Journal of Biological Methods

Mouse models with humanized immune systems are becoming increasingly prevalent in pharmaceutical research as a platform for preclinical testing with potential for greater translatability to clinical applications. However, the presence of both mouse and human cells that respond to TLR ligands poses a challenge for investigating therapeutic modalities targeting TLR signaling. AZ617 is a human TLR4 agonist, which has been shown in vitro to preferentially induce human cytokines via the TLR4 signaling pathway. We sought to examine the ability of AZ617 to preferentially induce human cytokines in CD34+ stem cell-engrafted NOG-EXL mice (huNOG-EXL), to determine its suitability as an in vivo human functional readout...

Over the last decades, PCR and molecular cloning have profoundly impacted various biological areas, from basic to pharmaceutical sciences. Presented in this study is a simple and step-by-step protocol that uses PCR to recover a poor-quality ligase product. In fact, a classic step that can be problematic in typical recombinant DNA manipulations can be the recovery of a product from a T4 DNA ligase reaction between two or more suitably prepared DNA fragments (sticky ends, blunt ends, TA cloning, etc.). This reaction can result in poor yields of the ligation product, due to various causes, mainly the preparation of the DNA fragments, and the poor yield can severely invalidate all subsequent steps...

Mycoplasmas, which are the smallest and simplest prokaryotes, lack a cell wall but possess the ability to undergo self-replication. Mycoplasma contamination is a common problem for laboratories engaging in cell culture. Due to their small size, Mycoplasmas can easily permeate filters designed to prevent bacterial and fungal contamination in cell culture. Although Mycoplasma contamination usually does not result in cell death, it can significantly affect cell proliferation, metabolism, and cause chromosomal aberrations...

Large-scale production of Arbuscular Mycorrhizal Fungi (AMF) consortia is a crucial stride in harnessing their potential for sustainable agriculture and plant growth enhancement. However, establishing optimal production conditions is challenging due to their obligate nature, variability, lack of standardized protocols, and limited understanding of their specific requirements. Previous attempts to standardize Root Organ Cultures (ROC) for AMF overlooked challenges related to viable inoculum production for field applications...

Millipedes are key players in recycling leaf litter into soil in tropical ecosystems. To elucidate their gut microbiota, we collected millipedes from different municipalities of Puerto Rico. Here we aim to benchmark which method is best for metagenomic skimming of this highly complex millipede microbiome. We sequenced the gut DNA with Oxford Nanopore Technologies' (ONT) MinION sequencer, then analyzed the data using MEGAN-LR , Kraken2 protein mode, Kraken2 nucleotide mode, GraphMap , and Minimap2 to classify these long ONT reads...

N6 -methyladenosine (m6 A), the most prevalent mRNA modification in eukaryotic cells, is known to play regulatory roles in a wide array of biological processes, including aging and cellular senescence. To investigate such roles, the m6 A modification can be identified across the entire transcriptome by immunoprecipitation of methylated RNA with an anti-m6 A antibody, followed by high-throughput sequencing (meRIP-seq or m6 A-seq). Presented here is a protocol for employing meRIP-seq to profile the RNA m6 A landscape in senescent human cells...

Serum contains proteins that possess important information about diseases and their progression. Unfortunately, these proteins, which carry the information in the serum are in low abundance and are masked by other serum proteins that are in high abundance. Such masking prevents their identification and quantification. Therefore, removal of high abundance proteins is required to enrich, identify, and quantify the low abundance proteins. Immunodepletion methods are often used for this purpose, but there are limitations in their use because of off-target effects and high costs...

To fully understand any cellular process, we not only need to identify the proteins implicated, but also how the protein network is structurally and spatially organized and changes over time. However, the dynamic nature of many protein interactions involved in cellular signaling pathways continues to be the bottleneck in mapping and studying protein networks. Fortunately, a recently developed proximity labeling method using engineered ascorbic acid peroxidase 2 (APEX2) in mammalian cells allows the identification of weak and/or transient protein interactions with spatial and temporal resolution...

Tape-stabilized cryohistology is a powerful histological method to reinforce tissue samples during and after sectioning, enhancing the overall image quality. This technique has widely been applied to section mineralized small animal ( i.e. , mice, rat, rabbit) specimens, but has only been sparsely implemented for large animal samples that have a greater tendency to tear due to their increased surface area. Here, we present an optimized protocol for tape-stabilized cryohistology of undecalcified minipig vertebral body, femoral head, and temporomandibular joint samples...

Spheroids and organoids are increasingly popular three-dimensional (3D) cell culture models. Spheroid models are more physiologically relevant to a tumor compared to two-dimensional (2D) cultures and organoids are a simplified version of an organ with similar composition. Spheroids are often only formed from a single cell type which does not represent the situation in vivo . However, despite this, both spheroids and organoids can be used in cell migration studies, disease modelling and drug discovery. A drawback of these models is, however, the lack of appropriate analytical tools for high throughput imaging and analysis over a time course...

Somatic mutations are evolutionarily important as determinants of individual organismal fitness, as well as being a focus of clinical research on age-related disease, such as cancer. Identifying somatic mutations and quantifying mutation rates, however, is extremely challenging and genome-wide somatic mutation rates have only been reported for a few model organisms. Here, we describe the application of Duplex Sequencing on bottlenecked WGS libraries to quantify somatic nuclear genome-wide base substitution rates in Daphnia magna ...

Skeletal muscle contractions stimulate glucose uptake into the working muscles during exercise. Because this signaling pathway is independent of insulin, exercise constitutes an important alternative pathway to increase glucose uptake, also in insulin-resistant muscle. Therefore, much effort is being put into understanding the molecular regulation of exercise-stimulated glucose uptake by skeletal muscle. To delineate the causal molecular mechanisms whereby muscle contraction or exercise regulate glucose uptake, the investigation of genetically manipulated rodents is necessary...

Cuttlefish are active carnivores that possess a wide repertoire of body patterns that can be changed within milliseconds for many types of camouflage and communication. The forms and functions of many body patterns are well known from ethological studies in the field and laboratory. Yet one aspect has not been reported in detail: the category of rapid, brief and high-contrast changes in body coloration ("Tentacle Shot Patterns" or TSPs) that always occur with the ejection of two ballistic tentacles to strike live moving prey ("Tentacles Go Ballistic" or TGB moment)...

The regulation of cellular energetics is a complex process that requires the coordinated function of multiple organelles. Historically, studies focused on understanding cellular energy utilization and production have been overwhelmingly concentrated on the mitochondria. While mitochondria account for the majority of intracellular energy production, they alone are incapable of maintaining the variable energetic demands of the cell. The peroxisome has recently emerged as a secondary metabolic organelle that complements and improves mitochondrial performance...

Severe burns are traumatic and physically debilitating injuries with a high rate of mortality. Bacterial infections often complicate burn injuries, which presents unique challenges for wound management and improved patient outcomes. Currently, pigs are used as the gold standard of pre-clinical models to study infected skin wounds due to the similarity between porcine and human skin in terms of structure and immunological response. However, utilizing this large animal model for wound infection studies can be technically challenging and create issues with data reproducibility...

Camelid single-domain antibody fragments, also called nanobodies, constitute a class of binders that are small in size (~15 kDa) and possess antigen-binding properties similar to their antibody counterparts. Facile production of recombinant nanobodies in several microorganisms has made this class of binders attractive within the field of molecular imaging. Particularly, their use in super-resolution microscopy has improved the spatial resolution of molecular targets due to a smaller linkage error. In single-molecule localization microscopy techniques, the effective spatial resolution can be further enhanced by site-specific fluorescent labeling of nanobodies owing to a more homogeneous protein-to-fluorophore stoichiometry, reduced background staining and a known distance between dye and epitope...

Surface modified microspheres have been leveraged as a useful way to immobilize antigen for serological studies. The use of carboxyl modified microspheres for this purpose is well-established, but commonly associated with technical challenges. Streptavidin modified microspheres require little technical expertise and thus address some of the shortcomings of carboxyl microspheres. An additional feature of streptavidin microspheres is the use of mono-biotinylated proteins, which contain a single biotinylation motif at the C-terminus...

No abstract text is available yet for this article.

Several murine models of corneal transplantation have been developed over the years to study the immunopathological processes that lead to the failure of grafted corneas. In all of them, the classic eight interrupted sutures technique is utilized for transplanting the donor cornea on the host bed. However, in clinical practice, a single continuous suture with a single knot is generally performed for corneal transplantation. Here, we describe the adaptation of the single continuous suture technique in a mouse model of corneal transplantation...

In late 2019, a novel coronavirus began spreading in Wuhan, China, causing a potentially lethal respiratory viral infection. By early 2020, the novel coronavirus, called SARS-CoV-2, had spread globally, causing the COVID-19 pandemic. The infection and mutation rates of SARS-CoV-2 make it amenable to tracking introduction, spread and evolution by viral genome sequencing. Efforts to develop effective public health policies, therapeutics, or vaccines to treat or prevent COVID-19 are also expected to benefit from tracking mutations of the SARS-CoV-2 virus...