Generation of Molecular Complexity from Cyclooctatetraene: Preparation of Aminobicyclo[5.1.0]octitols

A series of eight stereoisomeric N-(tetrahydroxy bicyclo-[5.1.0]oct-2S*-yl)phthalimides were prepared in one to four steps from N-(bicyclo[5.1.0]octa-3,5-dien-2-yl)phthalimide (±)-7, which is readily available from cyclooctatetraene (62 % yield). The structural assignments of the stereoisomers were established by 1H NMR spectral data as well as X-ray crystal structures for certain members. The outcomes of several epoxydiol hydrolyses, particularly ring contraction and enlargement, are of note. The isomeric phthalimides as well as the free amines did not exhibit β-glucosidase inhibitory activity at a concentration of less than 100 μM

Cellular imitations

Synthetic biologists typically construct new pathways within existing cells. While useful, this approach in many ways ignores the undefined but necessary components of life. A growing number of laboratories have begun to try to remove some of the mysteries of cellular life by building life-like systems from non-living component parts. Some of these attempts rely on purely chemical and physical forces alone without the aid of biological molecules, while others try to build artificial cells from the parts of life, such as nucleic acids, proteins, and lipids. Both bottom-up strategies suffer from the complication of trying to build something that remains undefined. The result has been the development of research programs that try to build systems that mimic in some way recognized living systems. Since it is difficult to quantify the mimicry of life, success often times is evaluated with a degree of subjectivity. Herein we highlight recent advances in mimicking the organization and behavior of cellular life from the bottom-up

UV-light-driven prebiotic synthesis of iron–sulfur clusters

Iron–sulfur clusters are ancient cofactors that play a fundamental role in metabolism and may have impacted the prebiotic chemistry that led to life. However, it is unclear whether iron–sulfur clusters could have been synthesized on prebiotic Earth. Dissolved iron on early Earth was predominantly in the reduced ferrous state, but ferrous ions alone cannot form polynuclear iron–sulfur clusters. Similarly, free sulfide may not have been readily available. Here we show that UV light drives the synthesis of [2Fe–2S] and [4Fe–4S] clusters through the photooxidation of ferrous ions and the photolysis of organic thiols. Iron–sulfur clusters coordinate to and are stabilized by a wide range of cysteine-containing peptides and the assembly of iron–sulfur cluster-peptide complexes can take place within model protocells in a process that parallels extant pathways. Our experiments suggest that iron–sulfur clusters may have formed easily on early Earth, facilitating the emergence of an iron–sulfur-cluster-dependent metabolism

Gene Position More Strongly Influences Cell-Free Protein Expression from Operons than T7 Transcriptional Promoter Strength

The cell-free transcription-translation of multiple proteins typically exploits genes placed behind strong transcriptional promoters that reside on separate pieces of DNA so that protein levels can be easily controlled by changing DNA template concentration. However, such systems are not amenable to the construction of artificial cells with a synthetic genome. Herein, we evaluated the activity of a series of T7 transcriptional promoters by monitoring the fluorescence arising from a genetically encoded Spinach aptamer. Subsequently the influences of transcriptional promoter strength on fluorescent protein synthesis from one, two, and three gene operons were assessed. It was found that transcriptional promoter strength was more effective at controlling RNA synthesis than protein synthesis in vitro with the PURE system. Conversely, the gene position within the operon strongly influenced protein synthesis but not RNA synthesis

Inexpensive and colorimetric RNA detection at ambient temperature with a cell-free protein synthesis platform

While many proposals of paper-based diagnostics utilize cell-free gene expression systems, these assays oftentimes suffer from the need for temperature cycling and high operational costs, particularly for developing countries. Here, we explore and report the experimental conditions for the colorimetric detection of viral RNA with an in vitro transcription/translation assay that uses crude E. coli extracts at room temperature where the signal amplification is aided by body heat. Clinically-relevant concentrations of RNA (ca. 600 copies/test) were detected from synthetic RNA samples. The activation of cell-free gene expression was achieved using toehold-switch-mediated riboregulatory elements that are specific to RNA sequences. The colorimetric output was generated by the α-complementation of β-galactosidase ω-fragment (LacZω) with cell-free expressed LacZα, using an X-gal analogue as a substrate. The estimated cost of a single reaction is as low as ~ 0.26 euro/test, which may help to facilitate the accessibility of the diagnostic kit in developing countries. With future optimizations and bacterial strain engineering, production costs can be even further brought down, and the test times can be shortened. Graphical Abstract

Intravesicle Isothermal DNA Replication

<p>Abstract</p> <p>Background</p> <p>Bacterial and viral DNA replication was previously reconstituted <it>in vitro </it>from component parts <abbrgrp><abbr bid="B1">1</abbr><abbr bid="B2">2</abbr><abbr bid="B3">3</abbr><abbr bid="B4">4</abbr></abbrgrp>. Significant advances in building minimal cell-like structures also have been made recently <abbrgrp><abbr bid="B5">5</abbr><abbr bid="B6">6</abbr><abbr bid="B7">7</abbr></abbrgrp>. Combining the two approaches would further attempts to build a minimal cell-like structure capable of undergoing evolution by combining membrane encapsulation and genome replication. Towards this end, we attempted to use purified genomic replication protein components from thermophilic bacterial sources to copy strands of DNA isothermally within lipid vesicles.</p> <p>Findings</p> <p>Bacterial replication components (such as helicases and DNA polymerases) are compatible with methods for the generation of lipid vesicles. Encapsulation inside phospholipid vesicles does not inhibit the activity of bacterial DNA genome replication machinery. Further the described system is efficient at isothermally amplifying short segments of DNA within phospholipid vesicles.</p> <p>Conclusions</p> <p>Herein we show that bacterial isothermal DNA replication machinery is functional inside of phospholipid vesicles, suggesting that replicating cellular mimics can be built from purified bacterial components.</p

Cyclophospholipids Increase Protocellular Stability to Metal Ions

Model protocells have long been constructed with fatty acids, because these lipids are prebiotically plausible and can, at least theoretically, support a protocell life cycle. However, fatty acid protocells are stable only within a narrow range of pH and metal ion concentration. This instability is particularly problematic as the early Earth would have had a range of conditions, and life as we know it is completely reliant on metal ions for catalysis and the folding and activity of biological polymers. Here we show that prebiotically plausible monoacyl cyclophospholipids form robust vesicles that survive a broad range of pH and high concentrations of Mg2+, Ca2+, and Na+. Importantly, stability to Mg2+ and Ca2+ is improved by the presence of environmental concentrations of Na+. These results suggest that cyclophospholipids, or lipids with similar characteristics, may have played a central role during the emergence of Darwinian evolution.</p

On RAF Sets and Autocatalytic Cycles in Random Reaction Networks

The emergence of autocatalytic sets of molecules seems to have played an important role in the origin of life context. Although the possibility to reproduce this emergence in laboratory has received considerable attention, this is still far from being achieved. In order to unravel some key properties enabling the emergence of structures potentially able to sustain their own existence and growth, in this work we investigate the probability to observe them in ensembles of random catalytic reaction networks characterized by different structural properties. From the point of view of network topology, an autocatalytic set have been defined either in term of strongly connected components (SCCs) or as reflexively autocatalytic and food-generated sets (RAFs). We observe that the average level of catalysis differently affects the probability to observe a SCC or a RAF, highlighting the existence of a region where the former can be observed, whereas the latter cannot. This parameter also affects the composition of the RAF, which can be further characterized into linear structures, autocatalysis or SCCs. Interestingly, we show that the different network topology (uniform as opposed to power-law catalysis systems) does not have a significantly divergent impact on SCCs and RAFs appearance, whereas the proportion between cleavages and condensations seems instead to play a role. A major factor that limits the probability of RAF appearance and that may explain some of the difficulties encountered in laboratory seems to be the presence of molecules which can accumulate without being substrate or catalyst of any reaction.Comment: pp 113-12

Preparation and in vivo Assessment of Nystatin-Loaded Solid Lipid Nanoparticles for Topical Delivery against Cutaneous Candidiasis

Solid lipid nanoparticles (SLNs) have gained great attention for the topical treatment of skin associated fungal infection as they facilitate the skin penetration of loaded drugs. Our work deals with the preparation of nystatin loaded solid lipid nanoparticles (NystSLNs) using the hot homogenization and ultrasonication method. The prepared NystSLNs were characterized in terms of entrapment efficiency, particle size, zeta potential, transmission electron microscopy, differential scanning calorimetry, rheological behavior and in vitro drug release. A stability study for 6 months was performed. A microbiological study was conducted in male rats infected with Candida albicans, by counting the colonies and examining the histopathological changes induced on the skin of infected rats. The results showed that SLNs dispersions are spherical in shape with particle size ranging from 83.26±11.33 to 955.04±1.09 nm. The entrapment efficiencies are ranging from 19.73±1.21 to 72.46±0.66% with zeta potential ranging from -18.9 to -38.8 mV and shear-thinning rheological Behavior. The stability studies done for 6 months showed that nystatin (Nyst) is a good candidate for topical SLN formulations. A least number of colony forming unit/ ml (cfu/ml) was recorded for the selected NystSLN compared to the drug solution and the commercial Nystatin® cream present in the market. It can be fulfilled from this work that SLNs provide a good skin targeting effect and may represent promising carrier for topical delivery of Nyst offering the sustained release and maintaining the localized effect, resulting in an effective treatment of cutaneous fungal infection