Lessons in "Slow" Engagement From Staff and Administrators at a Prebooking Jail Diversion Program.

Objective: In this study, the authors elicited the perspectives of criminal justice and mental health stakeholders about a prebooking jail diversion program, the Judge Ed Emmett Mental Health Diversion Center, serving primarily individuals experiencing chronic homelessness and diagnosed as having a serious mental illness. Methods: The authors analyzed semistructured interviews with 19 participants and observational fieldnotes from 60 hours of ethnographic fieldwork, conducted from January to July 2020 and including five administrative-level meetings. They used qualitative coding to develop themes. Administrative data were also reviewed. Results: Engagement of clients in the program was a major theme. Barriers to engagement included clients' fear of police involvement and strict rules around smoking. Facilitators to engagement included "slow" engagement, or gradual, gentle microengagements over time and across multiple visits, ideally with peer counselors. Conclusions: To promote client use of services at this critical point of care, jail diversion programs might consider ongoing negotiations with clients to balance expectations between the criminal justice and mental health systems of care by using "slow" client engagement, limiting police involvement, and adopting trauma-informed and harm-reduction approaches.

Construction of a Chassis for a Tripartite Protein-Based Molecular Motor.

Improving our understanding of biological motors, both to fully comprehend their activities in vital processes, and to exploit their impressive abilities for use in bionanotechnology, is highly desirable. One means of understanding these systems is through the production of synthetic molecular motors. We demonstrate the use of orthogonal coiled-coil dimers (including both parallel and antiparallel coiled coils) as a hub for linking other components of a previously described synthetic molecular motor, the Tumbleweed. We use circular dichroism, analytical ultracentrifugation, dynamic light scattering, and disulfide rearrangement studies to demonstrate the ability of this six-peptide set to form the structure designed for the Tumbleweed motor. The successful formation of a suitable hub structure is both a test of the transferability of design rules for protein folding as well as an important step in the production of a synthetic protein-based molecular motor.

Squaring the circle in peptide assembly: from fibers to discrete nanostructures by de novo design.

The design of bioinspired nanostructures and materials of defined size and shape is challenging as it pushes our understanding of biomolecular assembly to its limits. In such endeavors, DNA is the current building block of choice because of its predictable and programmable self-assembly. The use of peptide- and protein-based systems, however, has potential advantages due to their more-varied chemistries, structures and functions, and the prospects for recombinant production through gene synthesis and expression. Here, we present the design and characterization of two complementary peptides programmed to form a parallel heterodimeric coiled coil, which we use as the building blocks for larger, supramolecular assemblies. To achieve the latter, the two peptides are joined via peptidic linkers of variable lengths to produce a range of assemblies, from flexible fibers of indefinite length, through large colloidal-scale assemblies, down to closed and discrete nanoscale objects of defined stoichiometry. We posit that the different modes of assembly reflect the interplay between steric constraints imposed by short linkers and the bulk of the helices, and entropic factors that favor the formation of many smaller objects as the linker length is increased. This approach, and the resulting linear and proteinogenic polypeptides, represents a new route for constructing complex peptide-based assemblies and biomaterials.

A basis set of de novo coiled-coil peptide oligomers for rational protein design and synthetic biology.

Protein engineering, chemical biology, and synthetic biology would benefit from toolkits of peptide and protein components that could be exchanged reliably between systems while maintaining their structural and functional integrity. Ideally, such components should be highly defined and predictable in all respects of sequence, structure, stability, interactions, and function. To establish one such toolkit, here we present a basis set of de novo designed α-helical coiled-coil peptides that adopt defined and well-characterized parallel dimeric, trimeric, and tetrameric states. The designs are based on sequence-to-structure relationships both from the literature and analysis of a database of known coiled-coil X-ray crystal structures. These give foreground sequences to specify the targeted oligomer state. A key feature of the design process is that sequence positions outside of these sites are considered non-essential for structural specificity; as such, they are referred to as the background, are kept non-descript, and are available for mutation as required later. Synthetic peptides were characterized in solution by circular-dichroism spectroscopy and analytical ultracentrifugation, and their structures were determined by X-ray crystallography. Intriguingly, a hitherto widely used empirical rule-of-thumb for coiled-coil dimer specification does not hold in the designed system. However, the desired oligomeric state is achieved by database-informed redesign of that particular foreground and confirmed experimentally. We envisage that the basis set will be of use in directing and controlling protein assembly, with potential applications in chemical and synthetic biology. To help with such endeavors, we introduce Pcomp, an on-line registry of peptide components for protein-design and synthetic-biology applications.

Alpha-helical peptide assemblies giving new function to designed structures.

The design of alpha-helical tectons for self-assembly is maturing as a science. We have now reached the point where many different coiled-coil topologies can be reliably produced and validated in synthetic systems and the field is now moving on towards more complex, discrete structures and applications. Similarly the design of infinite or fiber assemblies has also matured, with the creation fibers that have been modified or functionalized in a variety of ways. This chapter discusses the progress made in both of these areas as well as outlining the challenges still to come.

Assembly pathway of a designed alpha-helical protein fiber.

Interest in the design of peptide-based fibrous materials is growing because it opens possibilities to explore fundamental aspects of peptide self-assembly and to exploit the resulting structures--for example, as scaffolds for tissue engineering. Here we investigate the assembly pathway of self-assembling fibers, a rationally designed alpha-helical coiled-coil system comprising two peptides that assemble on mixing. The dimensions spanned by the peptides and final structures (nanometers to micrometers), and the timescale over which folding and assembly occur (seconds to hours), necessitate a multi-technique approach employing spectroscopy, analytical ultracentrifugation, electron and light microscopy, and protein design to produce a physical model. We show that fibers form via a nucleation and growth mechanism. The two peptides combine rapidly (in less than seconds) to form sticky ended, partly helical heterodimers. A lag phase follows, on the order of tens of minutes, and is concentration-dependent. The critical nucleus comprises six to eight partially folded dimers. Growth is then linear in dimers, and subsequent fiber growth occurs in hours through both elongation and thickening. At later times (several hours), fibers grow predominantly through elongation. This kinetic, biomolecular description of the folding-and-assembly process allows the self-assembling fiber system to be manipulated and controlled, which we demonstrate through seeding experiments to obtain different distributions of fiber lengths. This study and the resulting mechanism we propose provide a potential route to achieving temporal control of functional fibers with future applications in biotechnology and nanoscale science and technology.

Talaporfin sodium.

IMPORTANCE OF THE FIELD: Despite therapeutic advances, cancer remains the cause of an estimated 23% of deaths in the USA. New treatments for malignancy are greatly needed. AREAS COVERED IN THIS REVIEW: Talaporfin sodium is a light-activated drug that causes tissue death through induction of apoptosis. Systemic antitumor effects mediated by CD8(+) T cells have been demonstrated in preclinical studies, providing a mechanism for distant response of tumors noted in clinical trials. Talaporfin sodium is approved in Japan for early-stage endobronchial cancer. Phase I and II studies in solid tumors have shown tumor regression in patients refractory to other therapies. Phase III pivotal studies against hepatocellular carcinoma as monotherapy and liver-metastatic colorectal cancer in combination with chemotherapy are ongoing. Talaporfin sodium is also in studies in men with symptomatic benign prostatic hyperplasia. Substantial safety data from clinical trials so far indicate that the drug is well tolerated. WHAT THE READER WILL GAIN: Talaporfin sodium has a broad safety profile and a mode of action that could affect growth in treated and untreated tumors. TAKE HOME MESSAGE: Clinical and preclinical studies indicate that talaporfin sodium treatment may offer a powerful option to synergize current therapies, as well as an alternative monotherapy in treating cancer.

Designed alpha-helical tectons for constructing multicomponent synthetic biological systems.

One possible route to develop new synthetic-biological systems is to assemble discrete nanoscale objects from programmed peptide-based building blocks. We describe an algorithm to design such blocks based on the coiled-coil protein-folding motif. The success of the algorithm is demonstrated by the production of six peptides that form three target parallel, blunted-ended heterodimers in preference to any of the other promiscuous pairings and alternate configurations, for example, homodimers, sticky-ended assemblies, and antiparallel arrangements. The peptides were linked to promote the assembly of larger, defined nanoscale rods, thus demonstrating that targeted peptide-peptide interactions can be specified in complex mixtures.

Rational design of peptide-based building blocks for nanoscience and synthetic biology.

The rational design of peptides that fold to form discrete nanoscale objects, and/ or self-assemble into nanostructured materials is an exciting challenge. Such efforts test and extend our understanding of sequence-to-structure relationships in proteins, and potentially provide materials for applications in bionanotechnology. Over the past decade or so, rules for the folding and assembly of one particular protein-structure motif--the alpha-helical coiled coil have advanced sufficiently to allow the confident design of novel peptides that fold to prescribed structures. Coiled coils are based on interacting alpha-helices, and guide and cement many protein-protein interactions in nature. As such, they present excellent starting points for building complex objects and materials that span the nano-to-micron scales from the bottom up. Along with others, we have translated and extended our understanding of coiled-coil folding and assembly to develop novel peptide-based biomaterials. Herein, we outline briefly the rules for the folding and assembly of coiled-coil motifs, and describe how we have used them in de novo design of discrete nanoscale objects and soft synthetic biomaterials. Moreover, we describe how the approach can be extended to other small, independently folded protein motifs--such as zinc fingers and EF-hands--that could be incorporated into more complex, multi-component synthetic systems and new hybrid and responsive biomaterials.

MagicWand: a single, designed peptide that assembles to stable, ordered alpha-helical fibers.

We describe a straightforward single-peptide design that self-assembles into extended and thickened nano-to-mesoscale fibers of remarkable stability and order. The basic chassis of the design is the well-understood dimeric alpha-helical coiled-coil motif. As such, the peptide has a heptad sequence repeat, abcdefg , with isoleucine and leucine residues at the a and d sites to ensure dimerization. In addition, to direct staggered assembly of peptides and to foster fibrillogenesisthat is, as opposed to blunt-ended discrete speciesthe terminal quarters of the peptide are cationic and the central half anionic with lysine and glutamate, respectively, at core-flanking e and g positions. This +,-,-,+ arrangement gives the peptide its name, MagicWand (MW). As judged by circular dichroism (CD) spectra, MW assembles to alpha-helical structures in the sub-micromolar range and above. The thermal unfolding of MW is reversible with a melting temperature >70 degrees C at 100 muM peptide concentration. Negative-stain transmission electron microscopy (TEM) of MW assemblies reveals stiff, straight, fibrous rods that extended for tens of microns. Moreover, different stains highlight considerable order both perpendicular and parallel to the fiber long axis. The dimensions of these features are consistent with bundles of long, straight coiled alpha-helical coiled coils with their axes aligned parallel to the long axis of the fibers. The fiber thickening indicates inter-coiled-coil interactions. Mutagenesis of the outer surface of the peptide i.e., at the b and f positionscombined with stability and microscopy measurements, highlights the role of electrostatic and cation-pi interactions in driving fiber formation, stability and thickening. These findings are discussed in the context of the growing number of self-assembling peptide-based fibrous systems.

Electrostatic control of thickness and stiffness in a designed protein fiber.

Attempts to design peptide-based fibers from first principles test our understanding of protein folding and assembly, and potentially provide routes to new biomaterials. Several groups have presented such designs based on alpha-helical and beta-strand building blocks. A key issue is this area now is engineering and controlling fiber morphology and related properties. Previously, we have reported the design and characterization of a self-assembling peptide fiber (SAF) system based on alpha-helical coiled-coil building blocks. With preceding designs, the SAFs are thickened, highly ordered structures in which many coiled coils are tightly bundled. As a result, the fibers behave as rigid rods. Here we report successful attempts to design new fibers that are thinner and more flexible by further programming at the amino-acid sequence level. This was done by introducing extended, or "smeared", electrostatic networks of arginine and glutamate residues to the surfaces of the coiled-coil building blocks. Furthermore, using arginine--rather than lysine--in these networks plays a major role in the fiber assembly, presumably by facilitating multidentate intra and intercoiled-coil salt bridges.

Peptide and protein building blocks for synthetic biology: from programming biomolecules to self-organized biomolecular systems.

There are several approaches to creating synthetic-biological systems. Here, we describe a molecular-design approach. First, we lay out a possible synthetic-biology space, which we define with a plot of complexity of components versus divergence from nature. In this scheme, there are basic units, which range from natural amino acids to totally synthetic small molecules. These are linked together to form programmable tectons, for example, amphipathic alpha-helices. In turn, tectons can interact to give self-assembled units, which can combine and organize further to produce functional assemblies and systems. To illustrate one path through this vast landscape, we focus on protein engineering and design. We describe how, for certain protein-folding motifs, polypeptide chains can be instructed to fold. These folds can be combined to give structured complexes, and function can be incorporated through computational design. Finally, we describe how protein-based systems may be encapsulated to control and investigate their functions.

L55P transthyretin accelerates subunit exchange and leads to rapid formation of hybrid tetramers.

Transthyretin is a tetrameric protein associated with the commonest form of systemic amyloid disease. Using isotopically labeled proteins and mass spectrometry, we compared subunit exchange in wild-type transthyretin with that of the variant associated with the most aggressive form of the disease, L55P. Wild-type subunit exchange occurs via both monomers and dimers, whereas exchange via dimers is the dominant mechanism for the L55P variant. Because patients with the L55P mutation are heterozygous, expressing both proteins simultaneously, we also analyzed the subunit exchange reaction between wild-type and L55P tetramers. We found that hybrid tetramers containing two or three L55P subunits dominate in the early stages of the reaction. Surprisingly, we also found that, in the presence of L55P transthyretin, the rate of dissociation of wild-type transthyretin is increased. This implies interactions between the two proteins that accelerate the formation of hybrid tetramers, a result with important implications for transthyretin amyloidosis.

The Trypanosoma cruzi enzyme TcGPXI is a glycosomal peroxidase and can be linked to trypanothione reduction by glutathione or tryparedoxin.

Trypanosoma cruzi glutathione-dependent peroxidase I (TcGPXI) can reduce fatty acid, phospholipid, and short chain organic hydroperoxides utilizing a novel redox cycle in which enzyme activity is linked to the reduction of trypanothione, a parasite-specific thiol, by glutathione. Here we show that TcGPXI activity can also be linked to trypanothione reduction by an alternative pathway involving the thioredoxin-like protein tryparedoxin. The presence of this new pathway was first detected using dialyzed soluble fractions of parasite extract. Tryparedoxin was identified as the intermediate molecule following purification, sequence analysis, antibody studies, and reconstitution of the redox cycle in vitro. The system can be readily saturated by trypanothione, the rate-limiting step being the interaction of trypanothione with the tryparedoxin. Both tryparedoxin and TcGPXI operate by a ping-pong mechanism. Overexpression of TcGPXI in transfected parasites confers increased resistance to exogenous hydroperoxides. TcGPXI contains a carboxyl-terminal tripeptide (ARI) that could act as a targeting signal for the glycosome, a kinetoplastid-specific organelle. Using immunofluorescence, tagged fluorescent proteins, and biochemical fractionation, we have demonstrated that TcGPXI is localized to both the glycosome and the cytosol. The ability of TcGPXI to use alternative electron donors may reflect their availability at the corresponding subcellular sites.