Fine-scale contemporary recombination variation and its fitness consequences in adaptively diverging stickleback fish.

Despite deep evolutionary conservation, recombination rates vary greatly across the genome and among individuals, sexes and populations. Yet the impact of this variation on adaptively diverging populations is not well understood. Here we characterized fine-scale recombination landscapes in an adaptively divergent pair of marine and freshwater populations of threespine stickleback from River Tyne, Scotland. Through whole-genome sequencing of large nuclear families, we identified the genomic locations of almost 50,000 crossovers and built recombination maps for marine, freshwater and hybrid individuals at a resolution of 3.8 kb. We used these maps to quantify the factors driving variation in recombination rates. We found strong heterochiasmy between sexes but also differences in recombination rates among ecotypes. Hybrids showed evidence of significant recombination suppression in overall map length and in individual loci. Recombination rates were lower not only within individual marine-freshwater-adaptive loci, but also between loci on the same chromosome, suggesting selection on linked gene 'cassettes'. Through temporal sampling along a natural hybrid zone, we found that recombinants showed traits associated with reduced fitness. Our results support predictions that divergence in cis-acting recombination modifiers, whose functions are disrupted in hybrids, may play an important role in maintaining differences among adaptively diverging populations.

Boundary stacking interactions enable cross-TAD enhancer-promoter communication during limb development.

Although promoters and their enhancers are frequently contained within a topologically associating domain (TAD), some developmentally important genes have their promoter and enhancers within different TADs. Hypotheses about molecular mechanisms enabling cross-TAD interactions remain to be assessed. To test these hypotheses, we used optical reconstruction of chromatin architecture to characterize the conformations of the Pitx1 locus on single chromosomes in developing mouse limbs. Our data support a model in which neighboring boundaries are stacked as a result of loop extrusion, bringing boundary-proximal cis-elements into contact. This stacking interaction also contributes to the appearance of architectural stripes in the population average maps. Through molecular dynamics simulations, we found that increasing boundary strengths facilitates the formation of the stacked boundary conformation, counter-intuitively facilitating border bypass. This work provides a revised view of the TAD borders' function, both facilitating and preventing cis-regulatory interactions, and introduces a framework to distinguish border-crossing from border-respecting enhancer-promoter pairs.

Multi-Functional Small Molecule Alleviates Fracture Pain and Promotes Bone Healing.

Bone injuries such as fractures are one major cause of morbidities worldwide. A considerable number of fractures suffer from delayed healing, and the unresolved acute pain may transition to chronic and maladaptive pain. Current management of pain involves treatment with NSAIDs and opioids with substantial adverse effects. Herein, we tested the hypothesis that the purine molecule, adenosine, can simultaneously alleviate pain and promote healing in a mouse model of tibial fracture by targeting distinctive adenosine receptor subtypes in different cell populations. To achieve this, a biomaterial-assisted delivery of adenosine is utilized to localize and prolong its therapeutic effect at the injury site. The results demonstrate that local delivery of adenosine inhibited the nociceptive activity of peripheral neurons through activation of adenosine A1 receptor (ADORA1) and mitigated pain as demonstrated by weight bearing and open field movement tests. Concurrently, local delivery of adenosine at the fracture site promoted osteogenic differentiation of mesenchymal stromal cells through adenosine A2B receptor (ADORA2B) resulting in improved bone healing as shown by histological analyses and microCT imaging. This study demonstrates the dual role of adenosine and its material-assisted local delivery as a feasible therapeutic approach to treat bone trauma and associated pain.

The genetic basis of novel trait gain in walking fish.

A major goal in biology is to understand how organisms evolve novel traits. Multiple studies have identified genes contributing to regressive evolution, the loss of structures that existed in a recent ancestor. However, fewer examples exist for genes underlying constructive evolution, the gain of novel structures and capabilities in lineages that previously lacked them. Sea robins are fish that have evolved enlarged pectoral fins, six mobile locomotory fin rays (legs) and six novel macroscopic lobes in the central nervous system (CNS) that innervate the corresponding legs. Here, we establish successful husbandry and use a combination of transcriptomics, CRISPR-Cas9 editing, and behavioral assays to identify key transcription factors that are required for leg formation and function in sea robins. We also generate hybrids between two sea robin species with distinct leg morphologies and use allele-specific expression analysis and gene editing to explore the genetic basis of species-specific trait diversity, including a novel sensory gain of function. Collectively, our study establishes sea robins as a new model for studying the genetic basis of novel organ formation, and demonstrates a crucial role for the conserved limb gene tbx3a in the evolution of chemosensory legs in walking fish.

Whole-genome Comparisons Identify Repeated Regulatory Changes Underlying Convergent Appendage Evolution in Diverse Fish Lineages.

Fins are major functional appendages of fish that have been repeatedly modified in different lineages. To search for genomic changes underlying natural fin diversity, we compared the genomes of 36 percomorph fish species that span over 100 million years of evolution and either have complete or reduced pelvic and caudal fins. We identify 1,614 genomic regions that are well-conserved in fin-complete species but missing from multiple fin-reduced lineages. Recurrent deletions of conserved sequences in wild fin-reduced species are enriched for functions related to appendage development, suggesting that convergent fin reduction at the organismal level is associated with repeated genomic deletions near fin-appendage development genes. We used sequencing and functional enhancer assays to confirm that PelA, a Pitx1 enhancer previously linked to recurrent pelvic loss in sticklebacks, has also been independently deleted and may have contributed to the fin morphology in distantly related pelvic-reduced species. We also identify a novel enhancer that is conserved in the majority of percomorphs, drives caudal fin expression in transgenic stickleback, is missing in tetraodontiform, syngnathid, and synbranchid species with caudal fin reduction, and alters caudal fin development when targeted by genome editing. Our study illustrates a broadly applicable strategy for mapping phenotypes to genotypes across a tree of vertebrate species and highlights notable new examples of regulatory genomic hotspots that have been used to evolve recurrent phenotypes across 100 million years of fish evolution.

Sea robins.

Allard et al. provide an overview of sea robins, a group of benthic fish that have evolved leg-like appendages that they use to walk on the sea floor and find prey.

Sophorolipid Candidates Demonstrate Cytotoxic Efficacy against 2D and 3D Breast Cancer Models.

Sophorolipids are biosurfactants derived from the nonpathogenic yeasts such as Starmerella bombicola with potential efficacy in anticancer applications. Simple and cost-effective synthesis of these drugs makes them a promising alternative to traditional chemotherapeutics, pending their success in preliminary drug-screening. Drug-screening typically utilizes 2D cell monolayers due to their simplicity and ease of high-throughput assessment. However, 2D assays fail to capture the complexity and 3D context of the tumor microenvironment and have consequently been implicated in the high percentage of drugs investigated in vitro that later fail in clinical trials. Herein, we screened two sophorolipid candidates and a clinically-used chemotherapeutic, doxorubicin, on in vitro breast cancer models ranging from 2D monolayers to 3D spheroids, employing optical coherence tomography to confirm these morphologies. We calculated corresponding IC50 values for these drugs and found one of the sophorolipids to have comparable toxicities to the chemotherapeutic control. Our findings show increased drug resistance associated with model dimensionality, such that all drugs tested showed that 3D spheroids exhibited higher IC50 values than their 2D counterparts. These findings demonstrate promising preliminary data to support the use of sophorolipids as a more affordable alternative to traditional clinical interventions and demonstrate the importance of 3D tumor models in assessing drug response.

Self-assembled innervated vasculature-on-a-chip to study nociception.

Nociceptor sensory neurons play a key role in eliciting pain. An active crosstalk between nociceptor neurons and the vascular system at the molecular and cellular level is required to sense and respond to noxious stimuli. Besides nociception, interaction between nociceptor neurons and vasculature also contributes to neurogenesis and angiogenesis.In vitromodels of innervated vasculature can greatly help delineate these roles while facilitating disease modeling and drug screening. Herein, we report the development of a microfluidic-assisted tissue model of nociception in the presence of microvasculature. The self-assembled innervated microvasculature was engineered using endothelial cells and primary dorsal root ganglion (DRG) neurons. The sensory neurons and the endothelial cells displayed distinct morphologies in presence of each other. The neurons exhibited an elevated response to capsaicin in the presence of vasculature. Concomitantly, increased transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor expression was observed in the DRG neurons in presence of vascularization. Finally, we demonstrated the applicability of this platform for modeling nociception associated with tissue acidosis. While not demonstrated here, this platform could also serve as a tool to study pain resulting from vascular disorders while also paving the way towards the development of innervated microphysiological models.

Whole-genome comparisons identify repeated regulatory changes underlying convergent appendage evolution in diverse fish lineages.

Fins are major functional appendages of fish that have been repeatedly modified in different lineages. To search for genomic changes underlying natural fin diversity, we compared the genomes of 36 wild fish species that either have complete or reduced pelvic and caudal fins. We identify 1,614 genomic regions that are well-conserved in fin-complete species but missing from multiple fin-reduced lineages. Recurrent deletions of conserved sequences (CONDELs) in wild fin-reduced species are enriched for functions related to appendage development, suggesting that convergent fin reduction at the organismal level is associated with repeated genomic deletions near fin-appendage development genes. We used sequencing and functional enhancer assays to confirm that PelA , a Pitx1 enhancer previously linked to recurrent pelvic loss in sticklebacks, has also been independently deleted and may have contributed to the fin morphology in distantly related pelvic-reduced species. We also identify a novel enhancer that is conserved in the majority of percomorphs, drives caudal fin expression in transgenic stickleback, is missing in tetraodontiform, s yngnathid, and synbranchid species with caudal fin reduction, and which alters caudal fin development when targeted by genome editing. Our study illustrates a general strategy for mapping phenotypes to genotypes across a tree of vertebrate species, and highlights notable new examples of regulatory genomic hotspots that have been used to evolve recurrent phenotypes during 100 million years of fish evolution.

Viscoelastic Properties of Bioprinted Alginate Microbeads Compared to Their Bulk Hydrogel Analogs.

Hydrogel microbeads are engineered spherical microgels widely used for biomedical applications in cell cultures, tissue engineering, and drug delivery. Their mechanical and physical properties (i.e., modulus, porosity, diffusion) heavily influence their utility by affecting encapsulated cellular behavior, biopayload elution kinetics, and stability for longer term cultures. There is a need to quantify these properties to guide microbead design for effective application. However, there are few techniques with the µN-level resolution required to evaluate these relatively small, compliant constructs. To circumvent mechanically testing individual microbeads, researchers often approximate microbead properties by characterizing larger bulk gel analogs of the same material formulation. This approach provides some insight into the hydrogel properties. However, bulk gels possess key structural and mechanical differences compared to their microbead equivalents, which may limit their accuracy and utility as analogs for estimating microbead properties. Herein, we explore how microbead properties are influenced by hydrogel formulation (i.e., alginate concentration, divalent cation crosslinker, and crosslinker concentration), and whether these trends are accurately reflected in bulk gel analogs. To accomplish this, we utilize laser direct-write bioprinting to create 12 × 12 arrays of alginate microbeads and characterize all 144 microbeads in parallel using a commercially available microcompression system. In this way, the compressive load is distributed across a large number of beads, thus amplifying sample signal. Comparing microbead properties to those of their bulk gel analogs, we found that their trends in modulus, porosity, and diffusion with hydrogel formulation are consistent, yet bulk gels exhibit significant discrepancies in their measured values.

Evolution of stickleback spines through independent cis-regulatory changes at HOXDB.

Understanding the mechanisms leading to new traits or additional features in organisms is a fundamental goal of evolutionary biology. We show that HOXDB regulatory changes have been used repeatedly in different fish genera to alter the length and number of the prominent dorsal spines used to classify stickleback species. In Gasterosteus aculeatus (typically 'three-spine sticklebacks'), a variant HOXDB allele is genetically linked to shortening an existing spine and adding an additional spine. In Apeltes quadracus (typically 'four-spine sticklebacks'), a variant HOXDB allele is associated with lengthening a spine and adding an additional spine in natural populations. The variant alleles alter the same non-coding enhancer region in the HOXDB locus but do so by diverse mechanisms, including single-nucleotide polymorphisms, deletions and transposable element insertions. The independent regulatory changes are linked to anterior expansion or contraction of HOXDB expression. We propose that associated changes in spine lengths and numbers are partial identity transformations in a repeating skeletal series that forms major defensive structures in fish. Our findings support the long-standing hypothesis that natural Hox gene variation underlies key patterning changes in wild populations and illustrate how different mutational mechanisms affecting the same region may produce opposite gene expression changes with similar phenotypic outcomes.

Stimulated Raman generation of aqueous singlet oxygen without photosensitizers.

Singlet oxygen is traditionally produced via photosensitizer molecules such as methylene blue, which function as catalysts. Here we investigate stimulated Raman generation of singlet oxygen from dissolved oxygen in both water (H2O) and heavy water (D2O) using nanosecond-pulsed visible blue laser light in the 400-440 nm spectral region without singlet oxygen photosensitizers. We report an oxygen-dependent Stokes peak in the red spectrum (600-670 nm) that is identical when produced in H2O and D2O. These red Stokes photons are not detected when an oxygen quencher is present. Temporal photodepletion of the uric acid absorbance peak at 294 nm is consistent with singlet oxygen generation. We postulate that a two-photon stimulated Raman process produces singlet oxygen from O2 dissolved within the solvents. We note that the energy difference between input and output photons of 0.97 eV is precisely the energy needed to excite O2 to its singlet state.

Genomic changes underlying repeated niche shifts in an adaptive radiation.

In adaptive radiations, single lineages rapidly diversify by adapting to many new niches. Little is known yet about the genomic mechanisms involved, that is, the source of genetic variation or genomic architecture facilitating or constraining adaptive radiation. Here, we investigate genomic changes associated with repeated invasion of many different freshwater niches by threespine stickleback in the Haida Gwaii archipelago, Canada, by resequencing single genomes from one marine and 28 freshwater populations. We find 89 likely targets of parallel selection in the genome that are enriched for old standing genetic variation. In contrast to theoretical expectations, their genomic architecture is highly dispersed with little clustering. Candidate genes and genotype-environment correlations match the three major environmental axes predation regime, light environment, and ecosystem size. In a niche space with these three dimensions, we find that the more divergent a new niche from the ancestral marine habitat, the more loci show signatures of parallel selection. Our findings suggest that the genomic architecture of parallel adaptation in adaptive radiation depends on the steepness of ecological gradients and the dimensionality of the niche space.

Debate: Child and adolescent mental health professionals have a responsibility to diagnose Personality Disorder.

In the United Kingdom, mental health professionals have been historically reluctant to diagnose Personality Disorder in young people over many decades, and there is little evidence that much has changed. However, there are consequences to this reluctance. Personality Disorders in young people are absent from national prevalence studies and research is sparse, as a lack of diagnosed patients makes it hard to recruit for studies. Consequently, national policy-makers lack an awareness of the scale of the problem and the focus on service provision to address it. Deliberate misdiagnosis of Personality Disorder due to professionals' unwillingness to use the diagnosis is not only unethical but can also cause harm as it stops young people from receiving the evidence-based interventions and support that they require. Young people and families often experience reassurance and relief when they receive an accurate diagnosis. Professionals need to reconsider whether their reluctance to diagnose is in their patients' best interests.

Financial and Quality Metrics of A Large, Publicly Traded U.S. Nursing Home Chain in the Age of Covid-19.

Nursing homes faced serious challenges with large COVID-19 resident infection rates and deaths during the pandemic. This descriptive case study examined the structure, operations, strategies, care outcomes, and owners of The Ensign Group Inc. the second largest U.S. for-profit chain, between 2007 and 2021. Ensign, as a holding company, has a complex organizational structure that uses more than 430 corporate entities to manage its 228 nursing homes and senior living facilities. With mostly Medicare and Medicaid revenues and favorable government COVID-19 relief, Ensign grew rapidly, even during the pandemic, to $2.5 billion (all amounts in U.S. Dollars) in revenues with a market capitalization of $4.5 billion and strong profits and financial metrics in 2020 to 2021. The company used real estate purchasing, debt financing, and spin-off companies, and tax arbitrage to optimize shareholder value. Before and during the pandemic, its 198 nursing homes had low registered nurse and total nurse staffing levels and regulatory violations with below-average ratings, and they had high COVID-19 infection rates during the pandemic. Ensign's small board, executives, and institutional investors protected and enhanced shareholder interests rather than ensuring that its nursing homes met professional standards and regulatory requirements.

Characterization of mouse Bmp5 regulatory injury element in zebrafish wound models.

Many key signaling molecules used to build tissues during embryonic development are re-activated at injury sites to stimulate tissue regeneration and repair. Bone morphogenetic proteins provide a classic example, but the mechanisms that lead to reactivation of BMPs following injury are still unknown. Previous studies have mapped a large "injury response element" (IRE) in the mouse Bmp5 gene that drives gene expression following bone fractures and other types of injury. Here we show that the large mouse IRE region is also activated in both zebrafish tail resection and mechanosensory hair cell injury models. Using the ability to test multiple constructs and image temporal and spatial dynamics following injury responses, we have narrowed the original size of the mouse IRE region by over 100 fold and identified a small 142 bp minimal enhancer that is rapidly induced in both mesenchymal and epithelial tissues after injury. These studies identify a small sequence that responds to evolutionarily conserved local signals in wounded tissues and suggest candidate pathways that contribute to BMP reactivation after injury.

Genetic studies of human-chimpanzee divergence using stem cell fusions.

Complete genome sequencing has identified millions of DNA changes that differ between humans and chimpanzees. Although a subset of these changes likely underlies important phenotypic differences between humans and chimpanzees, it is currently difficult to distinguish causal from incidental changes and to map specific phenotypes to particular genome locations. To facilitate further genetic study of human-chimpanzee divergence, we have generated human and chimpanzee autotetraploids and allotetraploids by fusing induced pluripotent stem cells (iPSCs) of each species. The resulting tetraploid iPSCs can be stably maintained and retain the ability to differentiate along ectoderm, mesoderm, and endoderm lineages. RNA sequencing identifies thousands of genes whose expression differs between humans and chimpanzees when assessed in single-species diploid or autotetraploid iPSCs. Analysis of gene expression patterns in interspecific allotetraploid iPSCs shows that human-chimpanzee expression differences arise from substantial contributions of both cis-acting changes linked to the genes themselves and trans-acting changes elsewhere in the genome. To enable further genetic mapping of species differences, we tested chemical treatments for stimulating genome-wide mitotic recombination between human and chimpanzee chromosomes, and CRISPR methods for inducing species-specific changes on particular chromosomes in allotetraploid cells. We successfully generated derivative cells with nested deletions or interspecific recombination on the X chromosome. These studies confirm an important role for the X chromosome in trans regulation of expression differences between species and illustrate the potential of this system for more detailed cis and trans mapping of the molecular basis of human and chimpanzee evolution.

Longer or shorter spines: Reciprocal trait evolution in stickleback via triallelic regulatory changes in Stanniocalcin2a.

Vertebrates have repeatedly modified skeletal structures to adapt to their environments. The threespine stickleback is an excellent system for studying skeletal modifications, as different wild populations have either increased or decreased the lengths of their prominent dorsal and pelvic spines in different freshwater environments. Here we identify a regulatory locus that has a major morphological effect on the length of stickleback dorsal and pelvic spines, which we term Maser (major spine enhancer). Maser maps in a closely linked supergene complex that controls multiple armor, feeding, and behavioral traits on chromosome IV. Natural alleles in Maser are differentiated between marine and freshwater sticklebacks; however, alleles found among freshwater populations are also differentiated, with distinct alleles found in short- and long-spined freshwater populations. The distinct freshwater alleles either increase or decrease expression of the bone growth inhibitor gene Stanniocalcin2a in developing spines, providing a simple genetic mechanism for either increasing or decreasing spine lengths in natural populations. Genomic surveys suggest many recurrently differentiated loci in sticklebacks are similarly specialized into three or more distinct alleles, providing multiple ancient standing variants in particular genes that may contribute to a range of phenotypes in different environments.

Predicting future from past: The genomic basis of recurrent and rapid stickleback evolution.

Similar forms often evolve repeatedly in nature, raising long-standing questions about the underlying mechanisms. Here, we use repeated evolution in stickleback to identify a large set of genomic loci that change recurrently during colonization of freshwater habitats by marine fish. The same loci used repeatedly in extant populations also show rapid allele frequency changes when new freshwater populations are experimentally established from marine ancestors. Marked genotypic and phenotypic changes arise within 5 years, facilitated by standing genetic variation and linkage between adaptive regions. Both the speed and location of changes can be predicted using empirical observations of recurrence in natural populations or fundamental genomic features like allelic age, recombination rates, density of divergent loci, and overlap with mapped traits. A composite model trained on these stickleback features can also predict the location of key evolutionary loci in Darwin's finches, suggesting that similar features are important for evolution across diverse taxa.