Biofoundry-Scale DNA Assembly Validation Using Cost-Effective High-Throughput Long-Read Sequencing.

Biofoundries are automated high-throughput facilities specializing in the design, construction, and testing of engineered/synthetic DNA constructs (plasmids), often from genetic parts. A critical step of this process is assessing the fidelity of the assembled DNA construct to the desired design. Current methods utilized for this purpose are restriction digest or PCR followed by fragment analysis and sequencing. The Edinburgh Genome Foundry (EGF) has recently established a single-molecule sequencing quality control step using the Oxford Nanopore sequencing technology, along with a companion Nextflow pipeline and a Python package, to perform in-depth analysis and generate a detailed report. Our software enables researchers working with plasmids, including biofoundry scientists, to rapidly analyze and interpret sequencing data. In conclusion, we have created a laboratory and software protocol that validates assembled, cloned, or edited plasmids, using Nanopore long-reads, which can serve as a useful resource for the genetics, synthetic biology, and sequencing communities.

Standardization of Synthetic Biology Tools and Assembly Methods for Saccharomyces cerevisiae and Emerging Yeast Species.

As redesigning organisms using engineering principles is one of the purposes of synthetic biology (SynBio), the standardization of experimental methods and DNA parts is becoming increasingly a necessity. The synthetic biology community focusing on the engineering of Saccharomyces cerevisiae has been in the foreground in this area, conceiving several well-characterized SynBio toolkits widely adopted by the community. In this review, the molecular methods and toolkits developed for S. cerevisiae are discussed in terms of their contributions to the required standardization efforts. In addition, the toolkits designed for emerging nonconventional yeast species including Yarrowia lipolytica, Komagataella phaffii, and Kluyveromyces marxianus are also reviewed. Without a doubt, the characterized DNA parts combined with the standardized assembly strategies highlighted in these toolkits have greatly contributed to the rapid development of many metabolic engineering and diagnostics applications among others. Despite the growing capacity in deploying synthetic biology for common yeast genome engineering works, the yeast community has a long journey to go to exploit it in more sophisticated and delicate applications like bioautomation.

Human anogenital monocyte-derived dendritic cells and langerin+cDC2 are major HIV target cells.

Tissue mononuclear phagocytes (MNP) are specialised in pathogen detection and antigen presentation. As such they deliver HIV to its primary target cells; CD4 T cells. Most MNP HIV transmission studies have focused on epithelial MNPs. However, as mucosal trauma and inflammation are now known to be strongly associated with HIV transmission, here we examine the role of sub-epithelial MNPs which are present in a diverse array of subsets. We show that HIV can penetrate the epithelial surface to interact with sub-epithelial resident MNPs in anogenital explants and define the full array of subsets that are present in the human anogenital and colorectal tissues that HIV may encounter during sexual transmission. In doing so we identify two subsets that preferentially take up HIV, become infected and transmit the virus to CD4 T cells; CD14+CD1c+ monocyte-derived dendritic cells and langerin-expressing conventional dendritic cells 2 (cDC2).

Developmental cell programs are co-opted in inflammatory skin disease.

The skin confers biophysical and immunological protection through a complex cellular network established early in embryonic development. We profiled the transcriptomes of more than 500,000 single cells from developing human fetal skin, healthy adult skin, and adult skin with atopic dermatitis and psoriasis. We leveraged these datasets to compare cell states across development, homeostasis, and disease. Our analysis revealed an enrichment of innate immune cells in skin during the first trimester and clonal expansion of disease-associated lymphocytes in atopic dermatitis and psoriasis. We uncovered and validated in situ a reemergence of prenatal vascular endothelial cell and macrophage cellular programs in atopic dermatitis and psoriasis lesional skin. These data illustrate the dynamism of cutaneous immunity and provide opportunities for targeting pathological developmental programs in inflammatory skin diseases.

Decoding human fetal liver haematopoiesis.

Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells and multipotent progenitors (HSC/MPPs) but remains poorly defined in humans. Here, using single-cell transcriptome profiling of approximately 140,000 liver and 74,000 skin, kidney and yolk sac cells, we identify the repertoire of human blood and immune cells during development. We infer differentiation trajectories from HSC/MPPs and evaluate the influence of the tissue microenvironment on blood and immune cell development. We reveal physiological erythropoiesis in fetal skin and the presence of mast cells, natural killer and innate lymphoid cell precursors in the yolk sac. We demonstrate a shift in the haemopoietic composition of fetal liver during gestation away from being predominantly erythroid, accompanied by a parallel change in differentiation potential of HSC/MPPs, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a reference for harnessing the therapeutic potential of HSC/MPPs.

Gene expression variability across cells and species shapes innate immunity.

As the first line of defence against pathogens, cells mount an innate immune response, which varies widely from cell to cell. The response must be potent but carefully controlled to avoid self-damage. How these constraints have shaped the evolution of innate immunity remains poorly understood. Here we characterize the innate immune response's transcriptional divergence between species and variability in expression among cells. Using bulk and single-cell transcriptomics in fibroblasts and mononuclear phagocytes from different species, challenged with immune stimuli, we map the architecture of the innate immune response. Transcriptionally diverging genes, including those that encode cytokines and chemokines, vary across cells and have distinct promoter structures. Conversely, genes that are involved in the regulation of this response, such as those that encode transcription factors and kinases, are conserved between species and display low cell-to-cell variability in expression. We suggest that this expression pattern, which is observed across species and conditions, has evolved as a mechanism for fine-tuned regulation to achieve an effective but balanced response.

The impact of single-cell RNA sequencing on understanding the functional organization of the immune system.

Application of single-cell genomics technologies has revolutionized our approach to study the immune system. Unravelling the functional diversity of immune cells and their coordinated response is key to understanding immunity. Single-cell transcriptomics technologies provide high-dimensional assessment of the transcriptional states of immune cells and have been successfully applied to discover new immune cell types, reveal haematopoietic lineages, identify gene modules dictating immune responses and investigate lymphocyte antigen receptor diversity. In this review, we discuss the impact and applications of single-cell RNA sequencing technologies in immunology.

MicroRNA profiling of the bovine alveolar macrophage response to Mycobacterium bovis infection suggests pathogen survival is enhanced by microRNA regulation of endocytosis and lysosome trafficking.

Mycobacterium bovis, the causative agent of bovine tuberculosis, a major problem for global agriculture, spreads via an airborne route and is taken up by alveolar macrophages (AM) in the lung. Here, we describe the first next-generation sequencing (RNA-seq) approach to temporally profile miRNA expression in primary bovine AMs post-infection with M. bovis. One, six, and forty miRNAs were identified as significantly differentially expressed at 2, 24 and 48 h post-infection, respectively. The differential expression of three miRNAs (bta-miR-142-5p, bta-miR-146a, and bta-miR-423-3p) was confirmed by RT-qPCR. Pathway analysis of the predicted mRNA targets of differentially expressed miRNAs suggests that these miRNAs preferentially target several pathways that are functionally relevant for mycobacterial pathogenesis, including endocytosis and lysosome trafficking, IL-1 signalling and the TGF-ß pathway. Over-expression studies using a bovine macrophage cell-line (Bomac) reveal the targeting of two key genes in the innate immune response to M. bovis, IL-1 receptor-associated kinase 1 (IRAK1) and TGF-ß receptor 2 (TGFBR2), by miR-146. Taken together, our study suggests that miRNAs play a key role in tuning the complex interplay between M. bovis survival strategies and the host immune response.

The Role of microRNAs in Bovine Infection and Immunity.

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that are recognized as critical regulators of immune gene expression during infection. Many immunologically significant human miRNAs have been found to be conserved in agriculturally important species, including cattle. Discovering how bovine miRNAs mediate the immune defense during infection is critical to understanding the etiology of the most prevalent bovine diseases. Here, we review current knowledge of miRNAs in the bovine genome, and discuss the advances in understanding of miRNAs as regulators of immune cell function, and bovine immune response activation, regulation, and resolution. Finally, we consider the future perspectives on miRNAs in bovine viral disease, their role as potential biomarkers and in therapy.

Profiling microRNA expression in bovine alveolar macrophages using RNA-seq.

MicroRNAs (miRNAs) are important regulators of gene expression and are known to play a key role in regulating both adaptive and innate immunity. Bovine alveolar macrophages (BAMs) help maintain lung homeostasis and constitute the front line of host defense against several infectious respiratory diseases, such as bovine tuberculosis. Little is known, however, about the role miRNAs play in these cells. In this study, we used a high-throughput sequencing approach, RNA-seq, to determine the expression levels of known and novel miRNAs in unchallenged BAMs isolated from lung lavages of eight different healthy Holstein-Friesian male calves. Approximately 80 million sequence reads were generated from eight BAM miRNA Illumina sequencing libraries, and 80 miRNAs were identified as being expressed in BAMs at a threshold of at least 100 reads per million (RPM). The expression levels of miRNAs varied over a large dynamic range, with a few miRNAs expressed at very high levels (up to 800,000RPM), and the majority lowly expressed. Notably, many of the most highly expressed miRNAs in BAMs have known roles in regulating immunity in other species (e.g. bta-let-7i, bta-miR-21, bta-miR-27, bta-miR-99b, bta-miR-146, bta-miR-147, bta-miR-155 and bta-miR-223). The most highly expressed miRNA in BAMs was miR-21, which has been shown to regulate the expression of antimicrobial peptides in Mycobacterium leprae-infected human monocytes. Furthermore, the predicted target genes of BAM-expressed miRNAs were found to be statistically enriched for roles in innate immunity. In addition to profiling the expression of known miRNAs, the RNA-seq data was also analysed to identify potentially novel bovine miRNAs. One putatively novel bovine miRNA was identified. To the best of our knowledge, this is the first RNA-seq study to profile miRNA expression in BAMs and provides an important reference dataset for investigating the regulatory roles miRNAs play in this important immune cell type.

Immune complex signatures of patients with active and inactive SLE revealed by multiplex protein binding analysis on antigen microarrays.

Systemic lupus erythematosus is characterized by dysfunctional clearance of apoptotic debris and the development of pathogenic autoantibodies. While the complement system is also involved in the disease no attempt has been made to generate a comprehensive view of immune complex formation from various autoantigens. We increased the complexity of autoantibody profiles by measuring the binding of two complement proteins, C3 and C4, in addition to two antibody classes, IgG and IgM, to a collection of autoantigens. These complement components covalently bind to those microarray features where antibodies and other serum components induce complement activation. Using this technology, we compared functional serum antibody profiles of control subjects (n = 31) and patients with lupus erythematosus (n = 61) in the active (n = 22) and inactive (n = 39) phase of the disease. Multivariate analysis was applied to identify contributions of binding data on 25 antigens to the discrimination of the study groups. Receiver operating characteristic analysis was used to portray the discriminative property of each measured parameter for each antigen in pairwise group comparisons. Complement C3 and C4 deposition increased on autoantibody targets in spite of the decreased serum complement concentrations, and decreased on other autoantigens, demonstrating the imbalance of complement function in patients with lupus erythematosus. Our observations confirmed previously known markers of disease and showed that C3 and C4 deposition data were at least as powerful as Ig binding data in separating the study groups.

Characterization of factors influencing on-chip complement activation to optimize parallel measurement of antibody and complement proteins on antigen microarrays.

Binding of immunoglobulins and complement fragments to targets of adaptive immune responses can be monitored using collections of arrayed antigens and is used to generate profiles of antibody binding and function. The collection of reliable data on these reactions on a large scale requires the establishment of criteria from sample collection through reaction conditions to normalization strategies. We characterized the detection of IgG, complement C3 and C4 under conditions that better resemble in vivo events than most serological assays and are also relevant for in vitro diagnostic purposes. Immune complex formation was modeled using nitrocellulose-based protein arrays and the effects of factors like anticoagulant use, serum dilution, time and bivalent cation concentrations were assessed. Blood samples from healthy controls (n=24) and patients with systemic autoimmune disease (n=60) were collected and correlations between classical laboratory tests and chip-based reference proteins were evaluated to optimize normalization schemes. Best signal-to-noise ratio and acceptable masking of IgG by complement C3 fragments was achieved at modest, five to ten-fold serum dilutions. C3 binding to captured human IgG was found to correlate best with serum C3 concentrations and C3 activity and is therefore an ideal reference feature for normalization of biological and methodological variations in complement activity and detection.

Progression of lupus-like disease drives the appearance of complement-activating IgG antibodies in MRL/lpr mice.

OBJECTIVES: Nucleic acids are known to induce complement activation, which results in the masking and removal of apoptotic cells exposing nuclear components. Dysregulation of these events is characteristic of SLE, a systemic autoimmune disease characterized by the appearance of ANAs. In this study, we aimed to investigate the relationship between development of ANAs and their effect on complement activation by nucleic acids. METHODS: We used protein array technology to characterize complement activation by murine mAbs and polyclonal antibodies against various forms of nucleic acid. Serum samples from MRL/lpr mice were collected, starting before the onset of the disease till 6 months of age. Binding of IgG and its subclasses to dsDNA, ssDNA, RNA, plasmid DNA and nucleosome complexes was determined, along with C3 fixation. RESULTS: We show that complement C3 binding to various forms of nucleic acid that serve as targets in lupus is absent in normal serum. The addition of dsDNA-specific mAbs to normal serum results in the deposition of complement C3 to nucleic acids. In MRL/lpr mice, IgG antibodies against various nuclear antigens appear with ageing and disease progression. C3 binding to the antigens is somewhat delayed and suggests that accumulation or maturation of pathogenic antibodies is required for inducing C3 binding to ICs containing nucleic acids. CONCLUSIONS: C3 deposition on nuclear antigens, therefore, reflects the state of disease progression in this murine model of SLE.

Detection of complement activation on antigen microarrays generates functional antibody profiles and helps characterization of disease-associated changes of the antibody repertoire.

Humoral immune responses are traditionally characterized by determining the presence and quality of Abs specific for certain Ags. Arraying of large numbers of Ags allows the parallel measurement of Abs, generating patterns called Ab profiles. Functional characterization of these Abs could help draw an even more informative map of an immune response. To generate functional Ab profiles we simultaneously tested not only IgM, IgG, and IgA binding to, but also complement activation by, a panel of endogenous and exogenous Ags printed as microarrays, using normal and autoimmune human sera. We show that complement activation by a particular Ag in a given individual cannot be predicted by the measurement of Ag-specific Abs, despite a general correlation between the amount of Ag-bound Ab and the deposited C3 fragments. This is due to both differences in the isotypes that dominate in the recognition of an Ag and individual variations for a given isotype, resulting in altered complement activation potential. Thus, Ag-specific C3 deposition can be used as an additional parameter in immune response monitoring. This is exemplified by comparing the coordinates of Ags, used for the diagnosis of systemic lupus erythematosus, of normal and autoimmune serum samples in a two-dimensional space derived from C3 deposition and Ab binding. Since cleavage fragments of C3 mediate important immunological processes, we propose that measurement of their deposition on Ag microarrays, in addition to Ab profiling, can provide useful functional signature about the tested serum.

B lymphocytes and macrophages release cell membrane deposited C3-fragments on exosomes with T cell response-enhancing capacity.

Recently exosomes have been shown to play important roles in several immune phenomena. These small vesicles contain MHC proteins along with co-stimulatory and adhesion molecules, and mediate antigen presentation to T cells. In the present study we show that upon incubation with autologous serum, murine macrophages and B cells--but not T lymphocytes--fix C3-fragments covalently to the cell membrane and release them on exosomes in a time dependent fashion. While in the case of human B lymphocytes CR2 has been shown to serve as the main C3b-acceptor site, here we clearly demonstrate that cells derived from CR1/2 KO animals also have the capacity to fix C3b covalently. This finding points to a major difference between human and murine systems, and suggests the existence of additional acceptor sites on the cell membrane. Here we show that C3-fragment containing exosomes derived from OVA loaded antigen presenting cells induce a significantly elevated T cell response in the presence of suboptimal antigen stimulus. These data reveal a novel function of cell surface-deposited C3-fragments and provide further evidence for the role of exosomes secreted by antigen presenting cells. Since fixation of C3b to plasma membranes can be substantial in the presence of pathogens; moreover tumor cells are also known to activate the complement system resulting in complement-deposition, C3-carrying exosomes released by these cells may play an important immunomodulatory role in vivo, as well.

Kinetic energy release of protonated methanol clusters using the low-temperature fast-atom bombardment: experiment and theory combined.

Low-temperature fast-atom bombardment was found to be an excellent method for generating large protonated methanol clusters, (CH(3)OH)(n)H(+) (n = 2 to 15). Metastable dissociations of these clusters, involving elimination of one methanol molecule, were studied using mass-analyzed ion kinetic energy spectra (MIKES). From metastable peak profiles kinetic energy release (KER) distributions were obtained, even for clusters as large as (CH(3)OH)(15)H(+). The results were analyzed by a simple thermal model, by the finite heat bath theory (FHBT) and by the RRKM-based MassKinetics algorithm. The KER distribution was shown to correspond to a three-dimensional translational energy distribution, implying statistical energy partitioning in the transition state. The mean KER values and transition state temperatures were found to increase with cluster size, reaching 25 meV and approximately 210 K for large clusters (n = 10).

Low-temperature SIMS mass spectra of diethyl ether.

For the first time a secondary ion mass spectrum of diethyl ether was obtained at low temperature. The spectrum recording became possible by carefully selecting the range of experimental conditions for the production of a cluster-type spectrum. This range is specified by the threshold for spectrum appearance above the melting temperature of the frozen sample and a fairly short time span of existence of the liquid estimated as only a few minutes. The latter necessitates rather rapid spectrum detection. In practice, about 1 min was available for recording of the cluster-type spectra. The secondary emission mass spectrum of diethyl ether appeared to be rich in peaks: along with abundant protonated clusters M(n).H(+) (n = 1-12), unusually intense [M(n) - H](+) and weaker M(n) (+.) peaks were present accompanied by several sets of fragmented clusters, [M(n) - 15](+), [M(n) - 29](+), [M(n) - 27](+), [M(n) - 45](+), and monohydrates, M(n).H(2)O.H(+). The analysis of all the peaks showed that the pattern of fragment clusters is qualitatively similar to the pattern of fragmentation of the diethyl ether molecular ion under high-energy electron impact. The general features of the behaviour of diethyl ether under low-temperature mass spectrometric conditions were similar to those observed earlier for some other organic solvents.