Lineage tracing reveals the phylodynamics, plasticity, and paths of tumor evolution.

Tumor evolution is driven by the progressive acquisition of genetic and epigenetic alterations that enable uncontrolled growth and expansion to neighboring and distal tissues. The study of phylogenetic relationships between cancer cells provides key insights into these processes. Here, we introduced an evolving lineage-tracing system with a single-cell RNA-seq readout into a mouse model of Kras;Trp53(KP)-driven lung adenocarcinoma and tracked tumor evolution from single-transformed cells to metastatic tumors at unprecedented resolution. We found that the loss of the initial, stable alveolar-type2-like state was accompanied by a transient increase in plasticity. This was followed by the adoption of distinct transcriptional programs that enable rapid expansion and, ultimately, clonal sweep of stable subclones capable of metastasizing. Finally, tumors develop through stereotypical evolutionary trajectories, and perturbing additional tumor suppressors accelerates progression by creating novel trajectories. Our study elucidates the hierarchical nature of tumor evolution and, more broadly, enables in-depth studies of tumor progression.

Single molecule infrared spectroscopy in the gas phase.

Spectroscopy is a key analytical tool that provides valuable insight into molecular structure and is widely used to identify chemical samples. Tagging spectroscopy is a form of action spectroscopy in which the absorption of a single photon by a molecular ion is detected via the loss of a weakly attached, inert 'tag' particle (for example, He, Ne, N2)1-3. The absorption spectrum is derived from the tag loss rate as a function of incident radiation frequency. So far, all spectroscopy of gas phase polyatomic molecules has been restricted to large molecular ensembles, thus complicating spectral interpretation by the presence of multiple chemical and isomeric species. Here we present a novel tagging spectroscopic scheme to analyse the purest possible sample: a single gas phase molecule. We demonstrate this technique with the measurement of the infrared spectrum of a single gas phase tropylium (C7H7+) molecular ion. The high sensitivity of our method revealed spectral features not previously observed using traditional tagging methods4. Our approach, in principle, enables analysis of multicomponent mixtures by identifying constituent molecules one at a time. Single molecule sensitivity extends action spectroscopy to rare samples, such as those of extraterrestrial origin5,6, or to reactive reaction intermediates formed at number densities that are too low for traditional action methods.

From Reductionism to Reintegration: Solving society's most pressing problems requires building bridges between data types across the life sciences.

Decades of reductionist approaches in biology have achieved spectacular progress, but the proliferation of subdisciplines, each with its own technical and social practices regarding data, impedes the growth of the multidisciplinary and interdisciplinary approaches now needed to address pressing societal challenges. Data integration is key to a reintegrated biology able to address global issues such as climate change, biodiversity loss, and sustainable ecosystem management. We identify major challenges to data integration and present a vision for a "Data as a Service"-oriented architecture to promote reuse of data for discovery. The proposed architecture includes standards development, new tools and services, and strategies for career-development and sustainability.

Coronary circulation enhances the aerobic performance of wild Pacific salmon.

Female Pacific salmon often experience higher mortality than males during their once-in-a-lifetime up-river spawning migration, particularly when exposed to secondary stressors (e.g. high temperatures). However, the underlying mechanisms remain unknown. One hypothesis is that female Pacific salmon hearts are more oxygen-limited than those of males and are less able to supply oxygen to the body's tissues during this demanding migration. Notably, female hearts have higher coronary blood flow, which could indicate a greater reliance on this oxygen source. Oxygen limitations can develop from naturally occurring coronary blockages (i.e. coronary arteriosclerosis) found in mature salmon hearts. If female hearts rely more heavily on coronary blood flow but experience similar arteriosclerosis levels as males, they will have disproportionately impaired aerobic performance. To test this hypothesis, we measured resting (RMR) and maximum metabolic rate (MMR), aerobic scope (AS) and acute upper thermal tolerance in coho salmon (Oncorhynchus kisutch) with an intact or artificially blocked coronary oxygen supply. We also assessed venous blood oxygen and chemistry (cortisol, ions and metabolite concentrations) at different time intervals during recovery from exhaustive exercise. We found that coronary blockage impaired MMR, AS and the partial pressure of oxygen in venous blood (PvO2) during exercise recovery but did not differ between sexes. Coronary ligation lowered acute upper thermal tolerance by 1.1°C. Although we did not find evidence of enhanced female reliance on coronary supply, our findings highlight the importance of coronary blood supply for mature wild salmon, where migration success may be linked to cardiac performance, particularly during warm water conditions.

Ecotoxicological soil risk assessment under the new soil exposure framework - an impact assessment.

For the European risk assessment (RA) for soil organisms exposed to plant protection products (PPPs) endpoints from ecotoxicological laboratory studies are compared with predicted environmental concentrations in soil (PECSOIL) at first tier. A safety margin must be met; otherwise, a higher tier RA is triggered (usually soil organism field studies). A new tiered exposure modeling guidance was published by EFSA to determine PECSOIL. This work investigates its potential impact on future soil RA. PECSOIL values for >50 active substances and metabolites were calculated and compared with the respective endpoints for soil organisms to calculate the RA failure rate. Compared to the current (FOCUS) exposure modeling, PECSOIL values for all EU regulatory zones considerably increased, e.g., resulting in active substance RA failure rates of 67%, 58% and 36% for modeling Tier-1, Tier-2 and Tier-3A, respectively. The main driving factors for elevated PECSOIL were soil bulk density, crop interception and wash-off, next to obligatory modeling and scenario adjustment factors. Spatial PECSOIL scenario selection procedures result in agronomically atypical soil characteristics (e.g., soil bulk density values in Tier-3A scenarios far below typical European agricultural areas). Consequently, exposure modeling and ecotoxicological study characteristics are inconsistent, which hinders scientifically reasonable comparison of both in the RA.

ZipSeq: barcoding for real-time mapping of single cell transcriptomes.

Spatial transcriptomics seeks to integrate single cell transcriptomic data within the three-dimensional space of multicellular biology. Current methods to correlate a cell's position with its transcriptome in living tissues have various limitations. We developed an approach, called 'ZipSeq', that uses patterned illumination and photocaged oligonucleotides to serially print barcodes ('zipcodes') onto live cells in intact tissues, in real time and with an on-the-fly selection of patterns. Using ZipSeq, we mapped gene expression in three settings: in vitro wound healing, live lymph node sections and a live tumor microenvironment. In all cases, we discovered new gene expression patterns associated with histological structures. In the tumor microenvironment, this demonstrated a trajectory of myeloid and T cell differentiation from the periphery inward. A combinatorial variation of ZipSeq efficiently scales in the number of regions defined, providing a pathway for complete mapping of live tissues, subsequent to real-time imaging or perturbation.

Low-Temperature Gas-Phase Kinetics of Ethanol-Methanol Heterodimer Formation.

The structures of gas-phase noncovalently bound clusters have long been studied in supersonic expansions. This method of study, while providing a wealth of information about the nature of noncovalent bonds, precludes observation of the formation of the cluster, as the clusters form just after the orifice of the pulsed valve. Here, we directly observe formation of ethanol-methanol dimers via microwave spectroscopy in a controlled cryogenic environment. Time profiles of the concentration of reagents in the cell yielded gas-phase reaction rate constants of kMe-g = (2.8 ± 1.4) × 10-13 cm3 molecule-1 s-1 and kMe-t = (1.6 ± 0.8) × 10-13 cm3 molecule-1 s-1 for the pseudo-second-order ethanol-methanol dimerization reaction at 8 K. The relaxation cross section between the gauche and trans conformers of ethanol was also measured using the same technique. In addition, thermodynamic relaxation between conformers of ethanol over time allowed for selection of conformer stoichiometry in the ethanol-methanol dimerization reaction, but no change in the ratio of dimer conformers was observed with changing ethanol monomer stoichiometry.

Metabolic constraints and individual variation shape the trade-off between physiological recovery and anti-predator responses in adult sockeye salmon.

Metabolic scope represents the aerobic energy budget available to an organism to perform non-maintenance activities (e.g., escape a predator, recover from a fisheries interaction, compete for a mate). Conflicting energetic requirements can give rise to ecologically relevant metabolic trade-offs when energy budgeting is constrained. The objective of this study was to investigate how aerobic energy is utilized when individual sockeye salmon (Oncorhynchus nerka) are exposed to multiple acute stressors. To indirectly assess metabolic changes in free-swimming individuals, salmon were implanted with heart rate biologgers. The animals were then exercised to exhaustion or briefly handled as a control, and allowed to recover from this stressor for 48 h. During the first 2 h of the recovery period, individual salmon were exposed to 90 ml of conspecific alarm cues or water as a control. Heart rate was recorded throughout the recovery period. Recovery effort and time was higher in exercised fish, relative to control fish, whereas exposure to an alarm cue had no effect on either of these metrics. Individual routine heart rate was negatively correlated with recovery time and effort. Together, these findings suggest that metabolic energy allocation towards exercise recovery (i.e., an acute stressor; handling, chase, etc.) trumps anti-predator responses in salmon, although individual variation may mediate this effect at the population level.

MULTI-seq: sample multiplexing for single-cell RNA sequencing using lipid-tagged indices.

Sample multiplexing facilitates scRNA-seq by reducing costs and identifying artifacts such as cell doublets. However, universal and scalable sample barcoding strategies have not been described. We therefore developed MULTI-seq: multiplexing using lipid-tagged indices for single-cell and single-nucleus RNA sequencing. MULTI-seq reagents can barcode any cell type or nucleus from any species with an accessible plasma membrane. The method involves minimal sample processing, thereby preserving cell viability and endogenous gene expression patterns. When cells are classified into sample groups using MULTI-seq barcode abundances, data quality is improved through doublet identification and recovery of cells with low RNA content that would otherwise be discarded by standard quality-control workflows. We use MULTI-seq to track the dynamics of T-cell activation, perform a 96-plex perturbation experiment with primary human mammary epithelial cells and multiplex cryopreserved tumors and metastatic sites isolated from a patient-derived xenograft mouse model of triple-negative breast cancer.

Kinopus chlorellivorus gen. nov., sp. nov. (Vampyrellida, Rhizaria), a New Algivorous Protist Predator Isolated from Large-Scale Outdoor Cultures of Chlorella sorokiniana.

The large-scale culture of low-cost algal biomass can be significantly affected by microbial grazing on the algae. To minimize the impact, it is necessary to manage the predators. In this study, we describe a new genus and species of vampyrellid amoeba, Kinopus chlorellivorus, which caused the loss of Chlorella sorokiniana in large-scale cultures. We assigned it to the family Leptophryidae (Vampyrellida) based on morphology and small-subunit (SSU) rRNA gene sequence comparisons. Using transmission electron microscopy, we found spherical lucent inclusions, which have not been reported for any leptophryids or other vampyrellids. The gene sequence of SSU rRNA did not match any recognized genera or species and contained four characteristic regions. K. chlorellivorus preys on algae by engulfment. Laboratory feeding experiments confirmed that its grazing rate was as high as 131 Chlorella cells day-1 individual-1. Results of prey-range experiments demonstrated that it could consume other chlorophyte microalgae (e.g., Scenedesmus, Coelastrella, and Haematococcus) but with a strong feeding ability on Chlorella spp., with ingestion rates ranging from 2.67 to 3.15 prey predator-1 h-1 and growth rates of the amoeba ranging from 0.039 to 0.045 h-1. On the basis of its high grazing ability on Chlorella, capacity to form large populations in a short period of time, and capacity to form resistant resting stages, this contaminant has the potential to cause serious problems in large-scale Chlorella culture and should be of concern to operators of algal production facilities. IMPORTANCE The vampyrellids (Vampyrellida, Rhizaria) are a major group of predatory amoebae that have attracted significant attention because of their diversity of feeding strategies. The crucial roles they play in important processes such as suppressing soil disease and controlling aquatic algae, and as microbial contaminants in outdoor large-scale algal cultures, have also received increasing attention. In this study, a new genus and species of algivorous vampyrellid amoeba, Kinopus chlorellivorus, is described as a significant grazer responsible for losses in outdoor industrial Chlorella cultures. We found that the amoeba's detrimental effects on Chlorella cultures may be related to its specific feeding characteristics. This study provides phenotypic and genetic information on a previously unknown vampyrellid, emphasizes the impact of contaminating vampyrellids in commercial microalgal cultures, and will contribute to the development of management strategies for predicting this kind of contaminant in large-scale microalgal cultivation.

Host-pathogen-environment interactions predict survival outcomes of adult sockeye salmon (Oncorhynchus nerka) released from fisheries.

Incorporating host-pathogen(s)-environment axes into management and conservation planning is critical to preserving species in a warming climate. However, the role pathogens play in host stress resilience remains largely unexplored in wild animal populations. We experimentally characterized how independent and cumulative stressors (fisheries handling, high water temperature) and natural infections affected the health and longevity of released wild adult sockeye salmon (Oncorhynchus nerka) in British Columbia, Canada. Returning adults were collected before and after entering the Fraser River, yielding marine- and river-collected groups, respectively (N = 185). Fish were exposed to a mild (seine) or severe (gill net) fishery treatment at collection, and then held in flow-through freshwater tanks for up to four weeks at historical (14°C) or projected migration temperatures (18°C). Using weekly nonlethal gill biopsies and high-throughput qPCR, we quantified loads of up to 46 pathogens with host stress and immune gene expression. Marine-collected fish had less severe infections than river-collected fish, a short migration distance (100 km, 5-7 days) that produced profound infection differences. At 14°C, river-collected fish survived 1-2 weeks less than marine-collected fish. All fish held at 18°C died within 4 weeks unless they experienced minimal handling. Gene expression correlated with infections in river-collected fish, while marine-collected fish were more stressor-responsive. Cumulative stressors were detrimental regardless of infections or collection location, probably due to extreme physiological disturbance. Because river-derived infections correlated with single stressor responses, river entry probably decreases stressor resilience of adult salmon by altering both physiology and pathogen burdens, which redirect host responses toward disease resistance.

Continuous probing of cold complex molecules with infrared frequency comb spectroscopy.

For more than half a century, high-resolution infrared spectroscopy has played a crucial role in probing molecular structure and dynamics. Such studies have so far been largely restricted to relatively small and simple systems, because at room temperature even molecules of modest size already occupy many millions of rotational/vibrational states, yielding highly congested spectra that are difficult to assign. Targeting more complex molecules requires methods that can record broadband infrared spectra (that is, spanning multiple vibrational bands) with both high resolution and high sensitivity. However, infrared spectroscopic techniques have hitherto been limited either by narrow bandwidth and long acquisition time, or by low sensitivity and resolution. Cavity-enhanced direct frequency comb spectroscopy (CE-DFCS) combines the inherent broad bandwidth and high resolution of an optical frequency comb with the high detection sensitivity provided by a high-finesse enhancement cavity, but it still suffers from spectral congestion. Here we show that this problem can be overcome by using buffer gas cooling to produce continuous, cold samples of molecules that are then subjected to CE-DFCS. This integration allows us to acquire a rotationally resolved direct absorption spectrum in the C-H stretching region of nitromethane, a model system that challenges our understanding of large-amplitude vibrational motion. We have also used this technique on several large organic molecules that are of fundamental spectroscopic and astrochemical relevance, including naphthalene, adamantane and hexamethylenetetramine. These findings establish the value of our approach for studying much larger and more complex molecules than have been probed so far, enabling complex molecules and their kinetics to be studied with orders-of-magnitude improvements in efficiency, spectral resolution and specificity.

Holocene shifts in the assembly of plant and animal communities implicate human impacts.

Understanding how ecological communities are organized and how they change through time is critical to predicting the effects of climate change. Recent work documenting the co-occurrence structure of modern communities found that most significant species pairs co-occur less frequently than would be expected by chance. However, little is known about how co-occurrence structure changes through time. Here we evaluate changes in plant and animal community organization over geological time by quantifying the co-occurrence structure of 359,896 unique taxon pairs in 80 assemblages spanning the past 300 million years. Co-occurrences of most taxon pairs were statistically random, but a significant fraction were spatially aggregated or segregated. Aggregated pairs dominated from the Carboniferous period (307 million years ago) to the early Holocene epoch (11,700 years before present), when there was a pronounced shift to more segregated pairs, a trend that continues in modern assemblages. The shift began during the Holocene and coincided with increasing human population size and the spread of agriculture in North America. Before the shift, an average of 64% of significant pairs were aggregated; after the shift, the average dropped to 37%. The organization of modern and late Holocene plant and animal assemblages differs fundamentally from that of assemblages over the past 300 million years that predate the large-scale impacts of humans. Our results suggest that the rules governing the assembly of communities have recently been changed by human activity.

Earliest known Oldowan artifacts at >2.58 Ma from Ledi-Geraru, Ethiopia, highlight early technological diversity.

The manufacture of flaked stone artifacts represents a major milestone in the technology of the human lineage. Although the earliest production of primitive stone tools, predating the genus Homo and emphasizing percussive activities, has been reported at 3.3 million years ago (Ma) from Lomekwi, Kenya, the systematic production of sharp-edged stone tools is unknown before the 2.58-2.55 Ma Oldowan assemblages from Gona, Ethiopia. The organized production of Oldowan stone artifacts is part of a suite of characteristics that is often associated with the adaptive grade shift linked to the genus Homo Recent discoveries from Ledi-Geraru (LG), Ethiopia, place the first occurrence of Homo ∼250 thousand years earlier than the Oldowan at Gona. Here, we describe a substantial assemblage of systematically flaked stone tools excavated in situ from a stratigraphically constrained context [Bokol Dora 1, (BD 1) hereafter] at LG bracketed between 2.61 and 2.58 Ma. Although perhaps more primitive in some respects, quantitative analysis suggests the BD 1 assemblage fits more closely with the variability previously described for the Oldowan than with the earlier Lomekwian or with stone tools produced by modern nonhuman primates. These differences suggest that hominin technology is distinctly different from generalized tool use that may be a shared feature of much of the primate lineage. The BD 1 assemblage, near the origin of our genus, provides a link between behavioral adaptations-in the form of flaked stone artifacts-and the biological evolution of our ancestors.

Sulfur monoxide thermal release from an anthracene-based precursor, spectroscopic identification, and transfer reactivity.

Sulfur monoxide (SO) is a highly reactive molecule and thus, eludes bulk isolation. We report here on synthesis and reactivity of a molecular precursor for SO generation, namely 7-sulfinylamino-7-azadibenzonorbornadiene (1). This compound has been shown to fragment readily driven by dinitrogen expulsion and anthracene formation on heating in the solid state and in solution, releasing SO at mild temperatures (<100 °C). The generated SO was detected in the gas phase by MS and rotational spectroscopy. In solution, 1 allows for SO transfer to organic molecules as well as transition metal complexes.

Longitudinal changes in blood-based biomarkers in chronic moderate to severe traumatic brain injury: preliminary findings.

Objectives: This longitudinal study aims at 1) providing preliminary evidence of changes in blood-based biomarkers across time in chronic TBI and 2) relating these changes to outcome measures and cerebral structure and activity.Methods: Eight patients with moderate-to-severe TBI (7 males, 35 ± 7.6 years old, 5 severe TBI, 17.52 ± 3.84 months post-injury) were evaluated at monthly intervals across 6 time-points using: a) Blood-based biomarkers (GFAP, NSE, S100A12, SDBP145, UCH-L1, T-tau, P-tau, P-tau/T-tau ratio); b) Magnetic Resonance Imaging to evaluate changes in brain structure; c) Resting-state electroencephalograms to evaluate changes in brain function; and d) Outcome measures to assess cognition, emotion, and functional recovery (MOCA, RBANS, BDI-II, and DRS).Results: Changes in P-tau levels were found across time [p = .007]. P-tau was positively related to functional [p < .001] and cognitive [p = .006] outcomes, and negatively related to the severity of depression, 6 months later [R = -0.901; p =.006]. P-tau and P-tau/T-tau ratio were also positively correlated to shape change in subcortical areas such as brainstem [T(7) = 4.71, p = .008] and putamen [T(7) = 3.25, p = .012].Conclusions: Our study provides preliminary findings that suggest a positive relationship between P-tau and the recovery of patients with chronic TBI.

Ultralong Spin-Coherence Times for Rubidium Atoms in Solid Parahydrogen via Dynamical Decoupling.

Coherence time is an essential parameter for quantum sensing, quantum information, and quantum computation. In this work, we demonstrate electron spin coherence times as long as 0.1 s for an ensemble of rubidium atoms trapped in a solid parahydrogen matrix. We explore the underlying physics limiting the coherence time. The properties of these matrix isolated atoms are very promising for future applications, including quantum sensing of nuclear spins. If combined with efficient single-atom readout, this would enable NMR and magnetic resonance imaging of single molecules cotrapped with alkali-metal atom quantum sensors within a parahydrogen matrix.

Using a Biopsychosocial Model to Understand Long-Term Outcomes in Persons With Burn Injuries.

OBJECTIVE: To determine the importance of preburn adjustment, injury-related variables, and selection of coping style in various outcome measures using a biopsychosocial model. DESIGN: Longitudinal study. SETTING: Outpatient burn clinics. PARTICIPANTS: Burn survivors (N=231) who participated in this study as part of a larger burn model system study of 645 patients with major burn injuries. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: The 36-Item Short-Form Health Survey was used to assess preburn adjustment. Other outcome measures entered into the model included the Ways of Coping Checklist Revised, the Brief Symptom Inventory, the Beck Depression Inventory-II, and the Davidson Trauma Scale. RESULTS: Correlational and mediational analyses revealed that preburn emotional health predicted better adjustment at year 1 and more posttraumatic stress disorder symptoms at year 2. Better preburn emotional health was also related to less use of avoidance coping strategies, which was found to be a mediator of the effect of preburn emotional health and posttraumatic stress disorder symptoms. Burn injury characteristics were not significantly associated with psychological adjustment at either year 1 or year 2. CONCLUSIONS: The results indicate that there is a complex relation between premorbid mental health and the selection of coping strategies that affect long-term adjustment in persons recovering from a burn injury. This relation seems to have greater effect on long-term outcomes than does preburn emotional or physical health alone or the severity of the burn.

The essential role of law in achieving the health-related Sustainable Development Goals.

In this article, we examine the essential role of law in achieving the health-related Sustainable Development Goals (SDGs). Following the World Health Organization's broad definition of health, all SDGs can be seen to impact on human health and hence the health goal (SDG3) should be right at the centre of the entire 2030 Agenda for Sustainable Development. We note recent research on the contribution of law, including international human rights law, to achieving health for all and discuss the role of law in addressing seven emerging health challenges. Law can and should play an important role in achieving all health-related SDGs, by respecting, protecting and fulfilling the right to health, ensuring that no one is left behind.