A standardized and reproducible method to measure decision-making in mice, bioRxiv, 2020-01-18

AbstractProgress in neuroscience is hindered by poor reproducibility of mouse behavior. Here we show that in a visual decision making task, reproducibility can be achieved by automating the training protocol and by standardizing experimental hardware, software, and procedures. We trained 101 mice in this task across seven laboratories at six different research institutions in three countries, and obtained 3 million mouse choices. In trained mice, variability in behavior between labs was indistinguishable from variability within labs. Psychometric curves showed no significant differences in visual threshold, bias, or lapse rates across labs. Moreover, mice across laboratories adopted similar strategies when stimulus location had asymmetrical probability that changed over time. We provide detailed instructions and open-source tools to set up and implement our method in other laboratories. These results establish a new standard for reproducibility of rodent behavior and provide accessible tools for the study of decision making in mice.

biorxiv neuroscience 200-500-users 2020

A simple, cost-effective, and robust method for rRNA depletion in RNA-sequencing studies, bioRxiv, 2020-01-07

AbstractThe profiling of gene expression by RNA-sequencing (RNA-seq) has enabled powerful studies of global transcriptional patterns in all organisms, including bacteria. Because the vast majority of RNA in bacteria is ribosomal RNA (rRNA), it is standard practice to deplete the rRNA from a total RNA sample such that the reads in an RNA-seq experiment derive predominantly from mRNA. One of the most commonly used commercial kits for rRNA depletion, the Ribo-Zero kit from Illumina, was recently discontinued. Here, we report the development a simple, cost-effective, and robust method for depleting rRNA that can be easily implemented by any lab or facility. We first developed an algorithm for designing biotinylated oligonucleotides that will hybridize tightly and specifically to the 23S, 16S, and 5S rRNAs from any species of interest. Precipitation of these oligonucleotides bound to rRNA by magnetic streptavidin beads then depletes rRNA from a complex, total RNA sample such that ~75-80% of reads in a typical RNA-seq experiment derive from mRNA. Importantly, we demonstrate a high correlation of RNA abundance or fold-change measurements in RNA-seq experiments between our method and the previously available Ribo-Zero kit. Complete details on the methodology are provided, including open-source software for designing oligonucleotides optimized for any bacterial species or metagenomic sample of interest.ImportanceThe ability to examine global patterns of gene expression in microbes through RNA-sequencing has fundamentally transformed microbiology. However, RNA-seq depends critically on the removal of ribosomal RNA from total RNA samples. Otherwise, rRNA would comprise upwards of 90% of the reads in a typical RNA-seq experiment, limiting the reads coming from messenger RNA or requiring high total read depth. A commonly used, kit for rRNA subtraction from Illumina was recently discontinued. Here, we report the development of a ‘do-it-yourself’ kit for rapid, cost-effective, and robust depletion of rRNA from total RNA. We present an algorithm for designing biotinylated oligonucleotides that will hybridize to the rRNAs from a target set of species. We then demonstrate that the designed oligos enable sufficient rRNA depletion to produce RNA-seq data with 75-80% of reads comming from mRNA. The methodology presented should enable RNA-seq studies on any species or metagenomic sample of interest.

biorxiv microbiology 200-500-users 2020

The predictive power of the microbiome exceeds that of genome-wide association studies in the discrimination of complex human disease, bioRxiv, 2020-01-02

AbstractOver the past decade, studies of the human genome and microbiome have deepened our understanding of the connections between human genes, environments, microbes, and disease. For example, the sheer number of indicators of the microbiome and human genetic common variants associated with disease has been immense, but clinical utility has been elusive. Here, we compared the predictive capabilities of the human microbiome versus human genomic common variants across 13 common diseases. We concluded that microbiomic indicators outperform human genetics in predicting host phenotype (overall Microbiome-Association-Study [MAS] area under the curve [AUC] = 0.79 [SE = 0.03], overall Genome-Wide-Association-Study [GWAS] AUC = 0.67 [SE = 0.02]). Our results, while preliminary and focused on a subset of the totality of disease, demonstrate the relative predictive ability of the microbiome, indicating that it may outperform human genetics in discriminating human disease cases and controls. They additionally motivate the need for population-level microbiome sequencing resources, akin to the UK Biobank, to further improve and reproduce metagenomic models of disease.

biorxiv bioinformatics 200-500-users 2020

Species-specific developmental timing is associated with global differences in protein stability in mouse and human, bioRxiv, 2020-01-01

ABSTRACTWhat determines the pace of embryonic development? Although many molecular mechanisms controlling developmental processes are evolutionarily conserved, the speed at which these operate can vary substantially between species. For example, the same genetic programme, comprising sequential changes in transcriptional states, governs the differentiation of motor neurons in mouse and human, but the tempo at which it operates differs between species. Using in vitro directed differentiation of embryonic stem cells to motor neurons, we show that the programme runs twice as fast in mouse as in human. We provide evidence that this is neither due to differences in signalling, nor the genomic sequence of genes or their regulatory elements. Instead, we find an approximately two-fold increase in protein stability and cell cycle duration in human cells compared to mouse. This can account for the slower pace of human development, indicating that global differences in key kinetic parameters play a major role in interspecies differences in developmental tempo.

biorxiv developmental-biology 200-500-users 2020

Perturbation of effector and regulatory T cell subsets in Myalgic EncephalomyelitisChronic Fatigue Syndrome (MECFS), bioRxiv, 2019-12-26

AbstractMyalgic encephalomyelitischronic fatigue syndrome (MECFS) is a debilitating disorder of unknown etiology, and diagnosis of the disease is largely based on clinical symptoms. We hypothesized that immunological disruption is the major driver of this disease and analyzed a large cohort of MECFS patient or control blood samples for differences in T cell subset frequencies and functions. We found that the ratio of CD4+ to CD8+ T cells and the proportion of CD8+ effector memory T cells were increased, whereas NK cells were reduced in MECFS patients younger than 50 years old compared to a healthy control group. Remarkably, major differences were observed in Th1, Th2, Th17 and mucosal-associated invariant T (MAIT) T cell subset functions across all ages of patients compared to healthy subjects. While CCR6+ Th17 cells in MECFS secreted less IL-17 compared to controls, their overall frequency was higher. Similarly, MAIT cells from patients secreted lower IFNγ, GranzymeA and IL-17 upon activation. Together, these findings suggest chronic stimulation of these T cell populations in MECFS patients. In contrast, the frequency of regulatory T cells (Tregs), which control excessive immune activation, was higher in MECFS patients. Finally, using a machine learning algorithm called random forest, we determined that the set of T cell parameters analyzed could identify more than 90% of the subjects in the MECFS cohort as patients (93% true positive rate or sensitivity). In conclusion, these multiple and major perturbations or dysfunctions in T cell subsets in MECFS patients suggest potential chronic infections or microbiome dysbiosis. These findings also have implications for development of MECFS specific immune biomarkers and reveal potential targets for novel therapeutic interventions.

biorxiv immunology 200-500-users 2019

On-demand spatiotemporal programming of collective cell migration via bioelectric stimulation, bioRxiv, 2019-12-23

Directed cell migration is critical across biological processes spanning healing to cancer invasion, yet no tools allow such migration to be interactively guided. We present a new bioreactor that harnesses electrotaxis—directed cell migration along electric field gradients—by integrating multiple independent electrodes under computer control to dynamically program electric field patterns, and hence steer cell migration. Using this platform, we programmed and characterized multiple precise, two-dimensional collective migration maneuvers in renal epithelia and primary skin keratinocyte ensembles. First, we demonstrated on-demand, 90-degree collective turning. Next, we developed a universal electrical stimulation scheme capable of programming arbitrary 2D migration maneuvers such as precise angular turns and directing cells to migrate in a complete circle. Our stimulation scheme proves that cells effectively timeaverage electric field cues, helping to elucidate the transduction time scales in electrotaxis. Together, this work represents a fundamentally different platform for controlling cell migration with broad utility across fields.

biorxiv bioengineering 200-500-users 2019