The call for the European Respiratory Society (ERS) to change their e-cigarette and vaping policy, from honourable people with decades of experience fighting the evils of tobacco, is unfortunately misconceived. The three issues of greatest concern are acute toxicity, chronic toxicity and, most importantly, the effects on children and young people. The efficacy of e-cigarettes as an adjunct to smoking cessation are outwith the expertise of paediatric specialists, but we would ask for assurances that any benefits really do outweigh the risks to children and young people (below). Our comments on these key issues are as follows:

We wish to thank J. Britton and co-workers for responding to our editorial and giving us an opportunity to clarify our position as well as correct a few misunderstandings. We definitely share the same goal, which is to relieve Europe and the rest of the world from the terrible results of the tobacco epidemic. We also do not "blankly oppose e-cigarettes"; however, we strongly advocate against a harm reduction strategy including e-cigarettes as well as heated tobacco products [1]. As clinicians we all see reluctant smokers where e-cigarettes can be tried as a last resort for getting off cigarette smoking, but that is of little relevance for a general harm reduction strategy. We also agree that the UK has achieved a lot in the area of smoking cessation but would argue that this has been achieved by impressive tobacco control, not by the use of e-cigarettes, and that a country such as Australia, which has banned nicotine-containing e-cigarettes, has achieved similar results.

The respiratory community is united in its desire to reduce and eliminate the harm caused by tobacco smoking, which is at present on course to kill one billion people in the 21st century. The stated policy of the European Respiratory Society is to strive "constantly to promote strong and evidence-based policies to reduce the burden of tobacco related diseases". In our view, the recent ERS Tobacco Control Committee statement on tobacco harm reduction [1], though well-intentioned, appears to be based on a number of false premises and draws its conclusions from a partial account of available data. It also presents a false dichotomy between the provision of "conventional" tobacco control and harm reduction approaches. We therefore respond, in turn, to the seven arguments presented against the adoption of harm reduction in the Committee's statement.

The World Health Organization (WHO) has listed moxifloxacin and linezolid among the preferred "group A" drugs in the treatment of multidrug-resistant (MDR)-tuberculosis (TB) [1]. Therapeutic drug monitoring (TDM) could potentially optimise MDR-TB therapy, since moxifloxacin and linezolid show large pharmacokinetic variability [1–4]. TDM of moxifloxacin focuses on identifying patients with low drug exposure who are at risk of treatment failure and acquired fluoroquinolone resistance [5, 6]. Alternatively, TDM of linezolid strives to reduce toxicity while ensuring an adequate drug exposure because of its narrow therapeutic index [1, 3, 7].

Objective

To use the variations in neurology consultations requested by emergency department (ED) physicians to identify opportunities to implement multidisciplinary interventions in an effort to reduce ED overcrowding.

Objective

To explore practice differences in the diagnosis and management of autoimmune encephalitis (AE), which is complicated by issues with sensitivity/specificity of antibody testing, nonspecific MRI/EEG/CSF findings, and competing differential diagnoses.

Multiple births are increasing in frequency related to advanced maternal age and fertility treatments, and they have an increased risk for congenital anomalies compared to singleton births. However, twins have the same congenital anomalies <15% of the time. Thus, having multiple births with discordant anomalies is a growing challenge for neonatologists. Although external anomalies can often be spotted quickly at delivery or sex differences between multiples can rapidly identify those with internal anomalies described on prenatal ultrasound, we present a case of male multiples, who would optimally receive different initial resuscitation strategies on the basis of the presence or absence of an internal anomaly. The similar size of 4 extremely preterm quadruplets raises concern for whether accurate, immediate identification of 1 neonate with a congenital diaphragmatic hernia will be reliable in the delivery room. Clinicians discuss the ethical considerations of an "all for one" approach to this resuscitation.

A 16-year-old girl presented to her primary care physician with a one-month history of decreased appetite and abdominal pain. She had normal bowel movements and no vomiting, but her periumbilical pain limited her ability to finish most meals. She had gradual weight loss over the previous 2 years, and during the previous 4 years, she intermittently received counseling for depression after the loss of her mother. Her initial physical examination and laboratory evaluation were unremarkable. She was referred to a nutritionist, adolescent medicine, and pediatric gastroenterology. Her presentation evolved over time, which ultimately led to a definitive diagnosis.

Causal analysis is a core function of safety programs. Although established protocols exist for conducting root cause analysis for serious safety events, there is limited guidance for apparent cause analysis (ACA) in health care. At our institution, through a novel facilitated ACA approach, we aim to improve safety culture and provide a clear approach to address precursor safety events and near-miss safety events. We define facilitated ACA as limited investigation (scope and duration) of a safety event that resulted in little to no harm. These investigations require fewer resources and focus on preventive strategies. Our facilitated ACA model, with an operational algorithm and structured process, was developed and implemented at our tertiary-care, freestanding, urban pediatric hospital in 2018. Sixty-four ACAs were completed, and 83% were identified with the algorithm. Process measures, including time from event reporting to ACA launch (median 3 days; interquartile range 2–6 days), are tracked. Patient safety consultants averaged 5 hours to complete a facilitated ACA. A median of 3 disciplines or departments participated in each facilitated ACA. Through an iterative process, we implemented a structured process for facilitated ACA, and the model’s strength includes (1) right event, (2) right team, (3) right analysis, and (4) right action plans. This novel facilitated ACA model may support organizational cause analysis and improve safety culture with higher-reliability processes.

As the technical ability for genetic diagnosis continues to improve, an increasing number of diagnoses are made in infancy or as early as the neonatal period. Many of these diagnoses are known to be associated with developmental delay and intellectual disability, features that would not be clinically detectable at the time of diagnosis. Others may be associated with cognitive impairment, but the incidence and severity are yet to be fully described. These neonates and infants with genetic diagnoses therefore represent an emerging group of patients who are at high risk for neurodevelopmental disabilities. Although there are well-established developmental supports for high-risk infants, particularly preterm infants, after discharge from the NICU, programs specifically for infants with genetic diagnoses are rare. And although previous research has demonstrated the positive effect of early developmental interventions on outcomes among preterm infants, the impact of such supports for infants with genetic disorders who may be born term, remains to be understood. We therefore review the literature regarding existing developmental assessment and intervention approaches for children with genetic disorders, evaluating these in the context of current developmental supports postdischarge for preterm infants. Further research into the role of developmental support programs for early assessment and intervention in high-risk neonates diagnosed with rare genetic disorders is needed.

The question of the relative evolutionary roles of adaptive and nonadaptive processes has been a central debate in population genetics for nearly a century. While advances have been made in the theoretical development of the underlying models, and statistical methods for estimating their parameters from large-scale genomic data, a framework for an appropriate null model remains elusive. A model incorporating evolutionary processes known to be in constant operation, genetic drift (as modulated by the demographic history of the population) and purifying selection, is lacking. Without such a null model, the role of adaptive processes in shaping within- and between-population variation may not be accurately assessed. Here, we investigate how population size changes and the strength of purifying selection affect patterns of variation at "neutral" sites near functional genomic components. We propose a novel statistical framework for jointly inferring the contribution of the relevant selective and demographic parameters. By means of extensive performance analyses, we quantify the utility of the approach, identify the most important statistics for parameter estimation, and compare the results with existing methods. Finally, we reanalyze genome-wide population-level data from a Zambian population of Drosophila melanogaster, and find that it has experienced a much slower rate of population growth than was inferred when the effects of purifying selection were neglected. Our approach represents an appropriate null model, against which the effects of positive selection can be assessed.

The Hippo pathway is an evolutionarily conserved signaling network that regulates organ size, cell fate, and tumorigenesis. In the context of organ size control, the pathway incorporates a large variety of cellular cues, such as cell polarity and adhesion, into an integrated transcriptional response. The central Hippo signaling effector is the transcriptional coactivator Yorkie, which controls gene expression in partnership with different transcription factors, most notably Scalloped. When it is not activated by Yorkie, Scalloped can act as a repressor of transcription, at least in part due to its interaction with the corepressor protein Tgi. The mechanism by which Tgi represses transcription is incompletely understood, and therefore we sought to identify proteins that potentially operate together with Tgi. Using an affinity purification and mass-spectrometry approach we identified Pits and CtBP as Tgi-interacting proteins, both of which have been linked to transcriptional repression. Both Pits and CtBP were required for Tgi to suppress the growth of the Drosophila melanogaster eye and CtBP loss suppressed the undergrowth of yorkie mutant eye tissue. Furthermore, as reported previously for Tgi, overexpression of Pits repressed transcription of Hippo pathway target genes. These findings suggest that Tgi might operate together with Pits and CtBP to repress transcription of genes that normally promote tissue growth. The human orthologs of Tgi, CtBP, and Pits (VGLL4, CTBP2, and IRF2BP2) have previously been shown to physically and functionally interact to control transcription, implying that the mechanism by which these proteins control transcriptional repression is conserved throughout evolution.

Replication initiation in eukaryotic cells occurs asynchronously throughout S phase, yielding early- and late-replicating regions of the genome, a process known as replication timing (RT). RT changes during development to ensure accurate genome duplication and maintain genome stability. To understand the relative contributions that cell lineage, cell cycle, and replication initiation regulators have on RT, we utilized the powerful developmental systems available in Drosophila melanogaster. We generated and compared RT profiles from mitotic cells of different tissues and from mitotic and endocycling cells of the same tissue. Our results demonstrate that cell lineage has the largest effect on RT, whereas switching from a mitotic to an endoreplicative cell cycle has little to no effect on RT. Additionally, we demonstrate that the RT differences we observed in all cases are largely independent of transcriptional differences. We also employed a genetic approach in these same cell types to understand the relative contribution the eukaryotic RT control factor, Rif1, has on RT control. Our results demonstrate that Rif1 can function in a tissue-specific manner to control RT. Importantly, the Protein Phosphatase 1 (PP1) binding motif of Rif1 is essential for Rif1 to regulate RT. Together, our data support a model in which the RT program is primarily driven by cell lineage and is further refined by Rif1/PP1 to ultimately generate tissue-specific RT programs.

Key Points

Efficient delivery of antigenic cargo to trigger protective immune responses is critical to the success of vaccination. Genetically engineered microorganisms, including virus, bacteria, and protozoa, can be modified to carry and deliver heterologous antigens to the host immune system. The biological vectors can induce a broad range of immune responses and enhance heterologous antigen-specific immunological outcomes. The protozoan genus Eimeria is widespread in domestic animals, causing serious coccidiosis. Eimeria parasites with strong immunogenicity are potent coccidiosis vaccine candidates and offer a valuable model of live vaccines against infectious diseases in animals. Eimeria parasites can also function as a vaccine vector. Herein, we review recent advances in design and application of recombinant Eimeria as a vaccine vector, which has been a topic of ongoing research in our laboratory. By recapitulating the establishment of an Eimeria transfection platform and its application, it will help lay the foundation for the future development of effective parasite-based vaccine delivery vectors and beyond.

Although substantial progress has been made in depicting the molecular pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection, the comprehensive mechanism of HIV-1 latency and the most promising therapeutic strategies to effectively reactivate the HIV-1 latent reservoir to achieve a functional cure for AIDS remain to be systematically illuminated. Here, we demonstrated that piwi (P element-induced Wimpy)-like RNA-mediated gene silencing 4 (PIWIL4) played an important role in suppressing HIV-1 transcription and contributed to the latency state in HIV-1-infected cells through its recruitment of various suppressive factors, including heterochromatin protein 1α/β/, SETDB1, and HDAC4. The knockdown of PIWIL4 enhanced HIV-1 transcription and reversed HIV-1 latency in both HIV-1 latently infected Jurkat T cells and primary CD4⁺ T lymphocytes and resting CD4⁺ T lymphocytes from HIV-1-infected individuals on suppressive combined antiretroviral therapy (cART). Furthermore, in the absence of PIWIL4, HIV-1 latently infected Jurkat T cells were more sensitive to reactivation with vorinostat (suberoylanilide hydroxamic acid, or SAHA), JQ1, or prostratin. These findings indicated that PIWIL4 promotes HIV-1 latency by imposing repressive marks at the HIV-1 5' long terminal repeat. Thus, the manipulation of PIWIL4 could be a novel strategy for developing promising latency-reversing agents (LRAs).

IMPORTANCE HIV-1 latency is systematically modulated by host factors and viral proteins. During this process, the suppression of HIV-1 transcription plays an essential role in promoting HIV-1 latency. In this study, we found that PIWIL4 repressed HIV-1 promoter activity and maintained HIV-1 latency. In particular, we report that PIWIL4 can regulate gene expression through its association with the suppressive activity of HDAC4. Therefore, we have identified a new function for PIWIL4: it is not only a suppressor of endogenous retrotransposons but also plays an important role in inhibiting transcription and leading to latent infection of HIV-1, a well-known exogenous retrovirus. Our results also indicate a novel therapeutic target to reactivate the HIV-1 latent reservoir.

Viruses commonly antagonize innate immune pathways that are primarily driven by nuclear factor kappa B (NF-B), interferon regulatory factor (IRF), and the signal transducer and activator of transcription proteins (STAT) family of transcription factors. Such a strategy allows viruses to evade immune surveillance and maximize their replication. Using an unbiased transcriptome sequencing (RNA-seq)-based approach to measure gene expression induced by transfected viral genomic RNA (vgRNA) and reovirus infection, we discovered that mammalian reovirus inhibits host cell innate immune signaling. We found that, while vgRNA and reovirus infection both induce a similar IRF-dependent gene expression program, gene expression driven by the NF-B family of transcription factors is lower in infected cells. Potent agonists of NF-B such as tumor necrosis factor alpha (TNF-α) and vgRNA failed to induce NF-B-dependent gene expression in infected cells. We demonstrate that NF-B signaling is blocked due to loss of critical members of the inhibitor of kappa B kinase (IKK) complex, NF-B essential modifier (NEMO), and IKKβ. The loss of the IKK complex components prevents nuclear translocation and phosphorylation of NF-B, thereby preventing gene expression. Our study demonstrates that reovirus infection selectively blocks NF-B, likely to counteract its antiviral effects and promote efficient viral replication.

IMPORTANCE Host cells mount a response to curb virus replication in infected cells and prevent spread of virus to neighboring, as yet uninfected, cells. The NF-B family of proteins is important for the cell to mediate this response. In this study, we show that in cells infected with mammalian reovirus, NF-B is inactive. Further, we demonstrate that NF-B is rendered inactive because virus infection results in reduced levels of upstream intermediaries (called IKKs) that are needed for NF-B function. Based on previous evidence that active NF-B limits reovirus infection, we conclude that inactivating NF-B is a viral strategy to produce a cellular environment that is favorable for virus replication.

The nuclear factor kappa B (NF-B) is a potent transcription factor, activation of which typically results in robust proinflammatory signaling and triggering of fast negative feedback modulators to avoid excessive inflammatory responses. Here, we report that infection of epithelial cells, including primary porcine respiratory epithelial cells, with the porcine alphaherpesvirus pseudorabies virus (PRV) results in the gradual and persistent activation of NF-B, illustrated by proteasome-dependent degradation of the inhibitory NF-B regulator IB and nuclear translocation and phosphorylation of the NF-B subunit p65. PRV-induced persistent activation of NF-B does not result in expression of negative feedback loop genes, like the gene for IBα or A20, and does not trigger expression of prototypical proinflammatory genes, like the gene for tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6). In addition, PRV infection inhibits TNF-α-induced canonical NF-B activation. Hence, PRV infection triggers persistent NF-B activation in an unorthodox way and dramatically modulates the NF-B signaling axis, preventing typical proinflammatory gene expression and the responsiveness of cells to canonical NF-B signaling, which may aid the virus in modulating early proinflammatory responses in the infected host.

IMPORTANCE The NF-B transcription factor is activated via different key inflammatory pathways and typically results in the fast expression of several proinflammatory genes as well as negative feedback loop genes to prevent excessive inflammation. In the current report, we describe that infection of cells with the porcine alphaherpesvirus pseudorabies virus (PRV) triggers a gradual and persistent aberrant activation of NF-B, which does not result in expression of hallmark proinflammatory or negative feedback loop genes. In addition, although PRV-induced NF-B activation shares some mechanistic features with canonical NF-B activation, it also shows remarkable differences; e.g., it is largely independent of the canonical IB kinase (IKK) and even renders infected cells resistant to canonical NF-B activation by the inflammatory cytokine TNF-α. Aberrant PRV-induced NF-B activation may therefore paradoxically serve as a viral immune evasion strategy and may represent an important tool to unravel currently unknown mechanisms and consequences of NF-B activation.

The rabbit hemorrhagic disease virus (RHDV), which belongs to the family Caliciviridae and the genus Lagovirus, causes lethal fulminant hepatitis in rabbits. RHDV decreases the activity of antioxidant enzymes regulated by Nrf2 in the liver. Antioxidants are important for the maintenance of cellular integrity and cytoprotection. However, the mechanism underlying the regulation of the Nrf2-antioxidant response element (ARE) signaling pathway by RHDV remains unclear. Using isobaric tags for relative and absolute quantification (iTRAQ) technology, the current study demonstrated that RHDV inhibits the induction of ARE-regulated genes and increases the expression of the p50 subunit of the NF-B transcription factor. We showed that RHDV replication causes a remarkable increase in reactive oxygen species (ROS), which is simultaneously accompanied by a significant decrease in Nrf2. It was found that nuclear translocation of Keap1 plays a key role in the nuclear export of Nrf2, leading to the inhibition of Nrf2 transcriptional activity. The p50 protein partners with Keap1 to form the Keap1-p50/p65 complex, which is involved in the nuclear translocation of Keap1. Moreover, upregulation of Nrf2 protein levels in liver cell nuclei by tert-butylhydroquinone (tBHQ) delayed rabbit deaths due to RHDV infection. Considered together, our findings suggest that RHDV inhibits the Nrf2-dependent antioxidant response via nuclear translocation of Keap1-NF-B complex and nuclear export of Nrf2 and provide new insight into the importance of oxidative stress during RHDV infection.

IMPORTANCE Recent studies have reported that rabbit hemorrhagic disease virus (RHDV) infection reduced Nrf2-related antioxidant function. However, the regulatory mechanisms underlying this process remain unclear. The current study showed that the NF-B p50 subunit partners with Keap1 to form the Keap1-NF-B complex, which plays a key role in the inhibition of Nrf2 transcriptional activity. More importantly, upregulated Nrf2 activity delayed the death of RHDV-infected rabbits, strongly indicating the importance of oxidative damage during RHDV infection. These findings may provide novel insights into the pathogenesis of RHDV.

IMMUNOLOGY AND INFLAMMATION Correction for “Treg-mediated prolonged survival of skin allografts without immunosuppression,” by Nina Pilat, Mario Wiletel, Anna M. Weijler, Romy Steiner, Benedikt Mahr, Joanna Warren, Theresa M. Corpuz, Thomas Wekerle, Kylie E. Webster, and Jonathan Sprent, which was first published June 13, 2019; 10.1073/pnas.1903165116 (Proc. Natl. Acad. Sci....

Isocitrate dehydrogenase (IDH) mutation is a common genetic abnormality in human malignancies characterized by remarkable metabolic reprogramming. Our present study demonstrated that IDH1-mutated cells showed elevated levels of reactive oxygen species and higher demands on Nrf2-guided glutathione de novo synthesis. Our findings showed that triptolide, a diterpenoid epoxide from Tripterygium...

Active matter, both synthetic and biological, demonstrates complex spatiotemporal self-organization and the emergence of collective behavior. A coherent rotational motion, the vortex phase, is of great interest because of its ability to orchestrate well-organized motion of self-propelled particles over large distances. However, its generation without geometrical confinement has been a...

A ubiquitous structural feature in biological systems is texture in extracellular matrix that gains functions when hardened, for example, cell walls, insect scales, and diatom tests. Here, we develop patterned liquid crystal elastomer (LCE) particles by recapitulating the biophysical patterning mechanism that forms pollen grain surfaces. In pollen grains, a...

Convection is buoyancy-driven flow resulting from unstable density stratification in the presence of a gravitational field. Beyond convection’s central role in myriad engineering heat transfer applications, it underlies many of nature’s dynamical designs on larger-than-human scales. For example, solar heating of Earth’s surface generates buoyancy forces that cause the winds...

Background:

Facilitation is an effective approach for helping practices implement sustainable evidence-based practice improvements. Few studies examine the facilitation infrastructure and support needed for large-scale dissemination and implementation initiatives.

Introduction:

Understanding patients’ perspectives about their diabetes and what causes those perspectives to shift is critical to building a treatment strategy with the patient and facilitating patient self-management behavior. Key "turning points" can provide crucial opportunities to enact a change in perspective. The goal of this study is to identify "turning points" that have significance to diabetes-related health.

Zhi-Jun Zhao, Davina Derous, Abby Gerrard, Jing Wen, Xue Liu, Song Tan, Catherine Hambly, and John R. Speakman

Lactating mice increase food intake 4- to 5-fold, reaching an asymptote in late lactation. A key question is whether this asymptote reflects a physiological constraint, or a maternal investment strategy (a ‘restraint’). We exposed lactating mice to periods of food restriction, hypothesizing that if the limit reflected restraint, they would compensate by breaching the asymptote when refeeding. In contrast, if it was a constraint, they would by definition be unable to increase their intake on refeeding days. Using isotope methods, we found that during food restriction, the females shut down milk production, impacting offspring growth. During refeeding, food intake and milk production rose again, but not significantly above unrestricted controls. These data provide strong evidence that asymptotic intake in lactation reflects a physiological/physical constraint, rather than restraint. Because hypothalamic neuropeptide Y (Npy) was upregulated under both states of restriction, this suggests the constraint is not imposed by limits in the capacity to upregulate hunger signalling (the saturated neural capacity hypothesis). Understanding the genetic basis of the constraint will be a key future goal and will provide us additional information on the nature of the constraining factors on reproductive output, and their potential links to life history strategies.

Wael Salem, Benjamin Cellini, Mark A. Frye, and Jean-Michel Mongeau

Most animals shift gaze by a ‘fixate and saccade’ strategy, where the fixation phase stabilizes background motion. A logical prerequisite for robust detection and tracking of moving foreground objects, therefore, is to suppress the perception of background motion. In a virtual reality magnetic tether system enabling free yaw movement, Drosophila implemented a fixate and saccade strategy in the presence of a static panorama. When the spatial wavelength of a vertical grating was below the Nyquist wavelength of the compound eyes, flies drifted continuously­ and gaze could not be maintained at a single location. Because the drift occurs from a motionless stimulus—thus any perceived motion stimuli are generated by the fly itself—it is illusory, driven by perceptual aliasing. Notably, the drift speed was significantly faster than under a uniform panorama suggesting perceptual enhancement due to aliasing. Under the same visual conditions in a rigid tether paradigm, wing steering responses to the unresolvable static panorama were not distinguishable from a resolvable static pattern, suggesting visual aliasing is induced by ego motion. We hypothesized that obstructing the control of gaze fixation also disrupts detection and tracking of objects. Using the illusory motion stimulus, we show that magnetically tethered Drosophila track objects robustly in flight even when gaze is not fixated as flies continuously drift. Taken together, our study provides further support for parallel visual motion processing and reveals the critical influence of body motion on visuomotor processing. Motion illusions can reveal important shared principles of information processing across taxa.

David E. Cade, J. Jacob Levenson, Robert Cooper, Rafael de la Parra, D. Harry Webb, and Alistair D. M. Dove

Whale sharks (Rhincodon typus Smith 1828) – the largest extant fish species – reside in tropical environments, making them an exception to the general rule that animal size increases with latitude. How this largest fish thrives in tropical environments that promote high metabolism but support less robust zooplankton communities has not been sufficiently explained. We used open-source inertial measurement units (IMU) to log 397 hours of whale shark behavior in Yucatan, Mexico, at a site of both active feeding and intense wildlife tourism. Here we show that the strategies employed by whale sharks to compensate for the increased drag of an open mouth are similar to ram-feeders five orders of magnitude smaller and one order of magnitude larger. Presumed feeding constituted 20% of the total time budget of four sharks, with individual feeding bouts lasting up to 11 consecutive hrs. Compared to normal, sub-surface swimming, three sharks increased their stroke rate and amplitude while surface feeding, while one shark that fed at depth did not demonstrate a greatly increased energetic cost. Additionally, based on time-depth budgets, we estimate that aerial surveys of shark populations should consider including a correction factor of 3 to account for the proportion of daylight hours that sharks are not visible at the surface. With foraging bouts generally lasting several hours, interruptions to foraging during critical feeding periods may represent substantial energetic costs to these endangered species, and this study presents baseline data from which management decisions affecting tourist interactions with whale sharks may be made.

Brad A. Seibel and Curtis Deutsch

The capacity to extract oxygen from the environment and transport it to respiring tissues in support of metabolic demand reportedly has implications for species’ thermal tolerance, body-size, diversity and biogeography. Here we derive a quantifiable linkage between maximum and basal metabolic rate and their oxygen, temperature and size dependencies. We show that, regardless of size or temperature, the physiological capacity for oxygen supply precisely matches the maximum evolved demand at the highest persistently available oxygen pressure and this is the critical PO₂ for the maximum metabolic rate. For most terrestrial and shallow-living marine species, this "P_crit-max" is the current atmospheric pressure, 21 kPa. Any reduction in oxygen partial pressure from current values will result in a calculable decrement in maximum metabolic performance. However, oxygen supply capacity has evolved to match demand across temperatures and body sizes and so does not constrain thermal tolerance or cause the well-known reduction in mass-specific metabolic rate with increasing body mass. The critical oxygen pressure for resting metabolic rate, typically viewed as an indicator of hypoxia tolerance, is, instead, simply a rate-specific reflection of the oxygen supply capacity. A compensatory reduction in maintenance metabolic costs in warm-adapted species constrains factorial aerobic scope and the critical PO₂ to a similar range, between ~2 and 6, across each species’ natural temperature range. The simple new relationship described here redefines many important physiological concepts and alters their ecological interpretation.

Respiratory support of the critically ill neonate has steadily shifted from invasive to noninvasive forms of support. There have recently been a number of important advances in our understanding of the changes to neonatal resuscitation practices as they pertain to clinically important outcomes, mechanisms of gas exchange for high-flow nasal cannula, and best use of noninvasive ventilation and predicting response. Although the proportion of infants requiring intubation and mechanical ventilation has decreased, the most severely ill often still require intubation and ventilation. Recently, volume-targeted ventilation, high-frequency ventilation, and different methods of assessing weaning and extubation have been investigated. This review summarizes a number of important advances that have been made in the management of prematurity and neonatal respiratory distress syndrome.

Arrestin-1 is the arrestin family member responsible for inactivation of the G protein–coupled receptor rhodopsin in photoreceptors. Arrestin-1 is also well-known to interact with additional protein partners and to affect other signaling cascades beyond phototransduction. In this study, we investigated one of these alternative arrestin-1 binding partners, the glycolysis enzyme enolase-1, to map the molecular contact sites between these two proteins and investigate how the binding of arrestin-1 affects the catalytic activity of enolase-1. Using fluorescence quench protection of strategically placed fluorophores on the arrestin-1 surface, we observed that arrestin-1 primarily engages enolase-1 along a surface that is opposite of the side of arrestin-1 that binds photoactivated rhodopsin. Using this information, we developed a molecular model of the arrestin-1–enolase-1 complex, which was validated by targeted substitutions of charge-pair interactions. Finally, we identified the likely source of arrestin's modulation of enolase-1 catalysis, showing that selective substitution of two amino acids in arrestin-1 can completely remove its effect on enolase-1 activity while still remaining bound to enolase-1. These findings open up opportunities for examining the functional effects of arrestin-1 on enolase-1 activity in photoreceptors and their surrounding cells.

Site-specific recombinases, such as Cre, are a widely used tool for genetic lineage tracing in the fields of developmental biology, neural science, stem cell biology, and regenerative medicine. However, nonspecific cell labeling by some genetic Cre tools remains a technical limitation of this recombination system, which has resulted in data misinterpretation and led to many controversies in the scientific community. In the past decade, to enhance the specificity and precision of genetic targeting, researchers have used two or more orthogonal recombinases simultaneously for labeling cell lineages. Here, we review the history of cell-tracing strategies and then elaborate on the working principle and application of a recently developed dual genetic lineage-tracing approach for cell fate studies. We place an emphasis on discussing the technical strengths and caveats of different methods, with the goal to develop more specific and efficient tracing technologies for cell fate mapping. Our review also provides several examples for how to use different types of DNA recombinase–mediated lineage-tracing strategies to improve the resolution of the cell fate mapping in order to probe and explore cell fate–related biological phenomena in the life sciences.

Premature infants have a higher incidence of indirect hyperbilirubinemia than term infants. Management of neonatal indirect hyperbilirubinemia in late preterm and term neonates has been well addressed by recognized, consensus-based guidelines. However, the extension of these guidelines to the preterm population has been an area of uncertainty because of limited evidence. This leads to variation in clinical practice and lack of recognition of the spectrum of bilirubin-induced neurologic dysfunction (BIND) in this population. Preterm infants are metabolically immature and at higher risk for BIND at lower bilirubin levels than their term counterparts. Early use of phototherapy to eliminate BIND and minimize the need for exchange transfusion is the goal of treatment in premature neonates. Although considered relatively safe, phototherapy does have side effects, and some NICUs tend to overuse phototherapy. In this review, we describe the epidemiology and pathophysiology of BIND in preterm neonates, and discuss our approach to standardized management of indirect hyperbilirubinemia in the vulnerable preterm population. The proposed treatment charts suggest early use of phototherapy in preterm neonates with the aim of reducing exposure to high irradiance levels, minimizing the need for exchange transfusions, and preventing BIND. The charts are pragmatic and have additional curves for stopping phototherapy and escalating its intensity. Having a standardized approach would support future research and quality improvement initiatives that examine dose and duration of phototherapy exposure with relation to outcomes.

The authors present results of the first high-resolution deep seismic reflection survey in the Pieniny Klippen Belt (PKB) in Poland. This survey sheds new light on the matter of olistostromes and the mélange character of the PKB. The sedimentary mass-transport deposits represented by olistoliths and olistostromes manifest themselves by different petrophysical parameters of rocks (velocity, density and resistivity) and seismic attributes. Seismic attributes are very effective in the interpretation of the geology of complex mélanges. The authors used selected attributes: low-pass filter, energy, energy gradient, dip-steered median filter, Prewitt filter, Laplacian edge enhancing filter and square root of the energy gradient. These attributes emphasize changes of the seismic image inside mélange zones. The distinguished olistoliths are now inside imbricated thrust structures and they are tectonically rearranged. Polygenetic mélanges in the PKB originated as a result of sedimentary and tectonic processes. The PKB in the investigated area forms several north-vergent thrust sheets belonging to the Złatne and Hulina nappes. Both nappes contain large chaotic, non-reflective olistoliths as well as the smaller mainly high-reflective olistoliths. Olistoliths are arranged parallel to the flysch layering and thrusts. The results presented confirm the postulated two olistostrome belts within the PKB structure.

Thematic collection: This article is part of the Polygenetic mélanges collection available at: https://www.lyellcollection.org/cc/polygenetic-melanges

Select agents (SA) pose unique challenges for licensing vaccines and therapies. In the case of toxin-mediated diseases, HHS assigns guidelines for SA use, oversees vaccine and therapy development, and approves animal models and approaches to identify mechanisms for toxin neutralization. In this commentary, we discuss next-generation vaccines and therapies against ricin toxin and botulinum toxin, which are regulated SA toxins that utilize structure-based approaches for countermeasures to guide rapid response to future biothreats.

The presence of antinuclear antibodies (ANAs) is a hallmark of a number of systemic autoimmune rheumatic diseases, and testing is usually performed as part of the initial diagnostic workup when suspicion of an underlying autoimmune disorder is high. The indirect immunofluorescence antibody (IFA) technique is the preferred method for detecting ANAs, as it demonstrates binding to specific intracellular structures within the cells, resulting in a number of staining patterns that are usually categorized based on the cellular components recognized and the degree of binding, as reflected by the fluorescence intensity or titer. As a screening tool, the ANA patterns can guide confirmatory testing useful in elucidating a specific clinical diagnosis or prognosis. However, routine use of ANA IFA testing as a global screening test is hampered by its labor-intensiveness, subjectivity, and limited diagnostic specificity, among other factors. This review focuses on current efforts to standardize the nomenclature of ANA patterns and on alternative methods for ANA determination, as well as on recent advances in image-based computer algorithms to automate IFA testing in clinical laboratories.

RiVax is a promising recombinant ricin toxin A subunit (RTA) vaccine antigen that has been shown to be safe and immunogenic in humans and effective at protecting rhesus macaques against lethal-dose aerosolized toxin exposure. We previously used a panel of RTA-specific monoclonal antibodies (MAbs) to demonstrate, by competition enzyme-linked immunosorbent assay (ELISA), that RiVax elicits similar serum antibody profiles in humans and macaques. However, the MAb binding sites on RiVax have yet to be defined. In this study, we employed hydrogen exchange-mass spectrometry (HX-MS) to localize the epitopes on RiVax recognized by nine toxin-neutralizing MAbs and one nonneutralizing MAb. Based on strong protection from hydrogen exchange, the nine MAbs grouped into four spatially distinct epitope clusters (namely, clusters I to IV). Cluster I MAbs protected RiVax's α-helix B (residues 94 to 107), a protruding immunodominant secondary structure element known to be a target of potent toxin-neutralizing antibodies. Cluster II consisted of two subclusters located on the "back side" (relative to the active site pocket) of RiVax. One subcluster involved α-helix A (residues 14 to 24) and α-helices F-G (residues 184 to 207); the other encompassed β-strand d (residues 62 to 69) and parts of α-helices D-E (154 to 164) and the intervening loop. Cluster III involved α-helices C and G on the front side of RiVax, while cluster IV formed a sash from the front to back of RiVax, spanning strands b, c, and d (residues 35 to 59). Having a high-resolution B cell epitope map of RiVax will enable the development and optimization of competitive serum profiling assays to examine vaccine-induced antibody responses across species.

Objective

To study the immunomodulatory effect of dimethyl fumarate (DF) on granulocyte macrophage colony-stimulating factor (GM-CSF) production in CD4⁺ T cells in experimental autoimmune encephalomyelitis (EAE) and human peripheral blood mononuclear cells (PBMCs).

A logistic regression model and a bivariate statistical analysis were used in this paper to evaluate the groundwater recharge susceptibility. The approach is based on the assessment of the relationship involving groundwater recharge and parameters that influence this hydrological process. Surface parameters and aquifer-related parameters were evaluated as thematic map layers using ArcGIS. Then, a weighted-rating method was adopted to categorize each parameter's map. To assess the role of each parameter in the aquifer recharge, a logistic regression model and a bivariate statistical analysis were applied to the Guenniche phreatic aquifer (Tunisia). Models are explored to establish a map showing the aquifer recharge susceptibility. The code Modflow was used to simulate the consequence of the recharge. The recharge amount was introduced in the model and was tested to verify the recharge effect on the hydraulic head for the two models. The obtained results reveal that the recharge as mapped in the bivariate statistical model has a minor impact on the hydraulic head. Results of the logistic regression model are more significant as the hydraulic head is widely affected. This model provides good results in mapping the spatial distribution of the aquifer recharge susceptibility.

Mutations in X-linked methyl-CpG-binding protein 2 (MECP2) cause Rett syndrome (RTT). To identify functional pathways that could inform therapeutic entry points, we carried out a genetic screen for secondary mutations that improved phenotypes in Mecp2/Y mice after mutagenesis with N-ethyl-N-nitrosourea (ENU). Here, we report the isolation of 106 founder animals that show suppression of Mecp2-null traits from screening 3177 Mecp2/Y genomes. Whole-exome sequencing, genetic crosses, and association analysis identified 22 candidate genes. Additional lesions in these candidate genes or pathway components associate variant alleles with phenotypic improvement in 30 lines. A network analysis shows that 63% of the genes cluster into the functional categories of transcriptional repression, chromatin modification, or DNA repair, delineating a pathway relationship with MECP2. Many mutations lie in genes that modulate synaptic signaling or lipid homeostasis. Mutations in genes that function in the DNA damage response (DDR) also improve phenotypes in Mecp2/Y mice. Association analysis was successful in resolving combinatorial effects of multiple loci. One line, which carries a suppressor mutation in a gene required for cholesterol synthesis, Sqle, carries a second mutation in retinoblastoma binding protein 8, endonuclease (Rbbp8, also known as CtIP), which regulates a DDR choice in double-stranded break (DSB) repair. Cells from Mecp2/Y mice have increased DSBs, so this finding suggests that the balance between homology-directed repair and nonhomologous end joining is important for neuronal cells. In this and other lines, two suppressor mutations confer greater improvement than one alone, suggesting that combination therapies could be effective in RTT.

Capicua (CIC) is a transcriptional repressor that counteracts activation of genes in response to receptor tyrosine kinase (RTK)/Ras/ERK signaling. Following activation of RTK, ERK enters the nucleus and serine-phosphorylates CIC, releasing it from its targets to permit gene expression. We recently showed that ERK triggers ubiquitin-mediated degradation of CIC in glioblastoma (GBM). In this study, we examined whether another important downstream effector of RTK/EGFR, the non-RTK c-Src, affects CIC repressor function in GBM. We found that c-Src binds and tyrosine-phosphorylates CIC on residue 1455 to promote nuclear export of CIC. On the other hand, CIC-mutant allele (CIC-Y1455F), that escapes c-Src–mediated tyrosine phosphorylation, remains localized to the nucleus and retains strong repressor function against CIC targets, the oncogenic transcription factors ETV1 and ETV5. Furthermore, we show that the orally available Src family kinase inhibitor, dasatinib, which prevents EGF-mediated tyrosine phosphorylation of CIC and attenuates elevated ETV1 and ETV5 levels, reduces viability of GBM cells and glioma stem cells (GSC), but not of their control cells with undetectable c-Src activity. In fact, GBM cells and GSC expressing the tyrosine-defective CIC mutant (Y1455F) lose sensitivity to dasatinib, further endorsing the effect of dasatinib on Src-mediated tyrosine phosphorylation of CIC. These findings elucidate important mechanisms of CIC regulation and provide the rationale to target c-Src alongside ERK pathway inhibitors as a way to fully restore CIC tumor suppressor function in neoplasms such as GBM.

In recent years, there have been intensive debates on whether healthy adults acquire new word knowledge through fast mapping (FM) by a different mechanism from explicit encoding (EE). In this study, we focused on this issue and investigated to what extent retention interval, prior knowledge (PK), and lure type modulated memory after FM and EE. Healthy young participants were asked to learn novel word-picture associations through both FM and EE. Half of the pictures were from familiar categories (i.e., high PK) and the other half were from unfamiliar categories (i.e., low PK). After 10 min and 1 wk, the participants were tested by forced-choice (FC) tasks, with lures from different categories (Experiment 1) or from the same categories of the target pictures (Experiment 2). Pseudowords were used to denote names of the novel pictures and baseline performance was controlled for each task. The results showed that in both Experiments 1 and 2, memory performance remained stable after FM, while it declined after EE from 10 min to 1 wk. Moreover, the effect of PK appeared at 10 min after FM while at 1 wk after EE in Experiment 2. PK enhanced memory of word-picture associations when the lures were from the same categories (Experiment 2), rather than from different categories (Experiment 1). These results were largely confirmed in Experiment 3 when encoding condition was manipulated as a between-subjects factor, while lure type as a within-subjects factor. The findings suggest that different from EE, FM facilitates rapid acquisition and consolidation of word-picture knowledge, and highlight that PK plays an important role in this process by enhancing access to detailed information.

Lisa Müller, Katrin Rietscher, Rene Keil, Marvin Neuholz, and Mechthild Hatzfeld

Desmosome remodeling is crucial for epidermal regeneration, differentiation and wound healing. It is mediated by adapting the composition, and by post-translational modifications, of constituent proteins. We have previously demonstrated in mouse suprabasal keratinocytes that plakophilin (PKP) 1 mediates strong adhesion, which is negatively regulated by insulin-like growth factor 1 (IGF1) signaling. The importance of PKP3 for epidermal adhesion is incompletely understood. Here, we identify a major role of epidermal growth factor (EGF), but not IGF1, signaling in PKP3 recruitment to the plasma membrane to facilitate desmosome assembly. We find that ribosomal S6 kinases (RSKs) associate with and phosphorylate PKP3, which promotes PKP3 association with desmosomes downstream of the EGF receptor. Knockdown of RSKs as well as mutation of an RSK phosphorylation site in PKP3 interfered with desmosome formation, maturation and adhesion. Our findings implicate a coordinate action of distinct growth factors in the control of adhesive properties of desmosomes through modulation of PKPs in a context-dependent manner.

Metabolism and development must be closely coupled to meet the changing physiological needs of each stage in the life cycle. The molecular mechanisms that link these pathways, however, remain poorly understood. Here we show that the Drosophila estrogen-related receptor (dERR) directs a transcriptional switch in mid-pupae that promotes glucose oxidation and lipogenesis in young adults. dERR mutant adults are viable but display reduced locomotor activity, susceptibility to starvation, elevated glucose, and an almost complete lack of stored triglycerides. Molecular profiling by RNA-seq, ChIP-seq, and metabolomics revealed that glycolytic and pentose phosphate pathway genes are induced by dERR, and their reduced expression in mutants is accompanied by elevated glycolytic intermediates, reduced TCA cycle intermediates, and reduced levels of long chain fatty acids. Unexpectedly, we found that the central pathways of energy metabolism, including glycolysis, the tricarboxylic acid cycle, and electron transport chain, are coordinately induced at the transcriptional level in mid-pupae and maintained into adulthood, and this response is partially dependent on dERR, leading to the metabolic defects observed in mutants. Our data support the model that dERR contributes to a transcriptional switch during pupal development that establishes the metabolic state of the adult fly.

Autophagy captures intracellular components and delivers them to lysosomes for degradation and recycling. Conditional autophagy deficiency in adult mice causes liver damage, shortens life span to 3 mo due to neurodegeneration, and is lethal upon fasting. As autophagy deficiency causes p53 induction and cell death in neurons, we sought to test whether p53 mediates the lethal consequences of autophagy deficiency. Here, we conditionally deleted Trp53 (p53 hereafter) and/or the essential autophagy gene Atg7 throughout adult mice. Compared with Atg7^/ mice, the life span of Atg7^/p53^/ mice was extended due to delayed neurodegeneration and resistance to death upon fasting. Atg7 also suppressed apoptosis induced by p53 activator Nutlin-3, suggesting that autophagy inhibited p53 activation. To test whether increased oxidative stress in Atg7^/ mice was responsible for p53 activation, Atg7 was deleted in the presence or absence of the master regulator of antioxidant defense nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2^–/–Atg7^/ mice died rapidly due to small intestine damage, which was not rescued by p53 codeletion. Thus, Atg7 limits p53 activation and p53-mediated neurodegeneration. In turn, NRF2 mitigates lethal intestine degeneration upon autophagy loss. These findings illustrate the tissue-specific roles for autophagy and functional dependencies on the p53 and NRF2 stress response mechanisms.

A multidisciplinary endocrinologist-led shared medical appointment (SMA) model showed statistically significant reductions in A1C from baseline over 3 years that were not significantly different from appointments with endocrinologists or primary care providers alone within a resource-poor population. Similarly, the SMA model achieved clinical outcomes on par with endocrinologist-only visits with the added benefit of improving endocrine provider productivity and specialty access for patients. Greater patient engagement with the SMA model was associated with significantly lower A1C.