Chapter 9: Life as We Don't Know It.

While Earth contains the only known example of life in the universe, it is possible that life elsewhere is fundamentally different from what we are familiar with. There is an increased recognition in the astrobiology community that the search for life should steer away from terran-specific biosignatures to those that are more inclusive to all life-forms. To start exploring the space of possibilities that life could occupy, we can try to dissociate life from the chemistry that composes it on Earth by envisioning how different life elsewhere could be in composition, lifestyle, medium, and form, and by exploring how the general principles that govern living systems on Earth might be found in different forms and environments across the Solar System. Exotic life-forms could exist on Mars or Venus, or icy moons like Europa and Enceladus, or even as a shadow biosphere on Earth. New perspectives on agnostic biosignature detection have also begun to emerge, allowing for a broader and more inclusive approach to seeking exotic life with unknown chemistry that is distinct from life as we know it on Earth.

The Prebiotic Kitchen: A Guide to Composing Prebiotic Soup Recipes to Test Origins of Life Hypotheses.

"Prebiotic soup" often features in discussions of origins of life research, both as a theoretical concept when discussing abiological pathways to modern biochemical building blocks and, more recently, as a feedstock in prebiotic chemistry experiments focused on discovering emergent, systems-level processes such as polymerization, encapsulation, and evolution. However, until now, little systematic analysis has gone into the design of well-justified prebiotic mixtures, which are needed to facilitate experimental replicability and comparison among researchers. This paper explores principles that should be considered in choosing chemical mixtures for prebiotic chemistry experiments by reviewing the natural environmental conditions that might have created such mixtures and then suggests reasonable guidelines for designing recipes. We discuss both "assembled" mixtures, which are made by mixing reagent grade chemicals, and "synthesized" mixtures, which are generated directly from diversity-generating primary prebiotic syntheses. We discuss different practical concerns including how to navigate the tremendous uncertainty in the chemistry of the early Earth and how to balance the desire for using prebiotically realistic mixtures with experimental tractability and replicability. Examples of two assembled mixtures, one based on materials likely delivered by carbonaceous meteorites and one based on spark discharge synthesis, are presented to illustrate these challenges. We explore alternative procedures for making synthesized mixtures using recursive chemical reaction systems whose outputs attempt to mimic atmospheric and geochemical synthesis. Other experimental conditions such as pH and ionic strength are also considered. We argue that developing a handful of standardized prebiotic recipes may facilitate coordination among researchers and enable the identification of the most promising mechanisms by which complex prebiotic mixtures were "tamed" during the origin of life to give rise to key living processes such as self-propagation, information processing, and adaptive evolution. We end by advocating for the development of a public prebiotic chemistry database containing experimental methods (including soup recipes), results, and analytical pipelines for analyzing complex prebiotic mixtures.

Chemical Ecosystem Selection on Mineral Surfaces Reveals Long-Term Dynamics Consistent with the Spontaneous Emergence of Mutual Catalysis.

How did chemicals first become organized into systems capable of self-propagation and adaptive evolution? One possibility is that the first evolvers were chemical ecosystems localized on mineral surfaces and composed of sets of molecular species that could catalyze each other's formation. We used a bottom-up experimental framework, chemical ecosystem selection (CES), to evaluate this perspective and search for surface-associated and mutually catalytic chemical systems based on the changes in chemistry that they are expected to induce. Here, we report the results of preliminary CES experiments conducted using a synthetic "prebiotic soup" and pyrite grains, which yielded dynamical patterns that are suggestive of the emergence of mutual catalysis. While more research is needed to better understand the specific patterns observed here and determine whether they are reflective of self-propagation, these results illustrate the potential power of CES to test competing hypotheses for the emergence of protobiological chemical systems.

Mineral surfaces select for longer RNA molecules.

We report empirically and theoretically that multiple prebiotic minerals can selectively accumulate longer RNAs, with selectivity enhanced at higher temperatures. We further demonstrate that surfaces can be combined with a catalytic RNA to form longer RNA polymers, supporting the potential of minerals to develop genetic information on the early Earth.

Co-Expression of a Chimeric Protease Inhibitor Secreted by a Tumor-Targeted Salmonella Protects Therapeutic Proteins from Proteolytic Degradation.

Sunflower trypsin inhibitor (SFTI) is a 14-amino-acid bicyclic peptide that contains a single internal disulfide bond. We initially constructed chimeras of SFTI with N-terminal secretion signals from the Escherichia coli OmpA and Pseudomonas aeruginosa ToxA, but only detected small amounts of protease inhibition resulting from these constructs. A substantially higher degree of protease inhibition was detected from a C-terminal SFTI fusion with E. coli YebF, which radiated more than a centimeter from an individual colony of E. coli using a culture-based inhibitor assay. Inhibitory activity was further improved in YebF-SFTI fusions by the addition of a trypsin cleavage signal immediately upstream of SFTI, and resulted in production of a 14-amino-acid, disulfide-bonded SFTI free in the culture supernatant. To assess the potential of the secreted SFTI to protect the ability of a cytotoxic protein to kill tumor cells, we utilized a tumor-selective form of the Pseudomonas ToxA (OTG-PE38K) alone and expressed as a polycistronic construct with YebF-SFTI in the tumor-targeted Salmonella VNP20009. When we assessed the ability of toxin-containing culture supernatants to kill MDA-MB-468 breast cancer cells, the untreated OTG-PE38K was able to eliminate all detectable tumor cells, while pretreatment with trypsin resulted in the complete loss of anticancer cytotoxicity. However, when OTG-PE38K was co-expressed with YebF-SFTI, cytotoxicity was completely retained in the presence of trypsin. These data demonstrate SFTI chimeras are secreted in a functional form and that co-expression of protease inhibitors with therapeutic proteins by tumor-targeted bacteria has the potential to enhance the activity of therapeutic proteins by suppressing their degradation within a proteolytic environment.

EGFR-targeted Chimeras of Pseudomonas ToxA released into the extracellular milieu by attenuated Salmonella selectively kill tumor cells.

Tumor-targeted Salmonella VNP20009 preferentially replicate within tumor tissue and partially suppress tumor growth in murine tumor models. These Salmonella have the ability to locally induce apoptosis when they are in direct contact with cancer cells but they lack significant bystander killing, which may correlate with their overall lack of antitumor activity in human clinical studies. In order to compensate for this deficiency without enhancing overall toxicity, we engineered the bacteria to express epidermal growth factor receptor (EGFR)-targeted cytotoxic proteins that are released into the extracellular milieu. In this study, we demonstrate the ability of the Salmonella strain VNP20009 to produce three different forms of the Pseudomonas exotoxin A (ToxA) chimeric with a tumor growth factor alpha (TGFα) which results in its producing culture supernatants that are cytotoxic and induce apoptosis in EGFR positive cancer cells as measured by the tetrazolium dye reduction, and Rhodamine 123 and JC-10 mitochondrial depolarization assays. In addition, exchange of the ToxA REDLK endoplasmic reticulum retention signal for KDEL and co-expression of the ColE3 lysis protein resulted in an overall increased cytotoxicity compared to the wild type toxin. This approach has the potential to significantly enhance the antitumor activity of VNP20009 while maintaining its previously established safety profile. Biotechnol. Bioeng. 2016;113: 2698-2711. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.