Towards an Ideal In Cell Hybridization-Based Strategy to Discover Protein Interactomes of Selected RNA Molecules

RNA-binding proteins are crucial to the function of coding and non-coding RNAs. The disruption of RNA–protein interactions is involved in many different pathological states. Several computational and experimental strategies have been developed to identify protein binders of selected RNA molecules. Amongst these, ‘in cell’ hybridization methods represent the gold standard in the field because they are designed to reveal the proteins bound to specific RNAs in a cellular context. Here, we compare the technical features of different ‘in cell’ hybridization approaches with a focus on their advantages, limitations, and current and potential future applications.

Mobility of kinetochore proteins measured by FRAP analysis in living cells

The kinetochore is essential for faithful chromosome segregation during mitosis and is assembled through dynamic processes involving numerous kinetochore proteins. Various experimental strategies have been used to understand kinetochore assembly processes. Fluorescence recovery after photobleaching (FRAP) analysis is also a useful strategy for revealing the dynamics of kinetochore assembly. In this study, we introduced fluorescence protein-tagged kinetochore protein cDNAs into each endogenous locus and performed FRAP analyses in chicken DT40 cells. Centromeric protein (CENP)-C was highly mobile in interphase, but immobile during mitosis. CENP-C mutants lacking the CENP-A-binding domain became mobile during mitosis. In contrast to CENP-C, CENP-T and CENP-H were immobile during both interphase and mitosis. The mobility of Dsn1, which is a component of the Mis12 complex and directly binds to CENP-C, depended on CENP-C mobility during mitosis. Thus, our FRAP assays provide dynamic aspects of how the kinetochore is assembled.

The Fundamental of TCP Techniques

Strategies for prioritizing test cases plan test cases to reduce the cost of retrospective testing and to enhance a specific objective function. Test cases are prioritized as those most important test cases under certain conditions are made before the re-examination process. There are many strategies available in the literature that focus on achieving various pre-test testing objectives and thus reduce their cost. In addition, inspectors often select a few well-known strategies for prioritizing trial cases. The main reason behind the lack of guidelines for the selection of TCP strategies. Therefore, this part of the study introduces the novel approach to TCP strategic planning using the ambiguous concept to support the effective selection of experimental strategies to prioritize experimental cases. This function is an extension of the already selected selection schemes for the prioritization of probation cases.

Identifying new molecular players in extracellular proteostasis

Proteostasis refers to a delicately tuned balance between the processes of protein synthesis, folding, localization, and the degradation of proteins found inside and outside cells. Our understanding of extracellular proteostasis is rather limited and largely restricted to knowledge of 11 currently established extracellular chaperones (ECs). This review will briefly outline what is known of the established ECs, before moving on to discuss experimental strategies used to identify new members of this growing family, and an examination of a group of putative new ECs identified using one of these approaches. An observation that emerges from an analysis of the expanding number of ECs is that all of these proteins are multifunctional. Strikingly, the armory of activities each possess uniquely suit them as a group to act together at sites of tissue damage, infection, and inflammation to restore homeostasis. Lastly, we highlight outstanding questions to guide future research in this field.

Comparison of Experimental Strategies to Study l-Type Amino Acid Transporter 1 (LAT1) Utilization by Ligands

l-Type amino acid transporter 1 (LAT1), expressed abundantly in the brain and placenta and overexpressed in several cancer cell types, has gained a lot of interest in drug research and development, as it can be utilized for brain-targeted drug delivery, as well as inhibiting the essential amino acid supply to cancer cells. The structure of LAT1 is today very well-known and the interactions of ligands at the binding site of LAT1 can be modeled and explained. However, less is known of LAT1′s life cycle within the cells. Moreover, the functionality of LAT1 can be measured by several different methods, which may vary between the laboratories and make the comparison of the results challenging. In the present study, the usefulness of indirect cis-inhibition methods and direct cellular uptake methods and their variations to interpret the interactions of LAT1-ligands were evaluated. Moreover, this study also highlights the importance of understanding the intracellular kinetics of LAT1-ligands, and how they can affect the regular function of LAT1 in critical tissues, such as the brain. Hence, it is discussed herein how the selected methodology influences the outcome and created knowledge of LAT1-utilizing compounds.

Experimental strategies to achieve efficient targeted knock-in via tandem paired nicking

Tandem paired nicking (TPN) is a method of genome editing that enables precise and relatively efficient targeted knock-in without appreciable restraint by p53-mediated DNA damage response. TPN is initiated by introducing two site-specific nicks on the same DNA strand using Cas9 nickases in such a way that the nicks encompass the knock-in site and are located within a homologous region between a donor DNA and the genome. This nicking design results in the creation of two nicks on the donor DNA and two in the genome, leading to relatively efficient homology-directed recombination between these DNA fragments. In this study, we sought to identify the optimal design of TPN experiments that would improve the efficiency of targeted knock-in, using multiple reporter systems based on exogenous and endogenous genes. We found that efficient targeted knock-in via TPN is supported by the use of 1700–2000-bp donor DNAs, exactly 20-nt-long spacers predicted to be efficient in on-target cleavage, and tandem-paired Cas9 nickases nicking at positions close to each other. These findings will help establish a methodology for efficient and precise targeted knock-in based on TPN, which could broaden the applicability of targeted knock-in to various fields of life science.

Perspectives in the Cell-Based Therapies of Various Aspects of the Spinal Cord Injury-Associated Pathologies: Lessons from the Animal Models

Traumatic injury of the spinal cord (SCI) is a devastating neurological condition often leading to severe dysfunctions, therefore an improvement in clinical treatment for SCI patients is urgently needed. The potential benefits of transplantation of various cell types into the injured spinal cord have been intensively investigated in preclinical SCI models and clinical trials. Despite the many challenges that are still ahead, cell transplantation alone or in combination with other factors, such as artificial matrices, seems to be the most promising perspective. Here, we reviewed recent advances in cell-based experimental strategies supporting or restoring the function of the injured spinal cord with a particular focus on the regenerative mechanisms that could define their clinical translation.

Leveraging Experimental Strategies to Capture Different Dimensions of Microbial Interactions

Microorganisms are a fundamental part of virtually every ecosystem on earth. Understanding how collectively they interact, assemble, and function as communities has become a prevalent topic both in fundamental and applied research. Owing to multiple advances in technology, answering questions at the microbial system or network level is now within our grasp. To map and characterize microbial interaction networks, numerous computational approaches have been developed; however, experimentally validating microbial interactions is no trivial task. Microbial interactions are context-dependent, and their complex nature can result in an array of outcomes, not only in terms of fitness or growth, but also in other relevant functions and phenotypes. Thus, approaches to experimentally capture microbial interactions involve a combination of culture methods and phenotypic or functional characterization methods. Here, through our perspective of food microbiologists, we highlight the breadth of innovative and promising experimental strategies for their potential to capture the different dimensions of microbial interactions and their high-throughput application to answer the question; are microbial interaction patterns or network architecture similar along different contextual scales? We further discuss the experimental approaches used to build various types of networks and study their architecture in the context of cell biology and how they translate at the level of microbial ecosystem.