Enabling inverse metabolic engineering through genomics

Inverse metabolic engineering (IME) is a powerful framework for engineering cellular phenotypes. Progress in this field has been limited by a lack of comprehensive methods for efficiently identifying the genetic basis of relevant phenotypes. Advances in genomics technologies, including DNA microarrays and gene sequencing, have dramatically improved our ability to relate changes in phenotype with associated changes in genotype. When applied in the context of IME, these tools should enable the integration of "evolutionary" and "direct" approaches to engineering cell physiology, which should improve our understanding of the complex interactions affecting the expression, evolution and engineering of traits in natural and industrial hosts.     

Mammalian cells as biopharmaceutical production hosts in the age of omics

Mammalian cells are important hosts for the production of a wide range of biopharmaceuticals due to their ability to produce correctly folded and glycosylated proteins. Compared to microbes and yeast, however, the productivity of mammalian cells is low because of their comparatively slow growth rate, tendency to undergo apoptosis, and low production capacities. While much effort has been invested in the engineering of mammalian cells with superior production characteristics, the success of these approaches has been limited to date. One factor responsible for this lack of success is our limited understanding of the cellular basis for high productivity, and of how discrete mechanisms within a cell contribute to the overall phenotype. Aiming to measure and characterize all cellular components at different functional levels, omics technologies have the potential to improve our understanding of mammalian cell physiology, elucidating new targets for the generation of a superior host cell line. This review provides a comprehensive analysis of recent examples of omics studies in the context of mammalian cells as production hosts, highlighting both the challenges and successes in the application of these powerful technologies.     

Applications of integrative OMICs approaches to gene regulation studies

Background: Functional genomics employs dozens of OMICs technologies to explore the functions of DNA, RNA and protein regulators in gene regulation processes. Despite each of these technologies being powerful tools on their own, like the parable of blind men and an elephant, any one single technology has a limited ability to depict the complex regulatory system. Integrative OMICS approaches have emerged and become an important area in biology and medicine. It provides a precise and effective way to study gene regulations.Results: This article reviews current popular OMICs technologies, OMICs data integration strategies, and bioinformatics tools used for multi-dimensional data integration. We highlight the advantages of these methods, particularly in elucidating molecular basis of biological regulatory mechanisms. Conclusions: To better understand the complexity of biological processes, we need powerful bioinformatics tools to integrate these OMICs data. Integrating multi-dimensional OMICs data will generate novel insights into system-level gene regulations and serves as a foundation for further hypothesis-driven research.     

Plant cell biology in the new millennium: new tools and new insights

The highly regulated structural components of the plant cell form the basis of its function. It is becoming increasingly recognized that cellular components are ordered into regulatory units ranging from the multienzyme complexes that allow metabolic channeling during primary metabolism to the "transducon" complexes of signal transduction elements that allow for the highly efficient transfer of information within the cell. Against this structural background the highly dynamic processes regulating cell function are played out. Recent technological advances in three areas have driven our understanding of the complexities of the structural and functional dynamics of the plant cell. First, microscope and digital camera technology has seen not only improvements in the resolution of the optics and sensitivity of detectors, but also the development of novel microscopy applications such as confocal and multiphoton microscopy. These technologies are allowing cell biologists to image the dynamics of living cells with unparalleled three-dimensional resolution. The second advance has been in the availability of increasingly powerful and affordable computers. The computer control/analysis required for many of the new microscopy techniques was simply unavailable until recently. Third, there have been dramatic advances in the available probes to use with these new microscopy approaches. Thus the plant cell biologist now has available a vast array of fluorescent probes that will report cell parameters as diverse as the pH of the cytosol, the oxygen level in a tissue, or the dynamics of the cytoskeleton. The combination of these new approaches has led to an increasingly detailed picture of how plant cells regulate their activities.     

Flow cytometry and FACS applied to filamentous fungi

Flow cytometry is an automated, laser- or impedance-based, high throughput method that allows very rapid analysis of multiple chemical and physical characteristics of single cells within a cell population. It is an extremely powerful technology that has been used for over four decades with filamentous fungi. Although single cells within a cell population are normally analysed rapidly on a cell-by-cell basis using the technique, flow cytometry can also be used to analyse cell (e.g. spore) aggregates or entire microcolonies. Living or fixed cells can be stained with a wide range of fluorescent reporters to label different cell components or measure different physiological processes. Flow cytometry is also suited for measurements of cell size, interaction, aggregation or shape using non-labelled cells by means of analysing their light scattering characteristics. Fluorescence-activated cell sorting (FACS) is a specialized form of flow cytometry that provides a method for sorting a heterogeneous mixture of cells into two or more containers based upon the fluorescence and/or light scattering properties of each cell. The major advantage of analysing cells by flow cytometry over microscopy is the speed of analysis: thousands of cells can be analysed per second or sorted in minutes. Drawbacks of flow cytometry are that specific cells cannot be followed in time and normally spatial information relating to individual cells is lacking. A big advantage over microscopy is when using FACS, cells with desired characteristics can be sorted for downstream experimentation (e.g. for growth, infection, enzyme production, gene expression assays or ‘omics’ approaches). In this review, we explain the basic concepts of flow cytometry and FACS, define its advantages and disadvantages in comparison with microscopy, and describe the wide range of applications in which these powerful technologies have been used with filamentous fungi.     

Advances in retroviral transduction of hematopoietic stem cells for the gene therapy of atherosclerosis

PURPOSE OF REVIEW: Atherosclerosis is a chronic inflammatory disease that is the primary cause of morbidity and mortality in the developed world. Many studies have shown that macrophages and T-cells play critical roles in multiple aspects of the pathogenesis of the disease. Given that these cells are ultimately derived from bone marrow precursors, the concept of performing gene therapy for atherosclerosis through the retroviral transduction of hematopoietic stem cells has received much attention. This review will highlight recent advances that will help bring this goal closer. RECENT FINDINGS: The clinical application of retroviral gene transfer into hematopoietic stem cells has been hampered, in part, by the absence of vectors that can direct long-lasting, cell-type specific gene expression. In this review we will detail recent developments in the design of novel retroviral and lentiviral vectors that appear to overcome these problems, offering approaches to express therapeutic genes in specific cell-types within atherosclerotic lesions. We will also highlight advances in our understanding of the pathogenesis of atherosclerosis that may offer new gene therapeutic targets. SUMMARY: The use of retroviral transduction of hematopoietic stem cells for treatment of patients with atherosclerosis still remains a long-term goal. However, the recent development of retroviral vectors capable of directing expression to specific cell types within the lesion will allow more targeted therapeutic strategies to be devised. In addition, these vectors will provide powerful experimental tools to further our understanding of the pathogenesis of the disease.     

单细胞转录组高通量测序分析新进展

细胞异质性是生物组织的普遍特征。常规转录组测序(RNA-Seq)技术需要上万个细胞,所测结果实际上是一群细胞基因表达的平均值,所以难以鉴别细胞之间基因表达的异质性。单细胞RNA-Seq技术的分辨率精确至单个细胞,为辨别异质性群体中各种细胞类型的转录组特征提供了有力的工具。近年来单细胞RNA-Seq技术发展迅速,在方法学上包括cDNA扩增方法的多样化、对灵敏度和技术噪声的定量分析、浅覆盖高通量单细胞RNA-Seq方法和原位RNA-Seq技术等;在技术应用方面应用范围从早期胚胎发育扩大到组织器官发育、免疫和肿瘤等多个领域。文章对单细胞RNA-Seq在方法学和技术应用两方面的研究进展进行了详细阐述。     

Life in the fast lane: mammalian disease models in the genomics era

Analyses of the human genome have proven extremely successful in identifying changes that contribute to human disease. Genetically engineered mice provide a powerful tool to analyze these changes, although they are slow and costly and do not always recapitulate human biology. Recent advances in genomic technologies, rodent-modeling approaches, and the production of patient-derived reprogrammed cell lines now provide a plethora of complementary systems to study disease states and test new therapies. Continued evolution and integration of these model systems will be the key to realizing the benefits of the genomic revolution and refining our understanding and treatment of human diseases.     

Emerging frontiers in immuno- and gene therapy for cancer

Background aimsThe field of cell and gene therapy in oncology has moved rapidly since 2017 when the first cell and gene therapies, Kymriah followed by Yescarta, were approved by the Food and Drug Administration in the United States, followed by multiple other countries. Since those approvals, several new products have gone on to receive approval for additional indications. Meanwhile, efforts have been made to target different cancers, improve the logistics of delivery and reduce the cost associated with novel cell and gene therapies. Here, we highlight various cell and gene therapy-related technologies and advances that provide insight into how these new technologies will speed the translation of these therapies into the clinic.ConclusionsIn this review, we provide a broad overview of the current state of cell and gene therapy-based approaches for cancer treatment – discussing various effector cell types and their sources, recent advances in both CAR and non-CAR genetic modifications, and highlighting a few promising approaches for increasing in vivo efficacy and persistence of therapeutic drug products.     

Ex vivo gene transfer and correction for cell-based therapies

Cell-based therapies are fast-growing forms of personalized medicine that make use of the steady advances in stem cell manipulation and gene transfer technologies. In this Review, I highlight the latest developments and the crucial challenges for this field, with an emphasis on haematopoietic stem cell gene therapy, which is taken as a representative example given its advanced clinical translation. New technologies for gene correction and targeted integration promise to overcome some of the main hurdles that have long prevented progress in this field. As these approaches marry with our growing capacity for genetic reprogramming of mammalian cells, they may fulfil the promise of safe and effective therapies for currently untreatable diseases.     

An overview of natural variation studies in the Arabidopsis thaliana circadian clock

Circadian clocks are ubiquitous mechanisms that provide an adaptive advantage by predicting subsequent environmental changes. In the model plant Arabidopsis thaliana (Arabidopsis), our understanding of the complex genetic network among clock components has considerably increased during these past years. Modeling has predicted the possibility of additional component to systematically and functionally complete the clock system. Mutagenesis screens have in the past been successfully employed to detect such novel components. With the advancement in sequencing technologies and improvements in statistical approaches, the extensive natural variation present in Arabidopsis accessions has emerged as a powerful alternative in functional gene discovery. In this review article, we review the previous efforts in mapping natural alleles affecting various clock parameters and will discuss further potentials of such natural-variation studies in physiological and ecological contexts.     

Tracking stem cell migration and survival in brain injury: Current approaches and future prospects

In recent years, stem cell-mediated therapies have gained considerable ground as potential treatments for a wide variety of brain pathologies including traumatic brain injury, stroke and neurodegenerative diseases. Despite extensive preclinical studies, many of these therapies have not been fully translated into viable clinical approaches. This is partly due to our inability to reliably track and monitor transplanted stem cells longitudinally over long periods of time in vivo. In this review, we discuss the predominant histological cell tracing methodologies, such as immunohistochemistry, and fluorescent cellular dyes and proteins, and compare them to emerging cellular imaging technologies. We show that advances in magnetic resonance imaging (MRI) have resulted in opportunities to use this technology to further our understanding of stem cell characteristics and behaviors in vivo. While MRI may not completely replace conventional cell tracking methods in pre-clinical, mechanistic work, it is clear that it has the potential to function as a powerful diagnostic tool for tracking stem cell migration and survival as well as for evaluating the efficacy of stem cell-mediated therapies.     

Z/AP, a double reporter for cre-mediated recombination

The Cre/loxP site-specific recombination system combined with embryonic stem cell-mediated technologies has greatly expanded our capability to address normal and disease development in mammals using genetic approaches. The success of this emerging technology hinges on the production of Cre-expressing transgenic lines that provide cell type-, tissue-, or developmental stage-specific recombination between loxP sites placed in the genome. Here we describe and characterize the production of a double-reporter mouse line that provides a convenient and reliable readout of Cre recombinase activity. Throughout all embryonic and adult stages, the transgenic animal expresses the lacZ reporter gene before Cre-mediated excision occurs. Cre excision, however, removes the lacZ gene, allowing expression of the second reporter, the human alkaline phosphatase gene. This double-reporter transgenic line is able to indicate the occurrence of Cre excision in an extremely widespread manner from early embryonic to adult lineages. It will be a valuable reagent for the increasing number of investigators taking advantage of the powerful tools provided by the Cre/loxP site-specific recombinase system.