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At least 19 records

Oxidative peptide /and amide/ formation from Schiff base complexes

One hypothesis of the origin of pre-modern forms of life is that the original replicating molecules were specific polypeptides which acted as templates for the assembly of poly-Schiff bases complementary to the template, and that these polymers were then oxidized to peptide linkages, probably by photo-produced oxidants. A double cycle of such anti-parallel complementary replication would yield the original peptide polymer. If this model were valid, the Schiff base between an N-acyl alpha mino aldehyde and an amino acid should yield a dipeptide in aqueous solution in the presence of an appropriate oxidant. In the present study it is shown that the substituted dipeptide, N-acetyl-tyrosyl-tyrosine, is produced in high yield in aqueous solution at pH 9 through the action of H2O2 on the Schiff-base complex between N-acetyl-tyrosinal and tyrosine and that a great variety of N-acyl amino acids are formed from amino acids and aliphatic aldehydes under similar conditions.

Strehler, B. L.↗

Molecular replication

The object of our research program is to understand how polynucleotide replication originated on the primitive Earth. This is a central issue in studies of the origins of life, since a process similar to modern DNA and RNA synthesis is likely to have formed the basis for the most primitive system of genetic information transfer. The major conclusion of studies so far is that a preformed polynucleotide template under many different experimental conditions will facilitate the synthesis of a new oligonucleotide with a sequence complementary to that of the template. It has been shown, for example, that poly(C) facilitates the synthesis of long oligo(G)s and that the short template CCGCC facilities the synthesis of its complement GGCGG. Very recently we have shown that template-directed synthesis is not limited to the standard oligonucleotide substrates. Nucleic acid-like molecules with a pyrophosphate group replacing the phosphate of the standard nucleic acid backbone are readily synthesized from deoxynucleotide 3'-5'-diphosphates on appropriate templates.

Orgel, L. E.↗

A theory for the origin of a self-replicating chemical system. I - Natural selection of the autogen from short, random oligomers

A general theory is presented for the origin of a self-replicating chemical system, termed an autogen, which is capable of both crude replication and translation (protein synthesis). The theory requires the availability of free energy and monomers to the system, a significant background low-yield synthesis of kinetically stable oligopeptides and oligonucleotides, the localization of the oligomers, crude oligonucleotide selectivity of amino acids during oligopeptide synthesis, crude oligonucleotide replication, and two short peptide families which catalyze replication and translation, to produce a localized group of at least one copy each of two protogenes and two protoenzymes. The model posits a process of random oligomerization, followed by the random nucleation of functional components and the rapid autocatalytic growth of the functioning autogen to macroscopic amounts, to account for the origin of the first self-replicating system. Such a process contains steps of such high probability and short time periods that it is suggested that the emergence of an autogen in a laboratory experiment of reasonable time scale may be possible.

White, D. H.↗

DNA-directed mutations. Leading and lagging strand specificity

The fidelity of replication has evolved to reproduce B-form DNA accurately, while allowing a low frequency of mutation. The fidelity of replication can be compromised, however, by defined order sequence DNA (dosDNA) that can adopt unusual or non B-DNA conformations. These alternative DNA conformations, including hairpins, cruciforms, triplex DNAs, and slipped-strand structures, may affect enzyme-template interactions that potentially lead to mutations. To analyze the effect of dosDNA elements on spontaneous mutagenesis, various mutational inserts containing inverted repeats or direct repeats were cloned in a plasmid containing a unidirectional origin of replication and a selectable marker for the mutation. This system allows for analysis of mutational events that are specific for the leading or lagging strands during DNA replication in Escherichia coli. Deletions between direct repeats, involving misalignment stabilized by DNA secondary structure, occurred preferentially on the lagging strand. Intermolecular strand switch events, correcting quasipalindromes to perfect inverted repeats, occurred preferentially during replication of the leading strand.

NASA Discipline Radiation Health↗

A theory for the origin of a self-replicating chemical system. II - Computer simulation of the autogen

In order to better understand the feasibility and limitations of the autogen (White 1980), a computer simulation based on the fluctuating clay environment was used to test whether autocatalytic growth would occur under various conditions. The results suggest that overall accuracies of replication and translation in the range of 90% and 10%, and protoenzyme turnover numbers of 10-120 monomers/protoenzyme/day are adequate for exponential growth. Nucleation of the components of the autogen from random background oligomers would be extremely rapid if oligomers lengths 2-6 were adequately functional, whereas oligomer lengths much greater than 10 are prohibited. The autogen would most likely nucleate and grow to dominance either rapidly (10-100 cycles of roughly 1 day each) or not at all.

White, D. H.↗

Effects of space environment on structural materials - A preliminary study and development of materials characterization protocols

A preliminary study of materials exposed in space in LEO for nearly six years in the NASA Long-Duration Exposure Facility is presented. It is demonstrated that it will be necessary to isolate surface debris and reaction products from materials exposed in space. Replication techniques originally designed for electron microscopy examination of surfaces can be applied to lift off and isolate such surface features. Debris and reaction products were examined through a variety of analytical techniques, including the surface morphology by SEM, and internal microstructures by STEM and TEM, EDS, and SAD. The results illustrate the role that atomic oxygen and micrometeorites play in surface alteration and reaction in LEO space environments, as well as the role of debris created from other proximate materials.

Miglionico, C.↗

Digital Synchronizer without Metastability

A proposed design for a digital synchronizing circuit would eliminate metastability that plagues flip-flop circuits in digital input/output interfaces. This metastability is associated with sampling, by use of flip-flops, of an external signal that is asynchronous with a clock signal that drives the flip-flops: it is a temporary flip-flop failure that can occur when a rising or falling edge of an asynchronous signal occurs during the setup and/or hold time of a flip-flop. The proposed design calls for (1) use of a clock frequency greater than the frequency of the asynchronous signal, (2) use of flip-flop asynchronous preset or clear signals for the asynchronous input, (3) use of a clock asynchronous recovery delay with pulse width discriminator, and (4) tying the data inputs to constant logic levels to obtain (5) two half-rate synchronous partial signals - one for the falling and one for the rising edge. Inasmuch as the flip-flop data inputs would be permanently tied to constant logic levels, setup and hold times would not be violated. The half-rate partial signals would be recombined to construct a signal that would replicate the original asynchronous signal at its original rate but would be synchronous with the clock signal.

Simle, Robert M.↗

X-Ray Imagery as the Record of All Data of Interest in Hypervelocity Impact Fragment Studies

Laboratory study of hypervelocity spacecraft fragmentation has traditionally involved the collection and analysis of fragments that were caught in deceleration material surrounding the impact. This process has typically involved the disintegration of the catchment material either through chemical dissolution, or through physical excavation to recover the fragments. Due to the scale of the three impact tests—the Satellite Orbital Debris Characterization Impact Test (SOCIT), the DebriSat satellite impact test, and the DebrisLV launch vehicle impact test—the latter two using more than 12 cubic meters of polyurethane foam to capture the fragments, hese projects have used x-ray imagery to precisely locate and thus, to more efficiently extract fragments in the soft-catch material. Three years into the DebriSat fragment extraction process, a side study was initiated to explore what additional information could be discerned from the x-rays, with significant results. This study was instrumental to a rapid replacement and retooling as the project was forced to replace the x-ray system around which the extraction process had been based. The revised process continues to map the debris for extraction. The project has, in parallel, systematically addressed the limits/tolerances of what x-rays can reveal about size, shape, density, mass, velocity, energy, and deformation/damage of the fragment during the deceleration in the catchment material while replicating the original extraction mapping function. All of these features have been optimized or have sufficient understanding to characterize the basic factors that will define a complete data set extracted solely from x-ray imagery. It is an ideal time to develop such a process, with extracted fragments providing “ground truth” against image-only data, and abundant available imagery of the same fragments under both the prior and replacement x-ray technologies, which have several fundamentally different characteristics. This paper addresses the types and quality of hypervelocity fragmentation data that can be and has been extracted from x-rays. It further addresses the question of whether and under what circumstances future hypervelocity experiments can use x-ray methods to largely—or to completely—avoid the extraction process in recording all appropriate results. Lastly, this paper addresses lessons learned and how future efforts can be further optimized.

John B. Bacon↗

Method Development for In-situ Detection of Latent Herpesvirus DNA from Saliva using Nanopore Sequencing

Research toward latent herpesvirus reactivation has been intensively addressed through Space Shuttle and International Space Station (ISS) investigations. This work has provided the understanding that persistent reactivation of herpesviruses from asymptomatic crew can be detected through viral shedding in saliva, urine, and blood. Occasionally, viral reactivation from the latency stage can pose a threat to crew health (clinical manifestation) before, during, and after flight missions. Furthermore, previous work detailing correlations with immunity indicate that monitoring viral reactivation could be implemented to assess potential immune dysfunction. While in-flight monitoring is desirable, there is no well-established procedure or method for real-time evaluations, and research to date has relied on postflight, ground-based analysis. The development of portable molecular technologies like the miniPCR™ (miniPCR Bio) thermal cycler and the MinION™ sequencer (Oxford Nanopore Technologies) have confirmed that real-time monitoring is possible in extreme and low resource environments. These devices, combined with simple sample preparation methods, have been used to demonstrate bacterial identification onboard the ISS, as well as rapid viral detection in remote locations on Earth. The work here builds upon previous molecular advancements onboard the ISS toward the development and validation of a spaceflight-compatible method for viral detection from crew samples. Several herpesviruses can be detected in saliva, which provides a non-invasive means to collect samples for monitoring. While the basis for this method lies in previous spaceflight investigations, key points for method optimization include DNA extraction from saliva, viral primer selection, and bioinformatic processes for data analysis. To increase viral yield, numerous DNA extraction methods have been evaluated and will be discussed in detail. For initial development and testing, varicella-zoster virus (VZV) is being targeted though open reading frame 51 and 63 (ORF51, ORF63), as the replication origin-binding protein is highly expressed during latency. Optimization of the thermal cycling parameters has resulted in the ability to test the entire process. The full method has been tested with both viral VZV DNA standards and saliva spiked with varying concentrations of VZV. Viral sequence data were mapped to the reference sequence using minimap2. Prior to mapping, DNA sequencing reads were filtered for length and quality, barcodes were removed, and alignment identity calculated. Following further assessments, statistics were compared across multiple sequencing experiments and are being used to determine the success of the protocols. Forward work will include the incorporation of herpes simplex virus 1 (HSV-1) and Epstein-Barr virus (EBV) primers as well as the validation of results to the terrestrial qPCR standard assay. Upon full validation of the developed method, saliva will be collected from 20 healthy subjects and spiked with viral DNA. These samples will be split and assayed with the MinION and standard qPCR assay.

Hang N Nguyen↗

Who or What? Self-Replication and Function-Reproduction in the Origin of Life

In this presentation, we will present results on the fundamental properties of two classes of replicating systems: autocatalytic replicators that reproduce exact copies of a template molecule, and function reproducers that maintain a set of essential functions without replicating the identities of the functional moieties. We will describe the stability and behavior in-the-large of autocatalytic replicators. Most importantly, we have found no sharp distinction between an autocatalytic and a non-autocatalytic domain. We will also present a new derivation of von Kiedrowski's square-root rate law. Function - reproducers are proposed as an important component of protocells and we will present theoretical results on a simple model system that incorporates known peptide biophysics. For a wide range of parameters, we have shown that this type of system can improve its overall performance, even in the absence of any method for information storage. This type of system improvement is defined to be non-genomic evolution.

New, Michael H.↗

Attempted nonenzymatic template-directed oligomerizations on a polyadenylic acid template: implications for the nature of the first genetic material

Previous attempts to produce nonenzymatic template-directed oligomerizations of activated pyrimidines on polypurine templates have been unsuccessful. The only efficient reactions are those where the template is composed primarily of pyrimidines, especially cytosine. Because molecular evolution requires that a synthesized daughter polynucleotide be capable of acting as a template for the synthesis of the original polynucleotide, the one-way replication achieved thus far is inadequate to initiate an evolving system. Several uracil analogs were used in this investigation in order to search for possible replacements for uracil. The monomers used in this investigation were the imidazolides of UMP, xanthosine 5'-monophosphate, the bis-monophosphates of the acyclic nucleosides of uracil, and 2,4-quinazolinedione. The concentrations of various salts, buffers, pH, and temperature were among the different variables investigated in attempts to find conditions that would permit template-directed oligomerizations. Although the different monomers in this study demonstrated varying abilities to form very short oligomers, we were unable to detect any enhancement of this oligomerization that could be attributed to the poly(A) template. Although special conditions might be found that would allow purine-rich templates to work, these reactions cannot be considered robust. The results of our experiments suggest that pyrimidines were not part of the original replicating system on the primitive Earth. It has already been shown that ribose is an unlikely component of the first replicating systems, and we now suggest that phosphate was absent as well. This is due to the low solubility of phosphate in the present ocean (3 x 10(-6) M), as well as the difficulty of prebiotic activation of phosphates.

Non-NASA Center↗

The limits of template-directed synthesis with nucleoside-5'-phosphoro(2-methyl)imidazolides

In earler work we have shown that C-rich templates containing isolated A, T or G residues and short oligo(G) sequences can be copied effectively using nucleoside-5'-phosphoro(2-methyl)imidazolides as substrates. We now show that isolated A or T residues within an oligo(G) sequence are a complete block to copying and that an isolated C residue is copied inefficiently. Replication is possible only if there are two complementary oligonucleotides each of which acts as a template to facilitate the synthesis of the other. We emphasize the severity of the problems that need to be overcome to make possible non-enzymatic replication in homogeneous aqueous solution. We conclude that an efficient catalyst was involved in the origin of polynucleotide replication.

Hill, Aubrey R.↗

The Limits of Template-Directed Synthesis with Nucleoside-5'-Phosphoro(2-Methyl) Imidazolides

In earlier work we have shown that C-rich templates containing isolated A, T or G residues and short oligo(G) sequences can be copied effectively using nucleoside-5'-phosphoro(2-methyl)imidazolides as substrates. We now show that isolated A or T residues within an oligo(G) sequence are a complete block to copying and that an isolated C residue is copied inefficiently. Replication is possible only if there are two complementary oligonucleotides each of which acts as a template to facilitate the synthesis of the other. We emphasize the severity of the problems that need to be overcome to make possible non-enzymatic replication in homogeneous aqueous solution. We conclude that an efficient catalyst was involved in the origin of polynucleotide replication.

Hill, Aubrey R., Jr.↗

Enantiomeric Cross-Inhibition in the Synthesis of Oligonucleotides on a Nonchiral Template

Prebiotic syntheses of chiral monomers always yield racemic mixtures. Living systems, however, utilize L-amino acids and D-nucleotides in their biopolymers. The generation of optical asymmetry by selection and amplification in an autocatalytic process is, therefore, an important element in many theories of the origin of life. Replication of polynucleotides in template-directed syntheses is an obvious candidate for such an amplification step in a pre-'RNA world'. A serious objection to this suggestion is the observation that the efficiency of template-directed syntheses of RNA is limited by enantiomeric cross-inhibition. Peptide Nucleic Acids (PNAs), amide-linked, nonchiral analogues of RNA, have been 'copied' into RNA and constitute an alternative to chiral polynucleotides as an informational replicating system. Here, we use PNA as model for a hypothetical, nonchiral precursor of RNA in experiments re-examining enantiomeric cross-inhibition. We find that enantiomeric cross-inhibition is as serious in the polymerization of nucleotides on a PNA template as it is on a conventional RNA or DNA template.

Schmidt, Jurgen G.↗

Paranoia.Ada: Sample output reports

Paranoia.Ada is a program to diagnose floating point arithmetic in the context of the Ada programming language. The program evaluates the quality of a floating point arithmetic implementation with respect to the proposed IEEE Standards P754 and P854. Paranoia.Ada is derived from the original BASIC programming language version of Paranoia. The Paranoia.Ada replicates in Ada the test algorithms originally implemented in BASIC and adheres to the evaluation criteria established by W. M. Kahan. Paranoia.Ada incorporates a major structural redesign and employs applicable Ada architectural and stylistic features.

Source record↗

Mach 5 electroformed nickel nozzle refurbishment FNAS investigation of ultra-smooth surfaces

This task is in support of the Quiet Hypersonic Wind Tunnel effort currently in effect at NASA Langley Research Center, VA. A laminar flow wind tunnel nozzle has been previously fabricated by electroforming pure nickel over a machined and polished two piece stainless steel mandrel. The mandrel was removed leaving the replicate nozzle surface. The nozzle was then pressed into a heavy stainless steel jacket for mounting features and rigidity. The original nickel surface was a replication of the polished mandrel but had degraded due to oxidation. The inside surface requirements are very stringent in order to achieve laminar or quiet flow at the desired pressure and temperature for the specific design of Mach 5. The throat area of the axisymmetric device must have a surface finish with no defects greater than 16 microinches. This requires an rms average background of about four microinches or better for inspection purposes. A coating of nickel-phosphorous alloy was applied by catalytic deposition and then the inside of the nozzle was polished retaining dimensional and surface finish tolerances as specified per drawings supplied. Since the unit is not an optical component, conventional optical inspection methods for surface finish and figure are not readily achieved. Measurements were made using surface profilometry.

Rood, Robert↗