Assembly of the Bacterial Ribosome with Circularly Permuted rRNA.

Co-transcriptional assembly is an integral feature of the formation of RNA-protein complexes that mediate translation. For ribosome synthesis, prior studies have indicated that the strict order of transcription of rRNA domains may not be obligatory during bacterial ribosome biogenesis, since a series of circularly permuted rRNAs are viable. In this work, we report the insights into assembly of the bacterial ribosome large subunit (LSU) based on cryo-EM density maps of intermediates that accumulate during in vitro ribosome synthesis using a set of circularly permuted (CiPer) rRNAs. The observed ensemble of twenty-three resolved ribosome large subunit intermediates reveals conserved assembly routes with an underlying hierarchy among cooperative assembly blocks. There are intricate interdependencies for the formation of key structural rRNA helices revealed from the circular permutation of rRNA. While the order of domain synthesis is not obligatory, the order of domain association does appear to proceed with a particular order, likely due to the strong evolutionary pressure on efficient ribosome synthesis. This work reinforces the robustness of the known assembly hierarchy of the bacterial large ribosomal subunit, and offers a coherent view of how efficient assembly of CiPer rRNAs can be understood in that context.

Tandem Mass Spectrometry across Platforms.

PubChem serves as a comprehensive repository, housing over 100 million unique chemical structures representing the breadth of our chemical knowledge across numerous fields including metabolism, pharmaceuticals, toxicology, cosmetics, agriculture, and many more. Rapid identification of these small molecules increasingly relies on electrospray ionization (ESI) paired with tandem mass spectrometry (MS/MS), particularly by comparison to genuine standard MS/MS data sets. Despite its widespread application, achieving consistency in MS/MS data across various analytical platforms remains an unaddressed concern. This study evaluated MS/MS data derived from one hundred molecular standards utilizing instruments from five manufacturers, inclusive of quadrupole time-of-flight (QTOF) and quadrupole orbitrap "exactive" (QE) mass spectrometers by Agilent (QTOF), Bruker (QTOF), SCIEX (QTOF), Waters (QTOF), and Thermo QE. We assessed fragment ion variations at multiple collisional energies (0, 10, 20, and 40 eV) using the cosine scoring algorithm for comparisons and the number of fragments observed. A parallel visual analysis of the MS/MS spectra across instruments was conducted, consistent with a standard procedure that is used to circumvent the still prevalent issue of mischaracterizations as shown for dimethyl sphingosine and C20 sphingosine. Our analysis revealed a notable consistency in MS/MS data and identifications, with fragment ions' m/z values exhibiting the highest concordance between instrument platforms at 20 eV, the other collisional energies (0, 10, and 40 eV) were significantly lower. While moving toward a standardized ESI MS/MS protocol is required for dependable molecular characterization, our results also underscore the continued importance of corroborating MS/MS data against standards to ensure accurate identifications. Our findings suggest that ESI MS/MS manufacturers, akin to the established norms for gas chromatography mass spectrometry instruments, should standardize the collision energy at 20 eV across different instrument platforms.

Assembly landscape for the bacterial large ribosomal subunit.

Assembly of ribosomes in bacteria is highly efficient, taking ~2-3 min, but this makes the abundance of assembly intermediates very low, which is a challenge for mechanistic understanding. Genetic perturbations of the assembly process create bottlenecks where intermediates accumulate, facilitating structural characterization. We use cryo-electron microscopy, with iterative subclassification to identify intermediates in the assembly of the 50S ribosomal subunit from E. coli. The analysis of the ensemble of intermediates that spans the entire biogenesis pathway for the 50 S subunit was facilitated by a dimensionality reduction and cluster picking approach using PCA-UMAP-HDBSCAN. The identity of the cooperative folding units in the RNA with associated proteins is revealed, and the hierarchy of these units reveals a complete assembly map for all RNA and protein components. The assembly generally proceeds co-transcriptionally, with some flexibility in the landscape to ensure efficiency for this central cellular process under a variety of growth conditions.

Inherited chitinases enable sustained growth and rapid dispersal of bacteria from chitin particles.

Many biogeochemical functions involve bacteria utilizing solid substrates. However, little is known about the coordination of bacterial growth with the kinetics of attachment to and detachment from such substrates. In this quantitative study of Vibrio sp. 1A01 growing on chitin particles, we reveal the heterogeneous nature of the exponentially growing culture comprising two co-existing subpopulations: a minority replicating on chitin particles and a non-replicating majority which was planktonic. This partition resulted from a high rate of cell detachment from particles. Despite high detachment, sustained exponential growth of cells on particles was enabled by the enrichment of extracellular chitinases excreted and left behind by detached cells. The 'inheritance' of these chitinases sustains the colonizing subpopulation despite its reduced density. This simple mechanism helps to circumvent a trade-off between growth and dispersal, allowing particle-associated marine heterotrophs to explore new habitats without compromising their fitness on the habitat they have already colonized.

Early HIV-1 Gag Assembly on Lipid Membrane with vRNA.

HIV-1 capsid assembly is an essential process in the virus infection cycle. Initiation of capsid assembly involves viral proteins, genomic RNA, and the inner leaflet of the plasma membrane, facilitated by a number of cellular factors1. The viral structural protein Gag plays a number of central roles in this process, including association with the membrane, selective binding of genomic RNA, and oligomerization and packaging to ultimately produce an immature budded pro-viral particle2. While there have been intensive studies regarding the early stages of Gag assembly, there is a lack of consensus on the mechanism for nucleation and growth of Gag complexes3-7. Here we show that myristoylated Gag forms a trimer nucleus in a model membrane that can selectively bind a dimeric RNA containing the packaging signal. Subsequent growth of myristoyl-Gag oligomers requires vRNA, and occurs by addition of 1 or 2 Gag monomers at a time from solution. These data support a model where the immature capsid lattice formation occurs by a gradual lattice edge expansion, following a trimeric nucleation event. The dynamic single molecule data that support this model were recorded using mass photometry, involving full length myristoylated protein, RNA, and lipid together. These data are the first to support a lattice edge expansion model of Gag during early stages of assembly in a biological-relevant setting, providing insights to the fundamental models of virus structural protein assembly process.

Near-physiological in vitro assembly of 50S ribosomes involves parallel pathways.

Understanding the assembly principles of biological macromolecular complexes remains a significant challenge, due to the complexity of the systems and the difficulties in developing experimental approaches. As a ribonucleoprotein complex, the ribosome serves as a model system for the profiling of macromolecular complex assembly. In this work, we report an ensemble of large ribosomal subunit intermediate structures that accumulate during synthesis in a near-physiological and co-transcriptional in vitro reconstitution system. Thirteen pre-50S intermediate maps covering the entire assembly process were resolved using cryo-EM single-particle analysis and heterogeneous subclassification. Segmentation of the set of density maps reveals that the 50S ribosome intermediates assemble based on fourteen cooperative assembly blocks, including the smallest assembly core reported to date, which is composed of a 600-nucleotide-long folded rRNA and three ribosomal proteins. The cooperative blocks assemble onto the assembly core following defined dependencies, revealing the parallel pathways at both early and late assembly stages of the 50S subunit.

Early HIV-1 Gag Assembly on Lipid Membrane with vRNA.

Mass photometry (MP) was used to investigate the assembly of myristoylated full-length HIV-1 Gag (myr-Gag) and vRNA 5’ UTR fragment in a supported lipid bilayer (SLB) model system. The MP trajectories demonstrated that Gag trimerization on the membrane is a key step of early Gag assembly in the presence of vRNA. Growth of myr-Gag oligomers requires vRNA, occuring by addition of 1 or 2 monomers at a time from solution. These data support a model where formation of the Gag hexamers characteristic of the immature capsid lattice occurs by a gradual edge expansion, following a trimeric nucleation event. These dynamic single molecule data involving protein, RNA, and lipid components together, provide novel and fundamental insights into the initiation of virus capsid assembly.

Using Body-worn Accelerometers to Detect Physiological Changes During Periods of Blast Overpressure Exposure.

Repetitive exposure to non-concussive blast expo-sure may result in sub-clinical neurological symptoms. These changes may be reflected in the neural control gait and balance. In this study, we collected body-worn accelerometry data on individuals who were exposed to repetitive blast overpressures as part of their occupation. Accelerometry features were gener-ated within periods of low-movement and gait. These features were the eigenvalues of high-dimensional correlation matrices, which were constructed with time-delay embedding at multiple delay scales. When focusing on the gait windows, there were significant correlations of the changes in features with the cumulative dose of blast exposure. When focusing on the low-movement frames, the correlation with exposure were lower than that of the gait frames and statistically insignificant. In a cross-validated model, the overpressure exposure was predicted from gait features alone. The model was statistically significant and yielded an RMSE of 1.27 dB. With continued development, the model may be used to assess the physiological effects of repetitive blast exposure and guide training procedures to minimize impact on the individual.

Growth rate-dependent coordination of catabolism and anabolism in the archaeon Methanococcus maripaludis under phosphate limitation.

Catabolic and anabolic processes are finely coordinated in microorganisms to provide optimized fitness under varying environmental conditions. Understanding this coordination and the resulting physiological traits reveals fundamental strategies of microbial acclimation. Here, we characterized the system-level physiology of Methanococcus maripaludis, a niche-specialized methanogenic archaeon, at different dilution rates ranging from 0.09 to 0.003 h-1 in chemostat experiments under phosphate (i.e., anabolic) limitation. Phosphate was supplied as the limiting nutrient, while formate was supplied in excess as the catabolic substrate and carbon source. We observed a decoupling of catabolism and anabolism resulting in lower biomass yield relative to catabolically limited cells at the same dilution rates. In addition, the mass abundance of several coarse-grained proteome sectors (i.e., combined abundance of proteins grouped based on their function) exhibited a linear relationship with growth rate, mostly ribosomes and their biogenesis. Accordingly, cellular RNA content also correlated with growth rate. Although the methanogenesis proteome sector was invariant, the metabolic capacity for methanogenesis, measured as methane production rates immediately after transfer to batch culture, correlated with growth rate suggesting translationally independent regulation that allows cells to only increase catabolic activity under growth-permissible conditions. These observations are in stark contrast to the physiology of M. maripaludis under formate (i.e., catabolic) limitation, where cells keep an invariant proteome including ribosomal content and a high methanogenesis capacity across a wide range of growth rates. Our findings reveal that M. maripaludis employs fundamentally different strategies to coordinate global physiology during anabolic phosphate and catabolic formate limitation.

Pytheas: a software package for the automated analysis of RNA sequences and modifications via tandem mass spectrometry.

Mass spectrometry is an important method for analysis of modified nucleosides ubiquitously present in cellular RNAs, in particular for ribosomal and transfer RNAs that play crucial roles in mRNA translation and decoding. Furthermore, modifications have effect on the lifetimes of nucleic acids in plasma and cells and are consequently incorporated into RNA therapeutics. To provide an analytical tool for sequence characterization of modified RNAs, we developed Pytheas, an open-source software package for automated analysis of tandem MS data for RNA. The main features of Pytheas are flexible handling of isotope labeling and RNA modifications, with false discovery rate statistical validation based on sequence decoys. We demonstrate bottom-up mass spectrometry characterization of diverse RNA sequences, with broad applications in the biology of stable RNAs, and quality control of RNA therapeutics and mRNA vaccines.

The Histone H1-Like Protein AlgP Facilitates Even Spacing of Polyphosphate Granules in Pseudomonas aeruginosa.

Synthesis of polyphosphate (polyP) is an ancient and universal stress and starvation response in bacteria. In many bacteria, polyP chains come together to form granular superstructures within cells. Some species appear to regulate polyP granule subcellular organization. Despite the critical role of polyP in starvation fitness, the composition of these structures, mechanism(s) underpinning their organization, and functional significance of such organization are poorly understood. We previously determined that granules become transiently evenly spaced on the cell's long axis during nitrogen starvation in the opportunistic human pathogen Pseudomonas aeruginosa. Here, we developed a granule-enrichment protocol to screen for polyP granule-localizing proteins. We identified AlgP as a protein that associates with polyP granules. We further discovered that AlgP is required for the even spacing of polyP granules. AlgP is a DNA-binding protein with a 154 amino acid C-terminal domain enriched in "KPAA" repeats and variants of this repeat, with an overall sequence composition similar to the C-terminal tail of eukaryotic histone H1. Granule size, number, and spacing are significantly perturbed in the absence of AlgP, or when AlgP is truncated to remove the C-terminus. The ΔalgP and algPΔCTD mutants have fewer, larger granules. We speculate that AlgP may contribute to spacing by tethering polyP granules to the chromosome, thereby inhibiting fusion with neighboring granules. Our discovery that AlgP facilitates granule spacing allows us for the first time to directly uncouple granule biogenesis from even spacing, and will inform future efforts to explore the functional significance of granule organization on fitness during starvation. IMPORTANCE The mechanisms underpinning polyP's pleiotropic effects on bacterial starvation physiology remain elusive. This simple polyanion's lack of protein binding specificity has impeded validation of bona fide polyP-binding proteins. However, polyP forms granule superstructures with spatial specificity. Our granule enrichment protocol identified a polyP granule-associated protein in Pseudomonas aeruginosa, AlgP. AlgP was originally reported as a regulator of alginate, an extracellular polysaccharide important in biofilm formation, including in cystic fibrosis (CF) chronic infections. AlgP's putative role in alginate biosynthesis has recently been called into question. We establish a distinct, previously unknown function for AlgP in modulating the subcellular organization of polyP, another polymer important for pathogenesis. In CF clinical isolates, the C-terminal repeat domain of AlgP is a hot spot for genetic rearrangements. Our finding that the C-terminus of AlgP is required for granule organization lays the groundwork for exploring the functional significance of these mutations in the evolutionary trajectory of chronic infections.

Quantitative mining of compositional heterogeneity in cryo-EM datasets of ribosome assembly intermediates.

Single-particle cryoelectron microscopy (cryo-EM) offers a unique opportunity to characterize macromolecular structural heterogeneity by virtue of its ability to place distinct particle populations into different groups through computational classification. However, there is a dearth of tools for surveying the heterogeneity landscape, quantitatively analyzing heterogeneous particle populations after classification, deciding how many unique classes are represented by the data, and accurately cross-comparing reconstructions. Here, we develop a workflow that contains discovery and analysis modules to quantitatively mine cryo-EM data for sets of structures with maximal diversity. This workflow was applied to a dataset of E. coli 50S ribosome assembly intermediates, which are characterized by significant structural heterogeneity. We identified more detailed branchpoints in the assembly process and characterized the interactions of an assembly factor with immature intermediates. While the tools described here were developed for ribosome assembly, they should be broadly applicable to the analysis of other heterogeneous cryo-EM datasets.

Gait instability and estimated core temperature predict exertional heat stroke.

OBJECTIVE: Exertional heat stroke (EHS), characterised by a high core body temperature (Tcr) and central nervous system (CNS) dysfunction, is a concern for athletes, workers and military personnel who must train and perform in hot environments. The objective of this study was to determine whether algorithms that estimate Tcr from heart rate and gait instability from a trunk-worn sensor system can forward predict EHS onset. METHODS: Heart rate and three-axis accelerometry data were collected from chest-worn sensors from 1806 US military personnel participating in timed 4/5-mile runs, and loaded marches of 7 and 12 miles; in total, 3422 high EHS-risk training datasets were available for analysis. Six soldiers were diagnosed with heat stroke and all had rectal temperatures of >41°C when first measured and were exhibiting CNS dysfunction. Estimated core temperature (ECTemp) was computed from sequential measures of heart rate. Gait instability was computed from three-axis accelerometry using features of pattern dispersion and autocorrelation. RESULTS: The six soldiers who experienced heat stroke were among the hottest compared with the other soldiers in the respective training events with ECTemps ranging from 39.2°C to 40.8°C. Combining ECTemp and gait instability measures successfully identified all six EHS casualties at least 3.5 min in advance of collapse while falsely identifying 6.1% (209 total false positives) examples where exertional heat illness symptoms were neither observed nor reported. No false-negative cases were noted. CONCLUSION: The combination of two algorithms that estimate Tcr and ataxic gate appears promising for real-time alerting of impending EHS.

Using Dynamics of Eye Movements, Speech Articulation and Brain Activity to Predict and Track mTBI Screening Outcomes.

Repeated subconcussive blows to the head during sports or other contact activities may have a cumulative and long lasting effect on cognitive functioning. Unobtrusive measurement and tracking of cognitive functioning is needed to enable preventative interventions for people at elevated risk of concussive injury. The focus of the present study is to investigate the potential for using passive measurements of fine motor movements (smooth pursuit eye tracking and read speech) and resting state brain activity (measured using fMRI) to complement existing diagnostic tools, such as the Immediate Post-concussion Assessment and Cognitive Testing (ImPACT), that are used for this purpose. Thirty-one high school American football and soccer athletes were tracked through the course of a sports season. Hypotheses were that (1) measures of complexity of fine motor coordination and of resting state brain activity are predictive of cognitive functioning measured by the ImPACT test, and (2) within-subject changes in these measures over the course of a sports season are predictive of changes in ImPACT scores. The first principal component of the six ImPACT composite scores was used as a latent factor that represents cognitive functioning. This latent factor was positively correlated with four of the ImPACT composites: verbal memory, visual memory, visual motor speed and reaction speed. Strong correlations, ranging between r = 0.26 and r = 0.49, were found between this latent factor and complexity features derived from each sensor modality. Based on a regression model, the complexity features were combined across sensor modalities and used to predict the latent factor on out-of-sample subjects. The predictions correlated with the true latent factor with r = 0.71. Within-subject changes over time were predicted with r = 0.34. These results indicate the potential to predict cognitive performance from passive monitoring of fine motor movements and brain activity, offering initial support for future application in detection of performance deficits associated with subconcussive events.

Detecting Parkinson's Disease from Wrist-Worn Accelerometry in the U.K. Biobank.

Parkinson's disease (PD) is a chronic movement disorder that produces a variety of characteristic movement abnormalities. The ubiquity of wrist-worn accelerometry suggests a possible sensor modality for early detection of PD symptoms and subsequent tracking of PD symptom severity. As an initial proof of concept for this technological approach, we analyzed the U.K. Biobank data set, consisting of one week of wrist-worn accelerometry from a population with a PD primary diagnosis and an age-matched healthy control population. Measures of movement dispersion were extracted from automatically segmented gait data, and measures of movement dimensionality were extracted from automatically segmented low-movement data. Using machine learning classifiers applied to one week of data, PD was detected with an area under the curve (AUC) of 0.69 on gait data, AUC = 0.84 on low-movement data, and AUC = 0.85 on a fusion of both activities. It was also found that classification accuracy steadily improved across the one-week data collection, suggesting that higher accuracy could be achievable from a longer data collection. These results suggest the viability of using a low-cost and easy-to-use activity sensor for detecting movement abnormalities due to PD and motivate further research on early PD detection and tracking of PD symptom severity.

An alternative resource allocation strategy in the chemolithoautotrophic archaeon Methanococcus maripaludis.

Most microorganisms in nature spend the majority of time in a state of slow or zero growth and slow metabolism under limited energy or nutrient flux rather than growing at maximum rates. Yet, most of our knowledge has been derived from studies on fast-growing bacteria. Here, we systematically characterized the physiology of the methanogenic archaeon Methanococcus maripaludis during slow growth. M. maripaludis was grown in continuous culture under energy (formate)-limiting conditions at different dilution rates ranging from 0.09 to 0.002 h-1, the latter corresponding to 1% of its maximum growth rate under laboratory conditions (0.23 h-1). While the specific rate of methanogenesis correlated with growth rate as expected, the fraction of cellular energy used for maintenance increased and the maintenance energy per biomass decreased at slower growth. Notably, proteome allocation between catabolic and anabolic pathways was invariant with growth rate. Unexpectedly, cells maintained their maximum methanogenesis capacity over a wide range of growth rates, except for the lowest rates tested. Cell size, cellular DNA, RNA, and protein content as well as ribosome numbers also were largely invariant with growth rate. A reduced protein synthesis rate during slow growth was achieved by a reduction in ribosome activity rather than via the number of cellular ribosomes. Our data revealed a resource allocation strategy of a methanogenic archaeon during energy limitation that is fundamentally different from commonly studied versatile chemoheterotrophic bacteria such as E. coli.

Using Oculomotor Features to Predict Changes in Optic Nerve Sheath Diameter and ImPACT Scores From Contact-Sport Athletes.

There is mounting evidence linking the cumulative effects of repetitive head impacts to neuro-degenerative conditions. Robust clinical assessment tools to identify mild traumatic brain injuries are needed to assist with timely diagnosis for return-to-field decisions and appropriately guide rehabilitation. The focus of the present study is to investigate the potential for oculomotor features to complement existing diagnostic tools, such as measurements of Optic Nerve Sheath Diameter (ONSD) and Immediate Post-concussion Assessment and Cognitive Testing (ImPACT). Thirty-one high school American football and soccer athletes were tracked through the course of a sports season. Given the high risk of repetitive head impacts associated with both soccer and football, our hypotheses were that (1) ONSD and ImPACT scores would worsen through the season and (2) oculomotor features would effectively capture both neurophysiological changes reflected by ONSD and neuro-functional status assessed via ImPACT. Oculomotor features were used as input to Linear Mixed-Effects Regression models to predict ONSD and ImPACT scores as outcomes. Prediction accuracy was evaluated to identify explicit relationships between eye movements, ONSD, and ImPACT scores. Significant Pearson correlations were observed between predicted and actual outcomes for ONSD (Raw = 0.70; Normalized = 0.45) and for ImPACT (Raw = 0.86; Normalized = 0.71), demonstrating the capability of oculomotor features to capture neurological changes detected by both ONSD and ImPACT. The most predictive features were found to relate to motor control and visual-motor processing. In future work, oculomotor models, linking neural structures to oculomotor function, can be built to gain extended mechanistic insights into neurophysiological changes observed through seasons of participation in contact sports.

Discrimination between Functional and Non-functional Cellular Gag Complexes involved in HIV-1 Assembly.

HIV-1 Gag and Gag-Pol are responsible for viral assembly and maturation and represent a major paradigm for enveloped virus assembly. Numerous intracellular Gag-containing complexes (GCCs) have been identified in cellular lysates using sucrose gradient ultracentrifugation. While these complexes are universally present in Gag-expressing cells, their roles in virus assembly are not well understood. Here we demonstrate that most GCC species are predominantly comprised of monomeric or dimeric Gag molecules bound to ribosomal complexes, and as such, are not on-pathway intermediates in HIV assembly. Rather, these GCCs represent a population of Gag that is not yet functionally committed for incorporation into a viable virion precursor. We hypothesize that these complexes act as a reservoir of monomeric Gag that can incorporate into assembling viruses, and serve to mitigate non-specific intracellular Gag oligomerization. We have identified a subset of large GCC complexes, comprising more than 20 Gag molecules, that may be equivalent to membrane-associated puncta previously shown to be bona fide assembling-virus intermediates. This work provides a clear rationale for the existence of diverse GCCs, and serves as the foundation for characterizing on-pathway intermediates early in virus assembly.

Audio, Visual, and Electrodermal Arousal Signals as Predictors of Mental Fatigue Following Sustained Cognitive Work.

Lapses in vigilance and slowed reactions due to mental fatigue can increase risk of accidents and injuries and degrade performance. This paper describes a method for rapid, unobtrusive detection of mental fatigue based on changes in electrodermal arousal (EDA), and changes in neuromotor coordination derived from speaking. Twenty-nine Soldiers completed a 2-hour battery of cognitive tasks intended to induce fatigue. Behavioral markers derived from audio and video during speech were acquired before and after the 2hour cognitive load tasks, as was EDA. Exposure to cognitive load produced detectable increases in neuromotor variability in speech and facial measures after load and even after a recovery period. A Gaussian mixture model classifier with crossvalidation and fusion across speech, video, and EDA produced an accuracy of AUC=0.99 in detecting a change in cognitive fatigue relative to a personalized baseline.

Estimating Sedentary Breathing Rate from Chest-Worn Accelerometry From Free-Living Data.

Breathing rate was estimated from chest-worn accelerometry collected from 1,522 servicemembers during training by a wearable physiological monitor. A total of 29,189 hours of training and sleep data were analyzed. The primary purpose of the monitor was to assess thermal-work strain and avoid heat injuries. The monitor design was thus not optimized to estimate breathing rate. Since breathing rate cannot be accurately estimated during periods of high activity, a qualifier was applied to identify sedentary time periods, totaling 8,867 hours. Breathing rate was estimated for a total of 4,179 hours, or 14% of the total collection and 47% of the sedentary total, primarily during periods of sleep. The breathing rate estimation method was compared to an FDA 510(K)-cleared criterion breathing rate sensor (Zephyr, Annapolis MD, USA) in a controlled laboratory experiment, which showed good agreement between the two techniques. Contributions of this paper are to: 1) provide the first analysis of accelerometry-derived breathing rate on free-living data including periods of high activity as well as sleep, along with a qualifier that effectively identifies sedentary periods appropriate for estimating breathing rate; 2) test breathing rate estimation on a data set with a total duration that is more than 60 times longer than that of the largest previously reported study, 3) test breathing rate estimation on data from a physiological monitor that has not been expressly designed for that purpose.