Recording gene expression order in DNA by CRISPR addition of retron barcodes.

Biological processes depend on the differential expression of genes over time, but methods to make physical recordings of these processes are limited. Here we report a molecular system for making time-ordered recordings of transcriptional events into living genomes. We do this through engineered RNA barcodes, based on prokaryotic retrons1, that are reverse transcribed into DNA and integrated into the genome using the CRISPR-Cas system2. The unidirectional integration of barcodes by CRISPR integrases enables reconstruction of transcriptional event timing based on a physical record through simple, logical rules rather than relying on pretrained classifiers or post hoc inferential methods. For disambiguation in the field, we will refer to this system as a Retro-Cascorder.

High-throughput functional variant screens via in vivo production of single-stranded DNA.

Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. RLR simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing. We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. We also perform RLR using the sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for causal variants, demonstrating that RLR is uniquely suited to utilize large pools of natural variation. Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome.

Fiber Optic Train Monitoring with Distributed Acoustic Sensing: Conventional and Neural Network Data Analysis.

Distributed acoustic sensing (DAS) over tens of kilometers of fiber optic cables is well-suited for monitoring extended railway infrastructures. As DAS produces large, noisy datasets, it is important to optimize algorithms for precise tracking of train position, speed, and the number of train cars. The purpose of this study is to compare different data analysis strategies and the resulting parameter uncertainties. We present data of an ICE 4 train of the Deutsche Bahn AG, which was recorded with a commercial DAS system. We localize the train signal in the data either along the temporal or spatial direction, and a similar velocity standard deviation of less than 5 km/h for a train moving at 160 km/h is found for both analysis methods. The data can be further enhanced by peak finding as well as faster and more flexible neural network algorithms. Then, individual noise peaks due to bogie clusters become visible and individual train cars can be counted. From the time between bogie signals, the velocity can also be determined with a lower standard deviation of 0.8 km/h. The analysis methods presented here will help to establish routines for near real-time train tracking and train integrity analysis.

Reduced Incidence of Intraoperative Femur Fracture With a Second-Generation Tapered Wedge Stem.

BACKGROUND: Intraoperative fractures during total hip arthroplasty (THA) are more common when using cementless stems. The purpose of this study was to investigate the impact of a new shorter second-generation cementless, tapered wedge stem with improved proximal femoral fit in reducing the incidence of intraoperative fracture. METHODS: A retrospective study was conducted on primary THA cases performed at a single institution using a first-generation or second-generation cementless stem from 2006-2016. All intraoperative femur fractures were identified, as well as early 30-day postoperative periprosthetic femur fractures, which could represent nondisplaced intraoperative fractures that were initially missed. Risk for intraoperative femur fracture was analyzed using logistic regression, accounting for demographic covariates and surgeon. RESULTS: Of 6473 primary THA performed with a cementless, tapered wedge stem during the study period, 3126 used a first-generation stem and 3347 used a second-generation stem. The incidence of intraoperative fracture was 1.79% for first-generation stems and 0.24% for second-generation stems, representing a 7.5-fold reduction of risk for fracture. After accounting for covariates, the odds of intraoperative fracture were 0.33 using the second-generation stem relative to the first-generation stem (P = .01). However, there was no significant difference in the odds of early 30-day postoperative fractures using the second-generation stem (odds ratio 0.93, P = .56). CONCLUSION: A new second-generation cementless stem resulted in a 7.5-fold decrease in the incidence of intraoperative femur fracture compared with the preceding stem.

Thanatin confers partial resistance against aflatoxigenic fungi in maize (Zea mays).

Aflatoxin-producing fungi can contaminate plants and plant-derived products with carcinogenic secondary metabolites that present a risk to human and animal health. In this study, we investigated the effect of antimicrobial peptides on the major aflatoxigenic fungi Aspergillus flavus and A. parasiticus. In vitro assays with different chemically-synthesized peptides demonstrated that the broad-spectrum peptide thanatin from the spined soldier bug (Podisus maculiventris) had the greatest potential to eliminate aflatoxigenic fungi. The minimal inhibitory concentrations of thanatin against A. flavus and A. parasiticus were 3.13 and 12.5 µM, respectively. A thanatin cDNA was subsequently cloned in a plant expression vector under the control of the ubiquitin-1 promoter allowing the recombinant peptide to be directed to the apoplast in transgenic maize plants. Successful integration of the thanatin expression cassette was confirmed by PCR and expression was demonstrated by semi-quantitative RT-PCR in transgenic maize kernels. Infection assays with maize kernels from T1 transgenic plants showed up to three-fold greater resistance against Aspergillus spp. infections compared to non-transgenic kernels. We demonstrated for the first time that heterologous expression of the antimicrobial peptide thanatin inhibits the growth of Aspergillus spp. in transgenic maize plants offering a solution to protect crops from aflatoxin-producing fungi and the resulting aflatoxin contamination in the field and under storage conditions.

Genome streamlining to improve performance of a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973.

Cyanobacteria are photosynthetic organisms that have garnered significant recognition as potential hosts for sustainable bioproduction. However, their complex regulatory networks pose significant challenges to major metabolic engineering efforts, thereby limiting their feasibility as production hosts. Genome streamlining has been demonstrated to be a successful approach for improving productivity and fitness in heterotrophs but is yet to be explored to its full potential in phototrophs. Here, we present the systematic reduction of the genome of the cyanobacterium exhibiting the fastest exponential growth, Synechococcus elongatus UTEX 2973. This work, the first of its kind in a photoautotroph, involved an iterative process using state-of-the-art genome-editing technology guided by experimental analysis and computational tools. CRISPR-Cas3 enabled large, progressive deletions of predicted dispensable regions and aided in the identification of essential genes. The large deletions were combined to obtain a strain with 55-kb genome reduction. The strains with streamlined genome showed improvement in growth (up to 23%) and productivity (by 22.7%) as compared to the wild type (WT). This streamlining strategy not only has the potential to develop cyanobacterial strains with improved growth and productivity traits but can also facilitate a better understanding of their genome-to-phenome relationships.IMPORTANCEGenome streamlining is an evolutionary strategy used by natural living systems to dispense unnecessary genes from their genome as a mechanism to adapt and evolve. While this strategy has been successfully borrowed to develop synthetic heterotrophic microbial systems with desired phenotype, it has not been extensively explored in photoautotrophs. Genome streamlining strategy incorporates both computational predictions to identify the dispensable regions and experimental validation using genome-editing tool, and in this study, we have employed a modified strategy with the goal to minimize the genome size to an extent that allows optimal cellular fitness under specified conditions. Our strategy has explored a novel genome-editing tool in photoautotrophs, which, unlike other existing tools, enables large, spontaneous optimal deletions from the genome. Our findings demonstrate the effectiveness of this modified strategy in obtaining strains with streamlined genome, exhibiting improved fitness and productivity.

Light and carbon: Synthetic biology toward new cyanobacteria-based living biomaterials.

Cyanobacteria are ideal candidates to use in developing carbon neutral and carbon negative technologies; they are efficient photosynthesizers and amenable to genetic manipulation. Over the past two decades, researchers have demonstrated that cyanobacteria can make sustainable, useful biomaterials, many of which are engineered living materials. However, we are only beginning to see such technologies applied at an industrial scale. In this review, we explore the ways in which synthetic biology tools enable the development of cyanobacteria-based biomaterials. First we give an overview of the ecological and biogeochemical importance of cyanobacteria and the work that has been done using cyanobacteria to create biomaterials so far. This is followed by a discussion of commonly used cyanobacteria strains and synthetic biology tools that exist to engineer cyanobacteria. Then, three case studies-bioconcrete, biocomposites, and biophotovoltaics-are explored as potential applications of synthetic biology in cyanobacteria-based materials. Finally, challenges and future directions of cyanobacterial biomaterials are discussed.

Antenna Modification in a Fast-Growing Cyanobacterium Synechococcus elongatus UTEX 2973 Leads to Improved Efficiency and Carbon-Neutral Productivity.

Our planet is sustained by sunlight, the primary energy source made accessible to all life forms by photoautotrophs. Photoautotrophs are equipped with light-harvesting complexes (LHCs) that enable efficient capture of solar energy, particularly when light is limiting. However, under high light, LHCs can harvest photons in excess of the utilization capacity of cells, causing photodamage. This damaging effect is most evident when there is a disparity between the amount of light harvested and carbon available. Cells strive to circumvent this problem by dynamically adjusting the antenna structure in response to the changing light signals, a process known to be energetically expensive. Much emphasis has been laid on elucidating the relationship between antenna size and photosynthetic efficiency and identifying strategies to synthetically modify antennae for optimal light capture. Our study is an effort in this direction and investigates the possibility of modifying phycobilisomes, the LHCs present in cyanobacteria, the simplest of photoautotrophs. We systematically truncate the phycobilisomes of Synechococcus elongatus UTEX 2973, a widely studied, fast-growing model cyanobacterium and demonstrate that partial truncation of its antenna can lead to a growth advantage of up to 36% compared to the wild type and an increase in sucrose titer of up to 22%. In contrast, targeted deletion of the linker protein which connects the first phycocyanin rod to the core proved detrimental, indicating that the core alone is not enough, and it is essential to maintain a minimal rod-core structure for efficient light harvest and strain fitness. IMPORTANCE Light energy is essential for the existence of life on this planet, and only photosynthetic organisms, equipped with light-harvesting antenna protein complexes, can capture this energy, making it readily accessible to all other life forms. However, these light-harvesting antennae are not designed to function optimally under extreme high light, a condition which can cause photodamage and significantly reduce photosynthetic productivity. In this study, we attempt to assess the optimal antenna structure for a fast-growing, high-light tolerant photosynthetic microbe with the goal of improving its productivity. Our findings provide concrete evidence that although the antenna complex is essential, antenna modification is a viable strategy to maximize strain performance under controlled growth conditions. This understanding can also be translated into identifying avenues to improve light harvesting efficiency in higher photoautotrophs.

Measuring ectomycorrhizal fungal dispersal: macroecological patterns driven by microscopic propagules.

Dispersal plays a prominent role in most conceptual models of community assembly. However, direct measurement of dispersal across a whole community is difficult at ecologically relevant spatial scales. For cryptic organisms, such as fungi and bacteria, the scale and importance of dispersal limitation has become a major point of debate. We use an experimental island biogeographic approach to measure the effects of dispersal limitation on the ecological dynamics of an important group of plant symbionts, ectomycorrhizal fungi. We manipulated the isolation of uncolonized host seedlings across a natural landscape and used a range of molecular techniques to measure the dispersal rates of ectomycorrhizal propagules and host colonization. Some species were prolific dispersers, producing annual spore loads on the order of trillions of spores per km(2). However, fungal propagules reaching host seedlings decreased rapidly with increasing distance from potential spore sources, causing a concomitant reduction in ectomycorrhizal species richness, host colonization and host biomass. There were also strong differences in dispersal ability across species, which correlated well with the predictable composition of ectomycorrhizal communities associated with establishing pine forest. The use of molecular tools to measure whole community dispersal provides a direct confirmation for a key mechanism underlying island biogeography theory and has the potential to make microbial systems a model for understanding the role of dispersal in ecological theory.

Development of Fast and Portable Frequency Magnetic Mixing-Based Serological SARS-CoV-2-Specific Antibody Detection Assay.

A novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged in China in December 2019, causing an ongoing, rapidly spreading global pandemic. Worldwide, vaccination is now expected to provide containment of the novel virus, resulting in an antibody-mediated immunity. To verify this, serological antibody assays qualitatively as well as quantitatively depicting the amount of generated antibodies are of great importance. Currently available test methods are either laboratory based or do not have the ability to indicate an estimation about the immune response. To overcome this, a novel and rapid serological magnetic immunodetection (MID) point-of-care (PoC) assay was developed, with sensitivity and specificity comparable to laboratory-based DiaSorin Liaison SARS-CoV-2 S1/S2 IgG assay. To specifically enrich human antibodies against SARS-CoV-2 in immunofiltration columns (IFCs) from patient sera, a SARS-CoV-2 S1 antigen was transiently produced in plants, purified and immobilized on the IFC. Then, an IgG-specific secondary antibody could bind to the retained antibodies, which was finally labeled using superparamagnetic nanoparticles. Based on frequency magnetic mixing technology (FMMD), the magnetic particles enriched in IFC were detected using a portable FMMD device. The obtained measurement signal correlates with the amount of SARS-CoV-2-specific antibodies in the sera, which could be demonstrated by titer determination. In this study, a MID-based assay could be developed, giving qualitative as well as semiquantitative results of SARS-CoV-2-specific antibody levels in patient's sera within 21 min of assay time with a sensitivity of 97% and a specificity of 92%, based on the analysis of 170 sera from hospitalized patients that were tested using an Food and Drug Administration (FDA)-certified chemiluminescence assay.

Recombineering and MAGE.

Recombination-mediated genetic engineering, also known as recombineering, is the genomic incorporation of homologous single-stranded or double-stranded DNA into bacterial genomes. Recombineering and its derivative methods have radically improved genome engineering capabilities, perhaps none more so than multiplex automated genome engineering (MAGE). MAGE is representative of a set of highly multiplexed single-stranded DNA-mediated technologies. First described in Escherichia coli, both MAGE and recombineering are being rapidly translated into diverse prokaryotes and even into eukaryotic cells. Together, this modern set of tools offers the promise of radically improving the scope and throughput of experimental biology by providing powerful new methods to ease the genetic manipulation of model and non-model organisms. In this Primer, we describe recombineering and MAGE, their optimal use, their diverse applications and methods for pairing them with other genetic editing tools. We then look forward to the future of genetic engineering.

Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells.

Understanding the evolutionary stability and possible context dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While synthetic auxotrophy previously prevented Escherichia coli from exhibiting detectable escape from batch cultures, its long-term effectiveness is unknown. Here, we report automated continuous evolution of a synthetic auxotroph while supplying a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution, triplicate populations exhibit no observable escape and exhibit normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction reveals the contribution of three genes to increased fitness at low BipA concentrations. Based on its evolutionary stability, we introduce the progenitor strain directly to mammalian cell culture and observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on time scales and in contexts that enable diverse applications.

A monoclonal antibody that specifically binds chitosan in vitro and in situ on fungal cell walls.

We report the generation of the first monoclonal antibody that specifically binds to the polysaccharide chitosan. Mice were immunized with a mixture of chitosans, and hybridoma clones were screened for specific binders resulting in the isolation of a single clone secreting a chitosan-specific IgM, mAbG7. In ELISAs, the antibody can bind to chitosans of varying composition, but demonstrates the highest affinity for chitosans with lower degrees of acetylation (DA) and very poor binding to chitin. We tested the ability of the antibody to bind to chitosan in situ, using preparations of fungal cell walls. Immunofluorescence microscopy confirmed that the antibody bound strongly to the cell walls of fungi with high levels of chitosan, whereas poor staining was observed in those species with cell walls of predominantly chitin or cellulose. The potential use of this antibody for the detection of fungal contamination and the protection of plants against fungal pathogens is discussed.

Monoclonal Antibody AP3 Binds Galactomannan Antigens Displayed by the Pathogens Aspergillus flavus, A. fumigatus, and A. parasiticus.

Aspergillus fumigatus and A. flavus are the fungal pathogens responsible for most cases of invasive aspergillosis (IA). Early detection of the circulating antigen galactomannan (GM) in serum allows the prompt application of effective antifungal therapy, thus improving the survival rate of IA patients. However, the use of monoclonal antibodies (mAbs) for the diagnosis of IA is often associated with false positives due to cross-reaction with bacterial polysaccharides. More specific antibodies are therefore needed. Here we describe the characterization of the Aspergillus-specific mAb AP3 (IgG1κ), including the precise identification of its corresponding antigen. The antibody was generated using A. parasiticus cell wall fragments and was shown to bind several Aspergillus species. Immunofluorescence microscopy revealed that AP3 binds a cell wall antigen, but immunoprecipitation and enzyme-linked immunosorbent assays showed that the antigen is also secreted into the culture medium. The inability of AP3 to bind the A. fumigatus galactofuranose (Galf )-deficient mutant ΔglfA confirmed that Galf residues are part of the epitope. Several lines of evidence strongly indicated that AP3 recognizes the Galf residues of O-linked glycans on Aspergillus proteins. Glycoarray analysis revealed that AP3 recognizes oligo-[ß-D-Galf-1,5] sequences containing four or more residues with longer chains more efficiently. We also showed that AP3 captures GM in serum, suggesting it may be useful as a diagnostic tool for patients with IA.

Method for Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas9.

CRISPR-Cas has proven to be a powerful tool for precision genetic engineering in a variety of difficult genetic systems. In the highly tractable yeast S. cerevisiae, CRISPR-Cas can be used to conduct multiple engineering steps in parallel, allowing for engineering of complex metabolic pathways at multiple genomic loci in as little as 1 week. In addition, CRISPR-Cas can be used to consolidate multiple causal alleles into a single strain, bypassing the laborious traditional methods using marked constructs, or mating. These tools compress the engineering timeline sixfold or more, greatly increasing the productivity of the strain engineer.

Aspergillus-specific antibodies - Targets and applications.

Aspergillus is a fungal genus comprising several hundred species, many of which can damage the health of plants, animals and humans by direct infection and/or due to the production of toxic secondary metabolites known as mycotoxins. Aspergillus-specific antibodies have been generated against polypeptides, polysaccharides and secondary metabolites found in the cell wall or secretions, and these can be used to detect and monitor infections or to quantify mycotoxin contamination in food and feed. However, most Aspergillus-specific antibodies are generated against heterogeneous antigen preparations and the specific target remains unknown. Target identification is important because this can help to characterize fungal morphology, confirm host penetration by opportunistic pathogens, detect specific disease-related biomarkers, identify new candidate targets for antifungal drug design, and qualify antibodies for diagnostic and therapeutic applications. In this review, we discuss how antibodies are raised against heterogeneous Aspergillus antigen preparations and how they can be characterized, focusing on strategies to identify their specific antigens and epitopes. We also discuss the therapeutic, diagnostic and biotechnological applications of Aspergillus-specific antibodies.

High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR-Cas9 in yeast.

Construction and characterization of large genetic variant libraries is essential for understanding genome function, but remains challenging. Here, we introduce a Cas9-based approach for generating pools of mutants with defined genetic alterations (deletions, substitutions, and insertions) with an efficiency of 80-100% in yeast, along with methods for tracking their fitness en masse. We demonstrate the utility of our approach by characterizing the DNA helicase SGS1 with small tiling deletion mutants that span the length of the protein and a series of point mutations against highly conserved residues in the protein. In addition, we created a genome-wide library targeting 315 poorly characterized small open reading frames (smORFs, <100 amino acids in length) scattered throughout the yeast genome, and assessed which are vital for growth under various environmental conditions. Our strategy allows fundamental biological questions to be investigated in a high-throughput manner with precision.

Galantamine improves cognition in schizophrenic patients stabilized on risperidone.

BACKGROUND: Cognition in schizophrenia is impaired in a variety of cognitive domains. Galantamine, a cholinesterase inhibitor with putative nicotinic agonist-like effects, improves cognition in Alzheimer's patients. METHODS: Sixteen schizophrenic or schizoaffective patients stabilized on risperidone were administered galantamine (n=8) or placebo (n=8) in a randomized, double-blind trial. The Repeatable Battery for Assessment of Neuropsychological Status (RBANS) assessed changes in cognitive performance over an eight-week treatment interval. RESULTS: Clinical symptoms improved in both groups during the trial with no evidence that galantamine exacerbated extrapyramidal symptoms. Patients treated with galantamine experienced an overall improvement in cognitive performance (RBANS Total scale score; galantamine = 12.1 +/- 12.8 SD, placebo = .5 +/- 13.5, t = 2.32, p < .04). Confidence intervals suggest that RBANS Attention and Delayed Memory subscale performance was robustly improved in galantamine patients by approximately one standard deviation, effectively normalizing cognitive performance in these domains. CONCLUSIONS: Adjunctive treatment with galantamine improves memory and attention in patients with schizophrenia who are stabilized on risperidone, providing the opportunity to improve functional outcome in these patients.

Genotype Specification Language.

We describe here the Genotype Specification Language (GSL), a language that facilitates the rapid design of large and complex DNA constructs used to engineer genomes. The GSL compiler implements a high-level language based on traditional genetic notation, as well as a set of low-level DNA manipulation primitives. The language allows facile incorporation of parts from a library of cloned DNA constructs and from the "natural" library of parts in fully sequenced and annotated genomes. GSL was designed to engage genetic engineers in their native language while providing a framework for higher level abstract tooling. To this end we define four language levels, Level 0 (literal DNA sequence) through Level 3, with increasing abstraction of part selection and construction paths. GSL targets an intermediate language based on DNA slices that translates efficiently into a wide range of final output formats, such as FASTA and GenBank, and includes formats that specify instructions and materials such as oligonucleotide primers to allow the physical construction of the GSL designs by individual strain engineers or an automated DNA assembly core facility.

Context-specific interactions between Notch and ALK1 cannot explain ALK1-associated arteriovenous malformations.

AIMS: Notch and activin receptor-like kinase 1 (ALK1) have been implicated in arterial specification, angiogenic tip/stalk cell differentiation, and development of arteriovenous malformations (AVMs), and ALK1 can cooperate with Notch to up-regulate expression of Notch target genes in cultured endothelial cells. These findings suggest that Notch and ALK1 might collaboratively program arterial identity and prevent AVMs. We therefore sought to investigate the interaction between Notch and Alk1 signalling in the developing vertebrate vasculature. METHODS AND RESULTS: We modulated Notch and Alk1 activities in zebrafish embryos and examined effects on Notch target gene expression and vascular morphology. Although Alk1 is not necessary for expression of Notch target genes in arterial endothelium, loss of Notch signalling unmasks a role for Alk1 in supporting hey2 and ephrinb2a expression in the dorsal aorta. In contrast, Notch and Alk1 play opposing roles in hey2 expression in cranial arteries and dll4 expression in all arterial endothelium, with Notch inducing and Alk1 repressing these genes. Although alk1 loss increases expression of dll4, AVMs in alk1 mutants could neither be phenocopied by Notch activation nor rescued by Dll4/Notch inhibition. CONCLUSION: Control of Notch targets in arterial endothelium is context-dependent, with gene-specific and region-specific requirements for Notch and Alk1. Alk1 is not required for arterial identity, and perturbations in Notch signalling cannot account for alk1 mutant-associated AVMs. These data suggest that AVMs associated with ALK1 mutation are not caused by defective arterial specification or altered Notch signalling.