Biological Sciences

In patients with chronic kidney disease, it is necessary to identify the etiology of glomerular disease. Renal biopsy is the gold standard for assessing the underlying pathology; however, it has the risk of potential complications. We have established a urinary fluorescence imaging technique to assess enzymatic activity using an activatable fluorescent probe targeting two enzymes: gamma-glutamyl transpeptidase and dipeptidyl-peptidase. The urinary fluorescence images can be easily obtained by adding an optical filter to the microscope with short incubation of the fluorescent probes. Urinary fluorescence imaging could help to assess underlying etiologies of kidney diseases and is a potential non-invasive qualitative assessment technique for kidney diseases in patients with diabetes.

Key features

• Non-invasive assessment of kidney disease.

• Urinary fluorescent imaging with enzyme-activatable fluorescent probes.

• Enables differentiation of diabetic kidney disease and glomerulonephritis.

Plant protoplasts are useful to study both transcriptional regulation and protein subcellular localization in rapid screens. Protoplast transformation can be used in automated platforms for design-build-test cycles of plant promoters, including synthetic promoters. A notable application of protoplasts comes from recent successes in dissecting synthetic promoter activity with poplar mesophyll protoplasts. For this purpose, we constructed plasmids with TurboGFP driven by a synthetic promoter together with TurboRFP constitutively controlled by a 35S promoter, to monitor transformation efficiency, allowing versatile screening of high numbers of cells by monitoring green fluorescent protein expression in transformed protoplasts. Herein, we introduce a protocol for poplar mesophyll protoplast isolation followed by protoplast transformation and image analysis for the selection of valuable synthetic promoters.

Graphical overview

Skeletal muscle disorders commonly affect the function and integrity of muscles. Novel interventions bring new potential to rescue or alleviate the symptoms associated with these disorders. In vivo and in vitro testing in mouse models allows quantitative evaluation of the degree of muscle dysfunction, and therefore, the level of potential rescue/restoration by the target intervention. Several resources and methods are available to assess muscle function and lean and muscle mass, as well as myofiber typing as separate concepts; however, a technical resource unifying these methods is missing. Here, we provide detailed procedures for analyzing muscle function, lean and muscle mass, and myofiber typing in a comprehensive technical resource paper.

Graphical abstract

Chloroplast movement has been observed and analyzed since the 19^th century. Subsequently, the phenomenon is widely observed in various plant species such as fern, moss, Marchantia polymorpha, and Arabidopsis. However, chloroplast movement in rice is less investigated, presumably due to the thick wax layer on its leaf surface, which reduces light sensitivity to the point that it was previously believed that there was no light-induced movement in rice. In this study, we present a convenient protocol suitable for observing chloroplast movement in rice only by optical microscopy without using special equipment. It will allow researchers to explore other signaling components involved in chloroplast movement in rice.

While wound healing in humans occurs primarily through re-epithelization, in rodents it also occurs through contraction of the panniculus carnosus, an underlying muscle layer that humans do not possess. Murine experimental models are by far the most convenient and inexpensive research model to study wound healing, as they offer great variability in genetic alterations and disease models. To overcome the obstacle of contraction biasing wound healing kinetics, our group invented the splinted excisional wound model. While other rodent wound healing models have been used in the past, the splinted excisional wound model has persisted as the most used model in the field of wound healing. Here, we present a detailed protocol of updated and refined techniques necessary to utilize this model, generate results with high validity, and accurately analyze the collected data. This model is simple to conduct and provides an easy, standardizable, and replicable model of human-like wound healing.

Anorexia nervosa (AN) is a devastating neuropsychiatric disease with a prevalence rate of approximately 0.3%–1% among women and morbidity and mortality rates among the highest of all neuropsychiatric disorders. The disease etiology is complex but primarily characterized by reduced food intake and body weight, and intense anxiety and fear associated with gaining weight. Existing rodent models of AN are useful and capture features of the disease, but either require specialized genetic mouse models or are difficult to implement in mice. Here, we describe two simple mouse models of stress-induced anorexia that are easy to implement in basic research labs, and capture core features associated with AN, such as reduced food intake in the context of social/physical stress and increased anxiety-related behavior. These protocols provide reproducible and robust assays for stress-induced anorexia and may be implemented with additional assays to probe the neural circuitry mediating the effects of psychological stress on feeding in mice.

Graphical abstract

Identifying genetic variations or treatments that confer greater resistance to drought is paramount to ensuring sustainable crop productivity. Accurate and reproducible measurement of drought stress symptoms can be achieved via automated, image-based phenotyping. Many phenotyping platforms are either cost-prohibitive, require specific technical expertise, or are simply more complex than necessary to effectively evaluate drought resistance. Certain mutations, allelic variations, or treatments result in plants that constitutively use less water. To accurately identify genetic differences or treatments that confer a drought phenotype, plants from all experimental groups must be subjected to equal levels of drought stress. This can be easily achieved by growing and imaging plants that are grown in the same pot. Here, we provide a detailed protocol to configure a Raspberry Pi computer and camera module to image seedlings of multiple genotypes growing in shared pots and to transfer images and metadata via the cloud for downstream analyses. Also detailed is a method to calculate percent soil water content of pots while being imaged to allow for comparison of stress symptoms with water availability. This protocol was recently used to uncouple differential water usage from drought resistance in a dwarf Arabidopsis thaliana mutant chiquita1-1/cost1 compared to the wild-type control. It is cost effective, suitable for any plant species, customizable to various biological questions, and requires no prior experience with electronics or basic software programming.

In nature, parasitoid wasp infections are a major cause of insect mortality. Parasitoid wasps attack a vast range of insect species to lay their eggs. As a defense, insects evolved survival strategies to protect themselves from parasitoid infection. While a growing number of studies reported both host defensive tactics and parasitoid counter-offensives, we emphasize that this parasite–host relationship presents a unique ecological and evolutionary relevant model that is often challenging to replicate in a laboratory. Although maintaining parasitoid wasp cultures in the laboratory requires meticulous planning and can be labor intensive, a diverse set of wasp species that target many different insect types can be maintained in similar culture conditions. Here, we describe the protocol for culturing parasitoid wasp species on Drosophila larvae and pupae in laboratory conditions. We also detail an egg-laying assay to assess the reproductive modification of Drosophila females in response to parasitoid wasps. This behavioral study is relatively simple and easily adaptable to study environmental or genetic influences on egg-laying, a readout for female germline development. Neither the parasitoid culture conditions or the behavioral assay require special supplies or equipment, making them a powerful and versatile approach in research or teaching laboratory settings.

Graphical abstract

Group A streptococcus (GAS) is a Gram-positive human pathogen that causes invasive infections with mild to life-threatening severity, like toxic shock syndrome, rheumatic heart disease, and necrotizing fasciitis (NF). NF is characterized by a clinical presentation of widespread tissue destruction due to the rapid spread of GAS infection into fascial planes. Despite quick medical interventions, mortality from NF is high. The early onset of the disease is difficult to diagnose because of non-specific clinical symptoms. Moreover, the unavailability of an effective vaccine against GAS warrants a genuine need for alternative treatments against GAS NF. One endoplasmic reticulum stress signaling pathway (PERK pathway) gets triggered in the host upon GAS infection. Bacteria utilize asparagine release as an output of this pathway for its pathogenesis. We reported that the combination of sub-cutaneous (SC) and intraperitoneal (IP) administration of PERK pathway inhibitors (GSK2656157 and ISRIB) cures local as well as systemic GAS infection in a NF murine model, by reducing asparagine release at the infection site. This protocol's methodology is detailed below.

Pathogen invasion of the central nervous system (CNS) is an important cause of infection-related mortality worldwide and can lead to severe neurological sequelae. To gain access to the CNS cells, pathogens have to overcome the blood–brain barrier (BBB), a protective fence from blood-borne factors. To study host–pathogen interactions, a number of cell culture and animal models were developed. However, in vitro models do not recapitulate the 3D architecture of the BBB and CNS tissue, and in vivo mammalian models present cellular and technical complexities as well as ethical issues, rendering systematic and genetic approaches difficult. Here, we present a two-pronged methodology allowing and validating the use of Drosophila larvae as a model system to decipher the mechanisms of infection in a developing CNS. First, an ex vivo protocol based on whole CNS explants serves as a fast and versatile screening platform, permitting the investigation of molecular and cellular mechanisms contributing to the crossing of the BBB and consequences of infection on the CNS. Then, an in vivo CNS infection protocol through direct pathogen microinjection into the fly circulatory system evaluates the impact of systemic parameters, including the contribution of circulating immune cells to CNS infection, and assesses infection pathogenicity at the whole host level. These combined complementary approaches identify mechanisms of BBB crossing and responses of a diversity of CNS cells contributing to infection, as well as novel virulence factors of the pathogen.

Graphical abstract

Procedures flowchart.
Mammalian neurotropic pathogens could be tested in two Drosophila central nervous system (CNS) infection setups (ex vivo and in vivo) for their ability to: (1) invade the CNS (pathogen quantifications), (2) disturb blood–brain barrier permeability, (3) affect CNS host cell behaviour (gene expression), and (4) alter host viability.