recombinant proteins and measuring permeabilization of liposomes and mitochondria in vitro. Our results demonstrate that Bcl-XL and Bax are functionally similar. Neither protein bound to membranes alone. However, the addition of tBid recruited molar excesses of either protein to membranes, indicating that tBid activates both pro- and antiapoptotic members of the Bcl-2 family. Bcl-XL competes with Bax for the activation of soluble, monomeric Bax through interaction with membranes, tBid, or t-Bid-activated Bax, thereby inhibiting Bax binding to membranes, oligomerization, and membrane permeabilization. Experiments in which individual interactions were abolished by mutagenesis indicate that both Bcl-XL–tBid and Bcl-XL–Bax binding contribute to the antiapoptotic function of Bcl-XL. By out-competing Bax for the interactions leading to membrane permeabilization, Bcl-XL ties up both tBid and Bax in nonproductive interactions and inhibits Bax binding to membranes. We propose that because Bcl-XL does not oligomerize it functions like a dominant-negative Bax in the membrane permeabilization process.

Bcl-2 family proteins can be classified into two subfamilies--anti-apoptotic members and pro-apoptotic members. Mechanistically, these two subfamilies can antagonize each other through heterodimerization while homodimerization has been proposed for each subfamily to carry out their corresponding anti-apoptotic or pro-apoptotic functions. To date, many small-molecule antagonists against anti-apoptotic Bcl-2 proteins have been developed, which are monomeric modulators. In this study, a series of BH3I-1 based dimeric modulators were developed with structure-activity relationship explored. Dimeric modulators compared to the monomeric antagonists have enhanced binding activity against anti-apoptotic Bcl-2 proteins. In addition, the acidic functional group was demonstrated to be critical for the binding interaction of the small-molecule antagonists with anti-apoptotic Bcl-2 proteins. Finally, the representative dimeric modulator revealed enhanced activity in inducing cytochrome c release from mitochondria compared to its monomeric counterpart. Taken together, dimerization of monomeric modulators is one practical approach to enhance the bioactivity of Bcl-2 antagonists.

Introduction Designing new healthcare facilities is complex and transitions to new clinical environments carry high risks, as unanticipated problems may arise resulting in inefficient care and patient harm. Design thinking, a human-centered design method, represents a unique framework to support the planning, testing, and evaluation of new clinical spaces throughout all phases of construction. Healthcare simulation has been used to test new clinical spaces, yet most report using simulation only in the late design stages. Moreover, healthcare design models have potentially underused human factors approaches calling for human-centered design. We applied a multimodal simulation-based approach underpinned by the principles of design thinking throughout the planning and construction stages of a newly renovated academic emergency department. Methods A multidisciplinary team developed and integrated 3 simulation strategies (table-top, mock-up, and in situ simulation) into the 5-step process of design thinking. Through end-user engagement, we identified potential challenges, prototyped solutions through table-top and mock-up simulations, and iteratively tested these solutions through in situ simulation within the actual clinical space. Results The team used end-user engagement and feedback to brainstorm and implement effective solutions to problems encountered before opening the new emergency department. The iterative steps and targeted use of simulation resulted in redesigning departmental processes and actual clinical space while mitigating anticipated safety threats and departmental deficiencies. Conclusions Design thinking coupled with multimodal simulation across all phases of construction enhanced the design and testing of new clinical infrastructure. Applying this approach early, thoroughly, and efficiently will help healthcare organizations plan changes to clinical spaces.