Residual OXPHOS is required to drive primary and metastatic lung tumours in an orthotopic breast cancer model.

Background: Fast adaptation of glycolytic and mitochondrial energy pathways to changes in the tumour microenvironment is a hallmark of cancer. Purely glycolytic ρ0 tumour cells do not form primary tumours unless they acquire healthy mitochondria from their micro-environment. Here we explored the effects of severely compromised respiration on the metastatic capability of 4T1 mouse breast cancer cells. Methods: 4T1 cell lines with different levels of respiratory capacity were generated; the Seahorse extracellular flux analyser was used to evaluate oxygen consumption rates, fluorescent confocal microscopy to assess the number of SYBR gold-stained mitochondrial DNA nucleoids, and the presence of the ATP5B protein in the cytoplasm and fluorescent in situ nuclear hybridization was used to establish ploidy. MinION nanopore RNA sequence analysis was used to compare mitochondrial DNA transcription between cell lines. Orthotopic injection was used to determine the ability of cells to metastasize to the lungs of female Balb/c mice. Results: OXPHOS-deficient ATP5B-KO3.1 cells did not generate primary tumours. Severely OXPHOS compromised ρ0D5 cells generated both primary tumours and lung metastases. Cells generated from lung metastasis of both OXPHOS-competent and OXPHOS-compromised cells formed primary tumours but no metastases when re-injected into mice. OXPHOS-compromised cells significantly increased their mtDNA content, but this did not result in increased OXPHOS capacity, which was not due to decreased mtDNA transcription. Gene set enrichment analysis suggests that certain cells derived from lung metastases downregulate their epithelial-to-mesenchymal related pathways. Conclusion: In summary, OXPHOS is required for tumorigenesis in this orthotopic mouse breast cancer model but even very low levels of OXPHOS are sufficient to generate both primary tumours and lung metastases.

Organic self-talk content categories and their relations to sport-specific contextual and personal factors.

While strategic self-talk interventions are well documented, less is known regarding organically occurring self-talk. Previously, eight organic self-talk content categories were identified, however contextual and personal factors relating to these categories is unexplored. The aim of the study was to explore the relationships between stress, coping, athletic identity, demographic and sport-related factors and the eight organic self-talk categories of anxiety control, confidence, disengagement, instruction, psych-up, somatic fatigue, worry, and irrelevant. 216 NCAA student-athletes were recruited. Regression was used to determine the relationships between the contextual and personal variables and organic self-talk. Stress, coping and self-talk categories were compared in and out-of-season. Stress was associated with all self-talk frequencies except confidence. Coping was associated with psych-up, confidence and worry. Athletic identity was associated with psych-up, confidence, instruction and disengagement. Age was associated with anxiety control and instruction. Lastly, sport type and division level were associated with disengagement and instruction self-talk respectively.

"Switching the mind off completely" - Understanding the psychology of rest in coaches.

OBJECTIVES: To better understand the psychology of rest in coaches. Rest appears to be important for coping, recovery, and well-being in coaches, yet there is limited research on and in turn understanding of this concept in this population. DESIGN: A qualitative description study design was employed. METHOD: 22 NCAA Division I coaches were interviewed about what rest means to them, key barriers to rest in coaching, and strategies employed to obtain rest in the face of these barriers. A codebook thematic analysis was undertaken to examine the analytical generalizability (Smith, 2018) to the coaching context of an extant model of the psychology of rest in athletes (Eccles & Kazmier, 2019). RESULTS: The Eccles-Kazmier model appears to offer some analytical generalizability to the coaching context. Consistent with the model, the process of resting in coaches involves both sleep and resting while awake. Resting while awake involves (a) a break from thinking about work, (b) a break from effortful thinking generally, and (c) engaging in life outside coaching. However, departures from the model were also observed; specifically, unlike for athletes, the wakeful resting process for coaches does not appear to involve assuming control or experiencing variety. CONCLUSION: The findings advance the current understanding of the constituents of rest in coaches and can inform coach education about how to obtain the rest needed to perform effectively and stay healthy.

Real-time genomics for One Health.

The ongoing degradation of natural systems and other environmental changes has put our society at a crossroad with respect to our future relationship with our planet. While the concept of One Health describes how human health is inextricably linked with environmental health, many of these complex interdependencies are still not well-understood. Here, we describe how the advent of real-time genomic analyses can benefit One Health and how it can enable timely, in-depth ecosystem health assessments. We introduce nanopore sequencing as the only disruptive technology that currently allows for real-time genomic analyses and that is already being used worldwide to improve the accessibility and versatility of genomic sequencing. We showcase real-time genomic studies on zoonotic disease, food security, environmental microbiome, emerging pathogens, and their antimicrobial resistances, and on environmental health itself - from genomic resource creation for wildlife conservation to the monitoring of biodiversity, invasive species, and wildlife trafficking. We stress why equitable access to real-time genomics in the context of One Health will be paramount and discuss related practical, legal, and ethical limitations.

Creation of High-Dimensional Reduction Analysis-Compatible Histocytometry Files from Images of Densely-Packed Cells and/or Variable Stain Intensity.

The power of high-dimensional reduction techniques using multiparameter images has been demonstrated across a variety of different publications. Recently, we published an end-to-end low-cost GUI-based protocol for performing histocytometric spatial analysis on images derived from the most common microscope image formats. However, this protocol is limited by the normalized marker intensity outputs and the difficulty in processing images of highly aggregated and/or exceptionally heterogenous cell populations. Here we present the basic protocols required to construct an advanced histocytometric data file using only freeware. This data file is compatible with images containing cell nuclei clusters that are difficult to segment, and results in histocytometry files retaining the original marker intensity values of the microscopic images they were derived from. This is especially useful in cells that are phenotyped based on relative marker expression levels. Histocytometry data files produced by these protocols are compatible with high-dimensional reduction analysis using marker intensity data, such as tSNEs. This methodology is showcased using stitched microscopic images of murine lymph nodes, complex organs with highly aggregated heterogenous cell populations, that are typically difficult to segment. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Image preprocessing and generation of nuclei marker probability maps Basic Protocol 2: Cell segmentation using ilastik-derived probability maps Basic Protocol 3: Generation of histocytometric .fcs files.

Bioenergetic and Metabolic Adaptation in Tumor Progression and Metastasis.

The ability of cancer cells to adjust their metabolism in response to environmental changes is a well-recognized hallmark of cancer. Diverse cancer and non-cancer cells within tumors compete for metabolic resources. Metabolic demands change frequently during tumor initiation, progression and metastasis, challenging our quest to better understand tumor biology and develop novel therapeutics. Vascularization, physical constraints, immune responses and genetic instability promote tumor evolution resulting in immune evasion, opportunities to breach basement membrane barriers and spread through the circulation and lymphatics. In addition, the unfolded protein response linked to the ubiquitin proteasome system is a key player in addressing stoichiometric imbalances between nuclear and mitochondrially-encoded protein subunits of respiratory complexes, and nuclear-encoded mitochondrial ribosomal protein subunits. While progressive genetic changes, some of which affect metabolic adaptability, contribute to tumorigenesis and metastasis through clonal expansion, epigenetic changes are also important and more dynamic in nature. Understanding the role of stromal and immune cells in the tumor microenvironment in remodeling cancer cell energy metabolism has become an increasingly important area of research. In this perspective, we discuss the adaptations made by cancer cells to balance mitochondrial and glycolytic energy metabolism. We discuss how hypoxia and nutrient limitations affect reductive and oxidative stress through changes in mitochondrial electron transport activity. We propose that integrated responses to cellular stress in cancer cells are central to metabolic flexibility in general and bioenergetic adaptability in particular and are paramount in tumor progression and metastasis.

Transcription Factors Active in the Anterior Blastema of Schmidtea mediterranea.

Regeneration, the restoration of body parts after injury, is quite widespread in the animal kingdom. Species from virtually all Phyla possess regenerative abilities. Human beings, however, are poor regenerators. Yet, the progress of knowledge and technology in the fields of bioengineering, stem cells, and regenerative biology have fostered major advancements in regenerative medical treatments, which aim to regenerate tissues and organs and restore function. Human induced pluripotent stem cells can differentiate into any cell type of the body; however, the structural and cellular complexity of the human tissues, together with the inability of our adult body to control pluripotency, require a better mechanistic understanding. Planarians, with their capacity to regenerate lost body parts thanks to the presence of adult pluripotent stem cells could help providing such an understanding. In this paper, we used a top-down approach to shortlist blastema transcription factors (TFs) active during anterior regeneration. We found 44 TFs-31 of which are novel in planarian-that are expressed in the regenerating blastema. We analyzed the function of half of them and found that they play a role in the regeneration of anterior structures, like the anterior organizer, the positional instruction muscle cells, the brain, the photoreceptor, the intestine. Our findings revealed a glimpse of the complexity of the transcriptional network governing anterior regeneration in planarians, confirming that this animal model is the perfect playground to study in vivo how pluripotency copes with adulthood.

The Modified Imitation Game: A Method for Measuring Interactional Expertise.

The study of the sociology of scientific knowledge distinguishes between contributory and interactional experts. Contributory experts have practical expertise-they can "walk the walk." Interactional experts have internalized the tacit components of expertise-they can "talk the talk" but are not able to reliably "walk the walk." Interactional expertise permits effective communication between contributory experts and others (e.g., laypeople), which in turn facilitates working jointly toward shared goals. Interactional expertise is attained through long-term immersion into the expert community in question. To assess interactional expertise, researchers developed the imitation game-a variant of the Turing test-to test whether a person, or a particular group, possesses interactional expertise of another. The imitation game, which has been used mainly in sociology to study the social nature of knowledge, may also be a useful tool for researchers who focus on cognitive aspects of expertise. In this paper, we introduce a modified version of the imitation game and apply it to examine interactional expertise in the context of blindness. Specifically, we examined blind and sighted individuals' ability to imitate each other in a street-crossing scenario. In Phase I, blind and sighted individuals provided verbal reports of their thought processes associated with crossing a street-once while imitating the other group (i.e., as a pretender) and once responding genuinely (i.e., as a non-pretender). In Phase II, transcriptions of the reports were judged as either genuine or imitated responses by a different set of blind and sighted participants, who also provided the reasoning for their decisions. The judges comprised blind individuals, sighted orientation-and-mobility specialists, and sighted individuals with infrequent socialization with blind individuals. Decision data were analyzed using probit mixed models for signal-detection-theory indices. Reasoning data were analyzed using natural-language-processing (NLP) techniques. The results revealed evidence that interactional expertise (i.e., relevant tacit knowledge) can be acquired by immersion in the group that possesses and produces the expert knowledge. The modified imitation game can be a useful research tool for measuring interactional expertise within a community of practice and evaluating practitioners' understanding of true experts.

Homeostatic IL-13 in healthy skin directs dendritic cell differentiation to promote TH2 and inhibit TH17 cell polarization.

The signals driving the adaptation of type 2 dendritic cells (DC2s) to diverse peripheral environments remain mostly undefined. We show that differentiation of CD11blo migratory DC2s-a DC2 population unique to the dermis-required IL-13 signaling dependent on the transcription factors STAT6 and KLF4, whereas DC2s in lung and small intestine were STAT6-independent. Similarly, human DC2s in skin expressed an IL-4 and IL-13 gene signature that was not found in blood, spleen and lung DCs. In mice, IL-13 was secreted homeostatically by dermal innate lymphoid cells and was independent of microbiota, TSLP or IL-33. In the absence of IL-13 signaling, dermal DC2s were stable in number but remained CD11bhi and showed defective activation in response to allergens, with diminished ability to support the development of IL-4+GATA3+ helper T cells (TH), whereas antifungal IL-17+RORγt+ TH cells were increased. Therefore, homeostatic IL-13 fosters a noninflammatory skin environment that supports allergic sensitization.

Expanding the conservation genomics toolbox: Incorporating structural variants to enhance genomic studies for species of conservation concern.

Structural variants (SVs) are large rearrangements (>50 bp) within the genome that impact gene function and the content and structure of chromosomes. As a result, SVs are a significant source of functional genomic variation, that is, variation at genomic regions underpinning phenotype differences, that can have large effects on individual and population fitness. While there are increasing opportunities to investigate functional genomic variation in threatened species via single nucleotide polymorphism (SNP) data sets, SVs remain understudied despite their potential influence on fitness traits of conservation interest. In this future-focused Opinion, we contend that characterizing SVs offers the conservation genomics community an exciting opportunity to complement SNP-based approaches to enhance species recovery. We also leverage the existing literature-predominantly in human health, agriculture and ecoevolutionary biology-to identify approaches for readily characterizing SVs and consider how integrating these into the conservation genomics toolbox may transform the way we manage some of the world's most threatened species.

The MinION as a cost-effective technology for diagnostic screening of the SCN1A gene in epilepsy patients.

The MinION is a portable DNA sequencer that allows real time sequencing at low capital cost investment. We assessed accuracy and cost-effectivess of the MinION for genetic diagnostic testing of known SCN1A mutations that cause Dravet Syndrome (DS). DNA samples (n = 7) from DS patients previously shown to carry SCN1A mutations via Ion Torrent and Sanger sequencing were sequenced using the MinION. SCN1A amplicons for 8 exons were sequenced using the MinION with 1D chemistry on an R9.4 flow cell. All known missense mutations were detected in all samples showing 100 % concordance with results from other methods. However, the MinION failed to detect the insertions/deletions (INDELs) present in these patients. Nevertheless, these results indicate that MinION is a cost-effective platform for use as an initial screening step in the detection of nucleotide substitution mutations in in SCN1A, especially in under-resourced laboratories or hospitals. Further improvements are required to reliably detect INDELS in this gene.

Capturing SNP Association across the NK Receptor and HLA Gene Regions in Multiple Sclerosis by Targeted Penalised Regression Models.

Conventional genome-wide association studies (GWASs) of complex traits, such as Multiple Sclerosis (MS), are reliant on per-SNP p-values and are therefore heavily burdened by multiple testing correction. Thus, in order to detect more subtle alterations, ever increasing sample sizes are required, while ignoring potentially valuable information that is readily available in existing datasets. To overcome this, we used penalised regression incorporating elastic net with a stability selection method by iterative subsampling to detect the potential interaction of loci with MS risk. Through re-analysis of the ANZgene dataset (1617 cases and 1988 controls) and an IMSGC dataset as a replication cohort (1313 cases and 1458 controls), we identified new association signals for MS predisposition, including SNPs above and below conventional significance thresholds while targeting two natural killer receptor loci and the well-established HLA loci. For example, rs2844482 (98.1% iterations), otherwise ignored by conventional statistics (p = 0.673) in the same dataset, was independently strongly associated with MS in another GWAS that required more than 40 times the number of cases (~45 K). Further comparison of our hits to those present in a large-scale meta-analysis, confirmed that the majority of SNPs identified by the elastic net model reached conventional statistical GWAS thresholds (p < 5 × 10-8) in this much larger dataset. Moreover, we found that gene variants involved in oxidative stress, in addition to innate immunity, were associated with MS. Overall, this study highlights the benefit of using more advanced statistical methods to (re-)analyse subtle genetic variation among loci that have a biological basis for their contribution to disease risk.

Mitochondrial DNA Affects the Expression of Nuclear Genes Involved in Immune and Stress Responses in a Breast Cancer Model.

Tumor cells without mitochondrial (mt) DNA (ρ0 cells) are auxotrophic for uridine, and their growth is supported by pyruvate. While ATP synthesis in ρ0 cells relies on glycolysis, they fail to form tumors unless they acquire mitochondria from stromal cells. Mitochondrial acquisition restores respiration that is essential for de novo pyrimidine biosynthesis and for mitochondrial ATP production. The physiological processes that underpin intercellular mitochondrial transfer to tumor cells lacking mtDNA and the metabolic remodeling and restored tumorigenic properties of cells that acquire mitochondria are not well understood. Here, we investigated the changes in mitochondrial and nuclear gene expression that accompany mtDNA deletion and acquisition in metastatic murine 4T1 breast cancer cells. Loss of mitochondrial gene expression in 4T1ρ0 cells was restored in cells recovered from subcutaneous tumors that grew from 4T1ρ0 cells following acquisition of mtDNA from host cells. In contrast, the expression of most nuclear genes that encode respiratory complex subunits and mitochondrial ribosomal subunits was not greatly affected by loss of mtDNA, indicating ineffective mitochondria-to-nucleus communication systems for these nuclear genes. Further, analysis of nuclear genes whose expression was compromised in 4T1ρ0 cells showed that immune- and stress-related genes were the most highly differentially expressed, representing over 70% of those with greater than 16-fold higher expression in 4T1 compared with 4T1ρ0 cells. The monocyte recruiting chemokine, Ccl2, and Psmb8, a subunit of the immunoproteasome that generates MHCI-binding peptides, were the most highly differentially expressed. Early monocyte/macrophage recruitment into the tumor mass was compromised in 4T1ρ0 cells but recovered before mtDNA could be detected. Taken together, our results show that mitochondrial acquisition by tumor cells without mtDNA results in bioenergetic remodeling and re-expression of genes involved in immune function and stress adaptation.

Metagenomic analysis of planktonic riverine microbial consortia using nanopore sequencing reveals insight into river microbe taxonomy and function.

BACKGROUND: Riverine ecosystems are biogeochemical powerhouses driven largely by microbial communities that inhabit water columns and sediments. Because rivers are used extensively for anthropogenic purposes (drinking water, recreation, agriculture, and industry), it is essential to understand how these activities affect the composition of river microbial consortia. Recent studies have shown that river metagenomes vary considerably, suggesting that microbial community data should be included in broad-scale river ecosystem models. But such ecogenomic studies have not been applied on a broad "aquascape" scale, and few if any have applied the newest nanopore technology. RESULTS: We investigated the metagenomes of 11 rivers across 3 continents using MinION nanopore sequencing, a portable platform that could be useful for future global river monitoring. Up to 10 Gb of data per run were generated with average read lengths of 3.4 kb. Diversity and diagnosis of river function potential was accomplished with 0.5-1.0 ⋅ 106 long reads. Our observations for 7 of the 11 rivers conformed to other river-omic findings, and we exposed previously unrecognized microbial biodiversity in the other 4 rivers. CONCLUSIONS: Deeper understanding that emerged is that river microbial consortia and the ecological functions they fulfil did not align with geographic location but instead implicated ecological responses of microbes to urban and other anthropogenic effects, and that changes in taxa manifested over a very short geographic space.

A semi-automated technique for adenoma quantification in the ApcMin mouse using FeatureCounter.

Colorectal cancer is a major contributor to death and disease worldwide. The ApcMin mouse is a widely used model of intestinal neoplasia, as it carries a mutation also found in human colorectal cancers. However, the method most commonly used to quantify tumour burden in these mice is manual adenoma counting, which is time consuming and poorly suited to standardization across different laboratories. We describe a method to produce suitable photographs of the small intestine of ApcMin mice, process them with an ImageJ macro, FeatureCounter, which automatically locates image features potentially corresponding to adenomas, and a machine learning pipeline to identify and quantify them. Compared to a manual method, the specificity (or True Negative Rate, TNR) and sensitivity (or True Positive Rate, TPR) of this method in detecting adenomas are similarly high at about 80% and 87%, respectively. Importantly, total adenoma area measures derived from the automatically-called tumours were just as capable of distinguishing high-burden from low-burden mice as those established manually. Overall, our strategy is quicker, helps control experimenter bias, and yields a greater wealth of information about each tumour, thus providing a convenient route to getting consistent and reliable results from a study.

Associations of autozygosity with a broad range of human phenotypes.

In many species, the offspring of related parents suffer reduced reproductive success, a phenomenon known as inbreeding depression. In humans, the importance of this effect has remained unclear, partly because reproduction between close relatives is both rare and frequently associated with confounding social factors. Here, using genomic inbreeding coefficients (FROH) for >1.4 million individuals, we show that FROH is significantly associated (p < 0.0005) with apparently deleterious changes in 32 out of 100 traits analysed. These changes are associated with runs of homozygosity (ROH), but not with common variant homozygosity, suggesting that genetic variants associated with inbreeding depression are predominantly rare. The effect on fertility is striking: FROH equivalent to the offspring of first cousins is associated with a 55% decrease [95% CI 44-66%] in the odds of having children. Finally, the effects of FROH are confirmed within full-sibling pairs, where the variation in FROH is independent of all environmental confounding.

Tree Lab: Portable genomics for Early Detection of Plant Viruses and Pests in Sub-Saharan Africa.

In this case study we successfully teamed the PDQeX DNA purification technology developed by MicroGEM, New Zealand, with the MinION and MinIT mobile sequencing devices developed by Oxford Nanopore Technologies to produce an effective point-of-need field diagnostic system. The PDQeX extracts DNA using a cocktail of thermophilic proteinases and cell wall-degrading enzymes, thermo-responsive extractor cartridges and a temperature control unit. This closed system delivers purified DNA with no cross-contamination. The MinIT is a newly released data processing unit that converts MinION raw signal output into nucleotide base called data locally in real-time, removing the need for high-specification computers and large file transfers from the field. All three devices are battery powered with an exceptionally small footprint that facilitates transport and setup. To evaluate and validate capability of the system for unbiased pathogen identification by real-time sequencing in a farmer's field setting, we analysed samples collected from cassava plants grown by subsistence farmers in three sub-Sahara African countries (Tanzania, Uganda and Kenya). A range of viral pathogens, all with similar symptoms, greatly reduce yield or destroy cassava crops. Eight hundred (800) million people worldwide depend on cassava for food and yearly income, and viral diseases are a significant constraint to its production. Early pathogen detection at a molecular level has great potential to rescue crops within a single growing season by providing results that inform decisions on disease management, use of appropriate virus-resistant or replacement planting. This case study presented conditions of working in-field with limited or no access to mains power, laboratory infrastructure, Internet connectivity and highly variable ambient temperature. An additional challenge is that, generally, plant material contains inhibitors of downstream molecular processes making effective DNA purification critical. We successfully undertook real-time on-farm genome sequencing of samples collected from cassava plants on three farms, one in each country. Cassava mosaic begomoviruses were detected by sequencing leaf, stem, tuber and insect samples. The entire process, from arrival on farm to diagnosis, including sample collection, processing and provisional sequencing results was complete in under 3 h. The need for accurate, rapid and on-site diagnosis grows as globalized human activity accelerates. This technical breakthrough has applications that are relevant to human and animal health, environmental management and conservation.

Harnessing the MinION: An example of how to establish long-read sequencing in a laboratory using challenging plant tissue from Eucalyptus pauciflora.

Long-read sequencing technologies are transforming our ability to assemble highly complex genomes. Realizing their full potential is critically reliant on extracting high-quality, high-molecular-weight (HMW) DNA from the organisms of interest. This is especially the case for the portable MinION sequencer which enables all laboratories to undertake their own genome sequencing projects, due to its low entry cost and minimal spatial footprint. One challenge of the MinION is that each group has to independently establish effective protocols for using the instrument, which can be time-consuming and costly. Here, we present a workflow and protocols that enabled us to establish MinION sequencing in our own laboratories, based on optimizing DNA extraction from a challenging plant tissue as a case study. Following the workflow illustrated, we were able to reliably and repeatedly obtain >6.5 Gb of long-read sequencing data with a mean read length of 13 kb and an N50 of 26 kb. Our protocols are open source and can be performed in any laboratory without special equipment. We also illustrate some more elaborate workflows which can increase mean and average read lengths if this is desired. We envision that our workflow for establishing MinION sequencing, including the illustration of potential pitfalls and suggestions of how to adapt it to other tissue types, will be useful to others who plan to establish long-read sequencing in their own laboratories.