Variety Is the Spice of Life: Diverse Social Networks Are Associated With Social Cohesion and Well-Being.

Both homophily and heterophily are observed in humans. Homophily reinforces homogeneous social networks, and heterophily creates new experiences and collaborations. However, at the extremes, high levels of homophily can cultivate prejudice toward out-groups, whereas high levels of heterophily can weaken in-group support. Using data from 24,726 adults (M = 46 years; selected from 10,398 English neighborhoods) and the composition of their social networks based on age, ethnicity, income, and education, we tested the hypothesis that a middle ground between homophily and heterophily could be the most beneficial for individuals. We found that network homophily, mediated by perceived social cohesion, is associated with higher levels of subjective well-being but that there are diminishing returns, because at a certain point increasing network homophily is associated with lower social cohesion and, in turn, lower subjective well-being. Our results suggest that building diverse social networks provides benefits that cannot be attained by homogeneous networks.

Insights Into Informal Caregivers' Well-being: A Longitudinal Analysis of Care Intensity, Care Location, and Care Relationship.

OBJECTIVES: This study investigates the psychological well-being of informal caregivers over time. It identifies the thresholds (or "tipping points") of caring intensity at which caregiving is associated with lower psychological well-being, and how this varies by care location and caregiver-care recipient relationships. It also examines how caring location and relationship are linked to informal caregivers' psychological well-being while controlling for caring intensity. METHODS: Waves 1-18 (1991-2009) of the harmonized British Household Panel Survey and Waves 1-8 (2009-2017) of the U.K. Household Longitudinal Study were analyzed. Psychological well-being was measured using the General Health Questionnaire (GHQ)-12 score. Care intensity was measured by the weekly hours of care provided. Fixed-effects estimators were applied to the GHQ-12 score of caregivers across different care intensities, caring locations, and caring relationships. RESULTS: All levels of informal care intensity are associated with lower psychological well-being among spousal caregivers. The thresholds to well-being are 5 hours per week when caring for a parent, and 50 hours per week when caring for a child (with a disability or long-term illness). Caring for "other relatives" or nonrelatives is not negatively associated with psychological well-being. The thresholds are 5 hours per week for both coresident and extraresident caregivers. Extraresident caregivers experience better psychological well-being compared to coresident caregivers, given relatively lower weekly care hours. Caring for primary kin (especially spouses) is linked to lower psychological well-being compared to other caregiving relationships, regardless of care intensity. DISCUSSION: Policy and practice responses should pay particular attention to spousal caregivers' well-being. Caregiving relationship has a stronger association with the caregiver's well-being than care location.

Tunable Dynamics in a Multistrain Transcriptional Pulse Generator.

One challenge in synthetic biology is the tuning of regulatory components within gene circuits to elicit a specific behavior. This challenge becomes more difficult in synthetic microbial consortia since each strain's circuit must function at the intracellular level and their combination must operate at the population level. Here we demonstrate that circuit dynamics can be tuned in synthetic consortia through the manipulation of strain fractions within the community. To do this, we construct a microbial consortium comprised of three strains of engineered Escherichia coli that, when cocultured, use homoserine lactone-mediated intercellular signaling to create a multistrain incoherent type-1 feedforward loop (I1-FFL). Like naturally occurring I1-FFL motifs in gene networks, this engineered microbial consortium acts as a pulse generator of gene expression. We demonstrate that the amplitude of the pulse can be easily tuned by adjusting the relative population fractions of the strains. We also develop a mathematical model for the temporal dynamics of the microbial consortium. This model allows us to identify population fractions that produced desired pulse characteristics, predictions that were confirmed for all but extreme fractions. Our work demonstrates that intercellular gene circuits can be effectively tuned simply by adjusting the starting fractions of each strain in the consortium.

Independent age estimates resolve the controversy of ancient human footprints at White Sands.

Human footprints at White Sands National Park, New Mexico, USA, reportedly date to between ~23,000 and 21,000 years ago according to radiocarbon dating of seeds from the aquatic plant Ruppia cirrhosa. These ages remain controversial because of potential old carbon reservoir effects that could compromise their accuracy. We present new calibrated 14C ages of terrestrial pollen collected from the same stratigraphic horizons as those of the Ruppia seeds, along with optically stimulated luminescence ages of sediments from within the human footprint-bearing sequence, to evaluate the veracity of the seed ages. The results show that the chronologic framework originally established for the White Sands footprints is robust and reaffirm that humans were present in North America during the Last Glacial Maximum.

A portable regulatory RNA array design enables tunable and complex regulation across diverse bacteria.

A lack of composable and tunable gene regulators has hindered efforts to engineer non-model bacteria and consortia. Toward addressing this, we explore the broad-host potential of small transcription activating RNA (STAR) and propose a design strategy to achieve tunable gene control. First, we demonstrate that STARs optimized for E. coli function across different Gram-negative species and can actuate using phage RNA polymerase, suggesting that RNA systems acting at the level of transcription are portable. Second, we explore an RNA design strategy that uses arrays of tandem and transcriptionally fused RNA regulators to precisely alter regulator concentration from 1 to 8 copies. This provides a simple means to predictably tune output gain across species and does not require access to large regulatory part libraries. Finally, we show RNA arrays can be used to achieve tunable cascading and multiplexing circuits across species, analogous to the motifs used in artificial neural networks.

Signaling in microbial communities with open boundaries.

Microbial communities such as swarms or biofilms often form at the interfaces of solid substrates and open fluid flows. At the same time, in laboratory environments these communities are commonly studied using microfluidic devices with media flows and open boundaries. Extracellular signaling within these communities is therefore subject to different constraints than signaling within classic, closed-boundary systems such as developing embryos or tissues, yet is understudied by comparison. Here, we use mathematical modeling to show how advective-diffusive boundary flows and population geometry impact cell-cell signaling in monolayer microbial communities. We reveal conditions where the intercellular signaling lengthscale depends solely on the population geometry and not on diffusion or degradation, as commonly expected. We further demonstrate that diffusive coupling with the boundary flow can produce signal gradients within an isogenic population, even when there is no flow within the population. We use our theory to provide new insights into the signaling mechanisms of published experimental results, and we make several experimentally verifiable predictions. Our research highlights the importance of carefully evaluating boundary dynamics and environmental geometry when modeling microbial cell-cell signaling and informs the study of cell behaviors in both natural and synthetic systems.

A portable regulatory RNA array design enables tunable and complex regulation across diverse bacteria.

A lack of composable and tunable gene regulators has hindered efforts to engineer non-model bacteria and consortia. To address this, we explore the broad-host potential of small transcription activating RNA (STAR) and propose a novel design strategy to achieve tunable gene control. First, we demonstrate that STARs optimized for E. coli function across different Gram-negative species and can actuate using phage RNA polymerase, suggesting that RNA systems acting at the level of transcription are portable. Second, we explore a novel RNA design strategy that uses arrays of tandem and transcriptionally fused RNA regulators to precisely alter regulator concentration from 1 to 8 copies. This provides a simple means to predictably tune output gain across species and does not require access to large regulatory part libraries. Finally, we show RNA arrays can be used to achieve tunable cascading and multiplexing circuits across species, analogous to the motifs used in artificial neural networks.

Indirect Enrichment of Desirable, but Less Fit Phenotypes, from a Synthetic Microbial Community Using Microdroplet Confinement.

Spatial structure within microbial communities can provide nearly limitless opportunities for social interactions and are an important driver for evolution. As metabolites are often molecular signals, metabolite diffusion within microbial communities can affect the composition and dynamics of the community in a manner that can be challenging to deconstruct. We used encapsulation of a synthetic microbial community within microdroplets to investigate the effects of spatial structure and metabolite diffusion on population dynamics and to examine the effects of cheating by one member of the community. The synthetic community was composed of three strains: a "Producer" that makes the diffusible quorum sensing molecule (N-(3-oxododecanoyl)-l-homoserine lactone, C12-oxo-HSL) or AHL; a "Receiver" that is killed by AHL; and a Non-Producer or "cheater" that benefits from the extinction of the Receivers, but without the costs associated with the AHL synthesis. We demonstrate that despite rapid diffusion of AHL between microdroplets, the spatial structure imposed by the microdroplets allows a more efficient but transient enrichment of more rare and slower-growing Producer subpopulations. Eventually, the Non-Producer population drove the Producers to extinction. By including fluorescence-activated microdroplet sorting and providing sustained competition by the Receiver strain, we demonstrate a strategy for indirect enrichment of a rare and unlabeled Producer. The ability to screen and enrich metabolite Producers from a much larger population under conditions of rapid diffusion provides an important framework for the development of applications in synthetic ecology and biotechnology.

Indirect enrichment of desirable, but less fit phenotypes, from a synthetic microbial community using microdroplet confinement.

Spatial structure within microbial communities can provide nearly limitless opportunities for social interactions and are an important driver for evolution. As metabolites are often molecular signals, metabolite diffusion within microbial communities can affect the composition and dynamics of the community in a manner that can be challenging to deconstruct. We used encapsulation of a synthetic microbial community within microdroplets to investigate the effects of spatial structure and metabolite diffusion on population dynamics and to examine the effects of cheating by one member of the community. The synthetic community was comprised of three strains: a 'Producer' that makes the diffusible quorum sensing molecule ( N -(3-Oxododecanoyl)-L-homoserine lactone, C12-oxo-HSL) or AHL; a 'Receiver' that is killed by AHL and a Non-Producer or 'cheater' that benefits from the extinction of the Receivers, but without the costs associated with the AHL synthesis. We demonstrate that despite rapid diffusion of AHL between microdroplets, the spatial structure imposed by the microdroplets allow a more efficient but transient enrichment of more rare and slower growing 'Producer' subpopulations. Eventually, the Non-Producer population drove the Producers to extinction. By including fluorescence-activated microdroplet sorting and providing sustained competition by the Receiver strain, we demonstrate a strategy for indirect enrichment of a rare and unlabeled Producer. The ability to screen and enrich metabolite Producers from a much larger population under conditions of rapid diffusion provides an important framework for the development of applications in synthetic ecology and biotechnology.

Signaling in microbial communities with open boundaries.

Microbial communities such as swarms or biofilms often form at the interfaces of solid substrates and open fluid flows. At the same time, in laboratory environments these communities are commonly studied using microfluidic devices with media flows and open boundaries. Extracellular signaling within these communities is therefore subject to different constraints than signaling within classic, closed-boundary systems such as developing embryos or tissues, yet is understudied by comparison. Here, we use mathematical modeling to show how advective-diffusive boundary flows and population geometry impact cell-cell signaling in monolayer microbial communities. We reveal conditions where the intercellular signaling lengthscale depends solely on the population geometry and not on diffusion or degradation, as commonly expected. We further demonstrate that diffusive coupling with the boundary flow can produce signal gradients within an isogenic population, even when there is no flow within the population. We use our theory to provide new insights into the signaling mechanisms of published experimental results, and we make several experimentally verifiable predictions. Our research highlights the importance of carefully evaluating boundary dynamics and environmental geometry when modeling microbial cell-cell signaling and informs the study of cell behaviors in both natural and synthetic systems.

Strategies for Improving Small-Molecule Biosensors in Bacteria.

In recent years, small-molecule biosensors have become increasingly important in synthetic biology and biochemistry, with numerous new applications continuing to be developed throughout the field. For many biosensors, however, their utility is hindered by poor functionality. Here, we review the known types of mechanisms of biosensors within bacterial cells, and the types of approaches for optimizing different biosensor functional parameters. Discussed approaches for improving biosensor functionality include methods of directly engineering biosensor genes, considerations for choosing genetic reporters, approaches for tuning gene expression, and strategies for incorporating additional genetic modules.

Response to Comment on "Evidence of humans in North America during the Last Glacial Maximum".

Madsen et al. question the reliability of calibrated radiocarbon ages associated with human footprints discovered recently in White Sands National Park, New Mexico, USA. On the basis of the geologic, hydrologic, stratigraphic, and chronologic evidence, we maintain that the ages are robust and conclude that the footprints date to between ~23,000 and 21,000 years ago.Madsen et al. (1) question the veracity of calibrated radiocarbon ages used to constrain the antiquity of human trackways discovered recently at White Sands National Park (WHSA) Locality 2, New Mexico, USA (2). The ages were derived from seeds of the aquatic plant Ruppia cirrhosa, which they suggest may suffer from hard-water (or reservoir) effects, making them too old, potentially by thousands of years. We were well aware of this possibility, investigated it, and presented several lines of evidence that argued against such a problem. Here we respond to each of their four primary points.

Allosteric regulation within the highly interconnected structural scaffold of AraC/XylS homologs tolerates a wide range of amino acid changes.

To create bacterial transcription "circuits" for biotechnology, one approach is to recombine natural transcription factors, promoters, and operators. Additional novel functions can be engineered from existing transcription factors such as the E. coli AraC transcriptional activator, for which binding to DNA is modulated by binding L-arabinose. Here, we engineered chimeric AraC/XylS transcription activators that recognized ara DNA binding sites and responded to varied effector ligands. The first step, identifying domain boundaries in the natural homologs, was challenging because (i) no full-length, dimeric structures were available and (ii) extremely low sequence identities (≤10%) among homologs precluded traditional assemblies of sequence alignments. Thus, to identify domains, we built and aligned structural models of the natural proteins. The designed chimeric activators were assessed for function, which was then further improved by random mutagenesis. Several mutational variants were identified for an XylS•AraC chimera that responded to benzoate; two enhanced activation to near that of wild-type AraC. For an RhaR•AraC chimera, a variant with five additional substitutions enabled transcriptional activation in response to rhamnose. These five changes were dispersed across the protein structure, and combinatorial experiments testing subsets of substitutions showed significant non-additivity. Combined, the structure modeling and epistasis suggest that the common AraC/XylS structural scaffold is highly interconnected, with complex intra-protein and inter-domain communication pathways enabling allosteric regulation. At the same time, the observed epistasis and the low sequence identities of the natural homologs suggest that the structural scaffold and function of transcriptional regulation are nevertheless highly accommodating of amino acid changes.

Longitudinal analysis of local government spending on adult social care and carers' subjective well-being in England.

OBJECTIVES: Reform of England's social care system is repeatedly discussed in the context of increasing demand, rising costs and austere policies that have decreased service provision. This study investigates the association between unpaid carers' subjective well-being and local government spending on adult social care (ASC). SETTING AND PARTICIPANTS: Our sample consists of 110 188 observations on 29 174 adults in England from the 2004-2007 British Household Panel Survey and the 2009-2018 UK Household Longitudinal Study. The data on local authorities' spending on ASC where participants live is derived from the publications Personal Social Care Expenditure and Unit Costs (2004-2016); and ASC Activity and Finance Report England (2016-2018). OUTCOME MEASURES: Subjective well-being is measured by the 12-item version of the General Health Questionnaire (GHQ-12) and 12-item version of the Mental Component Summary (MCS-12). We applied fixed-effects linear models to investigate the moderating effect of ASC spending on the association between subjective well-being and caring, controlling for a range of socioeconomic and demographic variables. RESULTS: Carers have a lower level of subjective well-being compared with non-carers, evident in their higher average GHQ-12 Likert score (ß=2.7277 95% CI 0.2547 to 5.2008). Differences in the subjective well-being of carers and non-carers decrease with local government spending on ASC. Subjective well-being for carers was at a similar level to that of non-carers in high ASC spending local authorities (GHQ-12: -0.0123 95% CI -0.2185 to 0.1938, MCS-12: 0.0347 95% CI -0.3403 to 0.4098) and lower in other areas (GHQ-12: 0.1893 95% CI 0.0680 to 0.3107, MCS-12: -0.2906 95% CI -0.5107 to -0.0705). The moderating effect of ASC spending is found among people who care for 35+ hours per week. CONCLUSION: Government spending on ASC protects unpaid carers' well-being, and people providing more than 35 weekly hours of unpaid care are more likely to benefit from the current social care system.

Emergent spatiotemporal population dynamics with cell-length control of synthetic microbial consortia.

The increased complexity of synthetic microbial biocircuits highlights the need for distributed cell functionality due to concomitant increases in metabolic and regulatory burdens imposed on single-strain topologies. Distributed systems, however, introduce additional challenges since consortium composition and spatiotemporal dynamics of constituent strains must be robustly controlled to achieve desired circuit behaviors. Here, we address these challenges with a modeling-based investigation of emergent spatiotemporal population dynamics using cell-length control in monolayer, two-strain bacterial consortia. We demonstrate that with dynamic control of a strain's division length, nematic cell alignment in close-packed monolayers can be destabilized. We find that this destabilization confers an emergent, competitive advantage to smaller-length strains-but by mechanisms that differ depending on the spatial patterns of the population. We used complementary modeling approaches to elucidate underlying mechanisms: an agent-based model to simulate detailed mechanical and signaling interactions between the competing strains, and a reductive, stochastic lattice model to represent cell-cell interactions with a single rotational parameter. Our modeling suggests that spatial strain-fraction oscillations can be generated when cell-length control is coupled to quorum-sensing signaling in negative feedback topologies. Our research employs novel methods of population control and points the way to programming strain fraction dynamics in consortial synthetic biology.

Evidence of humans in North America during the Last Glacial Maximum.

Archaeologists and researchers in allied fields have long sought to understand human colonization of North America. Questions remain about when and how people migrated, where they originated, and how their arrival affected the established fauna and landscape. Here, we present evidence from excavated surfaces in White Sands National Park (New Mexico, United States), where multiple in situ human footprints are stratigraphically constrained and bracketed by seed layers that yield calibrated radiocarbon ages between ~23 and 21 thousand years ago. These findings confirm the presence of humans in North America during the Last Glacial Maximum, adding evidence to the antiquity of human colonization of the Americas and providing a temporal range extension for the coexistence of early inhabitants and Pleistocene megafauna.

Sexing white 2D footprints using convolutional neural networks.

Footprints are left, or obtained, in a variety of scenarios from crime scenes to anthropological investigations. Determining the sex of a footprint can be useful in screening such impressions and attempts have been made to do so using single or multi landmark distances, shape analyses and via the density of friction ridges. Here we explore the relative importance of different components in sexing two-dimensional foot impressions namely, size, shape and texture. We use a machine learning approach and compare this to more traditional methods of discrimination. Two datasets are used, a pilot data set collected from students at Bournemouth University (N = 196) and a larger data set collected by podiatrists at Sheffield NHS Teaching Hospital (N = 2677). Our convolutional neural network can sex a footprint with accuracy of around 90% on a test set of N = 267 footprint images using all image components, which is better than an expert can achieve. However, the quality of the impressions impacts on this success rate, but the results are promising and in time it may be possible to create an automated screening algorithm in which practitioners of whatever sort (medical or forensic) can obtain a first order sexing of a two-dimensional footprint.

Improved pyrrolysine biosynthesis through phage assisted non-continuous directed evolution of the complete pathway.

Pyrrolysine (Pyl, O) exists in nature as the 22nd proteinogenic amino acid. Despite being a fundamental building block of proteins, studies of Pyl have been hindered by the difficulty and inefficiency of both its chemical and biological syntheses. Here, we improve Pyl biosynthesis via rational engineering and directed evolution of the entire biosynthetic pathway. To accommodate toxicity of Pyl biosynthetic genes in Escherichia coli, we also develop Alternating Phage Assisted Non-Continuous Evolution (Alt-PANCE) that alternates mutagenic and selective phage growths. The evolved pathway provides 32-fold improved yield of Pyl-containing reporter protein compared to the rationally engineered ancestor. Evolved PylB mutants are present at up to 4.5-fold elevated levels inside cells, and show up to 2.2-fold increased protease resistance. This study demonstrates that Alt-PANCE provides a general approach for evolving proteins exhibiting toxic side effects, and further provides an improved pathway capable of producing substantially greater quantities of Pyl-proteins in E. coli.

Technological innovation in the recovery and analysis of 3D forensic footwear evidence: Structure from motion (SfM) photogrammetry.

The recovery of three-dimensional footwear impressions at crime scenes can be a challenge but can also yield important investigative data. Traditional methods involve casting 3D impressions but these methods have limitations: the trace is usually destroyed during capture; the process can be time consuming, with a risk of failure; and the resultant cast is bulky and therefore difficult to share and store. The use of Structure from Motion (SfM) photogrammetry has been used widely to capture fossil footprints in the geological record and while there is a small body of work advocating its use in forensic practice the full potential of this technique has yet to be realised in an operational context. The availability of affordable software is one limiting factor and here we report the availability of a bespoke freeware for SfM recovery and subsequent analysis of for footwear evidence (DigTrace). Our aim here is not to provide a rigorous comparison of SfM methods to other recovery methods, but more to illustrate the potential while also documenting the typical workflows and potential errors associated with an SfM based approach. By doing so we hope to encourage further research, experimentation and ultimately adoption by practitioners.

Control of synthetic microbial consortia in time, space, and composition.

While synthetic microbial systems are becoming increasingly complicated, single-strain systems cannot match the complexity of their multicellular counterparts. Such complexity, however, is much more difficult to control. Recent advances have increased our ability to control temporal, spatial, and community compositional organization, including modular adhesive systems, strain growth relationships, and asymmetric cell division. While these systems generally work independently, combining them into unified systems has proven difficult. Once such unification is proven successful we will unlock a new frontier of synthetic biology and open the door to the creation of synthetic biological systems with true multicellularity.