Direct Identification of Hundreds of Expression-Modulating Variants using a Multiplexed Reporter Assay.

Although studies have identified hundreds of loci associated with human traits and diseases, pinpointing causal alleles remains difficult, particularly for non-coding variants. To address this challenge, we adapted the massively parallel reporter assay (MPRA) to identify variants that directly modulate gene expression. We applied it to 32,373 variants from 3,642 cis-expression quantitative trait loci and control regions. Detection by MPRA was strongly correlated with measures of regulatory function. We demonstrate MPRA's capabilities for pinpointing causal alleles, using it to identify 842 variants showing differential expression between alleles, including 53 well-annotated variants associated with diseases and traits. We investigated one in detail, a risk allele for ankylosing spondylitis, and provide direct evidence of a non-coding variant that alters expression of the prostaglandin EP4 receptor. These results create a resource of concrete leads and illustrate the promise of this approach for comprehensively interrogating how non-coding polymorphism shapes human biology.

High-throughput Oligopaint screen identifies druggable 3D genome regulators.

The human genome functions as a three-dimensional chromatin polymer, driven by a complex collection of chromosome interactions1-3. Although the molecular rules governing these interactions are being quickly elucidated, relatively few proteins regulating this process have been identified. Here, to address this gap, we developed high-throughput DNA or RNA labelling with optimized Oligopaints (HiDRO)-an automated imaging pipeline that enables the quantitative measurement of chromatin interactions in single cells across thousands of samples. By screening the human druggable genome, we identified more than 300 factors that influence genome folding during interphase. Among these, 43 genes were validated as either increasing or decreasing interactions between topologically associating domains. Our findings show that genetic or chemical inhibition of the ubiquitous kinase GSK3A leads to increased long-range chromatin looping interactions in a genome-wide and cohesin-dependent manner. These results demonstrate the importance of GSK3A signalling in nuclear architecture and the use of HiDRO for identifying mechanisms of spatial genome organization.

How deregulation, drought and increasing fire impact Amazonian biodiversity.

Biodiversity contributes to the ecological and climatic stability of the Amazon Basin1,2, but is increasingly threatened by deforestation and fire3,4. Here we quantify these impacts over the past two decades using remote-sensing estimates of fire and deforestation and comprehensive range estimates of 11,514 plant species and 3,079 vertebrate species in the Amazon. Deforestation has led to large amounts of habitat loss, and fires further exacerbate this already substantial impact on Amazonian biodiversity. Since 2001, 103,079-189,755 km2 of Amazon rainforest has been impacted by fires, potentially impacting the ranges of 77.3-85.2% of species that are listed as threatened in this region5. The impacts of fire on the ranges of species in Amazonia could be as high as 64%, and greater impacts are typically associated with species that have restricted ranges. We find close associations between forest policy, fire-impacted forest area and their potential impacts on biodiversity. In Brazil, forest policies that were initiated in the mid-2000s corresponded to reduced rates of burning. However, relaxed enforcement of these policies in 2019 has seemingly begun to reverse this trend: approximately 4,253-10,343 km2 of forest has been impacted by fire, leading to some of the most severe potential impacts on biodiversity since 2009. These results highlight the critical role of policy enforcement in the preservation of biodiversity in the Amazon.

Sex-dependent phenological responses to climate vary across species' ranges.

Anthropogenic climate change has significantly altered the flowering times (i.e., phenology) of plants worldwide, affecting their reproduction, survival, and interactions. Recent studies utilizing herbarium specimens have uncovered significant intra- and inter-specific variation in flowering phenology and its response to changes in climate but have mostly been limited to animal-pollinated species. Thus, despite their economic and ecological importance, variation in phenological responses to climate remain largely unexplored among and within wind-pollinated dioecious species and across their sexes. Using both herbarium specimens and volunteer observations of cottonwood (Populus) species, we examined how phenological sensitivity to climate varies across species, their ranges, sexes, and phenophases. The timing of flowering varied significantly across and within species, as did their sensitivity to spring temperature. In particular, male flowering generally happened earlier in the season and was more sensitive to warming than female flowering. Further, the onset of flowering was more sensitive to changes in temperature than leaf out. Increased temporal gaps between male and female flowering time and between the first open flower date and leaf out date were predicted for the future under two climate change scenarios. These shifts will impact the efficacy of sexual reproduction and gene flow among species. Our study demonstrates significant inter- and intra-specific variation in phenology and its responses to environmental cues, across species' ranges, phenophases, and sex, in wind-pollinated species. These variations need to be considered to predict accurately the effects of climate change and assess their ecological and evolutionary consequences.

Co-depletion of NIPBL and WAPL balance cohesin activity to correct gene misexpression.

The relationship between cohesin-mediated chromatin looping and gene expression remains unclear. NIPBL and WAPL are two opposing regulators of cohesin activity; depletion of either is associated with changes in both chromatin folding and transcription across a wide range of cell types. However, a direct comparison of their individual and combined effects on gene expression in the same cell type is lacking. We find that NIPBL or WAPL depletion in human HCT116 cells each alter the expression of ~2,000 genes, with only ~30% of the genes shared between the conditions. We find that clusters of differentially expressed genes within the same topologically associated domain (TAD) show coordinated misexpression, suggesting some genomic domains are especially sensitive to both more or less cohesin. Finally, co-depletion of NIPBL and WAPL restores the majority of gene misexpression as compared to either knockdown alone. A similar set of NIPBL-sensitive genes are rescued following CTCF co-depletion. Together, this indicates that altered transcription due to reduced cohesin activity can be functionally offset by removal of either its negative regulator (WAPL) or the physical barriers (CTCF) that restrict loop-extrusion events.

The Invisible University Is COVID-19 Positive.

Within the ivory tower of academia, the coronavirus disease 2019 (COVID-19) pandemic stands to disproportionately impact the invisible workforce of postdoctoral researchers (postdocs). Faced with university closures, hiring freezes, and a general lack of support and benefits, an entire generation of postdocs and their knowledge and skills may be lost to academia without intervention.

Complex climate-mediated effects of urbanization on plant reproductive phenology and frost risk.

Urbanization can affect the timing of plant reproduction (i.e. flowering and fruiting) and associated ecosystem processes. However, our knowledge of how plant phenology responds to urbanization and its associated environmental changes is limited. Herbaria represent an important, but underutilized source of data for investigating this question. We harnessed phenological data from herbarium specimens representing 200 plant species collected across 120 yr from the eastern US to investigate the spatiotemporal effects of urbanization on flowering and fruiting phenology and frost risk (i.e. time between the last frost date and flowering). Effects of urbanization on plant reproductive phenology varied significantly in direction and magnitude across species ranges. Increased urbanization led to earlier flowering in colder and wetter regions and delayed fruiting in regions with wetter spring conditions. Frost risk was elevated with increased urbanization in regions with colder and wetter spring conditions. Our study demonstrates that predictions of phenological change and its associated impacts must account for both climatic and human effects, which are context dependent and do not necessarily coincide. We must move beyond phenological models that only incorporate temperature variables and consider multiple environmental factors and their interactions when estimating plant phenology, especially at larger spatial and taxonomic scales.

Darwin's naturalization conundrum can be explained by spatial scale.

Darwin proposed two seemingly contradictory hypotheses regarding factors influencing the outcome of biological invasions. He initially posited that nonnative species closely related to native species would be more likely to successfully establish, because they might share adaptations to the local environment (preadaptation hypothesis). However, based on observations that the majority of naturalized plant species in the United States belonged to nonnative genera, he concluded that the lack of competitive exclusion would facilitate the establishment of alien invaders phylogenetically distinct from the native flora (competition-relatedness hypothesis). To date, no consensus has been reached regarding these opposing hypotheses. Here, following Darwin, we use the flora of the United States to examine patterns of taxonomic and phylogenetic relatedness between native and nonnative taxa across thousands of nested locations ranging in size and extent, from local to regional scales. We find that the probability of observing the signature of environmental filtering over that of competition increases with spatial scale. Further, native and nonnative species tended to be less related in warm, humid environments. Our work provides an empirical assessment of the role of observation scale and climate in biological invasions and demonstrates that Darwin's two opposing hypotheses need not be mutually exclusive.

The ecological implications of intra- and inter-species variation in phenological sensitivity.

Plant-pollinator mutualisms rely upon the synchrony of interacting taxa. Climate change can disrupt this synchrony as phenological responses to climate vary within and across species. However, intra- and interspecific variation in phenological responses is seldom considered simultaneously, limiting our understanding of climate change impacts on interactions among taxa across their ranges. We investigated how variation in phenological sensitivity to climate can alter ecological interactions simultaneously within and among species using natural history collections and citizen science data. We focus on a unique system, comprising a wide-ranged spring ephemeral with varying color morphs (Claytonia virginica) and its specialist bee pollinator (Andrena erigeniae). We found strongly opposing trends in the phenological sensitivities of plants vs their pollinators. Flowering phenology was more sensitive to temperature in warmer regions, whereas bee phenology was more responsive in colder regions. Phenological sensitivity varied across flower color morphs. Temporal synchrony between flowering and pollinator activity was predicted to change heterogeneously across the species' ranges in the future. Our work demonstrates the complexity and fragility of ecological interactions in time and the necessity of incorporating variation in phenological responses across multiple axes to understand how such interactions will change in the future.

Phenological displacement is uncommon among sympatric angiosperms.

Interactions between species can influence successful reproduction, resulting in reproductive character displacement, where the similarity of reproductive traits - such as flowering time - among close relatives growing together differ from when growing apart. Evidence for the overall prevalence and direction of this phenomenon, and its stability under environmental change, remains untested across large scales. Using the power of crowdsourcing, we gathered phenological information from over 40 000 herbarium specimens, and investigated displacement in flowering time across 110 animal-pollinated species in the eastern USA. Overall, flowering time displacement is not common across large scales. However, displacement is generally greater among species pairs that flower close in time, regardless of direction. Furthermore, with climate change, the flowering times of closely related species are predicted, on average, to shift further apart by the mid-21st century. We demonstrate that the degree and direction of phenological displacement among co-occurring closely related species pairs varies tremendously. However, future climate change may alter the differences in reproductive timing among many of these species pairs, which may have significant consequences for species interactions and gene flow. Our study provides one promising path towards understanding how the phenological landscape is structured and may respond to future environmental change.

Air bubble removal: Wettability contrast enabled microfluidic interconnects.

The presence of air bubbles boosts the shear resistance and causes pressure fluctuation within fluid-perfused microchannels, resulting in possible cell damage and even malfunction of microfluidic devices. Eliminating air bubbles is especially challenging in microscale where the adhesive surface tension force is often dominant over other forces. Here, we present an air bubble removal strategy from a novel surface engineering perspective. A microfluidic port-to-port interconnect was fabricated by modifying the peripheral of the microfluidic ports superhydrophobic, while maintaining the inner polymer microchannels hydrophilic. Such a sharp wettability contrast enabled a preferential fluidic entrance into the easy-wetting microchannels over the non-wetting boundaries of the microfluidic ports, while simultaneously filtering out any incoming air bubbles owing to the existence of port-to-port gaps. This bubble-eliminating capability was consistently demonstrated at varying flow rates and liquid analytes. Compared to equipment-intensive techniques and porous membrane-venting strategies, our wettability contrast-governed strategy provides a simple yet effective route for eliminating air bubbles and simultaneously sealing microfluidic interconnects.

Targeted DNA methylation of neurodegenerative disease genes via homology directed repair.

DNA methyltransferases (DNMTs) are thought to be involved in the cellular response to DNA damage, thus linking DNA repair mechanisms with DNA methylation. In this study we present Homology Assisted Repair Dependent Epigenetic eNgineering (HARDEN), a novel method of targeted DNA methylation that utilizes endogenous DNA double strand break repair pathways. This method allows for stable targeted DNA methylation through the process of homology directed repair (HDR) via an in vitro methylated exogenous repair template. We demonstrate that HARDEN can be applied to the neurodegenerative disease genes C9orf72 and APP, and methylation can be induced via HDR with both single and double stranded methylated repair templates. HARDEN allows for higher targeted DNA methylation levels than a dCas9-DNMT3a fusion protein construct at C9orf72, and genome-wide methylation analysis reveals no significant off-target methylation changes when inducing methylation via HARDEN, whereas the dCas9-DNMT3a fusion construct causes global off-target methylation. HARDEN is applied to generate a patient derived iPSC model of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) that recapitulates DNA methylation patterns seen in patients, demonstrating that DNA methylation of the 5' regulatory region directly reduces C9orf72 expression and increases histone H3K9 tri-methylation levels.

Idiopathic Intracranial Hypertension in Pregnancy.

Idiopathic intracranial hypertension (IIH) is a syndrome characterized by elevated intracranial pressure without an identifiable underlying cause. Pregnancy has unique and important diagnostic and therapeutic implications for patients with IIH. Despite these implications, there are no guidelines to assist clinicians in managing IIH during pregnancy. Our review aims to summarize the key considerations related to the diagnosis and management of IIH during pregnancy, to optimize the care of these patients and mitigate the risk of disease-related complications. The optimal management of IIH in pregnancy should include a multidisciplinary team, including an obstetrician (or maternal-fetal medicine specialist), a neurologist, and an ophthalmologist (or neuro-ophthalmologist).

Machine learning predicts large scale declines in native plant phylogenetic diversity.

Though substantial effort has gone into predicting how global climate change will impact biodiversity patterns, the scarcity of taxon-specific information has hampered the efficacy of these endeavors. Further, most studies analyzing spatiotemporal patterns of biodiversity focus narrowly on species richness. We apply machine learning approaches to a comprehensive vascular plant database for the United States and generate predictive models of regional plant taxonomic and phylogenetic diversity in response to a wide range of environmental variables. We demonstrate differences in predicted patterns and potential drivers of native vs nonnative biodiversity. In particular, native phylogenetic diversity is likely to decrease over the next half century despite increases in species richness. We also identify that patterns of taxonomic diversity can be incongruent with those of phylogenetic diversity. The combination of macro-environmental factors that determine diversity likely varies at continental scales; thus, as climate change alters the combinations of these factors across the landscape, the collective effect on regional diversity will also vary. Our study represents one of the most comprehensive examinations of plant diversity patterns to date and demonstrates that our ability to predict future diversity may benefit tremendously from the application of machine learning.

Microfluidic gasketless interconnects sealed by superhydrophobic surfaces.

Existing methods for sealing chip-to-chip (or module-to-motherboard) microfluidic interconnects commonly use additional interconnect components (O-rings, gaskets, and tubing), and manual handling expertise for assembly. Novel gasketless superhydrophobic fluidic interconnects (GSFIs) sealed by transparent superhydrophobic surfaces, forming liquid bridges between the fluidic ports for fluidic passages were demonstrated. Two test platforms were designed, fabricated, and evaluated, a multi-port chip system (ten interconnects) and a modules-on-a-motherboard system (four interconnects). System assembly in less than 3 sec was done by embedded magnets and pin-in-V-groove structures. Flow tests with deionized (DI) water, ethanol/water mixture, and plasma confirmed no leakage through the gasketless interconnects up to a maximum flow rate of 100 µL/min for the multi-port chip system. The modules-on-a-motherboard system showed no leakage of water at a flow rate of 20 µL/min and a pressure drop of 3.71 psi. Characterization of the leakage pressure as a function of the surface tension of the sample liquid in the multi-port chip system revealed that lower surface tension of the liquid led to lower static water contact angles on the superhydrophobic-coated substrate and lower leakage pressures. The high-density, rapidly assembled, gasketless interconnect technology will open up new avenues for chip-to-chip fluid transport in complex microfluidic modular systems.

Managing mental health: why we need to redress the balance between healthcare spending and social spending.

BACKGROUND: Mental health outcomes vary widely among high-income countries, although mental health problems represent an increasing proportion of the burden of disease for all countries. This has led to increased demand for healthcare services, but mental health outcomes may also be particularly sensitive to the availability of social services. This paper examines the variation in the absolute and relative amounts that high-income countries spend on healthcare and social services to determine whether increased expenditure on social services relative to healthcare expenditure might be associated with better mental health outcomes. METHODS: This paper estimates the association between patterns of government spending and population mental health, as measured by the death rate resulting from mental and behavioural disorders, across member countries of the Organisation for Economic Cooperation and Development (OECD). We use country-level repeated measures multivariable modelling for the period from 1995 to 2016 with region and time effects, adjusted for total spending and demographic and economic characteristics. Healthcare spending includes all curative services, long-term care, ancillary services, medical goods, preventative care and administration whilst social spending consists of all transfer payments made to individuals and families as part of the welfare state. RESULTS: We find that a higher ratio of social to healthcare expenditure is associated with significantly better mental health outcomes for OECD populations, as measured by the death rate resulting from mental and behavioural disorders. We also find that there is no statistically significant association between healthcare spending and population mental health when we do not control for social spending. CONCLUSION: This study suggests that OECD countries can have a significant impact on population mental health by investing a greater proportion of total expenditure in social services.

Genome and evolution of the shade-requiring medicinal herb Panax ginseng.

Panax ginseng C. A. Meyer, reputed as the king of medicinal herbs, has slow growth, long generation time, low seed production and complicated genome structure that hamper its study. Here, we unveil the genomic architecture of tetraploid P. ginseng by de novo genome assembly, representing 2.98 Gbp with 59 352 annotated genes. Resequencing data indicated that diploid Panax species diverged in association with global warming in Southern Asia, and two North American species evolved via two intercontinental migrations. Two whole genome duplications (WGD) occurred in the family Araliaceae (including Panax) after divergence with the Apiaceae, the more recent one contributing to the ability of P. ginseng to overwinter, enabling it to spread broadly through the Northern Hemisphere. Functional and evolutionary analyses suggest that production of pharmacologically important dammarane-type ginsenosides originated in Panax and are produced largely in shoot tissues and transported to roots; that newly evolved P. ginseng fatty acid desaturases increase freezing tolerance; and that unprecedented retention of chlorophyll a/b binding protein genes enables efficient photosynthesis under low light. A genome-scale metabolic network provides a holistic view of Panax ginsenoside biosynthesis. This study provides valuable resources for improving medicinal values of ginseng either through genomics-assisted breeding or metabolic engineering.

Widespread sampling biases in herbaria revealed from large-scale digitization.

Nonrandom collecting practices may bias conclusions drawn from analyses of herbarium records. Recent efforts to fully digitize and mobilize regional floras online offer a timely opportunity to assess commonalities and differences in herbarium sampling biases. We determined spatial, temporal, trait, phylogenetic, and collector biases in c. 5 million herbarium records, representing three of the most complete digitized floras of the world: Australia (AU), South Africa (SA), and New England, USA (NE). We identified numerous shared and unique biases among these regions. Shared biases included specimens collected close to roads and herbaria; specimens collected more frequently during biological spring and summer; specimens of threatened species collected less frequently; and specimens of close relatives collected in similar numbers. Regional differences included overrepresentation of graminoids in SA and AU and of annuals in AU; and peak collection during the 1910s in NE, 1980s in SA, and 1990s in AU. Finally, in all regions, a disproportionately large percentage of specimens were collected by very few individuals. We hypothesize that these mega-collectors, with their associated preferences and idiosyncrasies, shaped patterns of collection bias via 'founder effects'. Studies using herbarium collections should account for sampling biases, and future collecting efforts should avoid compounding these biases to the extent possible.

Taxon sampling effects on the quantification and comparison of community phylogenetic diversity.

Ecologists are increasingly making use of molecular phylogenies, especially in the fields of community ecology and conservation. However, these phylogenies are often used without full appreciation of their underlying assumptions and uncertainties. A frequent practice in ecological studies is inferring a phylogeny with molecular data from taxa only within the community of interest. These "inferred community phylogenies" are inherently biased in their taxon sampling. Despite the importance of comprehensive sampling in constructing phylogenies, the implications of using inferred community phylogenies in ecological studies have not been examined. Here, we evaluate how taxon sampling affects the quantification and comparison of community phylogenetic diversity using both simulated and empirical data sets. We demonstrate that inferred community trees greatly underestimate phylogenetic diversity and that the probability of incorrectly ranking community diversity can reach up to 25%, depending on the dating methods employed. We argue that to reach reliable conclusions, ecological studies must improve their taxon sampling and generate the best phylogeny possible.