A comprehensive platform for analyzing longitudinal multi-omics data.

Longitudinal bulk and single-cell omics data is increasingly generated for biological and clinical research but is challenging to analyze due to its many intrinsic types of variations. We present PALMO ( https://github.com/aifimmunology/PALMO ), a platform that contains five analytical modules to examine longitudinal bulk and single-cell multi-omics data from multiple perspectives, including decomposition of sources of variations within the data, collection of stable or variable features across timepoints and participants, identification of up- or down-regulated markers across timepoints of individual participants, and investigation on samples of same participants for possible outlier events. We have tested PALMO performance on a complex longitudinal multi-omics dataset of five data modalities on the same samples and six external datasets of diverse background. Both PALMO and our longitudinal multi-omics dataset can be valuable resources to the scientific community.

Optimized workflow for human PBMC multiomic immunosurveillance studies.

Deep immune profiling is essential for understanding the human immune system in health and disease. Successful biological interpretation of this data requires consistent laboratory processing with minimal batch-to-batch variation. Here, we detail a robust pipeline for the profiling of human peripheral blood mononuclear cells by both high-dimensional flow cytometry and single-cell RNA-seq. These protocols reduce batch effects, generate reproducible data, and increase throughput. For complete details on the use and execution of this protocol, please refer to Savage et al. (2021).

Multimodal analysis for human ex vivo studies shows extensive molecular changes from delays in blood processing.

Multi-omic profiling of human peripheral blood is increasingly utilized to identify biomarkers and pathophysiologic mechanisms of disease. The importance of these platforms in clinical and translational studies led us to investigate the impact of delayed blood processing on the numbers and state of peripheral blood mononuclear cells (PBMC) and on the plasma proteome. Similar to previous studies, we show minimal effects of delayed processing on the numbers and general phenotype of PBMC up to 18 hours. In contrast, profound changes in the single-cell transcriptome and composition of the plasma proteome become evident as early as 6 hours after blood draw. These reflect patterns of cellular activation across diverse cell types that lead to progressive distancing of the gene expression state and plasma proteome from native in vivo biology. Differences accumulating during an overnight rest (18 hours) could confound relevant biologic variance related to many underlying disease states.

Simultaneous trimodal single-cell measurement of transcripts, epitopes, and chromatin accessibility using TEA-seq.

Single-cell measurements of cellular characteristics have been instrumental in understanding the heterogeneous pathways that drive differentiation, cellular responses to signals, and human disease. Recent advances have allowed paired capture of protein abundance and transcriptomic state, but a lack of epigenetic information in these assays has left a missing link to gene regulation. Using the heterogeneous mixture of cells in human peripheral blood as a test case, we developed a novel scATAC-seq workflow that increases signal-to-noise and allows paired measurement of cell surface markers and chromatin accessibility: integrated cellular indexing of chromatin landscape and epitopes, called ICICLE-seq. We extended this approach using a droplet-based multiomics platform to develop a trimodal assay that simultaneously measures transcriptomics (scRNA-seq), epitopes, and chromatin accessibility (scATAC-seq) from thousands of single cells, which we term TEA-seq. Together, these multimodal single-cell assays provide a novel toolkit to identify type-specific gene regulation and expression grounded in phenotypically defined cell types.

Whole exome sequencing in the rat.

BACKGROUND: The rat genome was sequenced in 2004 with the aim to improve human health altered by disease and environmental influences through gene discovery and animal model validation. Here, we report development and testing of a probe set for whole exome sequencing (WES) to detect sequence variants in exons and UTRs of the rat genome. Using an in-silico approach, we designed probes targeting the rat exome and compared captured mutations in cancer-related genes from four chemically induced rat tumor cell lines (C6, FAT7, DSL-6A/C1, NBTII) to validated cancer genes in the human database, Catalogue of Somatic Mutations in Cancer (COSMIC) as well as normal rat DNA. Paired, fresh frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) liver tissue from naive rats were sequenced to confirm known dbSNP variants and identify any additional variants. RESULTS: Informatics analysis of available gene annotation from rat RGSC6.0/rn6 RefSeq and Ensembl transcripts provided 223,636 unique exons representing a total of 26,365 unique genes and untranslated regions. Using this annotation and the Rn6 reference genome, an in-silico probe design generated 826,878 probe sequences of which 94.2% were uniquely aligned to the rat genome without mismatches. Further informatics analysis revealed 25,249 genes (95.8%) covered by at least one probe and 23,603 genes (93.5%) had every exon covered by one or more probes. We report high performance metrics from exome sequencing of our probe set and Sanger validation of annotated, highly relevant, cancer gene mutations as cataloged in the human COSMIC database, in addition to several exonic variants in cancer-related genes. CONCLUSIONS: An in-silico probe set was designed to enrich the rat exome from isolated DNA. The platform was tested on rat tumor cell lines and normal FF and FFPE liver tissue. The method effectively captured target exome regions in the test DNA samples with exceptional sensitivity and specificity to obtain reliable sequencing data representing variants that are likely chemically induced somatic mutations. Genomic discovery conducted by means of high throughput WES queries should benefit investigators in discovering rat genomic variants in disease etiology and in furthering human translational research.

Firefighter feedback during active cooling: a useful tool for heat stress management?

Monitoring an individual's thermic state in the workplace requires reliable feedback of their core temperature. However, core temperature measurement technology is expensive, invasive and often impractical in operational environments, warranting investigation of surrogate measures which could be used to predict core temperature. This study examines an alternative measure of an individual's thermic state, thermal sensation, which presents a more manageable and practical solution for Australian firefighters operating on the fireground. Across three environmental conditions (cold, warm, hot & humid), 49 Australian volunteer firefighters performed a 20-min fire suppression activity, immediately followed by 20 min of active cooling using hand and forearm immersion techniques. Core temperature (Tc) and thermal sensation (TS) were measured across the rehabilitation period at five minute intervals. Despite the decline in Tc and TS throughout the rehabilitation period, there was little similarity in the magnitude or rate of decline between each measure in any of the ambient conditions. Moderate to strong correlations existed between Tc and TS in the cool (0.41, p<0.05) and hot & humid (0.57, p<0.05) conditions, however this was resultant in strong correlation during the earlier stages of rehabilitation (first five minutes), which were not evident in the latter stages. Linear regression revealed TS to be a poor predictor of Tc in all conditions (SEE=0.45-0.54°C) with a strong trend for TS to over-predict Tc (77-80% of the time). There is minimal evidence to suggest that ratings of thermal sensation, which represent a psychophysical assessment of an individual's thermal comfort, are an accurate reflection of the response of an individual's core temperature. Ratings of thermal sensation can be highly variable amongst individuals, likely moderated by local skin temperature. In account of these findings, fire managers require a more reliable source of information to guide decisions of heat stress management.

Muscle activation during the Pack Hike test and a critical wildfire fighting task.

The aim of this study was to examine the muscle activation of six global muscles during the successful completion of the Pack Hike test (PHT) and compare this to muscle activations during a critical wildfire fighting task. In-field surface electromyography was recorded from eight male wildfire fighters during the PHT and the rakehoe task - a critical wildfire suppression activity. All participants successfully completed the PHT within the 45-min time limit. No significant changes in peak muscle activation levels as well as no significant shifts in median frequency in the six muscle analysed were recorded during the 4.83-km hike. Significantly different peak muscle activation levels were recorded in four of the six muscles tested when the PHT was compared to the rakehoe task. These results suggest the PHT should not be administered in isolation and other tests that specifically challenge upper body muscle endurance should be incorporated into a battery that accurately assesses the job-specific fitness of wildfire fighters.

Validating 'fit for duty' tests for Australian volunteer fire fighters suppressing bushfires.

INTRODUCTION: This study compared fire fighter's work-rates and performance between the Pack Hike Test (PHT), Field Walk Test (FWT) and critical bushfire suppression tasks. METHODS: Nineteen volunteer fire fighters undertook the PHT, whilst a further 11 also performed the FWT. All 30 fire fighters completed four critical tasks. Physical, physiological and subjective ratings were measured during tasks and tests. RESULTS: Peak and mean heart rate during the Hose Drag was lower than during the FWT. Mean velocities for the PHT and FWT were higher than in all tasks except Hose Drag. Finishing times in the PHT and FWT were strongly correlated with finishing times for three and four of the critical tasks. CONCLUSIONS: The PHT and FWT may be valid fit for duty tests for Australian rural fire fighters though the clear differences between the tests and some tasks may prompt fire agencies to consider other tests that more accurately simulate bushfire suppression work.

Identification of physically demanding tasks performed during bushfire suppression by Australian rural firefighters.

PURPOSE: To identify and characterize the physically demanding tasks undertaken during multi-day wildfire (known as bushfire in Australia) suppression by Australian rural firefighters. METHODS: During semi-structured group interviews, thirty-one experienced male firefighters reviewed 53 firefighting tasks that could be performed during tanker-based bushfire suppression. Participants were asked to nominate the most physically demanding tasks and then define their typical frequencies, durations, operational importance and the dominant actions and activity types in each task. RESULTS: Seven tasks were identified as physically demanding. They were further categorized into three hose and four handtool (e.g., rakehoe) related activities. These tasks were assessed as moderately important to critical and were thought to occur less than one up to 700 times in a four-month bushfire 'season'. Each task's duration was estimated to last approximately 2-30 min depending on the task. Dominant actions were carry, drag, dig/rake actions in seven, three and four of the demanding tasks, respectively. 'Strength-endurance' was the dominant activity type for five of the seven tasks. CONCLUSION: Seven fireground tasks, three using a hose and four using handtools were classified as physically demanding by incumbent firefighters. The combination of hose and handtool work indicates that the tanker-based bushfire suppression tactics used by Australian rural firefighters appears to be a hybrid of structural and wildfire firefighting techniques and may require a dedicated physiological analyses before the job demands for these firefighters can be quantified.