Does anyone still remember how we perform researches before the emergence of CRISPR-Cas9 genome editing, cryo-EM, and even high-throughput sequencing? In fact, it will not cost much time to recall, because this is still fresh to us. In the past few years, the pace of technological advances in biology research is incredibly fast.
Molecular Cell magazine, belonging to Cell Press’s, has released technology special issue focusing on the development of new technologies and their impact on research and clinical treatment. The content of the entire magazine is used to describe the powerful emerging technical tools, look back on the road we have been through and the future journey we are going to set foot on. There are eleven review articles introducing the mighty technologies in recent years.
High-throughput sequencing technologies
Human genome sequencing technology is revolutionizing our understanding of biology, human diversity, and disease. From the first sequencing sketch to the arrival of the individual genome and the genomics era, almost all of these are due to the rapid development of DNA sequencing technology over the past decade. High-Throughput Sequencing Technologies discusses the development of commonly-used high-throughput sequencing platforms, the associated detection methods as well as the problems with which the current sequencing platform and its clinical application is confronted.
What is particularly noteworthy among these technologies is the early trial result of pocket sequencer—MinION from Oxford Nanopore Company. Some university research teams have published research results by using MinION. For instance, researchers from the University of East Anglia have identified the resistance genes of a kind of multiple drug-resistant bacteria with MinION. This study shows that this technique can be well used for the diagnosis of infectious diseases.
Researchers pointed out that the combination of low-cost but fast nanobase sequencing and short read sequencing can provide a fully-assembled bacterial genome for scientists, and it is believed that public health institutions and clinical laboratories will soon benefit from it. Additionally, after further improvements in the nanopore sequencing system, short read data will no longer be required.
Advances and application of single cell sequencing technology
Single cell sequencing (SCS) is an important tool for studying scarce cells and analyzing complex components. Over the past five years, SCS technology in DNA and RNA aspects has had a wide impact on many areas, including microbiology, neurobiology, developmental biology, tissue inlay, immunology, cancer research, etc. Advances and applications of Single-Cell Sequencing Technologies discusses SCS technology and its application in transformational medicine.
Researchers from the University of California, for example, used the latest technology to analyze the gene activity in a single cell and determined the unique characteristics of cells in the development of the brain. The techniques they used are focused on a “microfluidic” device, in which individual cells are captured and flowed into nanoscale chambers, where they perform the necessary chemical reactions needed by DNA sequencing effectively and accurately. Studies have shown that the number of reading steps for identifying and spelling unique sequences and determining cell types successfully is 100 times less than originally-estimated steps. This technique is developed by Fluidigm Inc., and can be used simultaneously for 96 cells in a separate process.
Single cell RNA sequencing
In sngle-celled organisms and multicellular organisms, differences between individual cell can have important functional effects. The recently-developed single-cell mRNA sequencing method can help scientists analyze these single cells in a non-biased, high-throughput and high-resolution way.
Compared to traditional methods for batch populations of cells, this approach presents more dimensions for transcriptomics. Recently, single cell RNA sequencing technology has revealed important new mechanism of biological tissue, transcription kinetics and gene regulation. The rapid development of cell capture, cell typing, molecular biology and bioinformatics has paved the way for future biology and medical applications.
Recently, EMBL has developed a new technology that can simultaneously detect thousands of genes in cell resolution by using single-cell RNA-seq to amplify the brain gene expression profile of the marine worm (Platynereis dumerilii). This method allows the researcher to match quantitative and spatial data, raising the tag by several orders of magnitude, this tag can display the specific cell type in a tissue. Thus, a broader pattern may begin to emerge.
At the same time, Affymetrix eBioscience also introduced the first technology platform to simultaneously detect single cell levels of RNA and protein expression—PrimeFlow RNA Assay. The main advantage of this technique is that when the performance of flow antibodies of the corresponding protein is poor or the flow antibodies can not be obtained, mRNA can also be detected with high sensitivity. In addition, as people gradually recognize the key function of non-coding RNA, direct determination of noncoding RNA in heterogeneous cell populations will also be a trend. At the meeting of the American Society of Neuroscience, Fluidigm also announced a new process to achieve high-throughput single cell mRNA sequencing. This process combined with significant advances in integrated fluid pathways (IFC) can dramatically increase throughput and ease of use while reducing the cost of single cell preparation.
Designing Cell-Type-Specific Genome-wide Experiments
Multicellular organisms require a variety of special cell types to differentiate to survive. The special developmental processes and different coping styles against environmental challenges of these cells are guided by the same genetic drawings. Thus a key issue in modern biology is to understand how these genes express the normal level at the right place and normal times, and in many diseases this fine gene expression regulation can be disrupted. So it is important to understand these.
Designing Cell-Type-Specific Genome-wide Experiments points out some recently-emerged technologies can use cell-type specific methods to capture gene expression within the whole genome, these new technologies help researchers understand the genetic regulation of the body with unprecedented clarity, give us a deeper understanding of how multicellular organisms adapt to the environment. This article explores how to design a specific cell type experiment through preparing the material, screening the appropriate controls, verifying the data.
Fluorescent protein probe
Advances in optical imaging and genetic engineering research have contributed to the development of new technologies for biological visualization. At present, some genetic coding indicator factors based on fluorescent protein FPs can be used to image the activities of ions, molecules and enzymes. As Qian Yongjian (Roger Tsien) said, “spy in the cell”, these indicators can enter special tissues, cell types or subcellular structures to report their special intracellular activities. Molecular Spies for Bioimaging-Fluorescent Protein-Based Probes summarizes the current single molecule reporter factors, which can convert the conformational changes of proteins into fluorescent signals, and many reporter factors can be used for fluorescence resonance energy transfer and single FP methods. During the same period of last year, a group of researchers from the Polytechnic Institute in Lausanne, Switzerland, developed a new fluorescent molecule that could easily enter living cells, this new kind of molecule is nontoxic and has a lasting signal, and most importantly, it can provide the unprecedented image resolution.
This probe, called as SiR-tubulin and SiR-actin, is used to visualize the cytoskeleton dynamics in human skin cells. Because the optical signal of the probe is emitted with far red light, it is easy to be separated from the background noise, when a super-resolution microscope technique is applied, an unprecedented high-resolution image is produced.
Imaging Live-Cell Dynamics and Structure at the Single-Molecule Level
Observation of living cell molecular processes is an important way to quantify the function of biological systems, especially to solve the complex behavior of cells at the single molecule level can help us understand the dynamics, transport, and self-assembly process.
Over the past decade, the rapid development of fluorescence microscopy, fluorescence correlation spectroscopy and fluorescent labeling allowed us to observe the complexity and randomness of different molecular mechanism at high resolution.
Imaging Live-Cell Dynamics and Structure at the Single-Molecule Level explores new concepts and techniques living cell structure and functional imaging at single molecule levels.
Positioning the ubiquitous signal
Ubiquitous (UB) signaling systems are common in organisms and often interact with other types of post-translational modifications (PTMs), such as phosphorylation. But so far we have little understanding of the dynamics of this signaling pathway and the rate-limiting intermediates.
Quantifying Ubiquitin Signaling reviews how to use quantitative proteomics tools, and enrichment strategies to analyze ubiquitous signal systems in the past, and how to connect this signal pathway with the regulation of phosphorylation events, the PINK1 / PARKIN pathway. At the same time, a key feature of ubiquitination is to find the way for ubiquitous pathway chain to regulate the downstream process. This article also describes how to use proteomics and enzymatic tools to identify and quantify the ubiquitin chain synthesis process as well as the associated tendencies. The article points out that the development of quantitative proteomics will be able to propose a new standard to resolve the ubiquitin signaling pathway biochemical mechanism.
This year, a new study found the new function of ubiquitin. Ubiquitination has another function that is independent of protein degradation and can help cells resist stress. Studies have shown that a special ubiquitination (K63) is capable of modifying and stabilizing ribosomes, which are protein synthesis engine. Researchers have found that preventing yeast from establishing K63 ubiquitin chain will result in the significant reduction of protein production, and the cells are highly sensitive to stress conditions. Researchers are studying K63 ubiquitin chains in yeast, but this kind of ubiquitination also appears in neurons of mice. This also indicates that the new mechanism of ubiquitination is also present in mammals and is likely to be associated with human health.
Cryo-EM: A Unique Tool for the Visualization of Macromolecular Complexity
3D cryo-EM (cryo-EM), although it is an important tool in structural biology research, but its potential has not been yet fully dug out. Recently, the technical resolution gained a leap in development and its application in biological systems is wider and wider. Because this technique does not require crystallization, the amount of required sample is small, and it can be imaged in the computer classification, so cryo-EM can be used to deal with mixture with many complex conformation.
Cryo-EM: A Unique Tool for the Visualization of Macromolecular Complexity introduces the main principle of this technique for single particle analysis and explores the key issues of recent applications. And the article also highlights some of the ongoing new technologies, many of which will be able to achieve the resolution—“Dream Team”, which can not be completed before.
National Institute of Cancer NCI recently has reached the highest resolution (2.2) of Cryo-EM imaging, which only X-ray crystal diffraction achieved before. This can provide people with enough structural information to conduct better drug development.
Defining the Metabolome: Size, Flux and Regulation
Over the past two decades, metabolic studies have contributed to the explosive development of metabolic technology, and the first two metabolomics methods to be noted are “untargeted metabolomics” and “exploratory”. The goal of this technique is to measure the overall metabolism, which involves a large number of undefined molecular groups. Since the full coverage of metabolomics, this should be the first choice for scientists looking for answers to a certain metabolic research question. However, finding non-scalable metabolomics is not necessarily the best experimental choice, and non-scalable metabolomics can only provide information on the relative differences in metabolic pool size in the traditional method. So it still depends on the respective research project, and stable isotope detection method might also be a good choice.
Defining the Metabolome: Size, Flux and Regulation introduces the information of this aspect.
The Cancer Cell Map Initiative: Defining the Hallmark Networks of Cancer
Progress in DNA sequencing technology has helped scientists discover thousands of somatic mutations in the cancer genome, demonstrating the surprising complexity of cancer cells. But it is unclear which are the key drivers involved in the development of cancer, which are passive mutations, and how these mutations affect the pathogenesis and response to treatment. Although similar types of tumors and clinical efficacy may tell us that the patterns of mutations are quite different, it is clear that these mutations do have some of the same molecular pathways and network interactions. Therefore, in order to successfully explain the cancer genome, it is necessary to fully understand the selective stress of the molecular network during the process of carcinogenesis.
The Cancer Cell Map Initiative: Defining the Hallmark Networks of Cancer announces another important step in this research—the Cancer Cell Atlas Project (CCMI), which aims to systematically detail the complex association between oncogenes, and their differences in disease and health. The article also explores recent achievements in this area and its impact on precision medicine.