Despite their crucial role in health and disease, our knowledge of immune cells within human tissues remains limited. We surveyed the immune compartment of 16 tissues from 12 adult donors by ...single-cell RNA sequencing and VDJ sequencing generating a dataset of ~360,000 cells. To systematically resolve immune cell heterogeneity across tissues, we developed CellTypist, a machine learning tool for rapid and precise cell type annotation. Using this approach, combined with detailed curation, we determined the tissue distribution of finely phenotyped immune cell types, revealing hitherto unappreciated tissue-specific features and clonal architecture of T and B cells. Our multitissue approach lays the foundation for identifying highly resolved immune cell types by leveraging a common reference dataset, tissue-integrated expression analysis, and antigen receptor sequencing.
The evolutionary origins of Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV) are unknown. Current evidence suggests that insectivorous bats are likely to be the original source, as ...several 2c CoVs have been described from various species in the family
Here, we describe a MERS-like CoV identified from a
bat sampled in Uganda (strain PREDICT/PDF-2180), further supporting the hypothesis that bats are the evolutionary source of MERS-CoV. Phylogenetic analysis showed that PREDICT/PDF-2180 is closely related to MERS-CoV across much of its genome, consistent with a common ancestry; however, the spike protein was highly divergent (46% amino acid identity), suggesting that the two viruses may have different receptor binding properties. Indeed, several amino acid substitutions were identified in key binding residues that were predicted to block PREDICT/PDF-2180 from attaching to the MERS-CoV DPP4 receptor. To experimentally test this hypothesis, an infectious MERS-CoV clone expressing the PREDICT/PDF-2180 spike protein was generated. Recombinant viruses derived from the clone were replication competent but unable to spread and establish new infections in Vero cells or primary human airway epithelial cells. Our findings suggest that PREDICT/PDF-2180 is unlikely to pose a zoonotic threat. Recombination in the S1 subunit of the spike gene was identified as the primary mechanism driving variation in the spike phenotype and was likely one of the critical steps in the evolution and emergence of MERS-CoV in humans.
Global surveillance efforts for undiscovered viruses are an important component of pandemic prevention initiatives. These surveys can be useful for finding novel viruses and for gaining insights into the ecological and evolutionary factors driving viral diversity; however, finding a viral sequence is not sufficient to determine whether it can infect people (i.e., poses a zoonotic threat). Here, we investigated the specific zoonotic risk of a MERS-like coronavirus (PREDICT/PDF-2180) identified in a bat from Uganda and showed that, despite being closely related to MERS-CoV, it is unlikely to pose a threat to humans. We suggest that this approach constitutes an appropriate strategy for beginning to determine the zoonotic potential of wildlife viruses. By showing that PREDICT/PDF-2180 does not infect cells that express the functional receptor for MERS-CoV, we further show that recombination was likely to be the critical step that allowed MERS to emerge in humans.
We describe the design and performance of the Medium Resolution Spectrometer (MRS) for the JWST-MIRI instrument. The MRS incorporates four coaxial spectral channels in a compact opto-mechanical ...layout that generates spectral images over fields of view up to 7.7 × 7.7″ in extent and at spectral resolving powers ranging from 1300 to 3700. Each channel includes an all-reflective integral field unit (IFU): an "image slicer" that reformats the input field for presentation to a grating spectrometer. Two 1024 × 1024 focal plane detector arrays record the output spectral images with an instantaneous spectral coverage of approximately one third of the full wavelength range of each channel. The full 5-28.5 μm spectrum is then obtained by making three exposures using gratings and pass-band-determining filters that are selected using just two three-position mechanisms. The expected on-orbit optical performance is presented, based on testing of the MIRI Flight Model and including spectral and spatial coverage and resolution. The point spread function of the reconstructed images is shown to be diffraction limited and the optical transmission is shown to be consistent with the design expectations.
Birth interval is an important and potentially modifiable factor that is associated with child health. Whether an association exists with longer-term outcomes in adults is less well known.
Using the ...1982 Pelotas (Brazil) Birth Cohort Study, the association of birth interval with markers of cardiovascular health at 30 years of age was examined. Multivariable linear regression was used with birth interval as a continuous variable and categorical variable, and effect modification by gender was explored.
Birth interval and cardiovascular data were present for 2,239 individuals. With birth interval as a continuous variable, no association was found but stratification by gender tended to show stronger associations for girls. When compared to birth intervals of <18 months, as binary variable, longer intervals were associated with increases in height (1.6 cm; 95% CI: 0.5, 2.8) and lean mass (1.7 kg; 95% CI: 0.2, 3.2). No difference was seen with other cardiovascular outcomes.
An association was generally not found between birth interval and cardiovascular outcomes at 30 years of age, though some evidence existed for differences between males and females and for an association with height and lean mass for birth intervals of 18 months and longer.
Our understanding of how enzymes work is coloured by static structure depictions where the enzyme scaffold is presented as either immobile, or in equilibrium between well‐defined static ...conformations. Proteins, however, exhibit a large degree of motion over a broad range of timescales and magnitudes and this is defined thermodynamically by the enzyme free energy landscape (FEL). The role and importance of enzyme motion is extremely contentious. Much of the challenge is in the experimental detection of so called ‘conformational sampling’ involved in enzyme turnover. Herein we apply combined pressure and temperature kinetics studies to elucidate the full suite of thermodynamic parameters defining an enzyme FEL as it relates to enzyme turnover. We find that the key thermodynamic parameters governing vibrational modes related to enzyme turnover are the isobaric expansivity term and the change in heat capacity for enzyme catalysis. Variation in the enzyme FEL affects these terms. Our analysis is supported by a range of biophysical and computational approaches that specifically capture information on protein vibrational modes and the FEL (all atom flexibility calculations, red edge excitation shift spectroscopy and viscosity studies) that provide independent evidence for our findings. Our data suggest that restricting the enzyme FEL may be a powerful strategy when attempting to rationally engineer enzymes, particularly to alter thermal activity. Moreover, we demonstrate how rational predictions can be made with a rapid computational approach.
The intramolecular motion of enzymes is potentially important for their function. We have used a range of biophysical approaches to understand the link between the energetics (free energy landscape) of the enzyme and catalysis. We find that the energetics is sensitive to differences in enzyme motions and could be a useful and predictable control parameter when designing enzymes.
There is an increasing realization that structure-based drug design may show improved success by understanding the ensemble of conformations accessible to an enzyme and how the environment affects ...this ensemble. Human monoamine oxidase B (MAO-B) catalyzes the oxidation of amines and is inhibited for the treatment of both Parkinson’s disease and depression. Despite its clinical importance, its catalytic mechanism remains unclear, and routes to drugging this target would be valuable. Evidence of a radical in either the transition state or the resting state of MAO-B is present throughout the literature and is suggested to be a flavin semiquinone, a tyrosyl radical, or both. Here we see evidence of a resting-state flavin semiquinone, via absorption redox studies and electron paramagnetic resonance, suggesting that the anionic semiquinone is biologically relevant. On the basis of enzyme kinetic studies, enzyme variants, and molecular dynamics simulations, we find evidence for the importance of the membrane environment in mediating the activity of MAO-B and that this mediation is related to the protein dynamics of MAO-B. Further, our MD simulations identify a hitherto undescribed entrance for substrate binding, membrane modulated substrate access, and indications for half-site reactivity: only one active site is accessible to binding at a time. Our study combines both experimental and computational evidence to illustrate the subtle interplay between enzyme activity and protein dynamics and the immediate membrane environment. Understanding key biomedical enzymes to this level of detail will be crucial to inform strategies (and binding sites) for rational drug design for these targets.
We present the detection of three exoplanets orbiting the early M dwarf TOI-663 (TIC 54962195; V = 13.7 mag, J = 10.4 mag, R ★ = 0.512 ± 0.015 R ⊙ , M ★ = 0.514 ± 0.012 M ⊙ , d = 64 pc). TOI-663 b, ...c, and d, with respective radii of 2.27 ± 0.10 R ⊕ , 2.26 ± 0.10 R ⊕ , and 1.92 ± 0.13 R ⊕ and masses of 4.45 ± 0.65 M ⊕ , 3.65 ± 0.97 M ⊕ , and <5.2 M ⊕ at 99%, are located just above the radius valley that separates rocky and volatile-rich exoplanets. The planet candidates are identified in two TESS sectors and are validated with ground-based photometric follow-up, precise radial-velocity measurements, and high-resolution imaging. We used the software package juliet to jointly model the photometric and radial-velocity datasets, with Gaussian processes applied to correct for systematics. The three planets discovered in the TOI-663 system are low-mass mini-Neptunes with radii significantly larger than those of rocky analogs, implying that volatiles, such as water, must predominate. In addition to this internal structure analysis, we also performed a dynamical analysis that confirmed the stability of the system. The three exoplanets in the TOI-663 system, similarly to other sub-Neptunes orbiting M dwarfs, have been found to have lower densities than planets of similar sizes orbiting stars of different spectral types.
Common bottlenose dolphins (Tursiops truncatus) have previously demonstrated exposure to phthalate esters. Phthalates and phthalate esters are commonly added to consumer goods to enhance desirable ...properties. As the amount of plastic marine debris increases, these chemicals can easily leach from these products into the surrounding environment. To evaluate demographic variability in exposure, eight phthalate metabolites were quantified in urine samples collected from free‐ranging bottlenose dolphins sampled in Sarasota Bay, FL, USA (2010–2019; n = 51). Approximately 75% of individual dolphins had detectable concentrations of at least one phthalate metabolite. The most frequently detected metabolites were mono(2‐ethylhexyl) phthalate (MEHP; n = 28; GM = 4.57 ng/mL; 95% CI = 2.37–8.80; KM mean = 7.95; s.d. = 15.88) and monoethyl phthalate (MEP; GM = 4.51 ng/mL; 95% CI = 2.77–7.34; ROS mean = 2.24; s.d. = 5.58). Urinary concentrations of MEHP and MEP were not significantly different between sex (MEHP p = 0.09; MEP p = 0.22) or age class (i.e., calf/juvenile vs. adult; MEHP p = 0.67; MEP p = 0.13). Additionally, there were no significant group differences in the likelihood of MEHP or MEP detection for any demographic as determined by a Peto‐Peto test. Frequency of detection was similar for both metabolites between males and females (MEHP p = 0.10; MEP p = 0.40) as well as between juveniles and adults (MEHP p = 0.50; MEP: p = 0.60). These findings suggest ubiquitous exposure risk for both sexes and age classes, warranting further investigation into potential sources and health implications.
Plain Language Summary
Previous studies have detected exposure to phthalates among bottlenose dolphins, demonstrating environmental contamination. Using archived samples from Sarasota Bay bottlenose dolphins (2010–2019), this study evaluated demographic differences in the magnitude and frequency of detection of phthalate metabolites. Unlike exposure patterns for other common environmental contaminants (e.g., polychlorinated biphenyls PCBs, polybrominated diphenyl ethers PBDEs) where adult male dolphins and first‐born calves have differentially higher concentrations, evidence from this study suggests equivalent phthalate exposure risk across sexes and age classes. Given phthalate‐associated health impacts observed in human studies and the ubiquity of phthalate use, additional research is warranted to better understand sources of exposure and potential implications for bottlenose dolphin health.
Key Points
Detectable phthalate metabolites found in 75% of bottlenose dolphin urine samples
No differences in magnitude or frequency of detectable mono(2‐ethylhexyl) phthalate/monoethyl phthalate concentrations among demographics
Potential hazard of phthalate exposure is not specific to any sex or age class
Atomic force microscopy (AFM) is a powerful imaging technique that allows for structural characterization of single biomolecules with nanoscale resolution. AFM has a unique capability to image ...biological molecules in their native states under physiological conditions without the need for labeling or averaging. DNA has been extensively imaged with AFM from early single-molecule studies of conformational diversity in plasmids, to recent examinations of intramolecular variation between groove depths within an individual DNA molecule. The ability to image dynamic biological interactions in situ has also allowed for the interaction of various proteins and therapeutic ligands with DNA to be evaluated—providing insights into structural assembly, flexibility, and movement. This review provides an overview of how innovation and optimization in AFM imaging have advanced our understanding of DNA structure, mechanics, and interactions. These include studies of the secondary and tertiary structure of DNA, including how these are affected by its interactions with proteins. The broader role of AFM as a tool in translational cancer research is also explored through its use in imaging DNA with key chemotherapeutic ligands, including those currently employed in clinical practice.
Among the major challenges in the development of biopharmaceuticals are structural heterogeneity and aggregation. The development of a successful therapeutic monoclonal antibody (mAb) requires both a ...highly active and also stable molecule. Whilst a range of experimental (biophysical) approaches exist to track changes in stability of proteins, routine prediction of stability remains challenging. The fluorescence red edge excitation shift (REES) phenomenon is sensitive to a range of changes in protein structure. Based on recent work, we have found that quantifying the REES effect is extremely sensitive to changes in protein conformational state and dynamics. Given the extreme sensitivity, potentially this tool could provide a 'fingerprint' of the structure and stability of a protein. Such a tool would be useful in the discovery and development of biopharamceuticals and so we have explored our hypothesis with a panel of therapeutic mAbs. We demonstrate that the quantified REES data show remarkable sensitivity, being able to discern between structurally identical antibodies and showing sensitivity to unfolding and aggregation. The approach works across a broad concentration range (µg-mg/ml) and is highly consistent. We show that the approach can be applied alongside traditional characterisation testing within the context of a forced degradation study (FDS). Most importantly, we demonstrate the approach is able to predict the stability of mAbs both in the short (hours), medium (days) and long-term (months). The quantified REES data will find immediate use in the biopharmaceutical industry in quality assurance, formulation and development. The approach benefits from low technical complexity, is rapid and uses instrumentation which exists in most biochemistry laboratories without modification.
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