Engineering a Model Cell for Rational Tuning of GPCR Signaling.

G protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond to specific cues in their environment. However, the relationship between stimulus and response for each GPCR is difficult to predict due to diversity in natural signal transduction architecture and expression. Using genome engineering in yeast, we constructed an insulated, modular GPCR signal transduction system to study how the response to stimuli can be predictably tuned using synthetic tools. We delineated the contributions of a minimal set of key components via computational and experimental refactoring, identifying simple design principles for rationally tuning the dose response. Using five different GPCRs, we demonstrate how this enables cells and consortia to be engineered to respond to desired concentrations of peptides, metabolites, and hormones relevant to human health. This work enables rational tuning of cell sensing while providing a framework to guide reprogramming of GPCR-based signaling in other systems.

A Public BCR Present in a Unique Dual-Receptor-Expressing Lymphocyte from Type 1 Diabetes Patients Encodes a Potent T Cell Autoantigen.

T and B cells are the two known lineages of adaptive immune cells. Here, we describe a previously unknown lymphocyte that is a dual expresser (DE) of TCR and BCR and key lineage markers of both B and T cells. In type 1 diabetes (T1D), DEs are predominated by one clonotype that encodes a potent CD4 T cell autoantigen in its antigen binding site. Molecular dynamics simulations revealed that this peptide has an optimal binding register for diabetogenic HLA-DQ8. In concordance, a synthetic version of the peptide forms stable DQ8 complexes and potently stimulates autoreactive CD4 T cells from T1D patients, but not healthy controls. Moreover, mAbs bearing this clonotype are autoreactive against CD4 T cells and inhibit insulin tetramer binding to CD4 T cells. Thus, compartmentalization of adaptive immune cells into T and B cells is not absolute, and violators of this paradigm are likely key drivers of autoimmune diseases.

Dual quantum spin Hall insulator by density-tuned correlations in TaIrTe4.

The convergence of topology and correlations represents a highly coveted realm in the pursuit of new quantum states of matter1. Introducing electron correlations to a quantum spin Hall (QSH) insulator can lead to the emergence of a fractional topological insulator and other exotic time-reversal-symmetric topological order2-8, not possible in quantum Hall and Chern insulator systems. Here we report a new dual QSH insulator within the intrinsic monolayer crystal of TaIrTe4, arising from the interplay of its single-particle topology and density-tuned electron correlations. At charge neutrality, monolayer TaIrTe4 demonstrates the QSH insulator, manifesting enhanced nonlocal transport and quantized helical edge conductance. After introducing electrons from charge neutrality, TaIrTe4 shows metallic behaviour in only a small range of charge densities but quickly goes into a new insulating state, entirely unexpected on the basis of the single-particle band structure of TaIrTe4. This insulating state could arise from a strong electronic instability near the van Hove singularities, probably leading to a charge density wave (CDW). Remarkably, within this correlated insulating gap, we observe a resurgence of the QSH state. The observation of helical edge conduction in a CDW gap could bridge spin physics and charge orders. The discovery of a dual QSH insulator introduces a new method for creating topological flat minibands through CDW superlattices, which offer a promising platform for exploring time-reversal-symmetric fractional phases and electromagnetism2-4,9,10.

Layer Hall effect in a 2D topological axion antiferromagnet.

Whereas ferromagnets have been known and used for millennia, antiferromagnets were only discovered in the 1930s1. At large scale, because of the absence of global magnetization, antiferromagnets may seem to behave like any non-magnetic material. At the microscopic level, however, the opposite alignment of spins forms a rich internal structure. In topological antiferromagnets, this internal structure leads to the possibility that the property known as the Berry phase can acquire distinct spatial textures2,3. Here we study this possibility in an antiferromagnetic axion insulator-even-layered, two-dimensional MnBi2Te4-in which spatial degrees of freedom correspond to different layers. We observe a type of Hall effect-the layer Hall effect-in which electrons from the top and bottom layers spontaneously deflect in opposite directions. Specifically, under zero electric field, even-layered MnBi2Te4 shows no anomalous Hall effect. However, applying an electric field leads to the emergence of a large, layer-polarized anomalous Hall effect of about 0.5e2/h (where e is the electron charge and h is Planck's constant). This layer Hall effect uncovers an unusual layer-locked Berry curvature, which serves to characterize the axion insulator state. Moreover, we find that the layer-locked Berry curvature can be manipulated by the axion field formed from the dot product of the electric and magnetic field vectors. Our results offer new pathways to detect and manipulate the internal spatial structure of fully compensated topological antiferromagnets4-9. The layer-locked Berry curvature represents a first step towards spatial engineering of the Berry phase through effects such as layer-specific moiré potential.

Rapid growth of new atmospheric particles by nitric acid and ammonia condensation.

A list of authors and their affiliations appears at the end of the paper New-particle formation is a major contributor to urban smog1,2, but how it occurs in cities is often puzzling3. If the growth rates of urban particles are similar to those found in cleaner environments (1-10 nanometres per hour), then existing understanding suggests that new urban particles should be rapidly scavenged by the high concentration of pre-existing particles. Here we show, through experiments performed under atmospheric conditions in the CLOUD chamber at CERN, that below about +5 degrees Celsius, nitric acid and ammonia vapours can condense onto freshly nucleated particles as small as a few nanometres in diameter. Moreover, when it is cold enough (below -15 degrees Celsius), nitric acid and ammonia can nucleate directly through an acid-base stabilization mechanism to form ammonium nitrate particles. Given that these vapours are often one thousand times more abundant than sulfuric acid, the resulting particle growth rates can be extremely high, reaching well above 100 nanometres per hour. However, these high growth rates require the gas-particle ammonium nitrate system to be out of equilibrium in order to sustain gas-phase supersaturations. In view of the strong temperature dependence that we measure for the gas-phase supersaturations, we expect such transient conditions to occur in inhomogeneous urban settings, especially in wintertime, driven by vertical mixing and by strong local sources such as traffic. Even though rapid growth from nitric acid and ammonia condensation may last for only a few minutes, it is nonetheless fast enough to shepherd freshly nucleated particles through the smallest size range where they are most vulnerable to scavenging loss, thus greatly increasing their survival probability. We also expect nitric acid and ammonia nucleation and rapid growth to be important in the relatively clean and cold upper free troposphere, where ammonia can be convected from the continental boundary layer and nitric acid is abundant from electrical storms4,5.

A mutagenesis study of autoantigen optimization for potential T1D vaccine design.

A previously reported autoreactive antigen, termed the X-idiotype, isolated from a unique cell population in Type 1 diabetes (T1D) patients, was found to stimulate their CD4+ T cells. This antigen was previously determined to bind more favorably than insulin and its mimic (insulin superagonist) to HLA-DQ8, supporting its strong role in CD4+ T cell activation. In this work, we probed HLA-X-idiotype-TCR binding and designed enhanced-reactive pHLA-TCR antigens using an in silico mutagenesis approach which we functionally validated by cell proliferation assays and flow cytometry. From a combination of single, double, and swap mutations, we identified antigen-binding sites p4 and p6 as potential mutation sites for HLA binding affinity enhancement. Site p6 is revealed to favor smaller but more hydrophobic residues than the native tyrosine, such as valine (Y6V) and isoleucine (Y6I), indicating a steric mechanism in binding affinity improvement. Meanwhile, site p4 methionine mutation to hydrophobic residues isoleucine (M4I) or leucine (M4L) modestly increases HLA binding affinity. Select p6 mutations to cysteine (Y6C) or isoleucine (Y6I) exhibit favorable TCR binding affinities, while a swap p5-p6 tyrosine-valine double mutant (V5Y_Y6V) and a p6-p7 glutamine-glutamine double mutant (Y6Q_Y7Q) exhibit enhanced HLA binding affinity but weakened TCR affinity. This work holds relevance to potential T1D antigen-based vaccine design and optimization.

Axion optical induction of antiferromagnetic order.

Using circularly polarized light to control quantum matter is a highly intriguing topic in physics, chemistry and biology. Previous studies have demonstrated helicity-dependent optical control of chirality and magnetization, with important implications in asymmetric synthesis in chemistry; homochirality in biomolecules; and ferromagnetic spintronics. We report the surprising observation of helicity-dependent optical control of fully compensated antiferromagnetic order in two-dimensional even-layered MnBi2Te4, a topological axion insulator with neither chirality nor magnetization. To understand this control, we study an antiferromagnetic circular dichroism, which appears only in reflection but is absent in transmission. We show that the optical control and circular dichroism both arise from the optical axion electrodynamics. Our axion induction provides the possibility to optically control a family of [Formula: see text]-symmetric antiferromagnets ([Formula: see text], inversion; [Formula: see text], time-reversal) such as Cr2O3, even-layered CrI3 and possibly the pseudo-gap state in cuprates. In MnBi2Te4, this further opens the door for optical writing of a dissipationless circuit formed by topological edge states.

The potential for improved outcomes in the prevention and therapy of diabetic kidney disease through 'stacking' of drugs from different classes.

Aggressive therapy of diabetic kidney disease (DKD) can not only slow the progression of DKD to renal failure but, if utilized at an early enough stage of DKD, can also stabilize and/or reverse the decline in renal function. The currently recognized standard of therapy for DKD is blockade of the renin-angiotensin system with angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers (ARBs). However, unless utilized at a very early stage, monotherapy with these drugs in DKD will only prevent or slow the progression of DKD and will neither stabilize nor reverse the progression of DKD to renal decompensation. Recently, the addition of a sodium-glucose cotransporter-2 inhibitor and/or a mineralocorticoid receptor blocker to ACE inhibitors or ARBs has been clearly shown to further decelerate the decline in renal function. The use of glucagon-like peptide-1 (GLP-1) agonists shown promise in decelerating the progression of DKD. Other drugs that may aid in the deceleration the progression of DKD are dipeptidyl peptidase-4 inhibitors, pentoxifylline, statins, and vasodilating beta blockers. Therefore, aggressive therapy with combinations of these drugs (stacking) should improve the preservation of renal function in DKD.

Blazing the trail for innovative tuberculosis diagnostics.

The COVID-19 pandemic brought diagnostics into the spotlight in an unprecedented way not only for case management but also for population health, surveillance, and monitoring. The industry saw notable levels of investment and accelerated research which sparked a wave of innovation. Simple non-invasive sampling methods such as nasal swabs have become widely used in settings ranging from tertiary hospitals to the community. Self-testing has also been adopted as standard practice using not only conventional lateral flow tests but novel and affordable point-of-care molecular diagnostics. The use of new technologies, including artificial intelligence-based diagnostics, have rapidly expanded in the clinical setting. The capacity for next-generation sequencing and acceptance of digital health has significantly increased. However, 4 years after the pandemic started, the market for SARS-CoV-2 tests is saturated, and developers may benefit from leveraging their innovations for other diseases; tuberculosis (TB) is a worthwhile portfolio expansion for diagnostics developers given the extremely high disease burden, supportive environment from not-for-profit initiatives and governments, and the urgent need to overcome the long-standing dearth of innovation in the TB diagnostics field. In exchange, the current challenges in TB detection may be resolved by adopting enhanced swab-based molecular methods, instrument-based, higher sensitivity antigen detection technologies, and/or artificial intelligence-based digital health technologies developed for COVID-19. The aim of this article is to review how such innovative approaches for COVID-19 diagnosis can be applied to TB to have a comparable impact.

Real-Time Identification of Aerosol-Phase Carboxylic Acid Production Using Extractive Electrospray Ionization Mass Spectrometry.

Comprehensive identification of aerosol sources and their constituent organic compounds requires aerosol-phase molecular-level characterization with a high time resolution. While real-time chemical characterization of aerosols is becoming increasingly common, information about functionalization and structure is typically obtained from offline methods. This study presents a method for determining the presence of carboxylic acid functional groups in real time using extractive electrospray ionization mass spectrometry based on measurements of [M - H + 2Na]+ adducts. The method is validated and characterized using standard compounds. A proof-of-concept application to α-pinene secondary organic aerosol (SOA) shows the ability to identify carboxylic acids even in complex mixtures. The real-time capability of the method allows for the observation of the production of carboxylic acids, likely formed in the particle phase on short time scales (<120 min). Our research explains previous findings of carboxylic acids being a significant component of SOA and a quick decrease in peroxide functionalization following SOA formation. We show that the formation of these acids is commensurate with the increase of dimers in the particle phase. Our results imply that SOA is in constant evolution through condensed-phase processes, which lower the volatility of the aerosol components and increase the available condensed mass for SOA growth and, therefore, aerosol mass loading in the atmosphere. Further work could aim to quantify the effect of particle-phase acid formation on the aerosol volatility distributions.

Nitrate Radicals Suppress Biogenic New Particle Formation from Monoterpene Oxidation.

Highly oxygenated organic molecules (HOMs) are a major source of new particles that affect the Earth's climate. HOM production from the oxidation of volatile organic compounds (VOCs) occurs during both the day and night and can lead to new particle formation (NPF). However, NPF involving organic vapors has been reported much more often during the daytime than during nighttime. Here, we show that the nitrate radicals (NO3), which arise predominantly at night, inhibit NPF during the oxidation of monoterpenes based on three lines of observational evidence: NPF experiments in the CLOUD (Cosmics Leaving OUtdoor Droplets) chamber at CERN (European Organization for Nuclear Research), radical chemistry experiments using an oxidation flow reactor, and field observations in a wetland that occasionally exhibits nocturnal NPF. Nitrooxy-peroxy radicals formed from NO3 chemistry suppress the production of ultralow-volatility organic compounds (ULVOCs) responsible for biogenic NPF, which are covalently bound peroxy radical (RO2) dimer association products. The ULVOC yield of α-pinene in the presence of NO3 is one-fifth of that resulting from ozone chemistry alone. Even trace amounts of NO3 radicals, at sub-parts per trillion level, suppress the NPF rate by a factor of 4. Ambient observations further confirm that when NO3 chemistry is involved, monoterpene NPF is completely turned off. Our results explain the frequent absence of nocturnal biogenic NPF in monoterpene (α-pinene)-rich environments.

Massive Dirac fermions in a ferromagnetic kagome metal.

The kagome lattice is a two-dimensional network of corner-sharing triangles that is known to host exotic quantum magnetic states. Theoretical work has predicted that kagome lattices may also host Dirac electronic states that could lead to topological and Chern insulating phases, but these states have so far not been detected in experiments. Here we study the d-electron kagome metal Fe3Sn2, which is designed to support bulk massive Dirac fermions in the presence of ferromagnetic order. We observe a temperature-independent intrinsic anomalous Hall conductivity that persists above room temperature, which is suggestive of prominent Berry curvature from the time-reversal-symmetry-breaking electronic bands of the kagome plane. Using angle-resolved photoemission spectroscopy, we observe a pair of quasi-two-dimensional Dirac cones near the Fermi level with a mass gap of 30 millielectronvolts, which correspond to massive Dirac fermions that generate Berry-curvature-induced Hall conductivity. We show that this behaviour is a consequence of the underlying symmetry properties of the bilayer kagome lattice in the ferromagnetic state and the atomic spin-orbit coupling. This work provides evidence for a ferromagnetic kagome metal and an example of emergent topological electronic properties in a correlated electron system. Our results provide insight into the recent discoveries of exotic electronic behaviour in kagome-lattice antiferromagnets and may enable lattice-model realizations of fractional topological quantum states.

Knockout of RSN1, TVP18 or CSC1-2 causes perturbation of Golgi cisternae in Pichia pastoris.

The structural organization of the Golgi stacks in mammalian cells is intrinsically linked to function, including glycosylation, but the role of morphology is less clear in lower eukaryotes. Here we investigated the link between the structural organization of the Golgi and secretory pathway function using Pichia pastoris as a model system. To unstack the Golgi cisternae, we disrupted 18 genes encoding proteins in the secretory pathway without loss of viability. Using biosensors, confocal microscopy and transmission electron microscopy we identified three strains with irreversible perturbations in the stacking of the Golgi cisternae, all of which had disruption in genes that encode proteins with annotated function as or homology to calcium/calcium permeable ion channels. Despite this, no variation in the secretory pathway for ER size, whole cell glycomics or recombinant protein glycans was observed. Our investigations showed the robust nature of the secretory pathway in P. pastoris and suggest that Ca2+ concentration, homeostasis or signalling may play a significant role for Golgi stacking in this organism and should be investigated in other organisms.

Sensitivity Constraints of Extractive Electrospray for a Model System and Secondary Organic Aerosol.

The quantification of an aerosol chemical composition is complicated by the uncertainty in the sensitivity of each species detected. Soft-ionization response factors can vary widely from molecule to molecule. Here, we have employed a method to separate molecules by their volatility through systematic evaporation with a thermal denuder (TD). The fraction remaining after evaporation is compared between an extractive electrospray ionization time-of-flight mass spectrometer (EESI-TOF) and a scanning mobility particle sizer (SMPS), which provides a comparison between a quantified mass loss by the SMPS and the signal loss in the EESI-TOF. The sensitivity of the EESI-TOF is determined for both a simplified complex mixture (PEG-300) and also for a complex mixture of α-pinene secondary organic aerosol (SOA). For PEG-300, separation is possible on a molecule-by-molecule level with the TD and provides insights into the molecule-dependent sensitivity of the EESI-TOF, showing a higher sensitivity toward the most volatile molecule. For α-pinene SOA, sensitivity determination for specific classes is possible because of the number of molecular formula observed by the EESI-TOF. These classes are separated by their volatility and are broken down into monomers (O3-5,6-7,8+), dimers (O4-7,8+), and higher order oligomers (e.g., trimers and tetramers). Here, we show that the EESI-TOF initially measures 60.1% monomers, 32.7% dimers, and 7.2% trimers and tetramers in α-pinene SOA, but after sensitivity correction, the distribution of SOA is 37.4% monomers, 56.1% dimers, and 6.4% trimers and tetramers. These results provide a path forward for the quantification of aerosol components with the EESI-TOF in other applications and potentially for atmospheric measurements.

Global estimates and determinants of antituberculosis drug pharmacokinetics in children and adolescents: a systematic review and individual patient data meta-analysis.

BACKGROUND: Suboptimal exposure to antituberculosis (anti-TB) drugs has been associated with unfavourable treatment outcomes. We aimed to investigate estimates and determinants of first-line anti-TB drug pharmacokinetics in children and adolescents at a global level. METHODS: We systematically searched MEDLINE, Embase and Web of Science (1990-2021) for pharmacokinetic studies of first-line anti-TB drugs in children and adolescents. Individual patient data were obtained from authors of eligible studies. Summary estimates of total/extrapolated area under the plasma concentration-time curve from 0 to 24 h post-dose (AUC0-24) and peak plasma concentration (C max) were assessed with random-effects models, normalised with current World Health Organization-recommended paediatric doses. Determinants of AUC0-24 and C max were assessed with linear mixed-effects models. RESULTS: Of 55 eligible studies, individual patient data were available for 39 (71%), including 1628 participants from 12 countries. Geometric means of steady-state AUC0-24 were summarised for isoniazid (18.7 (95% CI 15.5-22.6) h·mg·L-1), rifampicin (34.4 (95% CI 29.4-40.3) h·mg·L-1), pyrazinamide (375.0 (95% CI 339.9-413.7) h·mg·L-1) and ethambutol (8.0 (95% CI 6.4-10.0) h·mg·L-1). Our multivariate models indicated that younger age (especially <2 years) and HIV-positive status were associated with lower AUC0-24 for all first-line anti-TB drugs, while severe malnutrition was associated with lower AUC0-24 for isoniazid and pyrazinamide. N-acetyltransferase 2 rapid acetylators had lower isoniazid AUC0-24 and slow acetylators had higher isoniazid AUC0-24 than intermediate acetylators. Determinants of C max were generally similar to those for AUC0-24. CONCLUSIONS: This study provides the most comprehensive estimates of plasma exposures to first-line anti-TB drugs in children and adolescents. Key determinants of drug exposures were identified. These may be relevant for population-specific dose adjustment or individualised therapeutic drug monitoring.

Detecting and treating the protean manifestations of diabetic autonomic neuropathy.

The manifestations of diabetic autonomic neuropathy (DAN) are protean and clinically involve multiple systems, including the cardiovascular system, the gastrointestinal system, the genitourinary system as well as the sweat glands (sudomotor dysfunction) and the gallbladder. In addition, cardiac autonomic neuropathy (CAN) is associated with a correctible inability to appreciate and correct hypoglycaemia. While not a clinical problem, pupillary involvement should be the clue and the catalyst to investigate for other manifestations of DAN. This review outlines a practical approach to detecting and investigating the manifestations of DAN. Of particular importance is early detection of cardiovascular involvement where prompt therapy through glycaemic control can decrease the severity of CAN and decelerate the frequency and severity of retinopathy and nephropathy in addition to decreasing cardiovascular events and mortality. CAN also plays a role in accelerating other diabetic complications such as acute ischaemic stroke, heart failure, medial artery calcinosis, foot ulcers, peripheral artery disease and Charcot joints. Many therapies of DAN are available, which should not only decrease morbidity and mortality from DAN, but also improve the patient's quality of life. However, the therapies available are largely symptomatic.

In praise of pioglitazone: An economically efficacious therapy for type 2 diabetes and other manifestations of the metabolic syndrome.

Pioglitazone improves glycaemic control, not only by lowering insulin resistance, but also by improving beta cell function. Because of the improved beta cell function the glycaemic control that occurs with pioglitazone is prolonged. Pioglitazone has positive effects not only on cardiac risk factors and surrogate measures of cardiovascular disease, it also lowers the incidence of cardiac events in patients with diabetes. The recurrence of transient ischaemic attack and ischaemic stroke is also reduced in non-diabetic, insulin-resistant subjects. Utilized at preclinical stages (but not later) of heart failure, pioglitazone improves diastolic function and avoids progression to heart failure. Pioglitazone, through suppression of atrial remodelling, also decreases the incidence of atrial fibrillation. The manifestations of diseases associated with insulin resistance (non-alcoholic steatohepatitis and polycystic ovary disease) are also improved with pioglitazone. Pioglitazone may possibly improve psoriasis and other dermopathies. Pioglitazone is therefore an inexpensive and efficacious drug for the insulin-resistant subject with diabetes that is underutilized because of biases that have evolved from the toxicities of other thiazolidinediones.

Management of the 'wicked' combination of heart failure and chronic kidney disease in the patient with diabetes.

Patients with type 2 diabetes are at an increased risk of developing heart failure and chronic kidney disease. The presence of these co-morbidities substantially increases the risk of morbidity as well as mortality in patients with diabetes. The clinical focus has historically centred around reducing the risk of cardiovascular disease by targeting hyperglycaemia, hyperlipidaemia and hypertension. Nonetheless, patients with type 2 diabetes who have well-controlled blood glucose, blood pressure and lipid levels may still go on to develop heart failure, kidney disease or both. Major diabetes and cardiovascular societies are now recommending the use of treatments such as sodium-glucose co-transporter-2 inhibitors and non-steroidal mineralocorticoid receptor antagonists, in addition to currently recommended therapies, to promote cardiorenal protection through alternative pathways as early as possible in individuals with diabetes and cardiorenal manifestations. This review examines the most recent recommendations for managing the risk of cardiorenal progression in patients with type 2 diabetes.

Heart failure and diabetes: Clinical significance and epidemiology of this two-way association.

People with type 2 diabetes (T2DM) and those with prediabetes have an increased risk of heart failure (HF). Longer duration of T2DM correlates with a greater risk of HF, but HF is also seen in patients with recent-onset diabetes. Insulin resistance is more likely to be present in patients with HF. The risk of HF persists even in the face of standard-of-care preventive treatments for atherosclerotic cardiovascular (CV) disease. HF is commonly the presenting symptom of CV disease in people with diabetes and is the most expensive complication of diabetes because of the high cost of hospitalizations. Recently hospitalization for HF has been included in CV outcome trials (CVOTs), including for medications that are used to treat T2DM, which has led to new therapies for all HF patients. In addition, these CVOTs have shown that many drugs used in the therapy of diabetes are either neutral or detrimental in the HF patient and should be used with caution in patients with existing HF or those at high risk of HF. Most recently, sodium-glucose cotransporter-2 receptor blockers have shown efficacy in both HF with reduced ejection fraction (EF) and HF with preserved EF. The only other oral or injectable diabetes agent shown to improve outcomes in both is metformin.

Comprehensive phylogenomic time tree of bryophytes reveals deep relationships and uncovers gene incongruences in the last 500 million years of diversification.

PREMISE: Bryophytes form a major component of terrestrial plant biomass, structuring ecological communities in all biomes. Our understanding of the evolutionary history of hornworts, liverworts, and mosses has been significantly reshaped by inferences from molecular data, which have highlighted extensive homoplasy in various traits and repeated bursts of diversification. However, the timing of key events in the phylogeny, patterns, and processes of diversification across bryophytes remain unclear. METHODS: Using the GoFlag probe set, we sequenced 405 exons representing 228 nuclear genes for 531 species from 52 of the 54 orders of bryophytes. We inferred the species phylogeny from gene tree analyses using concatenated and coalescence approaches, assessed gene conflict, and estimated the timing of divergences based on 29 fossil calibrations. RESULTS: The phylogeny resolves many relationships across the bryophytes, enabling us to resurrect five liverwort orders and recognize three more and propose 10 new orders of mosses. Most orders originated in the Jurassic and diversified in the Cretaceous or later. The phylogenomic data also highlight topological conflict in parts of the tree, suggesting complex processes of diversification that cannot be adequately captured in a single gene-tree topology. CONCLUSIONS: We sampled hundreds of loci across a broad phylogenetic spectrum spanning at least 450 Ma of evolution; these data resolved many of the critical nodes of the diversification of bryophytes. The data also highlight the need to explore the mechanisms underlying the phylogenetic ambiguity at specific nodes. The phylogenomic data provide an expandable framework toward reconstructing a comprehensive phylogeny of this important group of plants.