Basic Biological Research Research Articles

National Effort to Re-Establish Heavy Ion Cancer Therapy in the United States.

In this review, we attempt to make a case for the establishment of a limited number of heavy ion cancer research and treatment facilities in the United States. Based on the basic physics and biology research, conducted largely in Japan and Germany, and early phase clinical trials involving a relatively small number of patients, we believe that heavy ions have a considerably greater potential to enhance the therapeutic ratio for many cancer types compared to conventional X-ray and proton radiotherapy. Moreover, with ongoing technological developments and with research in physical, biological, immunological, and clinical aspects, it is quite plausible that cost effectiveness of radiotherapy with heavier ions can be substantially improved.

Modulation of Local Cellular Activities using a Photothermal Dye-Based Subcellular-Sized Heat Spot.

Thermal engineering at the microscale, such as the regulation and precise evaluation of the temperature within cellular environments, is a major challenge for basic biological research and biomaterials development. We engineered a polymeric nanoparticle having a fluorescent temperature sensory dye and a photothermal dye embedded in the polymer matrix, named nanoheater-thermometer (nanoHT). When nanoHT is illuminated with a near-infrared laser at 808 nm, a subcellular-sized heat spot is generated in a live cell. Fluorescence thermometry allows the temperature increment to be read out concurrently at individual heat spots. Within a few seconds of an increase in temperature by approximately 11.4 °C from the base temperature (37 °C), we observed the death of HeLa cells. The cell death was observed to be triggered from the exact local heat spot at the subcellular level under the fluorescence microscope. Furthermore, we demonstrate the application of nanoHT for the induction of muscle contraction in C2C12 myotubes by heat release. We successfully showed heat-induced contraction to occur in a limited area of a single myotube based on the alteration of protein–protein interactions related to the contraction event. These results demonstrate that even a single heat spot provided by a photothermal material can be extremely effective in altering cellular functions.

Water-soluble improved NIR ratiometric fluorescent probe for specific imaging sulfur dioxide in cancer cell membranes and living mice

The comprehensive understanding of the mechanism between gas messenger molecules and cell membrane microenvironment is of great significance for basic biology research. Sulfur dioxide (SO2), as a vital gas molecule, is of great value in the permeability and fluidity of cell membranes. However, monitoring of sulfur dioxide in cell membranes with high temporal and spatial resolution remains a challenge. Herein, we designed and synthesized a ratiometric probe CMCB based on coumarin and indole sulfonate platform. The unsaturated CC is selected as the reaction site of SO2, utilizing sulfonic acid groups to increase CMCB hydrophilicity to achieve efficient targeting of cell membranes. The probe CMCB features a good cells membranes targeting and a NIR emission (720 nm). Interestingly, the probe showed low detection limit, high selectivity and specificity of targeting cell membranes. We expect that this probe will serve as an ideal tool in studying gas messenger molecule on cytomembrane in vivo.

Cross-Species Analysis Reveals Co-Expressed Genes Regulating Antler Development in Cervidae.

Antlers constitute an interesting model for basic research in regenerative biology. Despite decades of being studied, much is still unknown about the genes related to antler development. Here, we utilized both the genome and antlerogenic periosteum (AP) transcriptome data of four deer species to reveal antler-related genes through cross-species comparative analysis. The results showed that the global gene expression pattern matches the status of antler phenotypes, supporting the fact that the genes expressed in the AP may be related to antler phenotypes. The upregulated genes of the AP in three-antlered deer showed evidence of co-expression, and their protein sequences were highly conserved. These genes were growth related and likely participated in antler development. In contrast, the upregulated genes in antler-less deer (Chinese water deer) were involved mainly in organismal death and growth failure, possibly related to the loss of antlers during evolution. Overall, this study demonstrates that the co-expressed genes in antlered deer may regulate antler development.

Ubiquitinomics: History, methods, and applications in basic research and drug discovery.

The ubiquitin-proteasome system (UPS) was discovered about 40 years ago and is known to regulate a multitude of cellular processes including protein homeostasis. Ubiquitylated proteins are recognized by downstream effectors, resulting in alterations of protein abundance, activity, or localization. Not surprisingly, the ubiquitylation machinery is dysregulated in numerous diseases, including cancers and neurodegeneration. Mass spectrometry (MS)-based proteomics has emerged as a transformative technology for characterizing protein ubiquitylation in an unbiased fashion. Here, we provide an overview of the different MS-based approaches for studying protein ubiquitylation. We review various methods for enriching and quantifying ubiquitin modifications at the peptide or protein level, outline MS acquisition, and data processing approaches and discuss key challenges. Finally, we examine how MS-based ubiquitinomics can aid both basic biology and drug discovery research.

Molecular Research on Oral Diseases and Related Biomaterials: A Journey from Oral Cell Models to Advanced Regenerative Perspectives.

Oral diseases such as gingivitis, periodontitis, and oral cancer affect millions of people worldwide. Much research has been conducted to understand the pathogenetic mechanisms of these diseases and translate this knowledge into therapeutics. This review aims to take the reader on a journey from the initial molecular discoveries to complex regenerative issues in oral medicine. For this, a semi-systematic literature search was carried out in Medline and Web of Science databases to retrieve the primary literature describing oral cell models and biomaterial applications in oral regenerative medicine. First, an in vitro cell model of gingival keratinocytes is discussed, which illustrates patho- and physiologic principles in the context of oral epithelial homeostasis and carcinogenesis and represents a cellular tool to understand biomaterial-based approaches for periodontal tissue regeneration. Consequently, a layered gradient nonwoven (LGN) is described, which demonstrates that the key features of biomaterials serve as candidates for oral tissue regeneration. LGN supports proper tissue formation and obeys the important principles for molecular mechanotransduction. Furthermore, current biomaterial-based tissue regeneration trends, including polymer modifications, cell-based treatments, antimicrobial peptides and optogenetics, are introduced to represent the full spectrum of current approaches to oral disease mitigation and prevention. Altogether, this review is a foray through established and new concepts in oral regenerative medicine and illustrates the process of knowledge translation from basic molecular and cell biological research to future clinical applications.

Gulo gene locus, a new gene editing locus for mammalian cells.

Transgene technology is valuable and helpful in many fields, including basic medical and biological research, biotechnology, and therapy. Recent advances in targeting technology accelerate the production of transgenic plants and animals and the application for gene therapy. To develop the technology, we examine the utility as the new safe harbor locus, l-gulono-γ-lactone oxidase (Gulo) locus in human and mice. We performed experiments in vitro and in vivo knockout and knockin mouse and cell lines to validate their applicability using these loci. The Gulo locus might be good candidates for safe harbor loci for transgenic research.

DNA-Lysozyme Nanoarchitectonics: Quantitative Investigation on Charge Inversion and Compaction.

The interaction between DNA and proteins is fundamentally important not only for basic research in biology, but also for potential applications in nanotechnology. In the present study, the complexes formed by λ DNA and lysozyme in a dilute aqueous solution have been investigated using magnetic tweezers (MT), dynamic light scattering (DLS), and atomic force microscopy (AFM). We found that lysozyme induced DNA charge inversion by measuring its electrophoretic mobility by DLS. Lysozyme is very effective at neutralizing the positive charge of DNA, and its critical charge ration to induce charge inversion in solution is only 2.26. We infer that the high efficiency of charge neutralization is due to the highly positively charged (+8 e) and compact structure of lysozyme. When increasing the concentration of lysozymes from 6 ng·µL−1 to 70 ng·µL−1, DNA mobility (at fixed concentration of 2 ng·µL−1) increases from −2.8 to 1.5 (in unit of 10−4 cm2·V−1·S), implying that the effective charge of DNA switches its sign from negative to positive in the process. The corresponding condensing force increased from 0 pN to its maximal value of about 10.7 pN at concentrations of lysozyme at 25 ng·µL−1, then decreases gradually to 3.8 pN at 200 ng·µL−1. The maximal condensing force occurs at the complete DNA charge neutralization point. The corresponding morphology of DNA–lysozyme complex changes from loosely extensible chains to compact globule, and finally to less compact flower-like structure due to the change of attached lysozyme particles as observed by AFM.

Review of Label-Free Monitoring of Bacteria: From Challenging Practical Applications to Basic Research Perspectives.

Novel biosensors already provide a fast way to detect the adhesion of whole bacteria (or parts of them), biofilm formation, and the effect of antibiotics. Moreover, the detection sensitivities of recent sensor technologies are large enough to investigate molecular-scale biological processes. Usually, these measurements can be performed in real time without using labeling. Despite these excellent capabilities summarized in the present work, the application of novel, label-free sensor technologies in basic biological research is still rare; the literature is dominated by heuristic work, mostly monitoring the presence and amount of a given analyte. The aims of this review are (i) to give an overview of the present status of label-free biosensors in bacteria monitoring, and (ii) to summarize potential novel directions with biological relevancies to initiate future development. Optical, mechanical, and electrical sensing technologies are all discussed with their detailed capabilities in bacteria monitoring. In order to review potential future applications of the outlined techniques in bacteria research, we summarize the most important kinetic processes relevant to the adhesion and survival of bacterial cells. These processes are potential targets of kinetic investigations employing modern label-free technologies in order to reveal new fundamental aspects. Resistance to antibacterials and to other antimicrobial agents, the most important biological mechanisms in bacterial adhesion and strategies to control adhesion, as well as bacteria-mammalian host cell interactions are all discussed with key relevancies to the future development and applications of biosensors.

The pathogenesis of endothelial dysfunction

<p indent=0mm>Endothelial dysfunction is the most common pathological feature of diabetes, hypertension and atherosclerosis. Endothelial dysfunction can occur not only in large conduit arteries such as aorta and renal artery, but also in microvascular circulation in the retina and kidney. Decreased production and secretion of endothelium-derived vasodilator factors (mainly nitric oxide) or increased synthesis and release of vasoconstrictor factors (such as angiotensin II, endothelin-1, reactive oxygen species and cyclooxygenase metabolites) are the main causes of endothelial dysfunction, which is closely associated with pathogenesis of hypertension, diabetes, atherosclerosis, myocardial ischemia, and heart failure. This review briefly summarizes the mechanisms underlying endothelial dysfunction in relation to activation of the renin angiotensin system, cyclooxygenase 2, and bone morphogenetic protein 4, as well as increased oxidative stress and inflammatory response in blood vessels, hoping to identify the key molecular mediators of endothelial dysfunction, thereby providing new clues for both basic and translational research in cardio-metabolic biology and medicine.

SPECT imaging properties of the CdTe-Timepix3 detector used in a new prototype FullSPECT 3D multimodal preclinical imaging system: Comparison with scintillation gamma camera

Small animal SPECT imaging represents a fast-evolving method for a noninvasive molecular imaging of metabolism, diseases, or malfunctions in live animal models. Preclinical imaging is important for translational research related to development and testing of new tracers, diagnostic and therapeutic methods for clinical application, and helps also in basic biological research. The small animal imaging requires better spatial resolution compared to human imaging. The improvement on the detector side should be implemented because the currently used scintillation gamma cameras are now touching their limits. The main goal of this work aims on introduction of a new concept of our prototype system (FullSPECT 3D) and comparison of the performance of its hybrid semiconductor Timepix3 based detector, and standard scintillation gamma camera in a commercially available device (Albira Preclinical Imaging System) for SPECT imaging on phantom and animal model. We compared both detector types on widely used 99mTc-HDP (for bone scan), and 125I labeled antibody biodistribution assays. The images taken with a single Timepix3 based detector were comparable and in some parameters exceeded standard detectors under similar geometry in 2D projections. The differences in 3D reconstructed images were not calculated because of the huge difference in the reconstruction algorithms, nevertheless the 3D images are shown as well. The future SPECT systems could benefit from the speed, energy resolution and size of Timepix3 based detectors and open the way to construction of very fast multimodal imagers requiring less ionizing radiation delivered to the patient.

OsGAMYBL2 is required for pollen maturation and germination in rice

The reproductive development process of rice is not only important for basic developmental biology research, but also of great benefit to production practice. However, current understanding of the reproductive growth process is relatively limited, especially in the later stages of development. Here we report that OsGAMYBL2, a homologous gene of GAMYB, is required for pollen maturation and germination in rice, and that loss of function of OsGAMYBL2 leads to male sterility in rice. We generated three alleles of OsGAMYBL2 knockout mutants using CRISPR/Cas9 system, all of them lost fertility when grown in the paddy field. Semi-section and electron microscopy observations revealed that there are no visible morphological changes in the outer and inner surfaces of anther and in the surface of mature pollens between mutants and wild type. However, pollens at late developmental stages exhibited much lighter pollen viability staining and some of them shriveled. In vitro and in vivo germination experiments showed that mutant pollens fail to germinate, leading to abnormal pollen tube formation and subsequent defective pollination. Furthermore, we observed that as compared with high temperature at 30 ​°C, low-temperature at 22 ​°C affects less on osgmybl2 fertility. Collectively, we proposed that OsGAMYBL2 is likely involved in the regulation of starch accumulation during later pollen development with a thermal-sensitive way, which improves our understanding on the reproductive development in plants.

Application Progress of Organoids in Colorectal Cancer.

Currently, colorectal cancer is still the third leading cause of cancer-related mortality, and the incidence is rising. It is a long time since the researchers used cancer cell lines and animals as the study subject. However, these models possess various limitations to reflect the cancer progression in the human body. Organoids have more clinical significance than cell lines, and they also bridge the gap between animal models and humans. Patient-derived organoids are three-dimensional cultures that simulate the tumor characteristics in vivo and recapitulate tumor cell heterogeneity. Therefore, the emergence of colorectal cancer organoids provides an unprecedented opportunity for colorectal cancer research. It retains the molecular and cellular composition of the original tumor and has a high degree of homology and complexity with patient tissues. Patient-derived colorectal cancer organoids, as personalized tumor organoids, can more accurately simulate colorectal cancer patients’ occurrence, development, metastasis, and predict drug response in colorectal cancer patients. Colorectal cancer organoids show great potential for application, especially preclinical drug screening and prediction of patient response to selected treatment options. Here, we reviewed the application of colorectal cancer organoids in disease model construction, basic biological research, organoid biobank construction, drug screening and personalized medicine, drug development, drug toxicity and safety, and regenerative medicine. In addition, we also displayed the current limitations and challenges of organoids and discussed the future development direction of organoids in combination with other technologies. Finally, we summarized and analyzed the current clinical trial research of organoids, especially the clinical trials of colorectal cancer organoids. We hoped to lay a solid foundation for organoids used in colorectal cancer research.

TC-hunter: identification of the insertion site of a transgenic gene within the host genome

BackgroundTransgenic animal models are crucial for the study of gene function and disease, and are widely utilized in basic biological research, agriculture and pharma industries. Since the current methods for generating transgenic animals result in the random integration of the transgene under study, the phenotype may be compromised due to disruption of known genes or regulatory regions. Unfortunately, most of the tools that predict transgene insertion sites from high-throughput data are not publicly available or not properly maintained.ResultsWe implemented TC-hunter, Transgene-Construct hunter, an open tool that identifies transgene insertion sites and provides simple reports and visualization aids. It relies on common tools used in the analysis of high-throughput data and makes use of chimeric reads and discordant read pairs to identify and support the transgenic insertion site. To demonstrate its applicability, we applied TC-hunter to four transgenic mice samples harboring the human PPM1D gene, a model used in the study of malignant tumor development. We identified the transgenic insertion site in each sample and experimentally validated them with Touchdown-polymerase chain reaction followed by Sanger sequencing.ConclusionsTC-hunter is an accessible bioinformatics tool that can automatically identify transgene insertion sites from DNA sequencing data with high sensitivity (98%) and precision (92.45%). TC-hunter is a valuable tool that can aid in evaluating any potential phenotypic complications due to the random integration of the transgene and can be accessed at https://github.com/bcfgothenburg/SSF.

A data management infrastructure for the integration of imaging and omics data in life sciences

BackgroundAs technical developments in omics and biomedical imaging increase the throughput of data generation in life sciences, the need for information systems capable of managing heterogeneous digital assets is increasing. In particular, systems supporting the findability, accessibility, interoperability, and reusability (FAIR) principles of scientific data management.ResultsWe propose a Service Oriented Architecture approach for integrated management and analysis of multi-omics and biomedical imaging data. Our architecture introduces an image management system into a FAIR-supporting, web-based platform for omics data management. Interoperable metadata models and middleware components implement the required data management operations. The resulting architecture allows for FAIR management of omics and imaging data, facilitating metadata queries from software applications. The applicability of the proposed architecture is demonstrated using two technical proofs of concept and a use case, aimed at molecular plant biology and clinical liver cancer research, which integrate various imaging and omics modalities.ConclusionsWe describe a data management architecture for integrated, FAIR-supporting management of omics and biomedical imaging data, and exemplify its applicability for basic biology research and clinical studies. We anticipate that FAIR data management systems for multi-modal data repositories will play a pivotal role in data-driven research, including studies which leverage advanced machine learning methods, as the joint analysis of omics and imaging data, in conjunction with phenotypic metadata, becomes not only desirable but necessary to derive novel insights into biological processes.

Protoplast Isolation, Transfection, and Gene Editing for Soybean (Glycine max ).

Protoplast is a versatile system for conducting cell-based assays, analyzing diverse signaling pathways, studying functions of cellular machineries, and functional genomics screening. Protoplast engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. This system allows the direct delivery of DNA, RNA, or proteins into plant cells and provides a high-throughput system to validate gene-editing reagents. It also facilitates the delivery of homology-directed repair templates (donor molecules) into plant cells, enabling precise DNA edits in the genome. There is a great deal of interest in the plant community to develop these precise edits, as they may expand the potential for developing value-added traits which may be difficult to achieve by other gene-editing applications and/or traditional breeding alone. This chapter provides improved working protocols for isolating and transforming protoplast from immature soybean seeds with 44% of transfection efficiency validated by the green fluorescent protein reporter. We also describe a method for gene editing in soybean protoplasts using single guide RNA molecules.

The Development of Mitochondrial Gene Editing Tools and Their Possible Roles in Crop Improvement for Future Agriculture.

We are living in the era of genome editing. Nowadays, targeted editing of the plant nuclear DNA is prevalent in basic biological research and crop improvement since its first establishment a decade ago. However, achieving the same accomplishment for the plant mitochondrial genome has long been deemed impossible. Recently, the pioneer studies on editing plant mitogenome have been done using the mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs) in rice, rapeseed, and Arabidopsis. It is well documented that mitochondria play essential roles in plant development and stress tolerance, particularly, in cytoplasmic male sterility widely used in production of hybrids. The success of mitochondrial genome editing enables studying the fundamentals of mitochondrial genome. Furthermore, mitochondrial RNA editing (mostly by nuclear-encoded pentatricopeptide repeat (PPR) proteins) in a sequence-specific manner can simultaneously change the production of translatable mitochondrial mRNA. Moreover, direct editing of the nuclear-encoding mitochondria-targeted factors required for plant mitochondrial genome dynamics and recombination may facilitate genetic manipulation of plant mitochondria. Here, the present state of knowledge on editing the plant mitochondrial genome is reviewed.

CRISPR-Cas9-Mediated Gene Therapy in Neurological Disorders.

Neurological disorders are primarily diseases with sophisticated etiology that are always refractory and recrudescent. The major obstruction to effective therapies for neurological disorders is the poor understanding of their pathogenic mechanisms. CRISPR-Cas9 technology, which allows precise and effective gene editing in almost any cell type and organism, is accelerating the pace of basic biological research. An increasing number of groups are focusing on uncovering the molecular mechanisms of neurological disorders and developing novel therapies using the CRISPR-Cas9 system. This review highlights the application of CRISPR-Cas9 technology in the treatment of neurological disorders, including Alzheimer's disease, amyotrophic lateral sclerosis and/or frontotemporal dementia, Duchenne muscular dystrophy, Dravet syndrome, epilepsy, Huntington's disease, and Parkinson's disease. Hopefully, it will improve our understanding of neurological disorders and give insights into future treatments for neurological disorders.

Discovering new biology with drug-resistance alleles.

Small molecule drugs form the backbone of modern medicine's therapeutic arsenal. Often less appreciated is the role that small molecules have had in advancing basic biology. In this Review, we highlight how resistance mutations have unlocked the potential of small molecule chemical probes to discover new biology. We describe key instances in which resistance mutations and related genetic variants yielded foundational biological insight and categorize these examples on the basis of their role in the discovery of novel molecular mechanisms, protein allostery, physiology and cell signaling. Next, we suggest ways in which emerging technologies can be leveraged to systematically introduce and characterize resistance mutations to catalyze basic biology research and drug discovery. By recognizing how resistance mutations have propelled biological discovery, we can better harness new technologies and maximize the potential of small molecules to advance our understanding of biology and improve human health.

Rare Does Not Mean Worthless: How Rare Diseases Have Shaped Neurodevelopment Research in the NGS Era.

The advent of next-generation sequencing (NGS) is heavily changing both the diagnosis of human conditions and basic biological research. It is now possible to dig deep inside the genome of hundreds of thousands or even millions of people and find both common and rare genomic variants and to perform detailed phenotypic characterizations of both physiological organs and experimental models. Recent years have seen the introduction of multiple techniques using NGS to profile transcription, DNA and chromatin modifications, protein binding, etc., that are now allowing us to profile cells in bulk or even at a single-cell level. Although rare and ultra-rare diseases only affect a few people, each of these diseases represent scholarly cases from which a great deal can be learned about the pathological and physiological function of genes, pathways, and mechanisms. Therefore, for rare diseases, state-of-the-art investigations using NGS have double valence: their genomic cause (new variants) and the characterize the underlining the mechanisms associated with them (discovery of gene function) can be found. In a non-exhaustive manner, this review will outline the main usage of NGS-based techniques for the diagnosis and characterization of neurodevelopmental disorders (NDDs), under whose umbrella many rare and ultra-rare diseases fall.