An updated view of the pathogenesis of steroid-sensitive nephrotic syndrome

Idiopathic nephrotic syndrome is the most common childhood glomerular disease. Most forms of this syndrome respond to corticosteroids at standard doses and are, therefore, defined as steroid-sensitive nephrotic syndrome (SSNS). Immunological mechanisms and subsequent podocyte disorders play a pivotal role in SSNS and have been studied for years; however, the precise pathogenesis remains unclear. With recent advances in genetic techniques, an exhaustive hypothesis-free approach called a genome-wide association study (GWAS) has been conducted in various populations. GWASs in pediatric SSNS peaked in the human leukocyte antigen class II region in various populations. Additionally, an association of immune-related CALHM6/FAM26F, PARM1, BTNL2, and TNFSF15 genes, as well as NPHS1, which encodes nephrin expressed in podocytes, has been identified as a locus that achieves genome-wide significance in pediatric SSNS. However, the specific mechanism of SSNS development requires elucidation. This review describes an updated view of SSNS pathogenesis from immunological and genetic aspects, including interactions with infections or allergies, production of circulating factors, and an autoantibody hypothesis.

Axonemal Symmetry Break, a New Ultrastructural Diagnostic Tool for Primary Ciliary Dyskinesia?

Diagnosis testing for primary ciliary dyskinesia (PCD) requires a combination of investigations that includes study of ciliary beat pattern by high-speed video-microscopy, genetic testing and assessment of the ciliary ultrastructure by transmission electron microscopy (TEM). Historically, TEM was considered to be the “gold standard” for the diagnosis of PCD. However, with the advances in molecular genetic techniques, an increasing number of PCD variants show normal ultrastructure and cannot be diagnosed by TEM. During ultrastructural assessment of ciliary biopsies of patients with suspicion of PCD, we observed an axonemal defect not previously described that affects peripheral doublets tilting. To further characterize this defect of unknown significance, we studied the ciliary axonemes by TEM from both PCD-confirmed patients and patients with other sino-pulmonary diseases. We detected peripheral doublets tilting in all the PCD patients, without any significant difference in the distribution of ciliary beat pattern or mutated gene. This defect was also present in those patients with normal ultrastructure PCD subtypes. We believe that the performance of axonemal asymmetry analysis would be helpful to enhance diagnosis of PCD.

Nomenclature standards and DNA barcoding of apple varieties originated by VIR Crimean Experimental Breeding Station

Current pace of breeding technologies relies on a tighter control of quality and identity of breeding achievements. Thereby, standardised variety nomenclature becomes of particular relevance to store original genetic information and warrant authenticity of the breeding product. A nomenclature standard regulates the variety’s name and habitus. A herbarium specimen is most appropriate for standardisation, since it defines a set of strongly inherited morphological characters for a variety. Nomenclature standards must be permanently stored in a scientific herbarium collection. The N. I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR) in alliance with national plant breeders initiated a first nomenclature standard collection of national-breeding cultivars. Aside to classical herbarising, the collection utilises molecular genetic techniques, particularly genotyping, to enable extended variety verification. The VIR team has developed the nomenclature standards of six apple varieties originated by the Crimean Experimental Breeding Station, a VIR Branch (Beloe Solntse, Zolotoy Potok, Kubanochka, Leto Krasnoe, Luchistoe and Shchedrost). The standards are supplemented with DNA barcodes obtained jointly with the N. I. Vavilov Institute of General Genetics. Fifteen microsatellite markers were used for barcoding. The herbarium nomenclature specimens adhered to the VIR-developed protocol in accordance with the International Code of Nomenclature for Cultivated Plants (ICNCP). The specimens are registered in the VIR Herbarium database and deposited in the World Crop Wild and Weed Relatives (WIR) Herbarium type collection.

Chemical Biology Tools to Study Lipids and their Metabolism with Increased Spatial and Temporal Resolution

Lipids are important cellular components providing many essential functions. To fulfill these various functions evolution has selected for a diverse set of lipids and this diversity is seen at the organismal, cellular and subcellular level. Understanding how cells maintain this complex lipid organization is a very challenging problem, which for lipids, is not easily addressed using biochemical and genetic techniques. Therefore, chemical tools have an important role to play in our quest to understand the complexities of lipid metabolism. Here we discuss new chemical tools to study lipids, their distribution and metabolism with increased spatial and temporal resolution.

Zebrafish Embryos and Larvae as Alternative Animal Models for Toxicity Testing

Prerequisite to any biological laboratory assay employing living animals is consideration about its necessity, feasibility, ethics and the potential harm caused during an experiment. The imperative of these thoughts has led to the formulation of the 3R-principle, which today is a pivotal scientific standard of animal experimentation worldwide. The rising amount of laboratory investigations utilizing living animals throughout the last decades, either for regulatory concerns or for basic science, demands the development of alternative methods in accordance with 3R to help reduce experiments in mammals. This demand has resulted in investigation of additional vertebrate species displaying favourable biological properties. One prominent species among these is the zebrafish (Danio rerio), as these small laboratory ray-finned fish are well established in science today and feature outstanding biological characteristics. In this review, we highlight the advantages and general prerequisites of zebrafish embryos and larvae before free-feeding stages for toxicological testing, with a particular focus on cardio-, neuro, hepato- and nephrotoxicity. Furthermore, we discuss toxicokinetics, current advances in utilizing zebrafish for organ toxicity testing and highlight how advanced laboratory methods (such as automation, advanced imaging and genetic techniques) can refine future toxicological studies in this species.

Advancing Genetic Methods in the Study of Parrot Biology and Conservation

Parrots (Psittaciformes) are a well-studied, diverse group of birds distributed mainly in tropical and subtropical regions. Today, one-third of their species face extinction, mainly due to anthropogenic threats. Emerging tools in genetics have made major contributions to understanding basic and applied aspects of parrot biology in the wild and in captivity. In this review, we show how genetic methods have transformed the study of parrots by summarising important milestones in the advances of genetics and their implementations in research on parrots. We describe how genetics helped to further knowledge in specific research fields with a wide array of examples from the literature that address the conservation significance of (1) deeper phylogeny and historical biogeography; (2) species- and genus-level systematics and taxonomy; (3) conservation genetics and genomics; (4) behavioural ecology; (5) molecular ecology and landscape genetics; and (6) museomics and historical DNA. Finally, we highlight knowledge gaps to inform future genomic research on parrots. Our review shows that the application of genetic techniques to the study of parrot biology has far-reaching implications for addressing diverse research aims in a highly threatened and charismatic clade of birds.

Strategies for genetic manipulation of adult stem cell-derived organoids

Organoid technology allows the expansion of primary epithelial cells from normal and diseased tissues, providing a unique model for human (patho)biology. In a three-dimensional environment, adult stem cells self-organize and differentiate to gain tissue-specific features. Accessibility to genetic manipulation enables the investigation of the molecular mechanisms underlying cell fate regulation, cell differentiation and cell interactions. In recent years, powerful methodologies using lentiviral transgenesis, CRISPR/Cas9 gene editing, and single-cell readouts have been developed to study gene function and carry out genetic screens in organoids. However, the multicellularity and dynamic nature of stem cell-derived organoids also present challenges for genetic experimentation. In this review, we focus on adult gastrointestinal organoids and summarize the state-of-the-art protocols for successful transgenesis. We provide an outlook on emerging genetic techniques that could further increase the applicability of organoids and enhance the potential of organoid-based techniques to deepen our understanding of gene function in tissue biology.

Generation of knock-in lampreys by CRISPR-Cas9-mediated genome engineering

The lamprey represents the oldest group of living vertebrates and has been a key organism in various research fields such as evolutionary developmental biology and neuroscience. However, no knock-in technique for this animal has been established yet, preventing application of advanced genetic techniques. Here, we report efficient generation of F0 knock-in lampreys by CRISPR-Cas9-mediated genome editing. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) for genome digestion, a sgRNA for donor plasmid digestion, and Cas9 mRNA. Targeting different genetic loci, we succeeded in generating knock-in lampreys expressing photoconvertible protein Dendra2 as well as those expressing EGFP. With its simplicity, design flexibility, and high efficiency, we propose that the present method has great versatility for various experimental uses in lamprey research and that it can also be applied to other “non-model” organisms.