Cellular and Molecular Probing of Intact Human Organs.

Optical tissue transparency permits scalable cellular and molecular investigation of complex tissues in 3D. Adult human organs are particularly challenging to render transparent because of the accumulation of dense and sturdy molecules in decades-aged tissues. To overcome these challenges, we developed SHANEL, a method based on a new tissue permeabilization approach to clear and label stiff human organs. We used SHANEL to render the intact adult human brain and kidney transparent and perform 3D histology with antibodies and dyes in centimeters-depth. Thereby, we revealed structural details of the intact human eye, human thyroid, human kidney, and transgenic pig pancreas at the cellular resolution. Furthermore, we developed a deep learning pipeline to analyze millions of cells in cleared human brain tissues within hours with standard lab computers. Overall, SHANEL is a robust and unbiased technology to chart the cellular and molecular architecture of large intact mammalian organs.

Agrochemicals interact synergistically to increase bee mortality.

Global concern over widely documented declines in pollinators1-3 has led to the identification of anthropogenic stressors that, individually, are detrimental to bee populations4-7. Synergistic interactions between these stressors could substantially amplify the environmental effect of these stressors and could therefore have important implications for policy decisions that aim to improve the health of pollinators3,8,9. Here, to quantitatively assess the scale of this threat, we conducted a meta-analysis of 356 interaction effect sizes from 90 studies in which bees were exposed to combinations of agrochemicals, nutritional stressors and/or parasites. We found an overall synergistic effect between multiple stressors on bee mortality. Subgroup analysis of bee mortality revealed strong evidence for synergy when bees were exposed to multiple agrochemicals at field-realistic levels, but interactions were not greater than additive expectations when bees were exposed to parasites and/or nutritional stressors. All interactive effects on proxies of fitness, behaviour, parasite load and immune responses were either additive or antagonistic; therefore, the potential mechanisms that drive the observed synergistic interactions for bee mortality remain unclear. Environmental risk assessment schemes that assume additive effects of the risk of agrochemical exposure may underestimate the interactive effect of anthropogenic stressors on bee mortality and will fail to protect the pollinators that provide a key ecosystem service that underpins sustainable agriculture.

Inherent mosaicism and extensive mutation of human placentas.

Placentas can exhibit chromosomal aberrations that are absent from the fetus1. The basis of this genetic segregation, which is known as confined placental mosaicism, remains unknown. Here we investigated the phylogeny of human placental cells as reconstructed from somatic mutations, using whole-genome sequencing of 86 bulk placental samples (with a median weight of 28 mg) and of 106 microdissections of placental tissue. We found that every bulk placental sample represents a clonal expansion that is genetically distinct, and exhibits a genomic landscape akin to that of childhood cancer in terms of mutation burden and mutational imprints. To our knowledge, unlike any other healthy human tissue studied so far, the placental genomes often contained changes in copy number. We reconstructed phylogenetic relationships between tissues from the same pregnancy, which revealed that developmental bottlenecks genetically isolate placental tissues by separating trophectodermal lineages from lineages derived from the inner cell mass. Notably, there were some cases with full segregation-within a few cell divisions of the zygote-of placental lineages and lineages derived from the inner cell mass. Such early embryonic bottlenecks may enable the normalization of zygotic aneuploidy. We observed direct evidence for this in a case of mosaic trisomic rescue. Our findings reveal extensive mutagenesis in placental tissues and suggest that mosaicism is a typical feature of placental development.

Extensive phylogenies of human development inferred from somatic mutations.

Starting from the zygote, all cells in the human body continuously acquire mutations. Mutations shared between different cells imply a common progenitor and are thus naturally occurring markers for lineage tracing1,2. Here we reconstruct extensive phylogenies of normal tissues from three adult individuals using whole-genome sequencing of 511 laser capture microdissections. Reconstructed embryonic progenitors in the same generation of a phylogeny often contribute to different extents to the adult body. The degree of this asymmetry varies between individuals, with ratios between the two reconstructed daughter cells of the zygote ranging from 60:40 to 93:7. Asymmetries pervade subsequent generations and can differ between tissues in the same individual. The phylogenies resolve the spatial embryonic patterning of tissues, revealing contiguous patches of, on average, 301 crypts in the adult colonic epithelium derived from a most recent embryonic cell and also a spatial effect in brain development. Using data from ten additional men, we investigated the developmental split between soma and germline, with results suggesting an extraembryonic contribution to primordial germ cells. This research demonstrates that, despite reaching the same ultimate tissue patterns, early bottlenecks and lineage commitments lead to substantial variation in embryonic patterns both within and between individuals.

The mutational landscape of human somatic and germline cells.

Over the course of an individual's lifetime, normal human cells accumulate mutations1. Here we compare the mutational landscape in 29 cell types from the soma and germline using multiple samples from the same individuals. Two ubiquitous mutational signatures, SBS1 and SBS5/40, accounted for the majority of acquired mutations in most cell types, but their absolute and relative contributions varied substantially. SBS18, which potentially reflects oxidative damage2, and several additional signatures attributed to exogenous and endogenous exposures contributed mutations to subsets of cell types. The rate of mutation was lowest in spermatogonia, the stem cells from which sperm are generated and from which most genetic variation in the human population is thought to originate. This was due to low rates of ubiquitous mutational processes and may be partially attributable to a low rate of cell division in basal spermatogonia. These results highlight similarities and differences in the maintenance of the germline and soma.

Cells of the human intestinal tract mapped across space and time.

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.

Rationally seeded computational protein design of ɑ-helical barrels.

Computational protein design is advancing rapidly. Here we describe efficient routes starting from validated parallel and antiparallel peptide assemblies to design two families of α-helical barrel proteins with central channels that bind small molecules. Computational designs are seeded by the sequences and structures of defined de novo oligomeric barrel-forming peptides, and adjacent helices are connected by loop building. For targets with antiparallel helices, short loops are sufficient. However, targets with parallel helices require longer connectors; namely, an outer layer of helix-turn-helix-turn-helix motifs that are packed onto the barrels. Throughout these computational pipelines, residues that define open states of the barrels are maintained. This minimizes sequence sampling, accelerating the design process. For each of six targets, just two to six synthetic genes are made for expression in Escherichia coli. On average, 70% of these genes express to give soluble monomeric proteins that are fully characterized, including high-resolution structures for most targets that match the design models with high accuracy.

An expandable, modular de novo protein platform for precision redox engineering.

The electron-conducting circuitry of life represents an as-yet untapped resource of exquisite, nanoscale biomolecular engineering. Here, we report the characterization and structure of a de novo diheme "maquette" protein, 4D2, which we subsequently use to create an expanded, modular platform for heme protein design. A well-folded monoheme variant was created by computational redesign, which was then utilized for the experimental validation of continuum electrostatic redox potential calculations. This demonstrates how fundamental biophysical properties can be predicted and fine-tuned. 4D2 was then extended into a tetraheme helical bundle, representing a 7 nm molecular wire. Despite a molecular weight of only 24 kDa, electron cryomicroscopy illustrated a remarkable level of detail, indicating the positioning of the secondary structure and the heme cofactors. This robust, expressible, highly thermostable and readily designable modular platform presents a valuable resource for redox protein design and the future construction of artificial electron-conducting circuitry.

Oxygenic Photosynthesis in Far-Red Light: Strategies and Mechanisms.

Oxygenic photosynthesis, the process that converts light energy into chemical energy, is traditionally associated with the absorption of visible light by chlorophyll molecules. However, recent studies have revealed a growing number of organisms capable of using far-red light (700-800 nm) to drive oxygenic photosynthesis. This phenomenon challenges the conventional understanding of the limits of this process. In this review, we briefly introduce the organisms that exhibit far-red photosynthesis and explore the different strategies they employ to harvest far-red light. We discuss the modifications of photosynthetic complexes and their impact on the delivery of excitation energy to photochemical centers and on overall photochemical efficiency. Finally, we examine the solutions employed to drive electron transport and water oxidation using relatively low-energy photons. The findings discussed here not only expand our knowledge of the remarkable adaptation capacities of photosynthetic organisms but also offer insights into the potential for enhancing light capture in crops.

Biomarker Predictors of Clinical Efficacy of the Anti-IgE Biologic Omalizumab in Severe Asthma in Adults: Results of the SoMOSA Study.

Background: The anti-IgE monoclonal antibody omalizumab is widely used for severe asthma. This study aimed to identify biomarkers that predict clinical improvement during 1 year of omalizumab treatment. Methods: One-year open-label Study of Mechanisms of action of Omalizumab in Severe Asthma (SoMOSA) involving 216 patients with severe (Global Initiative for Asthma step 4/5) uncontrolled atopic asthma (at least two severe exacerbations in the previous year) taking high-dose inhaled corticosteroids and long-acting ß-agonists with or without maintenance oral corticosteroids. It had two phases: 0-16 weeks, to assess early clinical improvement by Global Evaluation of Therapeutic Effectiveness (GETE); and 16-52 weeks, to assess late responses based on ⩾50% reduction in exacerbations or mOCS dose. All participants provided samples (exhaled breath, blood, sputum, urine) before and after 16 weeks of omalizumab treatment. Measurements and Main Results: A total of 191 patients completed phase 1; 63% had early improvement. Of 173 who completed phase 2, 69% had reduced exacerbations by ⩾50% and 57% (37 of 65) taking mOCSs had reduced their dose by ⩾50%. The primary outcomes 2,3-dinor-11-ß-PGF2α, GETE score, and standard clinical biomarkers (blood and sputum eosinophils, exhaled nitric oxide, serum IgE) did not predict either clinical response. Five volatile organic compounds and five plasma lipid biomarkers strongly predicted the ⩾50% reduction in exacerbations (receiver operating characteristic areas under the curve of 0.780 and 0.922, respectively) and early responses (areas under the curve of 0.835 and 0.949, respectively). In an independent cohort, gas chromatography/mass spectrometry biomarkers differentiated between severe and mild asthma. Conclusions: This is the first discovery of omics biomarkers that predict improvement in asthma with biologic agent treatment. Prospective validation and development for clinical use is justified.

Understanding the impact of children's and young people's self-harm on parental well-being: a systematic literature review of qualitative and quantitative findings.

BACKGROUND: Self-harm in children and young people is increasing. Parents are vital in supporting young people; however, parents may experience distress linked to the self-harm. Previous reviews have highlighted the emotional impact and need for information and support, however, have not elucidated the relationships between these themes, nor examined the quantitative data examining parents' well-being. METHODS: We conducted a mixed methods review, with qualitative meta-synthesis focusing on links between themes and quantitative synthesis of parental well-being findings, including pooled means. PsycInfo, Medline, EMBASE, AMED, CINHAL and Web of Science were searched to identify relevant records. References of included studies were also searched. Every abstract was screened by two authors. Data were extracted by one author and checked by another. RESULTS: We identified 39 reports of 32 studies: 16 with qualitative data and 17 with quantitative data (one had both). Qualitative findings showed how parents' emotions were associated to their knowledge and beliefs about self-harm. Parents' emotions often evidenced the need to self-care, but emotions of guilt reduced engagement in self-care. How parents supported their young person was linked to their knowledge, and the management of their own emotions, and influenced if they could engage in self-care. Quantitative findings were mixed, however suggested poor general mental health amongst these parents. CONCLUSIONS: Further good quality quantitative studies are needed, with measurement of psychological mechanisms that may underpin parental distress. Current evidence supports peer-support and interventions that go beyond information provision to address the connected factors of knowledge, emotion, self-care, and parenting behaviours.

Trade-offs across values in cesspool management highlight challenges to policy making.

On-site Sewage Disposal Systems (OSDS) are globally common, and in Hawai'i they present a risk of contamination to drinking water sources and nearshore waters. State legislation has commanded that all cesspools are to be banned by 2050, thus requiring tens of thousands of systems to be converted in the coming decades. This project followed a participatory structured decision-making (SDM) approach to collaboratively design cost-effective and equitable solutions for thousands of cesspools in the high elevation areas of north Maui, Hawai'i. Participatory workshops with a diverse group of stakeholders set ten objectives and brainstormed 33 alternatives, for which the technical team then modeled groundwater nutrients, costs, and equity. All alternatives posed trade-offs, though composting toilets performed best across most objectives, albeit with high maintenance burden. Discounting innovative toilets, the multi-objective analysis suggests that the state should invest in cluster sewering of high-density communities, followed by incentivizing septic tank solutions in properties with the highest effluent flow first, then expanding across the area. The total project cost (installation and operation/maintenance) would be $183-258 million, depending upon the sewer-septic combination. An efficiency frontier reveals sub-par combinations, including aerobic treatment units and passive absorption systems, which cost much more and deliver lower mass flux reduction than more cost-effective alternatives. This study contributes a novel case of rural sanitation to the literature in which decision support tools are used to facilitate evidence-based, collaborative decision-making for sanitation planning. The state could use a similar participatory SDM process when approaching other communities to discuss their cesspool upgrade strategies. Broadening the use of decision analytic techniques can have wider ecological, economic, and social benefits for the state and contexts beyond Hawai'i, as SDM provides a transparent and rigorous, evidence-based decision-theoretic framework to explore multiple values and strategies to address difficult resource management problems.

Survival and Incidence Patterns of Pancreatic Neuroendocrine Tumors Over the Last 2 Decades: A SEER Database Analysis.

BACKGROUND: Pancreatic neuroendocrine tumors (pNETs) are rare cancers with outcomes determined by multiple factors including grade, stage, and clinical presentation. In this study, we aimed to determine the prognosis of patients with pNETs using a large population-based database. MATERIALS AND METHODS: In this population-based study, we identified patients with pNETs from the SEER 18 registry (2000-2016) using a combination of ICD-O-3 and histology codes. We calculated age-adjusted incidence rates using SEER*Stat 8.3.5. In addition, we analyzed overall survival (OS) using the Kaplan-Meier method, and investigated prognostic factors using a multivariable Cox proportional hazards model. RESULTS: A total of 8944 pNETs patients were identified. Annual incidence rates increased from 0.27 to 1.00 per 100 000. This was largely explained by an increase in number of patients diagnosed with localized disease in more recent years (2012-2016). Median OS was 68 months (95% CI [64, 73]) and 5-year OS rates in localized, regional, and metastatic disease were 83%, 67%, and 28%, respectively. There was a significant improvement in OS for patients diagnosed between 2009 and 2016 (median OS 85 months) compared with those diagnosed between 2000 and 2008 (median OS 46 months) (HR 0.66; 95% CI [0.62, 0.70]). This improvement in OS was consistent across all stages. CONCLUSIONS AND RELEVANCE: This study shows a steady increase pNETs incidence with notable stage migration to earlier stages in recent years. This increase in incidence is accompanied by a significant improvement in survival across different disease stages.

Probing the electronic structure and photophysics of thiophene-diketopyrrolopyrrole derivatives in solution.

Diketopyrrolopyrroles are a popular class of electron-withdrawing unit in optoelectronic materials. When combined with electron donating side-chain functional groups such as thiophenes, they form a very broad class of donor-acceptor molecules: thiophene-diketopyrrolopyrroles (TDPPs). Despite their widescale use in biosensors and photovoltaic materials, studies have yet to establish the important link between the electronic structure of the specific TDPP and the critical optical properties. To bridge this gap, ultrafast transient absorption with 22 fs time resolution has been used to explore the photophysics of three prototypical TDPP molecules: a monomer, dimer and polymer in solution. Interpretation of experimental data was assisted by a recent high-level theoretical study, and additional density functional theory calculations. These studies show that the photophysics of these molecular prototypes under visible photoexcitation are determined by just two excited electronic states, having very different electronic characters (one is optically bright, the other dark), their relative energetic ordering and the timescales for internal conversion from one to the other and/or to the ground state. The underlying difference in electronic structure alters the branching between these excited states and their associated dynamics. In turn, these factors dictate the fluorescence quantum yields, which are shown to vary by ∼1-2 orders of magnitude across the TDPP prototypes investigated here. The fast non-radiative transfer of molecules from the bright to dark states is mediated by conical intersections. Remarkably, wavepacket signals in the measured transient absorption data carry signatures of the nuclear motions that enable mixing of the electronic-nuclear wavefunction and facilitate non-adiabatic coupling between the bright and dark states.

Singlet and Triplet Contributions to the Excited-State Activities of Dihydrophenazine, Phenoxazine, and Phenothiazine Organocatalysts Used in Atom Transfer Radical Polymerization.

The photochemical dynamics of three classes of organic photoredox catalysts employed in organocatalyzed atom-transfer radical polymerization (O-ATRP) are studied using time-resolved optical transient absorption and fluorescence spectroscopy. The nine catalysts selected for study are examples of N-aryl and core-substituted dihydrophenazine, phenoxazine and phenothiazine compounds with varying propensities for control of polymerization outcomes. Excited singlet-state lifetimes extracted from the spectroscopic measurements are reported in N,N-dimethylformamide (DMF), dichloromethane (DCM), and toluene. Ultrafast (<200 fs to 3 ps) electronic relaxation of the photocatalysts after photoexcitation at near-UV wavelengths (318-390 nm) populates the first singlet excited state (S1). The S1-state lifetimes range from 130 ps to 40 ns with a considerable dependence on the photocatalyst structure and the solvent. The competition between ground electronic state recovery and intersystem crossing controls triplet state populations and is a minor pathway in the dihydrophenazine derivatives but is of greater importance for phenoxazine and phenothiazine catalysts. A comparison of our results with previously reported O-ATRP performances of the various photoredox catalysts shows that high triplet-state quantum yields are not a prerequisite for controlling polymer dispersity. For example, the photocatalyst 5,10-bis(4-cyanophenyl)-5,10-dihydrophenazine, shown previously to exert good polymerization control, possesses the shortest S1-state lifetime (135 ps in DMF and 180 ps in N,N-dimethylacetamide) among the nine examples reported here and a negligible triplet-state quantum yield. The results call for a re-evaluation of the excited-state properties of most significance in governing the photocatalytic behavior of organic photoredox catalysts in O-ATRP reactions.

Photosynthesis and crop productivity are enhanced by glucose-functionalised carbon dots.

From global food security to textile production and biofuels, the demands currently made on plant photosynthetic productivity will continue to increase. Enhancing photosynthesis using designer, green and sustainable materials offers an attractive alternative to current genetic-based strategies and promising work with nanomaterials has recently started to emerge. Here we describe the in planta use of carbon-based nanoparticles produced by low-cost renewable routes that are bioavailable to mature plants. Uptake of these functionalised nanoparticles directly from the soil improves photosynthesis and also increases crop production. We show for the first time that glucose functionalisation enhances nanoparticle uptake, photoprotection and pigment production, unlocking enhanced yields. This was demonstrated in Triticum aestivum 'Apogee' (dwarf bread wheat) and resulted in an 18% increase in grain yield. This establishes the viability of a functional nanomaterial to augment photosynthesis as a route to increased crop productivity.

Insights on Geochemical, Isotopic, and Volumetric Compositions of Produced Water from Hydraulically Fractured Williston Basin Oil Wells.

Tracing produced water origins from wells hydraulically fractured with freshwater-based fluids is sometimes predicated on assumptions that (1) each geological formation contains compositionally unique brine and (2) produced water from recently hydraulically fractured wells resembles fresher meteoric water more so than produced water from older wells. These assumptions are not valid in Williston Basin oil wells sampled in this study. Although distinct average 228Ra/226Ra ratios were found in water produced from the Bakken and Three Forks Formations, average δ2H, δ18O, specific gravity, and conductivity were similar but exhibited significant variability across five oil fields within each formation. Furthermore, initial produced water ("flowback") was operationally defined based on the presence of glycol ether compounds and water from wells that had produced <56% of the amount of fluids injected and sampled within 160 days of fracturing. Flowback unexpectedly exhibited higher temperature, specific gravity, conductivity, δ2H, and δ18O, but lower oxidation-reduction potential and δ11B, relative to the wells thought to be producing formation brines (from wells with a produced-to-injected water ratio [PIWR] > 0.84 and sampled more than 316 days after fracturing). As such, establishing an overall geochemical and isotopic signature of produced water compositions based solely on chemical similarity to meteoric water and formation without the consideration of well treatments, well completion depth, or lateral location across the basin could be misleading if these signatures are assumed to be applicable across the entire basin. These findings have implications for using produced water compositions to understand the interbasin fluid flow and trace sources of hydraulic fracturing fluids.

Bridging the Gap Among Clinical Practice Guidelines for Pain Management in Cancer and Sickle Cell Disease.

Opioids are a critical component of pain relief strategies for the management of patients with cancer and sickle cell disease. The escalation of opioid addiction and overdose in the United States has led to increased scrutiny of opioid prescribing practices. Multiple reports have revealed that regulatory and coverage policies, intended to curb inappropriate opioid use, have created significant barriers for many patients. The Centers for Disease Control and Prevention, National Comprehensive Cancer Network, and American Society of Clinical Oncology each publish clinical practice guidelines for the management of chronic pain. A recent JAMA Oncology article highlighted perceived variability in recommendations among these guidelines. In response, leadership from guideline organizations, government representatives, and authors of the original article met to discuss challenges and solutions. The meeting featured remarks by the Commissioner of Food and Drugs, presentations on each clinical practice guideline, an overview of the pain management needs of patients with sickle cell disease, an overview of perceived differences among guidelines, and a discussion of differences and commonalities among the guidelines. The meeting revealed that although each guideline varies in the intended patient population, target audience, and methodology, there is no disagreement among recommendations when applied to the appropriate patient and clinical situation. It was determined that clarification and education are needed regarding the intent, patient population, and scope of each clinical practice guideline, rather than harmonization of guideline recommendations. Clinical practice guidelines can serve as a resource for policymakers and payers to inform policy and coverage determinations.