FLU-LISA (fluorescence-linked immunosorbent assay): high-throughput antibody profiling using antigen microarrays.

Vaccination and natural infection both elicit potent humoral responses that provide protection from subsequent infections. The immune history of an individual following such exposures is in part encoded by antibodies. While there are multiple immunoassays for measuring antibody responses, the majority of these methods measure responses to a single antigen. A commonly used method for measuring antibody responses is ELISA-a semiquantitative assay that is simple to perform in research and clinical settings. Here, we present FLU-LISA (fluorescence-linked immunosorbent assay)-a novel antigen microarray-based assay for rapid high-throughput antibody profiling. The assay can be used for profiling immunoglobulin (Ig) G, IgA and IgM responses to multiple antigens simultaneously, requiring minimal amounts of sample and antigens. Using several influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen microarrays, we demonstrated the specificity and sensitivity of our novel assay and compared it with the traditional ELISA, using samples from mice, chickens and humans. We also showed that our assay can be readily used with dried blood spots, which can be collected from humans and wild birds. FLU-LISA can be readily used to profile hundreds of samples against dozens of antigens in a single day, and therefore offers an attractive alternative to the traditional ELISA.

Micellar Composition Affects Lipid Accretion Kinetics in Molecular Dynamics Simulations: Support for Lipid Network Reproduction.

Mixed lipid micelles were proposed to facilitate life through their documented growth dynamics and catalytic properties. Our previous research predicted that micellar self-reproduction involves catalyzed accretion of lipid molecules by the residing lipids, leading to compositional homeostasis. Here, we employ atomistic Molecular Dynamics simulations, beginning with 54 lipid monomers, tracking an entire course of micellar accretion. This was done to examine the self-assembly of variegated lipid clusters, allowing us to measure entry and exit rates of monomeric lipids into pre-micelles with different compositions and sizes. We observe considerable rate-modifications that depend on the assembly composition and scrutinize the underlying mechanisms as well as the energy contributions. Lastly, we describe the measured potential for compositional homeostasis in our simulated mixed micelles. This affirms the basis for micellar self-reproduction, with implications for the study of the origin of life.

A Similarity-Based Method for Predicting Enzymatic Functions in Yeast Uncovers a New AMP Hydrolase.

Despite decades of research and the availability of the full genomic sequence of the baker's yeast Saccharomyces cerevisiae, still a large fraction of its genome is not functionally annotated. This hinders our ability to fully understand cellular activity and suggests that many additional processes await discovery. The recent years have shown an explosion of high-quality genomic and structural data from multiple organisms, ranging from bacteria to mammals. New computational methods now allow us to integrate these data and extract meaningful insights into the functional identity of uncharacterized proteins in yeast. Here, we created a database of sensitive sequence similarity predictions for all yeast proteins. We use this information to identify candidate enzymes for known biochemical reactions whose enzymes are unidentified, and show how this provides a powerful basis for experimental validation. Using one pathway as a test case we pair a new function for the previously uncharacterized enzyme Yhr202w, as an extra-cellular AMP hydrolase in the NAD degradation pathway. Yhr202w, which we now term Smn1 for Scavenger MonoNucleotidase 1, is a highly conserved protein that is similar to the human protein E5NT/CD73, which is associated with multiple cancers. Hence, our new methodology provides a paradigm, that can be adopted to other organisms, for uncovering new enzymatic functions of uncharacterized proteins.

Dynamic lipid aptamers: non-polymeric chemical path to early life.

A widespread dogma asserts that life could not have emerged without biopolymers - RNA and proteins. However, the widely acknowledged implausibility of a spontaneous appearance and proliferation of these complex molecules in primordial messy chemistry casts doubt on this scenario. A proposed alternative is "Lipid-First", based on the evidence that lipid assemblies may spontaneously emerge in heterogeneous environments, and are shown to undergo growth and fission, and to portray autocatalytic self-copying. What seems undecided is whether lipid assemblies have protein-like capacities for stereospecific interactions, a sine qua non of life processes. This Viewpoint aims to alleviate such doubts, pointing to growing experimental evidence that lipid aggregates possess dynamic surface configurations capable of stereospecific molecular recognition. Such findings help support a possible key role of lipids in seeding life's origin.

Self-reproducing catalytic micelles as nanoscopic protocell precursors.

Protocells at life's origin are often conceived as bilayer-enclosed precursors of life, whose self-reproduction rests on the early advent of replicating catalytic biopolymers. This Perspective describes an alternative scenario, wherein reproducing nanoscopic lipid micelles with catalytic capabilities were forerunners of biopolymer-containing protocells. This postulate gains considerable support from experiments describing micellar catalysis and autocatalytic proliferation, and, more recently, from reports on cross-catalysis in mixed micelles that lead to life-like steady-state dynamics. Such results, along with evidence for micellar prebiotic compatibility, synergize with predictions of our chemically stringent computer-simulated model, illustrating how mutually catalytic lipid networks may enable micellar compositional reproduction that could underlie primal selection and evolution. Finally, we highlight studies on how endogenously catalysed lipid modifications could guide further protocellular complexification, including micelle to vesicle transition and monomer to biopolymer progression. These portrayals substantiate the possibility that protocellular evolution could have been seeded by pre-RNA lipid assemblies.

Polyamines Mediate Folding of Primordial Hyperacidic Helical Proteins.

Polyamines are known to mediate diverse biological processes, and specifically to bind and stabilize compact conformations of nucleic acids, acting as chemical chaperones that promote folding by offsetting the repulsive negative charges of the phosphodiester backbone. However, whether and how polyamines modulate the structure and function of proteins remain unclear. In particular, early proteins are thought to have been highly acidic, like nucleic acids, due to a scarcity of basic amino acids in the prebiotic context. Perhaps polyamines, the abiotic synthesis of which is simple, could have served as chemical chaperones for such primordial proteins? We replaced all lysines of an ancestral 60-residue helix-bundle protein with glutamate, resulting in a disordered protein with 21 glutamates in total. Polyamines efficiently induce folding of this hyperacidic protein at submillimolar concentrations, and their potency scaled with the number of amine groups. Compared to cations, polyamines were several orders of magnitude more potent than Na+, while Mg2+ and Ca2+ had an effect similar to that of a diamine, inducing folding at approximately seawater concentrations. We propose that (i) polyamines and dications may have had a role in promoting folding of early proteins devoid of basic residues and (ii) coil-helix transitions could be the basis of polyamine regulation in contemporary proteins.

Twenty Years of "Lipid World": A Fertile Partnership with David Deamer.

"The Lipid World" was published in 2001, stemming from a highly effective collaboration with David Deamer during a sabbatical year 20 years ago at the Weizmann Institute of Science in Israel. The present review paper highlights the benefits of this scientific interaction and assesses the impact of the lipid world paper on the present understanding of the possible roles of amphiphiles and their assemblies in the origin of life. The lipid world is defined as a putative stage in the progression towards life's origin, during which diverse amphiphiles or other spontaneously aggregating small molecules could have concurrently played multiple key roles, including compartment formation, the appearance of mutually catalytic networks, molecular information processing, and the rise of collective self-reproduction and compositional inheritance. This review brings back into a broader perspective some key points originally made in the lipid world paper, stressing the distinction between the widely accepted role of lipids in forming compartments and their expanded capacities as delineated above. In the light of recent advancements, we discussed the topical relevance of the lipid worldview as an alternative to broadly accepted scenarios, and the need for further experimental and computer-based validation of the feasibility and implications of the individual attributes of this point of view. Finally, we point to possible avenues for exploring transition paths from small molecule-based noncovalent structures to more complex biopolymer-containing proto-cellular systems.

Enceladus: First Observed Primordial Soup Could Arbitrate Origin-of-Life Debate.

A recent breakthrough publication has reported complex organic molecules in the plumes emanating from the subglacial water ocean of Saturn's moon Enceladus (Postberg et al., 2018, Nature 558:564-568). Based on detailed chemical scrutiny, the authors invoke primordial or endogenously synthesized carbon-rich monomers (<200 u) and polymers (up to 8000 u). This appears to represent the first reported extraterrestrial organics-rich water body, a conceivable milieu for early steps in life's origin ("prebiotic soup"). One may ask which origin-of-life scenario appears more consistent with the reported molecular configurations on Enceladus. The observed monomeric organics are carbon-rich unsaturated molecules, vastly different from present-day metabolites, amino acids, and nucleotide bases, but quite chemically akin to simple lipids. The organic polymers are proposed to resemble terrestrial insoluble kerogens and humic substances, as well as refractory organic macromolecules found in carbonaceous chondritic meteorites. The authors posit that such polymers, upon long-term hydrous interactions, might break down to micelle-forming amphiphiles. In support of this, published detailed analyses of the Murchison chondrite are dominated by an immense diversity of likely amphiphilic monomers. Our specific quantitative model for compositionally reproducing lipid micelles is amphiphile-based and benefits from a pronounced organic diversity. It thus contrasts with other origin models, which require the presence of very specific building blocks and are expected to be hindered by excess of irrelevant compounds. Thus, the Enceladus finds support the possibility of a pre-RNA Lipid World scenario for life's origin.

Protobiotic Systems Chemistry Analyzed by Molecular Dynamics.

Systems chemistry has been a key component of origin of life research, invoking models of life's inception based on evolving molecular networks. One such model is the graded autocatalysis replication domain (GARD) formalism embodied in a lipid world scenario, which offers rigorous computer simulation based on defined chemical kinetics equations. GARD suggests that the first pre-RNA life-like entities could have been homeostatically-growing assemblies of amphiphiles, undergoing compositional replication and mutations, as well as rudimentary selection and evolution. Recent progress in molecular dynamics has provided an experimental tool to study complex biological phenomena such as protein folding, ligand-receptor interactions, and micellar formation, growth, and fission. The detailed molecular definition of GARD and its inter-molecular catalytic interactions make it highly compatible with molecular dynamics analyses. We present a roadmap for simulating GARD's kinetic and thermodynamic behavior using various molecular dynamics methodologies. We review different approaches for testing the validity of the GARD model by following micellar accretion and fission events and examining compositional changes over time. Near-future computational advances could provide empirical delineation for further system complexification, from simple compositional non-covalent assemblies towards more life-like protocellular entities with covalent chemistry that underlies metabolism and genetic encoding.