Journal of biological methods最新文献

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. From our two samples, we obtained a total of 87,110 and 99,749 ONT reads, respectively. Kraken2 nucleotide mode classified the most reads compared to all other methods at the phylum and class taxonomic level, classifying 75% of the reads in the two samples, the other methods failed to assign enough reads to either phylum or class to yield asymptotes in the taxa rarefaction curves indicating that they required more sequencing depth to fully classify this community. The community is hyper diverse with all methods classifying 20-50 phyla in the two samples. There was significant overlap in the reads used and phyla classified between the five methods benchmarked. Our results suggest that Kraken2 nucleotide mode is the most appropriate tool for the application of metagenomic skimming of this highly complex community.

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. Here, we describe a protocol for successfully using the APEX2-proximity labeling method in Dictyostelium, using the cAMP receptor cAR1 as example. Coupled to the identification of the labeled proteins by mass spectrometry, this method expands Dictyostelium's proteomics toolbox and should be widely useful for identifying interacting partners involved in a variety of biological processes in Dictyostelium.

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. Here we presented a robust, reproducible and cost-effective experimental workflow to remove immunoglobulins and albumin from serum with high efficiency. The workflow did not suffer from such limitations and enabled identification of 681 low abundance proteins that were otherwise undetectable in the serum. The identified low abundance proteins belonged to 21 different protein classes, namely the immunity-related proteins, modulators of protein-binding activity, and protein-modifying enzymes. They also played roles in various metabolic events, such as integrin signalling, inflammation-mediated signalling, and cadherin signalling. The presented workflow can be adapted to remove abundant proteins from other types of biological material and to provide considerable enrichment for low-abundance proteins.

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. Daphnia, historically an ecological model system, has more recently been the focus of mutation studies, in part because of its high germline mutation rates. Using our protocol and pipeline, we estimate a somatic mutation rate of 5.6 × 10^-7 substitutions per site (in a genotype where the germline rate is 3.60 × 10^-9 substitutions per site per generation). To obtain this estimate, we tested multiple dilution levels to maximize sequencing efficiency and developed bioinformatic filters needed to minimize false positives when a high-quality reference genome is not available. In addition to laying the groundwork for estimating genotypic variation in rates of somatic mutations within D. magna, we provide a framework for quantifying somatic mutations in other non-model systems, and also highlight recent innovations to single molecule sequencing that will help to further refine such estimates.

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. However, the relative performance of streptavidin and carboxyl microspheres is unknown. Here, we performed a head-to-head comparison of streptavidin and carboxyl microspheres. We compared antigen binding, orientation, and staining quality and found that both microspheres perform similarly based on these defined parameters. We also evaluated the utility of streptavidin microspheres bound to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD), to reliably detect RBD-specific IgG1, IgG3, and IgA1 produced in individuals recently immunized with Pfizer/BioNTech mRNA coronavirus disease (COVID) vaccine as 'proof-of-concept'. We provide evidence that each of the antibody targets are detectable in serum using RBD-coated microspheres, Ig-specific 'detector' monoclonal antibodies (mAbs), and flow cytometry. We found that cross-reactivity of the detector mAbs can be minimized by antibody titration to improve differentiation between IgG1 and IgG3. We also coated streptavidin microspheres with SARS-CoV-2 delta variant RBD to determine if the streptavidin microsphere approach revealed any differences in binding of immune serum antibodies to wild-type (Wuhan) versus variant RBD (Delta). Overall, our results show that streptavidin microspheres loaded with mono-biotinylated antigen is a robust alternative to chemically cross-linking antigen to carboxyl microspheres for use in serological assays.

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. Presented here is a modified and optimized protocol assessing exercise-induced muscle glucose uptake in mice in response to acute treadmill running. Using this high-throughput protocol, running capacity can accurately and reproducibly be determined in mice, and basal- and exercise-stimulated skeletal muscle glucose uptake and intracellular signaling can precisely and dose-dependently be measured in awake mice in vivo without the need for catheterization and with minimal loss of blood.

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). We designed and tested a mechanical device that presented prey in a controlled manner, taking advantage of a key stimulus for feeding: motion of the prey. High-speed video recordings show a rapid transition into TSPs starting 114 ms before TGB (N = 114). TSPs are then suppressed as early as 470-500 ms after TGB (P < 0.05) in unsuccessful hunts, while persisting for at least 3 s after TGB in successful hunts. A granularity analysis revealed significant differences in the large-scale high-contrast body patterning present in TSPs compared to the camouflage body pattern deployed beforehand. TSPs best fit the category of secondary defense called deimatic displaying, meant to briefly startle predators and interrupt their attack sequence while cuttlefish are distracted by striking prey. We characterize TSPs as a pattern category for which the main distinguishing feature is a high-contrast signaling pattern with aspects of Acute Conflict Mottle or Acute Disruptive Pattern. The data and methodology presented here open opportunities for quantifying the rapid neural responses in this visual sensorimotor set of behaviors.