Seeing beyond the blot: A critical look at assumptions and raw data interpretation in Western blotting.

Rapid advancements in technology refine our understanding of intricate biological processes, but a crucial emphasis remains on understanding the assumptions and sources of uncertainty underlying biological measurements. This is particularly critical in cell signaling research, where a quantitative understanding of the fundamental mechanisms governing these transient events is essential for drug development, given their importance in both homeostatic and pathogenic processes. Western blotting, a technique developed decades ago, remains an indispensable tool for investigating cell signaling, protein expression, and protein-protein interactions. While improvements in statistical analysis and methodology reporting have undoubtedly enhanced data quality, understanding the underlying assumptions and limitations of visual inspection in Western blotting can provide valuable additional information for evaluating experimental conclusions. Using the example of agonist-induced receptor post-translational modification, we highlight the theoretical and experimental assumptions associated with Western blotting and demonstrate how raw blot data can offer clues to experimental variability that may not be fully captured by statistical analyses and reported methodologies. This article is not intended as a comprehensive technical review of Western blotting. Instead, we leverage an illustrative example to demonstrate how assumptions about experimental design and data normalization can be revealed within raw data and subsequently influence data interpretation.

Training Background and Demographic Characteristics of Primary Care Team Physicians in Professional Sports.

Background: Previous studies have shown that most professional head and orthopaedic team physicians are men, and most orthopaedic team physicians are fellowship-trained. It is unknown whether this holds true for primary care team physicians. Purpose: To evaluate the residency and fellowship training background as well as the demographic characteristics of primary care team physicians in professional sports. Study Design: Cross-sectional study. Methods: Publicly available information was used to determine the lead and supporting primary care team physicians for every US-based team in Major League Baseball, Major League Soccer, National Basketball Association, National Football League, National Hockey League, National Women's Soccer League, and Women's National Basketball Association. Data regarding training background and sex were obtained using internet-based sources. Results: We identified 310 primary care team physicians from all 165 US-based teams in the 7 leagues included in the study. Female physicians comprised 11.5% (19/165) of the lead primary care team physicians and 14.2% (44/310) of all primary care team physicians. Overall, 66.7% (110/165) of lead primary care team physicians and 75.5% (234/310) of all primary care team physicians were sports medicine fellowship-trained. There was a higher proportion of female (37.5%) and fellowship-trained (93.8%) physicians in women's professional sports leagues. Most primary care team physicians (244/310 [78.7%]) were trained in family medicine or internal medicine. Conclusion: Women constituted a small minority of primary care team physicians in professional sports. Most primary care team physicians were residency trained in family medicine or internal medicine and were sports medicine fellowship-trained. The proportion of female and fellowship-trained primary care team physicians was highest in the National Women's Soccer League and the Women's National Basketball Association.

Optimizing Tissue Engineering for Clinical Relevance in Rotator Cuff Repair.

Rotator cuff tear (RCT) is the most common cause of disability in the upper extremity. It results in 4.5 million physician visits in the United States every year and is the most common etiology of shoulder conditions evaluated by orthopedic surgeons. Over 460,000 RCT repair surgeries are performed in the United States annually. Rotator cuff (RC) retear and failure to heal remain significant postoperative complications. Literature suggests that the retear rates can range from 29.5% to as high as 94%. Weakened and irregular enthesis regeneration is a crucial factor in postsurgical failure. Although commercially available RC repair grafts have been introduced to augment RC enthesis repair, they have been associated with mixed clinical outcomes. These grafts lack appropriate biological cues such as stem cells and signaling molecules at the bone-tendon interface. In addition, they do little to prevent fibrovascular scar tissue formation, which causes the RC to be susceptible to retear. Advances in tissue engineering have demonstrated that mesenchymal stem cells (MSCs) and growth factors (GFs) enhance RC enthesis regeneration in animal models. These models show that delivering MSCs and GFs to the site of RCT enhances native enthesis repair and leads to greater mechanical strength. In addition, these models demonstrate that MSCs and GFs may be delivered through a variety of methods including direct injection, saturation of repair materials, and loaded microspheres. Grafts that incorporate MSCs and GFs enhance anti-inflammation, osteogenesis, angiogenesis, and chondrogenesis in the RC repair process. It is crucial that the techniques that have shown success in animal models are incorporated into the clinical setting. A gap currently exists between the promising biological factors that have been investigated in animal models and the RC repair grafts that can be used in the clinical setting. Future RC repair grafts must allow for stable implantation and fixation, be compatible with current arthroscopic techniques, and have the capability to deliver MSCs and/or GFs.

Public Health Dashboards in Overdose Prevention: The Rhode Island Approach to Public Health Data Literacy, Partnerships, and Action.

As the field of public health rises to the demands of real-time surveillance and rapid data-sharing needs in a postpandemic world, it is time to examine our approaches to the dissemination and accessibility of such data. Distinct challenges exist when working to develop a shared public health language and narratives based on data. It requires that we assess our understanding of public health data literacy, revisit our approach to communication and engagement, and continuously evaluate our impact and relevance. Key stakeholders and cocreators are critical to this process and include people with lived experience, community organizations, governmental partners, and research institutions. In this viewpoint paper, we offer an instructive approach to the tools we used, assessed, and adapted across 3 unique overdose data dashboard projects in Rhode Island, United States. We are calling this model the "Rhode Island Approach to Public Health Data Literacy, Partnerships, and Action." This approach reflects the iterative lessons learned about the improvement of data dashboards through collaboration and strong partnerships across community members, state agencies, and an academic research team. We will highlight key tools and approaches that are accessible and engaging and allow developers and stakeholders to self-assess their goals for their data dashboards and evaluate engagement with these tools by their desired audiences and users.

Mesozoic evolution of cicadas and their origins of vocalization and root feeding.

Extant cicada (Hemiptera: Cicadoidea) includes widely distributed Cicadidae and relictual Tettigarctidae, with fossils ascribed to these two groups based on several distinct, minimally varying morphological differences that define their extant counterparts. However, directly assigning Mesozoic fossils to modern taxa may overlook the role of unique and transitional features provided by fossils in tracking their early evolutionary paths. Here, based on adult and nymphal fossils from mid-Cretaceous Kachin amber of Myanmar, we explore the phylogenetic relationships and morphological disparities of fossil and extant cicadoids. Our results suggest that Cicadidae and Tettigarctidae might have diverged at or by the Middle Jurassic, with morphological evolution possibly shaped by host plant changes. The discovery of tymbal structures and anatomical analysis of adult fossils indicate that mid-Cretaceous cicadas were silent as modern Tettigarctidae or could have produced faint tymbal-related sounds. The discovery of final-instar nymphal and exuviae cicadoid fossils with fossorial forelegs and piercing-sucking mouthparts indicates that they had most likely adopted a subterranean lifestyle by the mid-Cretaceous, occupying the ecological niche of underground feeding on root. Our study traces the morphological, behavioral, and ecological evolution of Cicadoidea from the Mesozoic, emphasizing their adaptive traits and interactions with their living environments.

An Analysis of Uncertainties and Data Collection Agreements in the Cancer Drugs Fund.

BACKGROUND: Managed Access Agreements (MAAs) are a commercial arrangement that provide patients earlier access to innovative health technologies while uncertainties in the evidence base are resolved through data collection. In the UK, data collection agreements (DCAs) outline the evidence that will be collected during the MAA period and are intended to resolve uncertainties in the clinical- and cost-effectiveness of a technology sufficient for the National Institute of Health and Care Excellence (NICE) committee to make a final decision on reimbursement. OBJECTIVE: The aim of this study was to identify the primary uncertainties leading to a recommendation for entry to the Cancer Drugs Fund (CDF) and evaluate how the corresponding DCAs attempt to address these. METHODS: A database of MAAs agreed within the CDF was compiled with coverage between July 2016 and December 2020 (the time during which evidence generation was routinely collected within the CDF up until the time of analysis). Uncertainties in the evidence base for technologies entering the CDF were analysed alongside the outcomes planned for data collection during the MAA. These data provide an overview of the key uncertainties surrounding health technologies in the CDF on entry and the types of evidence targeted by DCAs. RESULTS: In the assessment of 39 Cancer Drugs Fund (CDF) cases, NICE committees identified a total of 108 key uncertainties in cost-effectiveness estimates. Overall survival was the most commonly identified uncertainty, followed by generalisability of the evidence to the target population. DCAs specified a range of outcomes relevant to understanding the clinical effectiveness of the technology, though fewer than half (43.6%) of the DCAs addressed all the key uncertainties identified by the NICE committee. CONCLUSION: The analysis indicated that data collection within the CDF is not sufficient to resolve all the uncertainties identified by the NICE committee, meaning that other approaches will be needed at re-appraisal to ensure that the NICE committee can reach a final decision on reimbursement.

A Small Multidrug Resistance Transporter in Pseudomonas aeruginosa Confers Substrate-Specific Resistance or Susceptibility.

Small Multidrug Resistance (SMR) transporters are key players in the defense of multidrug-resistant pathogens to toxins and other homeostasis-perturbing compounds. However, recent evidence demonstrates that EmrE, an SMR from Escherichia coli and a model for understanding transport, can also induce susceptibility to some compounds by drug-gated proton leak. This runs down the ∆pH component of the Proton Motive Force (PMF), reducing viability of the affected bacteria. Proton leak may provide an unexplored drug target distinct from the targets of most known antibiotics. Activating proton leak requires an SMR to be merely present, rather than be the primary resistance mechanism, and dissipates the energy source for many other efflux pumps. PAsmr, an EmrE homolog from P. aeruginosa, transports many EmrE substrates in cells and purified systems. We hypothesized that PAsmr, like EmrE, may confer susceptibility to some compounds via drug-gated proton leak. Growth assays of E. coli expressing PAsmr displayed substrate-dependent resistance and susceptibility phenotypes, and in vitro solid-supported membrane electrophysiology experiments revealed that PAsmr performs both antiport and substrate-gated proton uniport, demonstrating the same functional promiscuity observed in EmrE. Growth assays of P. aeruginosa strain PA14 demonstrated that PAsmr contributes resistance to some antimicrobial compounds, but no growth defect is observed with susceptibility substrates, suggesting P. aeruginosa can compensate for the proton leak occurring through PAsmr. These phenotypic differences between P. aeruginosa and E. coli advance our understanding of underlying resistance mechanisms in P. aeruginosa and prompt further investigation into the role that SMRs play in antibiotic resistance in pathogens.

C-N Bond Formation at Discrete CuIII-Aryl Complexes.

Copper(III) aryl species are widely proposed as intermediates in Cu-catalyzed C-C and C-heteroatom bond formation reactions. Despite their wide utility, mechanistic aspects of C-heteroatom formation at CuIII centers as well as factors that lead to byproducts, e.g., Ar-H, Ar-Ar, remain elusive due to the rarity of discrete CuIII-Ar complexes. Herein, we report the synthesis and reactivity of a series of CuII and CuIII aryl complexes that closely mimic the intermediates in Cu-catalyzed C-N coupling reactions. Copper(II) aryl complexes [TBA][LCuII-ArR] were synthesized via the treatment of CuII with a range of aryl donors, such as ZnAr2R, TMS-ArR, and ArR-Bpin. Oxidation of [TBA][LCuII-ArR] produces formal copper(III) aryl complexes LCuIII-ArR. Treatment of copper(III) aryl complexes with neutral nitrogen nucleophiles produces the C-N coupling product in up to 64% yield, along with commonly observed byproducts, such as Ar-H and Ar-Ar. Hammett analysis of the C-N bond formation performed with various N-nucleophiles shows a ρ value of -1.66, consistent with the electrophilic character of LCuIII-ArR. We propose mechanisms for common side reactions in Cu-catalyzed coupling reactions that lead to the formation of Ar-Ar and Ar-H.

A realist synthesis to develop an explanatory model of how policy instruments impact child and maternal health outcomes.

BACKGROUND: Child and maternal health, a key marker of overall health system performance, is a policy priority area by the World Health Organization and the United Nations, including the Sustainable Development Goals. Previous realist work has linked child and maternal health outcomes to globalization, political tradition, and the welfare state. It is important to explore the role of other key policy-related factors. This paper presents a realist synthesis, categorising policy instruments according to the established NATO model, to develop an explanatory model of how policy instruments impact child and maternal health outcomes. METHODS: A systematic literature search was conducted to identify studies assessing the relationships between policy instruments and child and maternal health outcomes. Data were analysed using a realist framework. The first stage of the realist analysis process was to generate micro-theoretical initial programme theories for use in the theory adjudication process. Proposed theories were then adjudicated iteratively to produce a set of final programme theories. FINDINGS: From a total of 43,415 unique records, 632 records proceeded to full-text screening and 138 papers were included in the review. Evidence from 132 studies was available to address this research question. Studies were published from 1995 to 2021; 76% assessed a single country, and 81% analysed data at the ecological level. Eighty-eight initial candidate programme theories were generated. Following theory adjudication, five final programme theories were supported. According to the NATO model, these were related to treasure, organisation, authority-treasure, and treasure-organisation instrument types. CONCLUSIONS: This paper presents a realist synthesis to develop an explanatory model of how policy instruments impact child and maternal health outcomes from a large, systematically identified international body of evidence. Five final programme theories were supported, showing how policy instruments play an important yet context-dependent role in influencing child and maternal health outcomes.

Vanadium Electronic Configuration Determination From L2,3 Transition in V-oxide Compounds and Roscoelite.

We report on the electronic structure of vanadium in synthetic V-oxides and in natural roscoelite (V-rich phyllosilicate). This study applied electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM), combined with first-principle calculations, to (1) establish relationships between the V oxidation state and EELS L2,3 features and (2) better constrain the oxidation state and crystallographic siting of V in roscoelite, with implications for other V-bearing phyllosilicates. Both EELS measurements and band structure calculations show that the EELS L2/L3 ratio increases as the oxidation state of V increases. We establish a quantitative relationship between the V L2,3 near-edge structure and the V oxidation state by normalizing the L2 maximum peak intensity to the L3 peak intensity. By applying this method to roscoelite, we find that it hosts a mix of trivalent and tetravalent V distributed between the octahedral and tetrahedral sites with a V4+/ΣV = 0.6 ± 0.1. This relationship is applicable to measurements of V oxidation states in oxide and phyllosilicate minerals, which is useful for constraining the conditions of rock and mineral formation and has potential implications for metal extraction from phyllosilicate ores.

Molecular Network Approach to Anisotropic Ising Lattices: Parsing Magnetization Dynamics in Er3+ Systems with 0-3-Dimensional Spin Interactivity.

We present a wide-ranging interrogation of the border between single-molecule and solid-state magnetism through a study of erbium-based Ising-type magnetic compounds with a fixed magnetic unit, using three different charge-balancing cations as the means to modulate the crystal packing environment. Properties rooted in the isolated spin Hamiltonian remain fixed, yet careful observation of the dynamics reveals the breakdown of this approximation in a number of interesting ways. First, differences in crystal packing lead to a striking 3 orders of magnitude suppression in magnetic relaxation rates, indicating a rich interplay between intermolecular interactions governed by the anisotropic Ising lattice stabilization and localized slow magnetic relaxation driven by the spin-forbidden nature of quantum tunneling of the f-electron-based magnetization. By means of diverse and rigorous physical methods, including temperature-dependent X-ray crystallography, field, temperature, and time-dependent magnetometry, and the application of a new magnetization fitting technique to quantify the magnetic susceptibility peakshape, we are able to construct a more nuanced view of the role nonzero-dimensional interactions can play in what are predominantly considered zero-dimensional magnetic materials. Specifically, we use low field susceptibility and virgin-curve analysis to isolate metamagnetic spin-flip transitions in each system with a field strength corresponding to the expected strength of the internal dipole-dipole lattice. This behavior is vital to a complete interpretation of the dynamics and is likely common for systems with such high anisotropy. This collective interactivity opens a new realm of possibility for molecular magnetic materials, where their unprecedented localized anisotropy is the determining factor in building higher dimensionality.

Early access schemes for innovative health technologies: the views of international stakeholders.

OBJECTIVES: Early access schemes (EASs) are approaches used by payers to balance and facilitate earlier patient access to innovative health technologies while evidence generation is ongoing. Schemes require investment from payers and are associated with significant risk since not all technologies will be routinely reimbursed. The purpose of this study was to gain the perspectives of policy experts about the key challenges for EASs and potential solutions for their optimal design and implementation. METHODS: Two virtual workshops were convened including (i) UK-based policy experts (England, Wales, and Scotland) and (ii) representatives from multiple healthcare systems (England, France, Sweden, Canada, Poland, and Norway). Participants were encouraged to share their experiences with EASs in their healthcare system and highlight key challenges for policy makers. Discussions were transcribed and analyzed using framework analysis. RESULTS: Participants agreed that EASs have value when targeted toward innovative technologies with the potential for significant clinical benefit in an area of high unmet need. Participants discussed potential solutions to the challenges faced by payers implementing EASs, including defining eligibility criteria, supporting evidence generation, and approaches to reimbursement. CONCLUSIONS: Participants agreed that EASs are one possible solution for their healthcare systems and have the potential to deliver significant clinical value to patients. However, widespread adoption of EASs is limited due to concerns about the risks for patients and healthcare budgets, further solutions are needed to deliver EASs for targeted therapies.

An evaluation of managed access agreements in England based on stakeholder experience.

OBJECTIVES: The objective of this research was to evaluate managed access policy in England, drawing upon the expertise of a range of stakeholders involved in its implementation. METHODS: Seven focus groups were conducted with payer and health technology assessment representatives, clinicians, and representatives from industry and patient/carer organizations within England. Transcripts were analyzed using framework analysis to identify stakeholders' views on the successes and challenges of managed access policy. RESULTS: Stakeholders discussed the many aims of managed access within the National Health Service in England, and how competing aims had affected decision making. While stakeholders highlighted a number of priorities within eligibility criteria for managed access agreements (MAAs), stakeholders agreed that strict eligibility criteria would be challenging to implement due to the highly variable nature of innovative technologies and their indications. Participants highlighted challenges faced with implementing MAAs, including evidence generation, supporting patients during and after the end of MAAs, and agreeing and reinforcing contractual agreements with industry. CONCLUSIONS: Managed access is one strategy that can be used by payers to resolve uncertainty for innovative technologies that present challenges for reimbursement and can also deliver earlier access to promising technologies for patients. However, participants cautioned that managed access is not a "silver bullet," and there is a need for greater clarity about the aims of managed access and how these should be prioritized in decision making. Discussions between key stakeholders involved in managed access identified challenges with implementing MAAs and these experiences should be used to inform future managed access policy.

Describing the landscape of integrated clinical educational programs for PAs and NPs.

PURPOSE: Postgraduate clinical training (PCT) has been available to PAs since the 1970s and to NPs since at least 2007. Some programs now enroll PAs and NPs. Although this new training model appears to be expanding, little data about integrated PA/NP programs are available. METHODS: This study examined the PA/NP PCT landscape in the United States. Programs were identified from membership rosters of the Association of Postgraduate Physician Assistant Programs and the Association of Post Graduate APRN Programs. Data (program name, sponsoring institution, location, specialty, accreditation status) were identified from programs' websites. RESULTS: We identified 106 programs at 42 sponsoring institutions. Various specialties, most commonly in emergency medicine, critical care, and surgery, were represented. Few were accredited. CONCLUSIONS: PA/NP PCT is now common, with about half of the programs accepting PAs and NPs. These programs represent a unique form of interprofessional education involving full integration of two professions in the same program and are worthy of further investigation.

Group-based active artistic interventions for adults with primary anxiety and depression: a systematic review.

OBJECTIVES: This systematic review examined the potential benefit of all group-based performing arts interventions for primary anxiety and/or depression. SETTING: Scholarly literature from any country or countries globally. DATA SOURCES: Three key bibliographic databases, Google Scholar and relevant citation chasing. PRIMARY AND SECONDARY OUTCOME MEASURES: Depression and/or anxiety symptom severity, well-being, quality of life, functional communication or social participation. RESULTS: Database searches returned a total of 63 678 records, of which 56 059 remained following dededuplication. From these database searches, a total of 153 records proceeded to full-text screening. These were supplemented by 18 additional unique full-text screening records from Google Scholar searches and citation chasing (12% of total). From a total of 171 records at the full-text screening stage, 12 publications (7%) were eligible for inclusion in this systematic review, each reporting on a separate study. Published from 2004 to 2021, these studies involved a total of 669 participants with anxiety and/or depression from nine countries and covered five broad artistic modalities: dance, music therapy, art therapy, martial arts and theatre. Dance was the most studied artistic modality (five studies), while there were three studies on art therapy, two on music therapy and one each on martial arts and theatre. The evidence was clearest for a benefit of arts therapies on depression and/or anxiety symptoms. CONCLUSIONS: This systematic review addresses all group-based active arts interventions in a focused population of primary anxiety and/or depression. The evidence suggests that the arts may be a useful therapeutic medium in this population. However, a substantial limitation of the evidence base is the lack of studies directly comparing different artistic modalities. Moreover, not all artistic modalities were assessed for all outcome domains. Therefore, it is not currently possible to determine which artistic modalities are most beneficial for which specific outcomes.

Intraoperative bowel perfusion assessment methods and their effects on anastomotic leak rates: meta-analysis.

BACKGROUND: Anastomotic leak is one of the most feared complications of colorectal surgery, and probably linked to poor blood supply to the anastomotic site. Several technologies have been described for intraoperative assessment of bowel perfusion. This systematic review and meta-analysis aimed to evaluate the most frequently used bowel perfusion assessment modalities in elective colorectal procedures, and to assess their associated risk of anastomotic leak. Technologies included indocyanine green fluorescence angiography, diffuse reflectance spectroscopy, laser speckle contrast imaging, and hyperspectral imaging. METHODS: The review was preregistered with PROSPERO (CRD42021297299). A comprehensive literature search was performed using Embase, MEDLINE, Cochrane Library, Scopus, and Web of Science. The final search was undertaken on 29 July 2022. Data were extracted by two reviewers and the MINORS criteria were applied to assess the risk of bias. RESULTS: Some 66 eligible studies involving 11 560 participants were included. Indocyanine green fluorescence angiography was most used with 10 789 participants, followed by diffuse reflectance spectroscopy with 321, hyperspectral imaging with 265, and laser speckle contrast imaging with 185. In the meta-analysis, the total pooled effect of an intervention on anastomotic leak was 0.05 (95 per cent c.i. 0.04 to 0.07) in comparison with 0.10 (0.08 to 0.12) without. Use of indocyanine green fluorescence angiography, hyperspectral imaging, or laser speckle contrast imaging was associated with a significant reduction in anastomotic leak. CONCLUSION: Bowel perfusion assessment reduced the incidence of anastomotic leak, with intraoperative indocyanine green fluorescence angiography, hyperspectral imaging, and laser speckle contrast imaging all demonstrating comparable results.

Extracellular histone proteins activate P2XR7 channel current.

Extracellular histone proteins are elevated in circulation after injury or activation of the innate immune response. In resistance-size arteries, extracellular histone proteins increased endothelial cell (EC) Ca2+ influx and propidium iodide (PI) labeling, but paradoxically decreased vasodilation. These observations could be explained by the activation of an EC resident non-selective cation channel. We tested the hypothesis that the ionotropic purinergic receptor 7 (P2XR7), a non-selective cation channel associated with cationic dye uptake, is activated by histone proteins. We expressed mouse P2XR7 (C57BL/6J variant 451L) in heterologous cells and measured inward cation current using two-electrode voltage clamp (TEVC). Cells expressing mouse P2XR7 had robust ATP- and histone-evoked inward cation currents. ATP- and histone-evoked currents reversed approximately at the same potential. Current decay with agonist removal was slower for histone-evoked than ATP- or BzATP-evoked currents. As with ATP-evoked P2XR7 currents, histone-evoked currents were inhibited by non-selective P2XR7 antagonists (Suramin, PPADS, and TNP-ATP). Selective P2XR7 antagonists, AZ10606120, A438079, GW791343, and AZ11645373, inhibited ATP-evoked P2XR7 currents but did not inhibit histone-evoked P2XR7 currents. As previously reported with ATP-evoked currents, histone-evoked P2XR7 currents were also increased in conditions of low extracellular Ca2+. These data demonstrate that P2XR7 is necessary and sufficient for histone-evoked inward cation currents in a heterologous expression system. These results provide insight into a new allosteric mechanism of P2XR7 activation by histone proteins.

The temporal structure of REM sleep shows minute-scale fluctuations across brain and body in mice and humans.

Rapid eye movement sleep (REM) is believed to have a binary temporal structure with "phasic" and "tonic" microstates, characterized by motoric activity versus quiescence, respectively. However, we observed in mice that the frequency of theta activity (a marker of rodent REM) fluctuates in a nonbinary fashion, with the extremes of that fluctuation correlating with phasic-type and tonic-type facial motricity. Thus, phasic and tonic REM may instead represent ends of a continuum. These cycles of brain physiology and facial movement occurred at 0.01 to 0.06 Hz, or infraslow frequencies, and affected cross-frequency coupling and neuronal activity in the neocortex, suggesting network functional impact. We then analyzed human data and observed that humans also demonstrate nonbinary phasic/tonic microstates, with continuous 0.01 to 0.04-Hz respiratory rate cycles matching the incidence of eye movements. These fundamental properties of REM can yield insights into our understanding of sleep health.