Myriam Franzke, Christian Kraus, David Dreyer, Keram Pfeiffer, M. Jerome Beetz, Anna L. Stöckl, James J. Foster, Eric J. Warrant, and Basil el Jundi

Monarch butterflies (Danaus plexippus) are prominent for their annual long-distance migration from North America to their overwintering area in Central Mexico. To find their way on this long journey, they use a sun compass as their main orientation reference but will also adjust their migratory direction with respect to mountain ranges. This indicates that the migratory butterflies also attend to the panorama to guide their travels. While the compass has been studied in detail in migrating butterflies, little is known about the orientation abilities of non-migrating butterflies. Here we studied if non-migrating butterflies - that stay in a more restricted area to feed and breed - also use a similar compass system to guide their flights. Performing behavioral experiments on tethered flying butterflies in an indoor LED flight simulator, we found that the monarchs fly along straight tracks with respect to a simulated sun. When a panoramic skyline was presented as the only orientation cue, the butterflies maintained their flight direction only during short sequences suggesting that they potentially use it for flight stabilization. We further found that when we presented the two cues together, the butterflies incorporate both cues in their compass. Taken together, we here show that non-migrating monarch butterflies can combine multiple visual cues for robust orientation, an ability that may also aid them during their migration.

Emily J. Lombardi, Candice L. Bywater, and Craig R. White

The Oxygen and Capacity-Limited Thermal Tolerance (OCLTT) hypothesis proposes that the thermal tolerance of an animal is shaped by its capacity to deliver oxygen in relation to oxygen demand. Studies testing this hypothesis have largely focused on measuring short-term performance responses in animals under acute exposure to critical thermal maximums. The OCLTT hypothesis, however, emphasises the importance of sustained animal performance over acute tolerance. The present study tested the effect of chronic hypoxia and hyperoxia during development on medium to long-term performance indicators at temperatures spanning the optimal temperature for growth in the speckled cockroach, Nauphoeta cinerea. In contrast to the predictions of the OCLTT hypothesis, development under hypoxia did not significantly reduce growth rate or running performance, and development under hyperoxia did not significantly increase growth rate or running performance. The effect of developmental temperature and oxygen on tracheal morphology and metabolic rate were also not consistent with OCLTT predictions, suggesting that oxygen delivery capacity is not the primary driver shaping thermal tolerance in this species. Collectively, these findings suggest that the OCLTT hypothesis does not explain moderate-to-long-term thermal performance in Nauphoeta cinerea, which raises further questions about the generality of the hypothesis.

Itsumi Nakamura, Rui Matsumoto, and Katsufumi Sato

It is generally assumed that the body temperature of large animals is less likely to change due to their large body size, resulting in a high thermal inertia and a smaller surface area to volume ratio. The goal of this study was to investigate the stability of body temperature in large fish using data from field experiments. We measured the muscle temperatures of free-ranging whale sharks (Rhincodon typus), the largest extant fish globally, and investigated their ectothermic physiology and the stability of their body temperatures. The measured muscle temperature of the whale sharks changed substantially more slowly than the water temperature fluctuations associated with vertical movements, and the whole-body heat-transfer coefficients (HTC) of whale sharks estimated using heat-budget models were lower than those of any other fish species measured to date. The heat-budget models also showed that internal heat production does not contribute to changes in muscle temperature. A comparative analysis showed that the HTC at cooling in various fish species including both ectothermic and endothermic species ranging from 10^–4 to 10³ kg was proportional to body mass^–0.63. This allometry was present regardless of whether the fish were ectothermic or endothermic, and was an extension of the relationship observed in previous studies on small fish. Thus, large fish have the advantage of body temperature stability while moving in environments with large temperature variations. Our results suggest that the large body size of whale sharks aids in preventing a decrease in body temperature during deep excursions to more than 1000 m depths without high metabolic costs of producing heat.

David J. Pritchard and Mario Vallejo-Marin

Vibrations play an important role in insect behaviour. In bees, vibrations are used in a variety of contexts including communication, as a warning signal to deter predators and during pollen foraging. However, little is known about how the biomechanical properties of bee vibrations vary across multiple behaviours within a species. In this study, we compared the properties of vibrations produced by Bombus terrestris audax (Hymenoptera: Apidae) workers in three contexts: during flight, during defensive buzzing, and in floral vibrations produced during pollen foraging on two buzz-pollinated plants (Solanum, Solanaceae). Using laser vibrometry, we were able to obtain contactless measures of both the frequency and amplitude of the thoracic vibrations of bees across the three behaviours. Despite all three types of vibrations being produced by the same power flight muscles, we found clear differences in the mechanical properties of the vibrations produced in different contexts. Both floral and defensive buzzes had higher frequency and amplitude velocity, acceleration, and displacement than the vibrations produced during flight. Floral vibrations had the highest frequency, amplitude velocity and acceleration of all the behaviours studied. Vibration amplitude, and in particular acceleration, of floral vibrations has been suggested as the key property for removing pollen from buzz-pollinated anthers. By increasing frequency and amplitude velocity and acceleration of their vibrations during vibratory pollen collection, foraging bees may be able to maximise pollen removal from flowers, although their foraging decisions are likely to be influenced by the presumably high cost of producing floral vibrations.

Nadia Vicenzi, Alejandro Laspiur, Paola L. Sassi, Ruben Massarelli, John Krenz, and Nora R. Ibargüengoytia

In ectotherms, temperature exerts a strong influence on the performance of physiological and ecological traits. One approach to understand the impact of rising temperatures on animals and their ability to cope with climate change is to quantify variation in thermal-sensitive traits. Here, we examined the thermal biology, the temperature dependence and the thermal plasticity of bite force (endurance and magnitude) in Diplolaemus leopardinus, an aggressive and territorial lizard, endemic to Mendoza province, Argentina. Our results indicated that this lizard behaves like a moderate thermoregulator which uses the rocks of its environment as the main heat source. Bite endurance was not influenced by head morphometry and body temperature, whereas bite force was influenced by head length and jaw length, and exhibited thermal dependence. Before thermal acclimation treatments, the maximum bite force for D. leopardinus occured at the lowest body temperature and fell sharply with increasing body temperature. After acclimation treatments, lizards acclimated at higher temperatures exhibited greater bite force. Bite force showed phenotypic plasticity, which reveals that leopard iguanas are able to maintain (and even improve) their bite force under a rising-temperature scenario.

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.

Jack Nguyen and Meir M. Barak

Cortical bone remodeling is an ongoing process triggered by microdamage, where osteoclasts resorb existing bone and osteoblasts deposit new bone in the form of secondary osteons (Haversian systems). Previous studies revealed regional variance in Haversian systems structure and possibly material, between opposite cortices of the same bone. As bone mechanical properties depend on tissue structure and material, it is predicted that bone mechanical properties will vary in accordance with structural and material regional heterogeneity. To test this hypothesis, we analyzed the structure, mineral content and compressive stiffness of secondary bone from the cranial and caudal cortices of the white-tailed deer proximal humerus. We found significantly larger Haversian systems and canals in the cranial cortex but no significant difference in mineral content between the two cortices. Accordingly, we found no difference in compressive stiffness between the two cortices and thus our working hypothesis was rejected. Seeing that the deer humerus is curved and thus likely subjected to bending during habitual locomotion, we expect that similar to other curved long bones, the cranial cortex of the deer humerus is likely subjected primarily to tensile strains and the caudal cortex is likely subject primarily to compressive strains. Consequently, our results suggest that strain magnitude (larger in compression) and sign (compression vs. tension) affect differently the osteoclasts and osteoblasts in the BMU. Our results further suggest that osteoclasts are inhibited in regions of high compressive strains (creating smaller Haversian systems) while osteoblasts’ osteoid deposition and mineralization is not affected by strain magnitude and sign.

Flemming Dahlke, Magnus Lucassen, Ulf Bickmeyer, Sylke Wohlrab, Velmurugu Puvanendran, Atle Mortensen, Melissa Chierici, Hans-Otto Pörtner, and Daniela Storch

The vulnerability of fish embryos and larvae to environmental factors is often attributed to a lack of adult-like organ systems (gills) and thus insufficient homeostatic capacity. However, experimental data supporting this hypothesis are scarce. Here, by using Atlantic cod (Gadus morhua) as a model, the relationship between embryo vulnerability (to projected ocean acidification and warming) and homeostatic capacity was explored through parallel analyses of stage-specific mortality and in vitro activity and expression of major ion pumps (ATP-Synthase, Na⁺/K⁺-ATPase, H⁺-ATPase) and co-transporters (NBC1, NKCC1). Immunolocalization of these transporters was used to study ionocyte morphology in newly-hatched larvae. Treatment-related embryo mortality until hatch (+20% due to acidification and warming) occurred primarily during an early period (gastrulation) characterized by extremely low ion transport capacities. Thereafter, embryo mortality decreased in parallel with an exponential increase in activity and expression of all investigated ion transporters. Significant changes in transporter activity and expression in response to acidification (+15% activity) and warming (-30% expression) indicate some potential for short-term acclimatization, although likely associated with energetic trade-offs. Interestingly, whole-larvae enzyme capacities (supported by abundant epidermal ionocytes) reached levels similar to those previously measured in gill tissue of adult cod, suggesting that early-life stages without functional gills are better equipped in terms of ion homeostasis than previously thought. This study implies that the gastrulation period represents a critical transition from inherited (maternal) defenses to active homeostatic regulation, which facilitates enhanced resilience of later stages to environmental factors.

Annette E. Allen, Joshua W. Mouland, Jessica Rodgers, Beatriz Bano-Otalora, Ronald H. Douglas, Glen Jeffery, Anthony A. Vugler, Timothy M. Brown, and Robert J. Lucas

An animal's temporal niche – the time of day at which it is active – is known to drive a variety of adaptations in the visual system. This includes variations in the topography, spectral sensitivity and density of retinal photoreceptors, and changes in the eye's gross anatomy and spectral transmission characteristics. We have characterised visual spectral sensitivity in the murid rodent Rhabdomys pumilio (‘the four-striped grass mouse’), which is the same family as (nocturnal) mice and rats, but exhibits a strong diurnal niche. As is common in diurnal species, the Rhabdomys lens acts as a long-pass spectral filter, providing limited transmission of light <400nm. Conversely, we found strong sequence homologies with the Rhabdomys SWS and MWS opsins and those of related nocturnal species (mice and rats) whose SWS opsins are maximally sensitive in the near UV. We continued to assess in vivo spectral sensitivity of cone vision using electroretinography and multi-channel recordings from the visual thalamus. These revealed that responses across the human visible range could be adequately described by those of a single pigment (assumed to be MWS opsin) maximally sensitive ~500nm, but that sensitivity in the near UV required inclusion of a second pigment whose peak sensitivity lay well into the UV range (_max <400nm, likely ~360nm). We therefore conclude that, despite the UV-filtering effects of the lens, the Rhabdomys retains an SWS pigment with a UV-A _max. In effect, this somewhat paradoxical combination of long-pass lens and UV-A _max results in narrow-band sensitivity for SWS cone pathways in the UV-A range.

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.

Maria Stager, Nathan R. Senner, Bret W. Tobalske, and Zachary A. Cheviron

Flexibility in heat generation and dissipation mechanisms provides endotherms the ability to match their thermoregulatory strategy with external demands. However, the degree to which these two mechanisms account for seasonal changes in body temperature regulation is little explored. Here we present novel data on the regulation of avian body temperature to investigate how birds alter mechanisms of heat production and heat conservation to deal with variation in ambient conditions. We subjected Dark-eyed Juncos (Junco hyemalis) to chronic cold acclimations of varying duration and subsequently quantified their metabolic rates, thermal conductance, and ability to maintain normothermia. Cold-acclimated birds adjusted traits related to both heat generation (increased summit metabolic rate) and heat conservation (decreased conductance) to improve their body temperature regulation. Increases in summit metabolic rate occurred rapidly, but plateaued after one week of cold exposure. In contrast, changes to conductance occurred only after nine weeks of cold exposure. Thus, the ability to maintain body temperature continued to improve throughout the experiment, but the mechanisms underlying this improvement changed through time. Our results demonstrate the ability of birds to adjust thermoregulatory strategies in response to thermal cues and reveal that birds may combine multiple responses to meet the specific demands of their environments.

Jessica M. Judson, Dawn M. Reding, and Anne M. Bronikowski

Immunosenescence is a well-known phenomenon in mammal systems, but its relevance in other long-lived vertebrates is less understood. Further, the influence of age and reproductive effort on immune function in long-lived species can be challenging to assess, as long-term data are scarce and it is often difficult to sample the oldest age classes. We used the painted turtle (Chrysemys picta) to test hypotheses of immunosenescence and a trade-off between reproductive output and immune function in a population of a long-lived vertebrate that has been monitored for over 30 years. These long-term data are utilized to employ a unique approach of aging turtles with mark-recapture data and population-specific growth modeling to obtain more accurate estimates of age. We analyzed natural antibodies, lysis ability, and bactericidal competence in 126 individuals from 1 to 33 years of age captured during May and June in 2011. Older turtles exhibited greater natural antibody levels than young individuals across sexes. Young females with large clutches exhibited greater lysis ability, while older females with large clutches had decreased lysis ability, suggesting a trade-off between reproductive output and immune function conditional upon age. However, bactericidal competence increased later in the nesting season for older females. Our study rejects the hypothesis of immunosenescence in a long-lived turtle, despite evidence of actuarial and reproductive senescence in this population. Additionally, we detected mixed evidence for a trade-off between reproduction and immune health.

Christine Elizabeth Cooper, Laura Leilani Hurley, Pierre Deviche, and Simon Charles Griffith

Desert birds inhabit hot, dry environments that are becoming hotter and drier as a consequence of climate change. Extreme weather such as heatwaves can cause mass-mortality events that may significantly impact populations and species. There are currently insufficient data concerning physiological plasticity to inform models of species’ response to extreme events and develop mitigation strategies. Consequently, we examine here the physiological plasticity of a small desert bird in response to hot (mean maximum ambient temperature=42.7°C) and cooler (mean maximum ambient temperature=31.4°C) periods during a single Austral summer. We measured body mass, metabolic rate, evaporative water loss, and body temperature, along with blood parameters (corticosterone, glucose, and uric acid) of wild zebra finches (Taeniopygia guttata; Gould 1837) to assess their physiological state and determine the mechanisms by which they respond to heatwaves. Hot days were not significant stressors; they did not result in modification of baseline blood parameters or an inability to maintain body mass, provided drinking water was available. During heatwaves, finches shifted their thermoneutral zone to higher temperatures. They reduced metabolic heat production, evaporative water loss and wet thermal conductance, and increased hyperthermia, especially when exposed to high ambient temperature. A consideration of the significant physiological plasticity that we have demonstrated to achieve more favourable heat and water balance is essential for effectively modelling and planning for the impacts of climate change on biodiversity.

In Arabidopsis, LTR retrotransposons are activated by mutations in the chromatin gene DECREASE in DNA METHYLATION 1 (DDM1), giving rise to 21- to 22-nt epigenetically activated siRNA (easiRNA) that depend on RNA DEPENDENT RNA POLYMERASE 6 (RDR6). We purified virus-like particles (VLPs) from ddm1 and ddm1rdr6 mutants in which genomic RNA is reverse transcribed into complementary DNA. High-throughput short-read and long-read sequencing of VLP DNA (VLP DNA-seq) revealed a comprehensive catalog of active LTR retrotransposons without the need for mapping transposition, as well as independent of genomic copy number. Linear replication intermediates of the functionally intact COPIA element EVADE revealed multiple central polypurine tracts (cPPTs), a feature shared with HIV in which cPPTs promote nuclear localization. For one member of the ATCOPIA52 subfamily (SISYPHUS), cPPT intermediates were not observed, but abundant circular DNA indicated transposon "suicide" by auto-integration within the VLP. easiRNA targeted EVADE genomic RNA, polysome association of GYPSY (ATHILA) subgenomic RNA, and transcription via histone H3 lysine-9 dimethylation. VLP DNA-seq provides a comprehensive landscape of LTR retrotransposons and their control at transcriptional, post-transcriptional, and reverse transcriptional levels.

The regulation of transposable element (TE) activity by small RNAs is a ubiquitous feature of germlines. However, despite the obvious benefits to the host in terms of ensuring the production of viable gametes and maintaining the integrity of the genomes they carry, it remains controversial whether TE regulation evolves adaptively. We examined the emergence and evolutionary dynamics of repressor alleles after P-elements invaded the Drosophila melanogaster genome in the mid-twentieth century. In many animals including Drosophila, repressor alleles are produced by transpositional insertions into piRNA clusters, genomic regions encoding the Piwi-interacting RNAs (piRNAs) that regulate TEs. We discovered that ~94% of recently collected isofemale lines in the Drosophila melanogaster Genetic Reference Panel (DGRP) contain at least one P-element insertion in a piRNA cluster, indicating that repressor alleles are produced by de novo insertion at an exceptional rate. Furthermore, in our sample of approximately 200 genomes, we uncovered no fewer than 80 unique P-element insertion alleles in at least 15 different piRNA clusters. Finally, we observe no footprint of positive selection on P-element insertions in piRNA clusters, suggesting that the rapid evolution of piRNA-mediated repression in D. melanogaster was driven primarily by mutation. Our results reveal for the first time how the unique genetic architecture of piRNA production, in which numerous piRNA clusters can encode regulatory small RNAs upon transpositional insertion, facilitates the nonadaptive rapid evolution of repression.

Recent progress has been made in identifying genomic regions implicated in trait evolution on a microevolutionary scale in many species, but whether these are relevant over macroevolutionary time remains unclear. Here, we directly address this fundamental question using bird beak shape, a key evolutionary innovation linked to patterns of resource use, divergence, and speciation, as a model trait. We integrate class-wide geometric-morphometric analyses with evolutionary sequence analyses of 10,322 protein-coding genes as well as 229,001 genomic regions spanning 72 species. We identify 1434 protein-coding genes and 39,806 noncoding regions for which molecular rates were significantly related to rates of bill shape evolution. We show that homologs of the identified protein-coding genes as well as genes in close proximity to the identified noncoding regions are involved in craniofacial embryo development in mammals. They are associated with embryonic stem cell pathways, including BMP and Wnt signaling, both of which have repeatedly been implicated in the morphological development of avian beaks. This suggests that identifying genotype-phenotype association on a genome-wide scale over macroevolutionary time is feasible. Although the coding and noncoding gene sets are associated with similar pathways, the actual genes are highly distinct, with significantly reduced overlap between them and bill-related phenotype associations specific to noncoding loci. Evidence for signatures of recent diversifying selection on our identified noncoding loci in Darwin finch populations further suggests that regulatory rather than coding changes are major drivers of morphological diversification over macroevolutionary times.

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.

Genome-wide association studies have implicated thousands of noncoding variants across common human phenotypes. However, they cannot directly inform the cellular context in which disease-associated variants act. Here, we use open chromatin profiles from discrete mouse cell populations to address this challenge. We applied stratified linkage disequilibrium score regression and evaluated heritability enrichment in 64 genome-wide association studies, emphasizing schizophrenia. We provide evidence that mouse-derived human open chromatin profiles can serve as powerful proxies for difficult to obtain human cell populations, facilitating the illumination of common disease heritability enrichment across an array of human phenotypes. We demonstrate that signatures from discrete subpopulations of cortical excitatory and inhibitory neurons are significantly enriched for schizophrenia heritability with maximal enrichment in cortical layer V excitatory neurons. We also show that differences between schizophrenia and bipolar disorder are concentrated in excitatory neurons in cortical layers II-III, IV, and V, as well as the dentate gyrus. Finally, we leverage these data to fine-map variants in 177 schizophrenia loci nominating variants in 104/177. We integrate these data with transcription factor binding site, chromatin interaction, and validated enhancer data, placing variants in the cellular context where they may modulate risk.

Cohesin is a ring-shaped multiprotein complex that is crucial for 3D genome organization and transcriptional regulation during differentiation and development. It also confers sister chromatid cohesion and facilitates DNA damage repair. Besides its core subunits SMC3, SMC1A, and RAD21, cohesin in somatic cells contains one of two orthologous STAG subunits, STAG1 or STAG2. How these variable subunits affect the function of the cohesin complex is still unclear. STAG1- and STAG2-cohesin were initially proposed to organize cohesion at telomeres and centromeres, respectively. Here, we uncover redundant and specific roles of STAG1 and STAG2 in gene regulation and chromatin looping using HCT116 cells with an auxin-inducible degron (AID) tag fused to either STAG1 or STAG2. Following rapid depletion of either subunit, we perform high-resolution Hi-C, gene expression, and sequential ChIP studies to show that STAG1 and STAG2 do not co-occupy individual binding sites and have distinct ways by which they affect looping and gene expression. These findings are further supported by single-molecule localizations via direct stochastic optical reconstruction microscopy (dSTORM) super-resolution imaging. Since somatic and congenital mutations of the STAG subunits are associated with cancer (STAG2) and intellectual disability syndromes with congenital abnormalities (STAG1 and STAG2), we verified STAG1-/STAG2-dependencies using human neural stem cells, hence highlighting their importance in particular disease contexts.

In the olfactory bulb, a cAMP/PKA/CREB-dependent form of learning occurs in the first week of life that provides a unique mammalian model for defining the epigenetic role of this evolutionarily ancient plasticity cascade. Odor preference learning in the week-old rat pup is rapidly induced by a 10-min pairing of odor and stroking. Memory is demonstrable at 24 h, but not 48 h, posttraining. Using this paradigm, pups that showed peppermint preference 30 min posttraining were sacrificed 20 min later for laser microdissection of odor-encoding mitral cells. Controls were given odor only. Microarray analysis revealed that 13 nonprotein-coding mRNAs linked to mRNA translation and splicing and 11 protein-coding mRNAs linked to transcription differed with odor preference training. MicroRNA23b, a translation inhibitor of multiple plasticity-related mRNAs, was down-regulated. Protein-coding transcription was up-regulated for Sec23b, Clic2, Rpp14, Dcbld1, Magee2, Mstn, Fam229b, RGD1566265, and Mgst2. Gng12 and Srcg1 mRNAs were down-regulated. Increases in Sec23b, Clic2, and Dcbld1 proteins were confirmed in mitral cells in situ at the same time point following training. The protein-coding changes are consistent with extracellular matrix remodeling and ryanodine receptor involvement in odor preference learning. A role for CREB and AP1 as triggers of memory-related mRNA regulation is supported. The small number of gene changes identified in the mitral cell input/output link for 24 h memory will facilitate investigation of the nature, and reversibility, of changes supporting temporally restricted long-term memory.

Behavioral flexibility is important in a changing environment. Previous research suggests that systems consolidation, a long-term poststorage process that alters memory traces, may reduce behavioral flexibility. However, exactly how systems consolidation affects flexibility is unknown. Here, we tested how systems consolidation affects: (1) flexibility in response to value changes and (2) flexibility in response to changes in the optimal sequence of actions. Mice were trained to obtain food rewards in a Y-maze by switching nose pokes between three arms. During initial training, all arms were rewarded and mice simply had to switch arms in order to maximize rewards. Then, after either a 1 or 28 d delay, we either devalued one arm, or we reinforced a specific sequence of pokes. We found that after a 1 d delay mice adapted relatively easily to the changes. In contrast, mice given a 28 d delay struggled to adapt, especially for changes to the optimal sequence of actions. Immediate early gene imaging suggested that the 28 d mice were less reliant on their hippocampus and more reliant on their medial prefrontal cortex. These data suggest that systems consolidation reduces behavioral flexibility, particularly for changes to the optimal sequence of actions.

In obsessive–compulsive disorder (OCD), functional behaviors such as checking that a door is locked become dysfunctional, maladaptive, and debilitating. However, it is currently unknown how aversive and appetitive motivations interact to produce functional and dysfunctional behavior in OCD. Here we show a double dissociation in the effects of anxiogenic cues and sensitivity to rewarding stimuli on the propensity to develop functional and dysfunctional checking behavior in a rodent analog of OCD, the observing response task (ORT). While anxiogenic manipulations of perceived threat (presentation of threat-associated contextual cues) and actual threat (punishment of incorrect responding on the ORT) enhanced functional checking, dysfunctional checking was unaffected. In contrast, rats that had previously been identified as "sign-trackers" on an autoshaping task—and therefore were highly sensitive to the incentive salience of appetitive environmental cues—selectively showed elevated levels of dysfunctional checking under a range of conditions, but particularly so under conditions of uncertainty. These data indicate that functional and dysfunctional checking are dissociable and supported by aversive and appetitive motivational processes, respectively. While functional checking is modulated by perceived and actual threat, dysfunctional checking recruits appetitive motivational processes, possibly akin to the "incentive habits" that contribute to drug-seeking in addiction.

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.

Xiaofei Cao, Sergio Lilla, Zhenbo Cao, Marie Anne Pringle, Ojore B. V. Oka, Philip J. Robinson, Tomasz Szmaja, Marcel van Lith, Sara Zanivan, and Neil J. Bulleid

Folding of proteins entering the mammalian secretory pathway requires the insertion of the correct disulfides. Disulfide formation involves both an oxidative pathway for their insertion and a reductive pathway to remove incorrectly formed disulfides. Reduction of these disulfides is crucial for correct folding and degradation of misfolded proteins. Previously, we showed that the reductive pathway is driven by NADPH generated in the cytosol. Here, by reconstituting the pathway using purified proteins and ER microsomal membranes, we demonstrate that the thioredoxin reductase system provides the minimal cytosolic components required for reducing proteins within the ER lumen. In particular, saturation of the pathway and its protease sensitivity demonstrates the requirement for a membrane protein to shuttle electrons from the cytosol to the ER. These results provide compelling evidence for the crucial role of the cytosol in regulating ER redox homeostasis, ensuring correct protein folding and facilitating the degradation of misfolded ER proteins.

Dalton Buysse, Anna-Katharina Pfitzner, Matt West, Aurelien Roux, and Greg Odorizzi

The ESCRT-III protein complex executes reverse-topology membrane scission. The scission mechanism is unclear but is linked to remodeling of ESCRT-III complexes at the membrane surface. At endosomes, ESCRT-III mediates the budding of intralumenal vesicles (ILVs). In Saccharomyces cerevisiae, ESCRT-III activity at endosomes is regulated through an unknown mechanism by Doa4, an ubiquitin hydrolase that deubiquitylates transmembrane proteins sorted into ILVs. We report that the non-catalytic N-terminus of Doa4 binds Snf7, the predominant ESCRT-III subunit. Through this interaction, Doa4 overexpression alters Snf7 assembly status and inhibits ILV membrane scission. In vitro, the Doa4 N-terminus inhibits association of Snf7 with Vps2, which functions with Vps24 to arrest Snf7 polymerization and remodel Snf7 polymer structure. In vivo, Doa4 overexpression inhibits Snf7 interaction with Vps2 and also with the ATPase Vps4, which is recruited by Vps2 and Vps24 to remodel ESCRT-III complexes by catalyzing subunit turnover. Our data suggest a mechanism by which the deubiquitylation machinery regulates ILV biogenesis by interfering with ESCRT-III remodeling.

Midori Ishii and Bungo Akiyoshi

The kinetochore is a macromolecular protein complex that drives chromosome segregation in eukaryotes. Unlike most eukaryotes that have canonical kinetochore proteins, evolutionarily divergent kinetoplastids, such as Trypanosoma brucei, have unconventional kinetochore proteins. T. brucei also lacks a canonical spindle checkpoint system, and it therefore remains unknown how mitotic progression is regulated in this organism. Here, we characterized, in the procyclic form of T. brucei, two paralogous kinetochore proteins with a CLK-like kinase domain, KKT10 and KKT19, which localize at kinetochores in metaphase but disappear at the onset of anaphase. We found that these proteins are functionally redundant. Double knockdown of KKT10 and KKT19 led to a significant delay in the metaphase to anaphase transition. We also found that phosphorylation of two kinetochore proteins, KKT4 and KKT7, depended on KKT10 and KKT19 in vivo. Finally, we showed that the N-terminal part of KKT7 directly interacts with KKT10 and that kinetochore localization of KKT10 depends not only on KKT7 but also on the KKT8 complex. Our results reveal that kinetochore localization of KKT10 and KKT19 is tightly controlled to regulate the metaphase to anaphase transition in T. brucei.

This article has an associated First Person interview with the first author of the paper.

Liling Niu, Fuyun Wu, Kaili Li, Jing Li, Shenyuan L. Zhang, Junjie Hu, and Qian Wang

Store-operated Ca²⁺ entry (SOCE) is critical for numerous Ca²⁺-related processes. The activation of SOCE requires engagement between stromal interaction molecule 1 (STIM1) molecules on the endoplasmic reticulum and Ca²⁺ release-activated channel (CRAC) Orai on the plasma membrane. However, the molecular details of their interactions remain elusive. Here, we analyzed STIM1-Orai interactions using synthetic peptides derived from the N- and C-termini of Orai channels (Orai-NT and Orai-CT, respectively) and purified fragments of STIM1. The binding of STIM1 to Orai-NT is hydrophilic based, whereas binding to the Orai-CT is mostly hydrophobic. STIM1 decreases its affinity for Orai-CT when Orai-NT is present, supporting a stepwise interaction. Orai3-CT exhibits stronger binding to STIM1 than Orai1-CT, largely due to the shortness of one helical turn. The role of newly identified residues was confirmed by co-immunoprecipitation and Ca²⁺ imaging using full-length molecules. Our results provide important insight into CRAC gating by STIM1.

Monica L. Salgado-Lucio, Danelia Ramirez-Ramirez, Coral Y. Jorge-Cruz, Ana L. Roa-Espitia, and Enrique O. Hernandez-Gonzalez

Actin polymerization is a crucial process during sperm capacitation. We have recently described the participation of FAK during actin polymerization in guinea pig spermatozoa. However, the mechanism by which FAK mediates these processes is unknown. Our previous data have shown that MAPK1 (hereafter referred to as ERK2) is activated during the first minutes of capacitation, and inhibition of ERK2 blocked actin polymerization and the acrosome reaction. In this current study, we found that FAK is involved in ERK2 activation – as FAK was phosphorylated at tyrosine residue 925 and bound to Grb2 – and that inhibition of FAK results in a significant decrease of ERK2 activation. We also confirmed the presence of Rho guanine nucleotide exchange factor 2 (ARHGEF2, hereafter referred to as GEF-H1), which is able to associate with RhoA during capacitation. RhoA activation and its participation in actin polymerization were also analyzed. Inhibition of FAK or ERK1/2 impeded GEF-H1 phosphorylation, RhoA activation, and the association between GEF-H1 and RhoA. Finally, we observed the presence of fibronectin on the sperm surface, its role in sperm–sperm interaction as well as participation of β-integrin in the activation of ERK2. Our results show that the signaling pathway downstream of fibronectin, via integrin, FAK, Grb2, MEK1/2, ERK2, GEF-H1 and RhoA regulates the actin polymerization associated with spermatozoa capacitation.

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.

Annelies Wouters, Jan-Pieter Ploem, Sabine A. S. Langie, Tom Artois, Aziz Aboobaker, and Karen Smeets

Pluripotent stem cells hold great potential for regenerative medicine. Increased replication and division, such is the case during regeneration, concomitantly increases the risk of adverse outcomes through the acquisition of mutations. Seeking for driving mechanisms of such outcomes, we challenged a pluripotent stem cell system during the tightly controlled regeneration process in the planarian Schmidtea mediterranea. Exposure to the genotoxic compound methyl methanesulfonate (MMS) revealed that despite a similar DNA-damaging effect along the anteroposterior axis of intact animals, responses differed between anterior and posterior fragments after amputation. Stem cell proliferation and differentiation proceeded successfully in the amputated heads, leading to regeneration of missing tissues. Stem cells in the amputated tails showed decreased proliferation and differentiation capacity. As a result, tails could not regenerate. Interference with the body-axis-associated component β-catenin-1 increased regenerative success in tail fragments by stimulating proliferation at an early time point. Our results suggest that differences in the Wnt signalling gradient along the body axis modulate stem cell responses to MMS.

Asja Guzman, Rachel C. Avard, Alexander J. Devanny, Oh Sang Kweon, and Laura J. Kaufman

The study of cancer cell invasion in 3D environments in vitro has revealed a variety of invasive modes, including amoeboid migration, characterized by primarily round cells that invade in a protease- and adhesion-independent manner. Here, we delineate a contractility-dependent migratory mode of primarily round breast cancer cells that is associated with extensive integrin-mediated extracellular matrix (ECM) reorganization that occurs at membrane blebs, with bleb necks sites of integrin clustering and integrin-dependent ECM alignment. We show that the spatiotemporal distribution of blebs and their utilization for ECM reorganization is mediated by functional β1 integrin receptors and other components of focal adhesions. Taken together, the work presented here characterizes a migratory mode of primarily round cancer cells in complex 3D environments and reveals a fundamentally new function for membrane blebs in cancer cell invasion.

Kristi McElmurry, Jessica E. Stone, Donghan Ma, Phillip Lamoureux, Yueyun Zhang, Michelle Steidemann, Lucas Fix, Fang Huang, Kyle E. Miller, and Daniel M. Suter

Previously, we have shown that bulk microtubule (MT) movement correlates with neurite elongation, and blocking either dynein activity or MT assembly inhibits both processes. However, whether the contributions of MT dynamics and dynein activity to neurite elongation are separate or interdependent is unclear. Here, we investigated the underlying mechanism by testing the roles of dynein and MT assembly in neurite elongation of Aplysia and chick neurites using time-lapse imaging, fluorescent speckle microscopy, super-resolution imaging and biophysical analysis. Pharmacologically inhibiting either dynein activity or MT assembly reduced neurite elongation rates as well as bulk and individual MT anterograde translocation. Simultaneously suppressing both processes did not have additive effects, suggesting a shared mechanism of action. Single-molecule switching nanoscopy revealed that inhibition of MT assembly decreased the association of dynein with MTs. Finally, inhibiting MT assembly prevented the rise in tension induced by dynein inhibition. Taken together, our results suggest that MT assembly is required for dynein-driven MT translocation and neurite outgrowth.

Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N⁶-methyladenosine (m⁶A) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes m⁶A in 18S rRNA at position A₁₈₃₂. We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in METTL5, thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of m⁶A in rRNA in stemness, differentiation, development, and diseases.

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.

The proteostasis network is regulated by transcellular communication to promote health and fitness in metazoans. In Caenorhabditis elegans, signals from the germline initiate the decline of proteostasis and repression of cell stress responses at reproductive maturity, indicating that commitment to reproduction is detrimental to somatic health. Here we show that proteostasis and stress resilience are also regulated by embryo-to-mother communication in reproductive adults. To identify genes that act directly in the reproductive system to regulate somatic proteostasis, we performed a tissue targeted genetic screen for germline modifiers of polyglutamine aggregation in muscle cells. We found that inhibiting the formation of the extracellular vitelline layer of the fertilized embryo inside the uterus suppresses aggregation, improves stress resilience in an HSF-1-dependent manner, and restores the heat-shock response in the somatic tissues of the parent. This pathway relies on DAF-16/FOXO activation in vulval tissues to maintain stress resilience in the mother, suggesting that the integrity of the embryo is monitored by the vulva to detect damage and initiate an organismal protective response. Our findings reveal a previously undescribed transcellular pathway that links the integrity of the developing progeny to proteostasis regulation in the parent.

Cell type-specific transcriptional programs that drive differentiation of specialized cell types are key players in development and tissue regeneration. One of the most dramatic changes in the transcription program in Drosophila occurs with the transition from proliferating spermatogonia to differentiating spermatocytes, with >3000 genes either newly expressed or expressed from new alternative promoters in spermatocytes. Here we show that opening of these promoters from their closed state in precursor cells requires function of the spermatocyte-specific tMAC complex, localized at the promoters. The spermatocyte-specific promoters lack the previously identified canonical core promoter elements except for the Inr. Instead, these promoters are enriched for the binding site for the TALE-class homeodomain transcription factors Achi/Vis and for a motif originally identified under tMAC ChIP-seq peaks. The tMAC motif resembles part of the previously identified 14-bp β2UE1 element critical for spermatocyte-specific expression. Analysis of downstream sequences relative to transcription start site usage suggested that ACA and CNAAATT motifs at specific positions can help promote efficient transcription initiation. Our results reveal how promoter-proximal sequence elements that recruit and are acted upon by cell type-specific chromatin binding complexes help establish a robust, cell type-specific transcription program for terminal differentiation.

Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural end of linear chromosomes. Most cells maintain telomere repeat lengths by using the enzyme telomerase, although there are some cancer cells that use a telomerase-independent mechanism of telomere extension, termed alternative lengthening of telomeres (ALT). Cells that use ALT are characterized, in part, by the presence of specialized PML nuclear bodies called ALT-associated PML bodies (APBs). APBs localize to and cluster telomeric ends together with telomeric and DNA damage factors, which led to the proposal that these bodies act as a platform on which ALT can occur. However, the necessity of APBs and their function in the ALT pathway has remained unclear. Here, we used CRISPR/Cas9 to delete PML and APB components from ALT-positive cells to cleanly define the function of APBs in ALT. We found that PML is required for the ALT mechanism, and that this necessity stems from APBs’ role in localizing the BLM–TOP3A–RMI (BTR) complex to ALT telomere ends. Strikingly, recruitment of the BTR complex to telomeres in a PML-independent manner bypasses the need for PML in the ALT pathway, suggesting that BTR localization to telomeres is sufficient to sustain ALT activity.

The emergence of drug resistance is a major obstacle for the success of targeted therapy in melanoma. Additionally, conventional chemotherapy has not been effective as drug-resistant cells escape lethal DNA damage effects by inducing growth arrest commonly referred to as cellular dormancy. We present a therapeutic strategy termed "targeted chemotherapy" by depleting protein phosphatase 2A (PP2A) or its inhibition using a small molecule inhibitor (1,10-phenanthroline-5,6-dione [phendione]) in drug-resistant melanoma. Targeted chemotherapy induces the DNA damage response without causing DNA breaks or allowing cellular dormancy. Phendione treatment reduces tumor growth of BRAF^V600E-driven melanoma patient-derived xenografts (PDX) and diminishes growth of NRAS^Q61R-driven melanoma, a cancer with no effective therapy. Remarkably, phendione treatment inhibits the acquisition of resistance to BRAF inhibition in BRAF^V600E PDX highlighting its effectiveness in combating the advent of drug resistance.

Peripheral somatosensory input is modulated in the dorsal spinal cord by a network of excitatory and inhibitory interneurons. PTF1A is a transcription factor essential in dorsal neural tube progenitors for specification of these inhibitory neurons. Thus, mechanisms regulating Ptf1a expression are key for generating neuronal circuits underlying somatosensory behaviors. Mutations targeted to distinct cis-regulatory elements for Ptf1a in mice, tested the in vivo contribution of each element individually and in combination. Mutations in an autoregulatory enhancer resulted in reduced levels of PTF1A, and reduced numbers of specific dorsal spinal cord inhibitory neurons, particularly those expressing Pdyn and Gal. Although these mutants survive postnatally, at ~3–5 wk they elicit a severe scratching phenotype. Behaviorally, the mutants have increased sensitivity to itch, but acute sensitivity to other sensory stimuli such as mechanical or thermal pain is unaffected. We demonstrate a requirement for positive transcriptional autoregulatory feedback to attain the level of the neuronal specification factor PTF1A necessary for generating correctly balanced neuronal circuits.

ABSTRACT

Members of family Coronaviridae cause a variety of diseases in birds and mammals. Porcine hemagglutinating encephalomyelitis virus (PHEV), a lesser-researched coronavirus, can infect naive pigs of any age, but clinical disease is observed in pigs ≤4 weeks of age. No commercial PHEV vaccines are available, and neonatal protection from PHEV-associated disease is presumably dependent on lactogenic immunity. Although subclinical PHEV infections are thought to be common, PHEV ecology in commercial swine herds is unknown. To begin to address this gap in knowledge, a serum IgG antibody enzyme-linked immunosorbent assay (ELISA) based on the S1 protein was developed and evaluated on known-status samples and then used to estimate PHEV seroprevalence in U.S. sow herds. Assessment of the diagnostic performance of the PHEV S1 ELISA using serum samples (n = 924) collected from 7-week-old pigs (n = 84; 12 pigs per group) inoculated with PHEV, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine respiratory coronavirus, or porcine deltacoronavirus showed that a sample-to-positive cutoff value of ≥0.6 was both sensitive and specific, i.e., all PHEV-inoculated pigs were seropositive from days postinoculation 10 to 42, and no cross-reactivity was observed in samples from other groups. The PHEV S1 ELISA was then used to estimate PHEV seroprevalence in U.S. sow herds (19 states) using 2,756 serum samples from breeding females (>28 weeks old) on commercial farms (n = 104) with no history of PHEV-associated disease. The overall seroprevalence was 53.35% (confidence interval [CI], ±1.86%) and herd seroprevalence was 96.15% (CI, ±3.70%).

IMPORTANCE There is a paucity of information concerning the ecology of porcine hemagglutinating encephalomyelitis virus (PHEV) in commercial swine herds. This study provided evidence that PHEV infection is endemic and highly prevalent in U.S. swine herds. These results raised questions for future studies regarding the impact of endemic PHEV on swine health and the mechanisms by which this virus circulates in endemically infected populations. Regardless, the availability of the validated PHEV S1 enzyme-linked immunosorbent assay (ELISA) provides the means for swine producers to detect and monitor PHEV infections, confirm prior exposure to the virus, and to evaluate the immune status of breeding herds.

Advances in small RNA sequencing have revealed the enormous diversity of small noncoding RNA (sRNA) classes in mammalian cells. At this point, most investigators in diabetes are aware of the success of microRNA (miRNA) research and appreciate the importance of posttranscriptional gene regulation in glycemic control. Nevertheless, miRNAs are just one of multiple classes of sRNAs and likely represent only a minor fraction of sRNA sequences in a given cell. Despite the widespread appreciation of sRNAs, very little research into non-miRNA sRNA function has been completed, likely due to some major barriers that present unique challenges for study. To emphasize the importance of sRNA research in cardiometabolic diseases, we highlight the success of miRNAs and competitive endogenous RNAs in cholesterol and glucose metabolism. Moreover, we argue that sequencing studies have demonstrated that miRNAs are just the tip of the iceberg for sRNAs. We are likely standing at the precipice of immense discovery for novel sRNA-mediated gene regulation in cardiometabolic diseases. To realize this potential, we must first address critical barriers with an open mind and refrain from viewing non-miRNA sRNA function through the lens of miRNAs, as they likely have their own set of distinct regulatory factors and functional mechanisms.

Nayeli G. Reyes-Nava, Hung-Chun Yu, Curtis R. Coughlin II, Tamim H. Shaikh, and Anita M. Quintana

We used whole-exome sequencing (WES) to determine the genetic etiology of a patient with a multi-system disorder characterized by a seizure phenotype. WES identified a heterozygous de novo missense mutation in the GABRA1 gene (c.875C>T). GABRA1 encodes the alpha subunit of the gamma-aminobutyric acid receptor A (GABA_AR). The GABA_AR is a ligand gated ion channel that mediates the fast inhibitory signals of the nervous system, and mutations in the subunits that compose the GABA_AR have been previously associated with human disease. To understand the mechanisms by which GABRA1 regulates brain development, we developed a zebrafish model of gabra1 deficiency. gabra1 expression is restricted to the nervous system and behavioral analysis of morpholino injected larvae suggests that the knockdown of gabra1 results in hypoactivity and defects in the expression of other subunits of the GABA_AR. Expression of the human GABRA1 protein in morphants partially restored the hypomotility phenotype. In contrast, the expression of the c.875C>T variant did not restore these behavioral deficits. Collectively, these results represent a functional approach to understand the mechanisms by which loss-of-function alleles cause disease.

Chalongrat Noree and Naraporn Sirinonthanawech

Previously, we have developed an extramitochondrial assembly system, where mitochondrial targeting signal (MTS) can be removed from a given mitochondrial enzyme, which could be used to characterize the regulatory factors involved in enzyme assembly/disassembly in vivo. Here, we demonstrate that addition of exogenous acetaldehyde can quickly induce the supramolecular assembly of MTS-deleted aldehyde dehydrogenase Ald4p in yeast cytoplasm. Also, by using PCR-based modification of the yeast genome, cytoplasmically targeted Ald4p cannot polymerize into long filaments when key functional amino acid residues are substituted, as shown by N192D, S269A, E290K and C324A mutations. This study has confirmed that extramitochondrial assembly could be a powerful external system for studying mitochondrial enzyme assembly, and its regulatory factors outside the mitochondria. In addition, we propose that mitochondrial enzyme assembly/disassembly is coupled to the regulation of a given mitochondrial enzyme activity.

Yuta Sakae, Akira Oikawa, Yuki Sugiura, Masatoshi Mita, Shuhei Nakamura, Toshiya Nishimura, Makoto Suematsu, and Minoru Tanaka

The teleost fish, medaka (Oryzias latipes), employs the XX/XY genetic sex determination system. We show here that the phenotypic sex of medaka is affected by changes in lipid metabolism. Medaka larvae subjected to 5 days of starvation underwent female-to-male sex reversal. Metabolomic and RT-qPCR analyses indicated that pantothenate metabolism was suppressed by starvation. Consistently, inhibiting the pantothenate metabolic pathway caused sex reversal. The final metabolite in this pathway is coenzyme A, an essential factor for lipogenesis. Inhibiting fatty acid synthesis, the first step of lipogenesis, also caused sex reversal. The expression of dmrt1, a critical gene for male development, was suppressed by starvation, and a dmrt1 (13) mutant did not show sex reversal under starvation. Collectively, these results indicate that fatty acid synthesis is involved in female-to-male sex reversal through ectopic expression of male gene dmrt1 under starvation.

Manoella Gemaque Cavalcante, Cleusa Yoshiko Nagamachi, Julio Cesar Pieczarka, and Renata Coelho Rodrigues Noronha

Eukaryotic genomes exhibit substantial accumulation of repetitive DNA sequences. These sequences can participate in chromosomal reorganization events and undergo molecular cooption to interfere with the function and evolution of genomes. In turtles, repetitive DNA sequences appear to be accumulated at probable break points and may participate in events such as non-homologous recombination and chromosomal rearrangements. In this study, repeated sequences of 5S rDNA, U2 snRNA and Tc1/Mariner transposons were amplified from the genomes of the turtles, Podocnemis expansa and Podocnemis unifilis, and mapped by fluorescence in situ hybridization. Our data confirm the 2n=28 chromosomes for these species (the second lowest 2n in the order Testudines). We observe high conservation of the co-located 5S rDNA and U2 snRNA genes on a small chromosome pair (pair 13), and surmise that this represents the ancestral condition. Our analysis reveals a wide distribution of the Tc1/Mariner transposons and we discuss how the mobility of these transposons can act on karyotypic reorganization events (contributing to the 2n decrease of those species). Our data add new information for the order Testudines and provide important insights into the dynamics and organization of these sequences in the chelonian genomes.

Yan Xing, Hongling Wei, Xiumei Xiao, Zekun Chen, Hui Liu, Xiaomei Tong, and Wei Zhou

Infants with intrauterine growth retardation (IUGR) have a high risk of developing bronchial asthma in childhood, but the underlying mechanisms remain unclear. This study aimed to disclose the role of vascular non-inflammatory molecule 1 (vannin-1, encoded by the Vnn1 gene) and its downstream signaling in IUGR asthmatic mice induced by ovalbumin. Significant histological alterations and an increase of vannin-1 expression were revealed in IUGR asthmatic mice, accompanied by elevated methylation of Vnn1 promoter regions. In IUGR asthmatic mice, we also found (i) a direct binding of HNF4α and PGC1α to Vnn1 promoter by ChIP assay; (ii) a direct interaction of HNF4α with PGC1α; (iii) upregulation of phospho-PI3K p85/p55 and phospho-Akt^Ser473 and downregulation of phospho-PTENT^yr366, and (iv) an increase in nuclear NFB p65 and a decrease in cytosolic IB-α. In primary cultured bronchial epithelial cells derived from the IUGR asthmatic mice, knockdown of Vnn1 prevented upregulation of phospho-Akt^Ser473 and an increase of reactive oxygen species (ROS) and TGF-β production. Taken together, we demonstrate that elevated vannin-1 activates the PI3K/Akt/NFB signaling pathway, leading to ROS and inflammation reactions responsible for asthma occurrence in IUGR individuals. We also disclose that interaction of PGC1α and HNF4α promotes methylation of Vnn1 promoter regions and then upregulates vannin-1 expression.

Yu Liu, Shuhua Qi, Fridtjof Thomas, Brittany L. Correia, Angela P. Taylor, Roy V. Sillitoe, and Detlef H. Heck

Respiration is controlled by central pattern generating circuits in the brain stem, whose activity can be modulated by inputs from other brain areas to adapt respiration to autonomic and behavioral demands. The cerebellum is known to be part of the neuronal circuitry activated during respiratory challenges, such as hunger for air, but has not been found to be involved in the control of spontaneous, unobstructed breathing (eupnea). Here we applied a measure of intrinsic rhythmicity, the CV2, which evaluates the similarity of subsequent intervals and is thus sensitive to changes in rhythmicity at the temporal resolution of individual respiratory intervals. The variability of intrinsic respiratory rhythmicity was reduced in a mouse model of cerebellar ataxia compared to their healthy littermates. Irrespective of that difference, the average respiratory rate and the average coefficient of variation (CV) were comparable between healthy and ataxic mice. We argue that these findings are consistent with a proposed role of the cerebellum in modulating the duration of individual respiratory intervals, which could serve the purpose of coordinating respiration with other rhythmic orofacial movements, such as fluid licking and swallowing.