Mammalian Cell-Free System Recapitulates the Early Events of Post-Fertilization Sperm Mitophagy.

Propagation of paternal sperm-contributed mitochondrial genes, resulting in heteroplasmy, is seldom observed in mammals due to post-fertilization degradation of sperm mitochondria, referred to as sperm mitophagy. Whole organelle sperm mitochondrion degradation is thought to be mediated by the interplay between the ubiquitin-proteasome system (UPS) and the autophagic pathway (Song et al., Proc. Natl. Acad. Sci. USA, 2016). Both porcine and primate post-fertilization sperm mitophagy rely on the ubiquitin-binding autophagy receptor, sequestosome 1 (SQSTM1), and the proteasome-interacting ubiquitinated protein dislocase, valosin-containing protein (VCP). Consequently, we anticipated that sperm mitophagy could be reconstituted in a cell-free system consisting of permeabilized mammalian spermatozoa co-incubated with porcine oocyte extracts. We found that SQSTM1 was detected in the midpiece/mitochondrial sheath of the sperm tail after, but not before, co-incubation with oocyte extracts. VCP was prominent in the sperm mitochondrial sheath both before and after the extract co-incubation and was also detected in the acrosome and postacrosomal sheath and the subacrosomal layer of the spermatozoa co-incubated with extraction buffer as control. Such patterns are consistent with our previous observation of SQSTM1 and VCP associating with sperm mitochondria inside the porcine zygote. In addition, it was observed that sperm head expansion mimicked the early stages of paternal pronucleus development in a zygote during prolonged sperm-oocyte extract co-incubation. Treatment with anti-SQSTM1 antibody during extract co-incubation prevented ooplasmic SQSTM1 binding to sperm mitochondria. Even in an interspecific cellular environment encompassing bull spermatozoa and porcine oocyte extract, ooplasmic SQSTM1 was recruited to heterospecific sperm mitochondria. Complementary with the binding of SQSTM1 and VCP to sperm mitochondria, two sperm-borne pro-mitophagy proteins, parkin co-regulated gene product (PACRG) and spermatogenesis associated 18 (SPATA18), underwent localization changes after extract coincubation, which were consistent with their degradation observed inside fertilized porcine oocytes. These results demonstrate that the early developmental events of post-fertilization sperm mitophagy observed in porcine zygote can be reconstituted in a cell-free system, which could become a useful tool for identifying additional molecules that regulate mitochondrial inheritance in mammals.

A Spider Toxin Exemplifies the Promises and Pitfalls of Cell-Free Protein Production for Venom Biodiscovery.

Arthropod venoms offer a promising resource for the discovery of novel bioactive peptides and proteins, but the limited size of most species translates into minuscule venom yields. Bioactivity studies based on traditional fractionation are therefore challenging, so alternative strategies are needed. Cell-free synthesis based on synthetic gene fragments is one of the most promising emerging technologies, theoretically allowing the rapid, laboratory-scale production of specific venom components, but this approach has yet to be applied in venom biodiscovery. Here, we tested the ability of three commercially available cell-free protein expression systems to produce venom components from small arthropods, using U2-sicaritoxin-Sdo1a from the six-eyed sand spider Hexophtalma dolichocephala as a case study. We found that only one of the systems was able to produce an active product in low amounts, as demonstrated by SDS-PAGE, mass spectrometry, and bioactivity screening on murine neuroblasts. We discuss our findings in relation to the promises and limitations of cell-free synthesis for venom biodiscovery programs in smaller invertebrates.

Microfluidic encapsulation of Xenopus laevis cell-free extracts using hydrogel photolithography.

Cell-free extract derived from the eggs of the African clawed frog Xenopus laevis is a well-established model system that has been used historically in bulk aliquots. Here, we describe a microfluidic approach for isolating discrete, biologically relevant volumes of cell-free extract, with more expansive and precise control of extract shape compared with extract-oil emulsions. This approach is useful for investigating the mechanics of intracellular processes affected by cell geometry or cytoplasmic volume, including organelle scaling and positioning mechanisms. For complete details on the use and execution of this protocol, please refer to Geisterfer et al. (2020).

Compartmentalizing Cell-Free Systems: Toward Creating Life-Like Artificial Cells and Beyond.

Building an artificial cell is a research area that is rigorously studied in the field of synthetic biology. It has brought about much attention with the aim of ultimately constructing a natural cell-like structure. In particular, with the more mature cell-free platforms and various compartmentalization methods becoming available, achieving this aim seems not far away. In this review, we discuss the various types of artificial cells capable of hosting several cellular functions. Different compartmental boundaries and the mature and evolving technologies that are used for compartmentalization are examined, and exciting recent advances that overcome or have the potential to address current challenges are discussed. Ultimately, we show how compartmentalization and cell-free systems have, and will, come together to fulfill the goal to assemble a fully synthetic cell that displays functionality and complexity as advanced as that in nature. The development of such artificial cell systems will offer insight into the fundamental study of evolutionary biology and the sea of applications as a result. Although several challenges remain, emerging technologies such as artificial intelligence also appear to help pave the way to address them and achieve the ultimate goal.

CTELS: A Cell-Free System for the Analysis of Translation Termination Rate.

Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3' UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a "leaky" stop codon context, which likely defines the basis of nonsense suppression.

Development and Characterization of an Acellular Porcine Small Intestine Submucosa Scaffold for Use in Corneal Epithelium Tissue Engineering.

Purpose: To produce an acellular small intestine submucosa (SIS) that would be a suitable scaffold for corneal epithelium tissue engineering.Methods: The SIS was decellularized by immersion in 0.1% (wt/vol) sodium dodecyl sulfate (SDS). The efficacy of acellularization was confirmed by histological observation and DNA quantification. The mechanical properties were evaluated by uniaxial tensile testing. ELISA was performed to assess the growth factor contents. The cytotoxicity of SIS scaffolds and extracts to rabbit corneal epithelial cells was determined by CCK-8 assay. We also investigated the inflammatory reaction of SIS implanted subcutaneously in a rat. The biocompatibility was studied by rabbit interlamellar corneal transplantation and reseeding assay with cornea-derived cells. Immunofluorescent staining was used to detect the expression of CK3, ZO-1 and K13.Results: Histological analyses showed that complete cell removal was achieved, and the DNA quantity, which reflects the presence of cellular materials, was significantly diminished in acellular SIS. Collagen fibers were properly preserved and appeared in an orderly fashion. The tissue structure, the mechanical properties and the growth factor contents within the acellular SIS were well retained. The CCK8 assay demonstrated that the acellular SIS scaffolds and extracts had no cytotoxicity to rabbit corneal epithelial cells. There was no sign that an immune reaction occurred with acellular SIS implanted subcutaneously in a rat. In fact, in vivo implantation to rabbit interlamellar stromal pockets showed good biocompatibility. We also observed that clusters of rabbit corneal epithelial cells were growing well on the surface of the SIS in vitro and the distinctive CK3, ZO-1 for corneal epithelial cells was detected.Conclusions: The decellularized SIS retained the major structural components. The matrix is biocompatible with cornea-derived cells and might be a suitable scaffold for corneal epithelium tissue engineering.

Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an E. coli Based Cell-Free Protein Synthesis System.

Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from Escherichia coli and Nicotiana tabacum using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and NtPIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.

Biological Analysis of Simvastatin-releasing Chitosan Scaffold as a Cell-free System for Pulp-dentin Regeneration.

INTRODUCTION: The improvement of biomaterials capable of driving the regeneration of the pulp-dentin complex mediated by resident cells is the goal of regenerative dentistry. In the present investigation, a chitosan scaffold (CHSC) that released bioactive concentrations of simvastatin (SIM) was tested, aimed at the development of a cell-free tissue engineering system. METHODS: First, we performed a dose-response assay to select the bioactive dose of SIM capable of inducing an odontoblastic phenotype in dental pulp cells (DPCs); after which we evaluated the synergistic effect of this dosage with the CHSC/DPC construct. SIM at 1.0 µmol/L (CHSC-SIM1.0) and 0.5 µmol/L were incorporated into the CHSC, and cell viability, adhesion, and calcium deposition were evaluated. Finally, we assessed the biomaterials in an artificial pulp chamber/3-dimensional culture model to simulate the cell-free approach in vitro. RESULTS: SIM at 0.1 µmol/L was selected as the bioactive dose. This drug was capable of strongly inducing an odontoblastic phenotype on the DPC/CHSC construct. The incorporation of SIM into CHSC had no deleterious effect on cell viability and adhesion to the scaffold structure. CHSC-SIM1.0 led to significantly higher calcium-rich matrix deposition on scaffold/dentin disc assay compared with the control (CHSC). This biomaterial induced the migration of DPCs from a 3-dimensional culture to its surface as well as stimulated significantly higher expressions of alkaline phosphatase, collagen type 1 alpha 1, dentin matrix acidic phosphoprotein 1, and dentin sialophosphoprotein on 3-dimensional-cultured DPCs than on those in contact with CHSC. CONCLUSIONS: CHSC-SIM1.0 scaffold was capable of increasing the chemotaxis and regenerative potential of DPCs.

Short DNA containing χ sites enhances DNA stability and gene expression in E. coli cell-free transcription-translation systems.

Escherichia coli cell-free transcription-translation (TXTL) systems offer versatile platforms for advanced biomanufacturing and for prototyping synthetic biological parts and devices. Production and testing could be accelerated with the use of linear DNA, which can be rapidly and cheaply synthesized. However, linear DNA is efficiently degraded in TXTL preparations from E. coli. Here, we show that double-stranded DNA encoding χ sites-eight base-pair sequences preferentially bound by the RecBCD recombination machinery-stabilizes linear DNA and greatly enhances the TXTL-based expression and activity of a fluorescent reporter gene, simple regulatory cascades, and T7 bacteriophage particles. The χ-site DNA and the DNA-binding λ protein Gam yielded similar enhancements, and DNA with as few as four χ sites was sufficient to ensure robust gene expression in TXTL. Given the affordability and scalability of producing the short χ-site DNA, this generalized strategy is expected to advance the broad use of TXTL systems across its many applications. Biotechnol. Bioeng. 2017;114: 2137-2141. © 2017 Wiley Periodicals, Inc.

Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements.

Cell-free transcription-translation systems were originally applied towards in vitro protein production. More recently, synthetic biology is enabling these systems to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and biosynthetic pathways. The Gram-positive soil bacterium, Bacillus subtilis, is an established model organism of industrial importance. To this end, we developed several B. subtilis-based cell-free systems. Our improved B. subtilis WB800N-based system was capable of producing 0.8µM GFP, which gave a ~72x fold-improvement when compared with a B. subtilis 168 cell-free system. Our improved system was applied towards the prototyping of a B. subtilis promoter library in which we engineered several promoters, derived from the wild-type Pgrac (σA) promoter, that display a range of comparable in vitro and in vivo transcriptional activities. Additionally, we demonstrate the cell-free characterisation of an inducible expression system, and the activity of a model enzyme - renilla luciferase.

Hybrid agent-based model for quantitative in-silico cell-free protein synthesis.

An advanced vision of the mRNA translation is presented through a hybrid modeling approach. The dynamics of the polysome formation was investigated by computer simulation that combined agent-based model and fine-grained Markov chain representation of the chemical kinetics. This approach allowed for the investigation of the polysome dynamics under non-steady-state and non-continuum conditions. The model is validated by the quantitative comparison of the simulation results and Luciferase protein production in cell-free system, as well as by testing of the hypothesis regarding the two possible mechanisms of the Edeine antibiotic. Calculation of the Hurst exponent demonstrated a relationship between the microscopic properties of amino acid elongation and the fractal dimension of the translation duration time series. The temporal properties of the amino acid elongation have indicated an anti-persistent behavior under low mRNA occupancy and evinced the appearance of long range interactions within the mRNA-ribosome system for high ribosome density. The dynamic and temporal characteristics of the polysomal system presented here can have a direct impact on the studies of the co-translation protein folding and provide a validated platform for cell-free system studies.

Single-molecule, antibody-free fluorescent visualisation of replication tracts along barcoded DNA molecules.

DNA combing is a standard technique to map DNA replication at the single molecule level. Typically, replicating DNA is metabolically labelled with nucleoside or nucleotide analogs, purified, stretched on coverslips and treated with fluorescent antibodies to reveal tracts of newly synthesized DNA. Fibres containing a locus of interest can then be identified by fluorescent in situ hybridization (FISH) with DNA probes. These steps are complex and the throughput is low. Here, we describe a simpler, antibody-free method to reveal replication tracts and identify the locus of origin of combed DNA replication intermediates. DNA was replicated in Xenopus egg extracts in the presence of a fluorescent dUTP. Purified DNA was barcoded by nicking with Nt.BspQI, a site-specific nicking endonuclease (NE), followed by limited nick-translation in the presence of another fluorescent dUTP. DNA was then stained with YOYO-1, a fluorescent DNA intercalator, and combed. Direct epifluorescence revealed the DNA molecules, their replication tracts and their Nt.BspQI sites in three distinct colours. Replication intermediates could thus be aligned to a reference genome map. In addition, replicated DNA segments showed a stronger YOYO-1 fluorescence than unreplicated segments. The entire length, replication tracts, and NE sites of combed DNA molecules can be simultaneously visualized in three distinct colours by standard epifluorescence microscopy, with no need for antibody staining and/or FISH detection. Furthermore, replication bubbles can be detected by quantitative YOYO-1 staining, eliminating the need for metabolic labelling. These results provide a starting point for genome-wide, single-molecule mapping of DNA replication in any organism.

Circulating Cell-Free DNA Differentiates Severity of Inflammation.

INTRODUCTION: As the U.S. population ages, the incidence of chronic disease will rise. Chronic diseases have been linked to chronic inflammation. The purpose of this review is to summarize the literature on cell-free DNA (cfDNA) in relation to inflammation. METHODS: PubMed, EMBASE, and Web of Science were searched. Inclusion criteria were noninterventional studies on acute and chronic inflammation, autoimmunity, and infection published in English after 2000, conducted in humans using the fluorescence method of quantifying DNA. Of the 442 articles retrieved, 83 were identified for full-text review and 13 remained after application of inclusion criteria. RESULTS: Of the reviewed studies, three involved acute inflammation, six involved chronic inflammation, and four involved infection. Healthy controls with interpretable results were included in six studies, three of which used the Quant-iT high-sensitivity DNA kit and found cfDNA quantities near 800 ng/ml, while the other three used other fluorescence methods and found quantities below 100 ng/ml. All 13 studies compared groups, and all but 1 found statistically significant differences between them. Among studies using the Quant-iT reagent, levels were higher in infection than in chronic inflammation. Among studies that used other reagents, levels increased from chronic to acute inflammation to severe infection. CfDNA levels were associated with mortality and with clinical outcomes in acute inflammation and infection. Most studies assessed cfDNA's correlation with other inflammation biomarkers and found inconclusive results. CONCLUSION: There appears to be an association between inflammation and cfDNA. Further research is necessary before cfDNA can be used clinically as a measure of inflammation.

Systems Biocatalysis: Development and engineering of cell-free "artificial metabolisms" for preparative multi-enzymatic synthesis.

Systems Biocatalysis is an emerging concept of organizing enzymes in vitro to construct complex reaction cascades for an efficient, sustainable synthesis of valuable chemical products. The strategy merges the synthetic focus of chemistry with the modular design of biological systems, which is similar to metabolic engineering of cellular production systems but can be realized at a far lower level of complexity from a true reductionist approach. Such operations are free from material erosion by competing metabolic pathways, from kinetic restrictions by physical barriers and regulating circuits, and from toxicity problems with reactive foreign substrates, which are notorious problems in whole-cell systems. A particular advantage of cell-free concepts arises from the inherent opportunity to construct novel biocatalytic reaction systems for the efficient synthesis of non-natural products ("artificial metabolisms") by using enzymes specifically chosen or engineered for non-natural substrate promiscuity. Examples illustrating the technology from our laboratory are discussed.

Preparation of efficient excision repair competent cell-free extracts from C. reinhardtii cells.

Chlamydomonas reinhardtii is a prospective model system for understanding molecular mechanisms associated with DNA repair in plants and algae. To explore this possibility, we have developed an in vitro repair system from C. reinhardtii cell-free extracts that can efficiently repair UVC damage (Thymine-dimers) in the DNA. We observed that excision repair (ER) synthesis based nucleotide incorporation, specifically in UVC damaged supercoiled (SC) DNA, was followed by ligation of nicks. Photoreactivation efficiently competed out the ER in the presence of light. In addition, repair efficiency in cell-free extracts from ER deficient strains was several fold lower than that of wild-type cell extract. Interestingly, the inhibitor profile of repair DNA polymerase involved in C. reinhardtii in vitro ER system was akin to animal rather than plant DNA polymerase. The methodology to prepare repair competent cell-free extracts described in the current study can aid further molecular characterization of ER pathway in C. reinhardtii.

Unreserved application of epigenetic methods to define differences of DNA methylation between urinary cellular and cell-free DNA.

Urinary DNA is increasingly gaining importance in diagnosis of urological malignancies. Especially cell-free DNA originating from apoptotic and necrotic cells of the early tumor could become a key target for early stage tumor diagnosis. Aberrant DNA methylation forms tumor cell characteristic epigenetic profiles which are covalently established before any tumor related aberration at transcriptional or protein level has occurred. In addition, these epigenetic signatures are alterably adapted to and accompanying the individual stages of multistep, progressive tumorigenesis. Hence, they seem very promising for diagnosis as well as for monitoring the patient's follow-up care and even for decisions regarding personalized therapeutic options. The essential prerequisite at this approach will be a reliable methodological handling of the biological material of interest. In this study we present detailed analyses of LINE-1 DNA methylation profiles and demonstrate the sensitive detection of LINE-1 DNA methylation differences as well as between cancer patients and healthy individuals, between urinary cellular and cell-free DNA. In addition, we show methylome differences between both DNA fractions from a healthy individual and bladder cancer patients. In conclusion, we demonstrate here the unrestricted amenability of urinary cell-free DNA for both, a detailed characterization of a distinct DNA methylation alteration and its sensitive detection and a comprehensive global, array-based screening for DNA methylation differences.

Clinical perspective of cell-free DNA testing for fetal aneuploidies.

Cell-free DNA testing in maternal blood provides the most effective method of screening for trisomy 21, with a reported detection rate of 99% and a false positive rate of less than 0.1%. After many years of research, this method is now commercially available and is carried out in an increasing number of patients, and there is an expanding number of conditions that can be screened for. However, the application of these methods in clinical practice requires a careful analysis. Current first-trimester screening strategies are based on a complex combination of tests, aiming at detecting fetal defects and predicting the risk of main pregnancy complications. It is therefore necessary to define the optimal way of combining cell-free DNA testing with current first-trimester screening methods. In this concise review we describe the basis of cell-free DNA testing and discuss the potential approaches for its implementation in combination with current tests in the first trimester.

An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins.

Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies.

Analysis of cell-free DNA in maternal blood in screening for aneuploidies: meta-analysis.

OBJECTIVE: To review clinical validation or implementation studies of maternal blood cell-free (cf) DNA analysis in screening for aneuploidies and to explore the potential use of this method in clinical practice. METHODS: Searches of PubMed and MEDLINE were performed to identify all peer-reviewed articles on cfDNA testing in screening for aneuploidies between 2011, when the first such study was published, and 20 December 2013. RESULTS: Weighted pooled detection rates (DR) and false-positive rates (FPR) in singleton pregnancies were 99.0% (95% CI 98.2­99.6) and 0.08% (95% CI0.03­0.14), respectively, for trisomy 21; 96.8% (95% CI 94.5­98.4) and 0.15% (95% CI 0.08­0.25) for trisomy 18; 92.1% (95% CI 85.9­96.7) and 0.20% (95% CI 0.04­0.46) for trisomy 13; 88.6% (95% CI 83.0­93.1) and 0.12% (95% CI 0.05­0.24) for monosomy X, and 93.8% (95% CI 85.9­98.7) and 0.12% (95% CI 0.02­0.28) for sex chromosome aneuploidies other than monosomy X. For twin pregnancies, the DR was 94.4% (95% 74.2­99.0) and the FPR was 0% (95% CI 0.00­1.84) for trisomy 21. CONCLUSION: An analysis of cfDNA in maternal blood provides effective screening for trisomies.

Replacing the combined test by cell-free DNA testing in screening for trisomies 21, 18 and 13: impact on the diagnosis of other chromosomal abnormalities.

OBJECTIVE: To estimate the proportion of other chromosomal abnormalities that could be missed if combined testing was replaced by cell-free (cf) DNA testing as the method of screening for trisomies 21, 18 and 13. METHODS: The prevalence of trisomies 21, 18 or 13, sex chromosome aneuploidies, triploidy and other chromosomal abnormalities was examined in pregnancies undergoing first-trimester combined screening and chorionic villus sampling (CVS). RESULTS: In 1,831 clinically significant chromosomal abnormalities in pregnancies with combined risk for trisomies 21, 18 and 13≥1:100, the contribution of trisomies 21, 18 or 13, sex chromosome aneuploidies, triploidy and other chromosomal abnormalities at high risk of adverse outcome was 82.9, 8.2, 3.9 and 5.0%, respectively. Combined screening followed by CVS for risk≥1:10 and cfDNA testing for risk 1:11-1:2,500 could detect 97% of trisomy 21 and 98% of trisomies 18 and 13. Additionally, 86% of monosomy X, half of 47,XXY, 47,XYY or 47,XXX, half of other chromosomal abnormalities and one third of triploidies, which are currently detected by combined screening and CVS for risk≥1:100, could be detected. CONCLUSIONS: Screening by cfDNA testing, contingent on results of combined testing, improves detection of trisomies, but misses a few of the other chromosomal abnormalities detected by screening with the combined test.