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Introduction: During the COVID-19 pandemic, ambulance divert in our EMS system reached critical levels. We hypothesized that eliminating ambulance divert would not be associated with an increase in the average number of daily ambulance arrivals. Our study objective was to quantify the EMS and emergency department (ED) effects of eliminating ambulance divert during the COVID-19 pandemic., Methods: Regional hospital divert data were obtained for the 10-county Twin Cities metro from MNTrac, a state-supported online system designed to allow hospitals to indicate their divert status to EMS. ED metrics are reported for a single Level I trauma center and were obtained by a deidentified data pull from our electronic medical record covering the 12 months prior to the elimination of divert (2021) and the 12 months after divert elimination (2022). The decision to eliminate divert occurred in November 2021, based on data available through October, with an implementation date of January 2022. The primary study outcome was to quantify the effect of the elimination of divert on the number of ambulances arriving per day at the study hospital., Results: Regional utilization of ambulance divert increased steadily by 859% from January to October 2021 when 355 individual divert events occurred, totaling 809 h (34 days). There was no significant difference in the number of ambulances that arrived to the study hospital in 2021 (30,774) vs 2022 (30,421) p  = 0.15. As compared to 2021, in 2022 there was no significant increase in mean ambulance arrivals per day (84/day vs 83/day, p  = 0.08), time to room Emergency Severity Index level 2 (ESI) patients (28 min vs 28 min, p  = 0.90), or time to obtain emergent head CT in acute "code stroke" patients (12 min vs 12 min, p = 0.15). Ambulance turnaround interval in the ED did not appreciably increase (16 min vs 17 min, p  = 0.15)., Conclusion: Elimination of ambulance divert was not associated with increases in the number of mean daily ambulance arrivals or EMS turnaround intervals, delays in ESI 2 patients being placed in beds, or prolonged time to head CT in stroke code patients.

Human genome variation contributes to diversity in neurodevelopmental outcomes and vulnerabilities; recognizing the underlying molecular and cellular mechanisms will require scalable approaches. Here, we describe a "cell village" experimental platform we used to analyze genetic, molecular, and phenotypic heterogeneity across neural progenitor cells from 44 human donors cultured in a shared in vitro environment using algorithms (Dropulation and Census-seq) to assign cells and phenotypes to individual donors. Through rapid induction of human stem cell-derived neural progenitor cells, measurements of natural genetic variation, and CRISPR-Cas9 genetic perturbations, we identified a common variant that regulates antiviral IFITM3 expression and explains most inter-individual variation in susceptibility to the Zika virus. We also detected expression QTLs corresponding to GWAS loci for brain traits and discovered novel disease-relevant regulators of progenitor proliferation and differentiation such as CACHD1. This approach provides scalable ways to elucidate the effects of genes and genetic variation on cellular phenotypes., Competing Interests: Declaration of interests K.E. is a founder of Q-State Biosciences, Quralis, and Enclear Therapies; an employee and shareholder of BioMarin; and a member of Cell Stem Cell’s advisory board., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: We describe implementation, evaluate performance, and report outcomes from the first program serving an entire metropolitan area designed to rapidly deliver extracorporeal membrane oxygenation (ECMO)-facilitated resuscitation to patients with refractory ventricular fibrillation/ventricular tachycardia (VF/VT) out-of-hospital cardiac arrest (OHCA)., Methods: This observational cohort study analyzed consecutive patients prospectively enrolled in the Minnesota Mobile Resuscitation Consortium's ECMO-facilitated resuscitation program. Entry criteria included: 1) adults (aged 18-75), 2) VF/VT OHCA, 3) no return of spontaneous circulation following 3 shocks, 4) automated cardiopulmonary resuscitation with a Lund University Cardiac Arrest System (LUCAS™), and 5) estimated transfer time of < 30 min. The primary endpoint was functionally favorable survival to hospital discharge with Cerebral Performance Category (CPC) 1 or 2. Secondary endpoints included 3-month functionally favorable survival, program benchmarks, ECMO cannulation rate, and safety. Essential program components included emergency medical services, 3 community ECMO Initiation Hospitals with emergency department ECMO cannulation sites and 24/7 cardiac catheterization laboratories, a 24/7 mobile ECMO cannulation team, and a single, centralized ECMO intensive care unit., Findings: From December 1, 2019 to April 1, 2020, 63 consecutive patients were transported and 58 (97%) met criteria and were treated by the mobile ECMO service. Mean age was 57 ± 1.8 years; 46/58 (79%) were male. Program benchmarks were variably met, 100% of patients were successfully cannulated, and no safety issues were identified. Of the 58 patients, 25/58 (43% [CI:31-56%]) were both discharged from the hospital and alive at 3 months with CPC 1 or 2., Interpretation: This first, community-wide ECMO-facilitated resuscitation program in the US demonstrated 100% successful cannulation, 43% functionally favorable survival rates at hospital discharge and 3 months, as well as safety. The program provides a potential model of this approach for other communities., Funding: The Helmsley Charitable Trust., Competing Interests: All authors report having received funding from the Leona M. and Harry B. Helmsley Charitable Trust. JAB, TPA, and DY also received funding from the National Heart, Lung, and Blood Institute., (© 2020 The Authors.)

A new catalytically active zeolite, designated EMM-17 (ExxonMobil Material-17), with a three-dimensional (3D) 11 × 10 × 10-ring topology has been discovered from high throughput experiments while evaluating a family of new organic structure directing agents (OSDAs), 1-alkyl-4-(pyrrolidin-1-yl)pyridin-1-ium hydroxide. The framework structure was determined by model building techniques and confirmed by diffraction calculations. The EMM-17 structure is a random intergrowth of two polymorphs which have a 3D arrangement of intersecting 11 × 10 × 10-ring pores. EMM-17 is stable to calcination to remove the OSDA and can be reproducibly synthesized in the presence of fluoride using common, inexpensive reagents over a wide Si/Al range from 15 to infinity, enabling the catalyst acidity to be tailored to almost any petrochemical application. Unlike OSDAs for many new zeolite structures, the OSDAs for EMM-17 are prepared in one simple alkylation step, making EMM-17 an easy to prepare, highly accessible, catalytically active zeolite. Zeolites containing odd numbered channel sizes are rare, and this is the first confirmed example of a 3D 11-ring aluminosilicate zeolite with a pore size in between those of the commercially important 10- and 12-ring zeolites such as ZSM-5 and Zeolite-Y, respectively. Catalysts prepared from EMM-17 exhibit significantly higher activity for catalytic isomerization with no loss in selectivity than current state of the art catalysts. Catalytic isomerization of linear to branched alkanes is a critical component of commercial dewaxing, allowing for the improvement of cold flow properties of hydrocarbon fuels and lubricants through selective hydroisomerization of normal paraffins.

mRNAs can fold into complex structures that regulate gene expression. Resolving such structures de novo has remained challenging and has limited our understanding of the prevalence and functions of mRNA structure. We use SHAPE-MaP experiments in living E. coli cells to derive quantitative, nucleotide-resolution structure models for 194 endogenous transcripts encompassing approximately 400 genes. Individual mRNAs have exceptionally diverse architectures, and most contain well-defined structures. Active translation destabilizes mRNA structure in cells. Nevertheless, mRNA structure remains similar between in-cell and cell-free environments, indicating broad potential for structure-mediated gene regulation. We find that the translation efficiency of endogenous genes is regulated by unfolding kinetics of structures overlapping the ribosome binding site. We discover conserved structured elements in 35% of UTRs, several of which we validate as novel protein binding motifs. RNA structure regulates every gene studied here in a meaningful way, implying that most functional structures remain to be discovered., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Study Objective: Invasively monitoring blood pressure through the IO device has not been thoroughly demonstrated. This study attempted to establish baseline values of IO pressure in a healthy human population., Methods: This was a prospective, healthy volunteer, observational study. Participants had two IO devices placed (humerus and tibia), and participant IO pressures, vital signs, and pain scores were monitored for up to 60 minutes. Participants were contacted at 24-hours and 7 days post-testing to assess for adverse events. Summary statistics were calculated for systolic, diastolic, and mean humeral and tibial IO pressure. The ratio of IO to non-invasive blood pressure was calculated, and Bland Altman plots were created. The slope (linear) of the mean humeral and the tibial IO pressures were also calculated., Results: Fifteen subjects were enrolled between April and July 2015. Fourteen of 15 humeral IOs were placed successfully (93.3%) and all 15 of the tibial IOs were placed successfully. Mean tibial systolic, diastolic, and mean IO pressure were 55.8 ± 27.9, 49.3 ± 27.1, and 48.4 ± 29.4 mm Hg, respectively. Humeral systolic, diastolic, and mean IO pressure were 32.9 ± 16.0, 27.4 ± 15.2, and 24.5 ± 14.3 mm Hg. The mean tibial IO pressure was 52.5% ± 32.0% of external cuff pressure ratio. The mean humeral IO pressure was 26.5% ± 15.2% of the external mean blood pressure. The Bland Altman plots showed an inconsistent relationship between the systolic and diastolic cuff pressure and the IO pressures. We observed a 1% per minute decrease in IO pressure from the initial placement until the final reading., Conclusions: Intraosseous pressure readings can be obtained in healthy human volunteers. However, absolute IOP values were not consistent between subjects. Future research may determine how IO pressure can be used to guide therapy in ill and injured patients.

Parental care is essential for the survival of mammals, yet the mechanisms underlying its evolution remain largely unknown. Here we show that two sister species of mice, Peromyscus polionotus and Peromyscus maniculatus, have large and heritable differences in parental behaviour. Using quantitative genetics, we identify 12 genomic regions that affect parental care, 8 of which have sex-specific effects, suggesting that parental care can evolve independently in males and females. Furthermore, some regions affect parental care broadly, whereas others affect specific behaviours, such as nest building. Of the genes linked to differences in nest-building behaviour, vasopressin is differentially expressed in the hypothalamus of the two species, with increased levels associated with less nest building. Using pharmacology in Peromyscus and chemogenetics in Mus, we show that vasopressin inhibits nest building but not other parental behaviours. Together, our results indicate that variation in an ancient neuropeptide contributes to interspecific differences in parental care.

Two-component systems are important regulatory systems that allow bacteria to adjust to environmental conditions, and in some bacteria are used in pathogenesis. We identified a novel two-component system in Burkholderia cenocepacia, an opportunistic pathogen that causes pneumonia in cystic fibrosis (CF) patients. The putative operon encodes BceS, a sensor kinase, and BceR, a response regulator. Our studies indicated that the bceR mutant showed a statistically significant decrease in protease, swimming motility, and quorum sensing when compared to the wild-type, but there was no significant difference in phospholipase C activity, swarming, and biofilm formation. In addition, the mutant showed a statistically significant reduction in virulence compared to the wild-type using the alfalfa plant model. Examination of the Burkholderia cepacia complex (a group of organisms that are phenotypically similar, but genotypically distinct) revealed that this system is prevalent in B. ambifaria, B. multivorans, B. vietnamiensis and B. dolosa. Interestingly, all these organisms have been associated with CF patients. The collective results indicate that BceSR influences various activities important in Burkholderia physiology and possibly pathogenesis. This information could be important in the design of novel therapeutics for Burkholderia infections.

Introduction: Intramuscular ketamine has become increasingly popular for prehospital chemical restraint of severely agitated or violent patients because of its favorable adverse effect profile, rapid onset, and wide therapeutic window. However, there is currently no literature quantifying the need for intubation or hospital admission for these patients once they reach the emergency department., Methods: Medical records for patients receiving prehospital ketamine who were transported to a single level 1 trauma center were abstracted. Ketamine dose, patient weight, final disposition, and presence of intubation were recorded. Exclusion criteria were missing dose or weight and ketamine given for an indication other than chemical restraint. Statistical analysis was preformed with unadjusted Student t test. Statistical significance was defined as P < .05., Results: A convenience sample of 51 consecutive patients was identified with 2 excluded because of missing data, leaving 49 for analysis. Ketamine dosing ranged from 2.25 to 9.42 mg/kg (mean, 5.26 ± 1.65 mg/kg). Significant differences were noted between those who required intubation (n = 14) and those who did not (n = 35) (6.16 ± 1.62 mg/kg vs 4.90 ± 1.54 mg/kg, P = .02). No patients were intubated prehospital. There was an increased dose in patients admitted to a medical ward (57%, 28/49) that approached statistical significance (5.62 ± 1.80 vs 4.78 ± 1.31, P = .06)., Conclusion: Intubation was observed in our emergency department in 29% of patients administered intramuscular ketamine for prehospital chemical restraint. There was a positive association between higher ketamine doses and both endotracheal intubation and hospital admission. Future research should aim to define the minimum effective ketamine dose for successful chemical restraint., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Surface populations of Astyanax mexicanus, living in rivers like their common ancestors, school, while several, independently derived cave populations of the same species have lost schooling behavior., Results: We quantify schooling behavior in individual A. mexicanus and identify quantitative trait loci (QTL) for this trait. We find that the evolutionary modulation of schooling has both vision-dependent and -independent components. We also quantify differences in the lateral line and vision between cavefish and surface fish and relate these differences to the evolutionary loss of schooling behavior. We provide evidence that a monoamine neurotransmitter may have played a role in the evolution of schooling behavior., Conclusions: We find that vision is essential for schooling tendency in A. mexicanus, while the lateral line has a small effect on this behavior. Schooling behavior in A. mexicanus has evolved both through changes in sensory systems and through changes in genetic loci that likely act downstream of sensory inputs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objectives: The primary aims of this study were to determine whether the frequency of placement, type of advanced airway, and settings of advanced airway placement (clinical vs. field) have changed for paramedic students over the last 11 years, and to describe regional differences regarding the same set of variables., Methods: This study was a retrospective review of prospectively reported airway procedures documented by paramedic students in Fisdap ( http://www.fisdap.net ). Students were included if they graduated from a paramedic program, had procedure entries verified by a preceptor, and provided consent for research. Exclusion criteria included students who had a total number of airway placements ≥2 standard deviations from the mean or had 0 airway placements recorded, and programs with <10 graduating students total over the study period. Airway device types and educational settings were descriptively compared over the 11-year study period by year and region., Results: A total of 8,934 paramedic student records were reviewed, with 2,811 excluded based on a priori criteria, leaving 6,123 records for analysis. In each year, the median number of airway devices placed per student was greater in the clinical setting. Endotracheal intubation (ETI) was more common than alternative airway placement in both the field and clinical settings. The median number of clinical ETIs per student has remained relatively constant at 7. The median number of field ETIs per student ranged from 0 to 1 over the study period, with a median alternative airway placement rate of 0 for both clinical and field settings. For all regions, the majority of procedures were performed in a clinical environment. The median number of clinical alternative airway device placements was 0 for all regions. The number of clinical ETIs ranged from 5 to 11 per student, with the highest number of ETIs per student in the West North Central and New England regions and the lowest in the West South Central and East South Central regions., Conclusion: Paramedic students gain the majority of their advanced airway experience in the clinical setting. ETI remains more common than alternative airway placement, although there is significant geographic variation in the number of ETIs per student. High rates of clinical intubations do not correlate with high rates of field intubations.

The identification of precise mutations is required for a complete understanding of the underlying molecular and evolutionary mechanisms driving adaptive phenotypic change. Using plasticine models in the field, we show that the light coat color of deer mice that recently colonized the light-colored soil of the Nebraska Sand Hills provides a strong selective advantage against visually hunting predators. Color variation in an admixed population suggests that this light Sand Hills phenotype is composed of multiple traits. We identified distinct regions within the Agouti locus associated with each color trait and found that only haplotypes associated with light trait values have evidence of selection. Thus, local adaptation is the result of independent selection on many mutations within a single locus, each with a specific effect on an adaptive phenotype, thereby minimizing pleiotropic consequences.

Relative to morphological traits, we know little about how genetics influence the evolution of complex behavioural differences in nature. It is unclear how the environment influences natural variation in heritable behaviour, and whether complex behavioural differences evolve through few genetic changes, each affecting many aspects of behaviour, or through the accumulation of several genetic changes that, when combined, give rise to behavioural complexity. Here we show that in nature, oldfield mice (Peromyscus polionotus) build complex burrows with long entrance and escape tunnels, and that burrow length is consistent across populations, although burrow depth varies with soil composition. This burrow architecture is in contrast with the small, simple burrows of its sister species, deer mice (P. maniculatus). When investigated under laboratory conditions, both species recapitulate their natural burrowing behaviour. Genetic crosses between the two species reveal that the derived burrows of oldfield mice are dominant and evolved through the addition of multiple genetic changes. In burrows built by first-generation backcross mice, entrance-tunnel length and the presence of an escape tunnel can be uncoupled, suggesting that these traits are modular. Quantitative trait locus analysis also indicates that tunnel length segregates as a complex trait, affected by at least three independent genetic regions, whereas the presence of an escape tunnel is associated with only a single locus. Together, these results suggest that complex behaviours--in this case, a classic 'extended phenotype'--can evolve through multiple genetic changes each affecting distinct behaviour modules.

To understand how organisms adapt to novel habitats, which involves both demographic and selective events, we require knowledge of the evolutionary history of populations and also selected alleles. There are still few cases in which the precise mutations (and hence, defined alleles) that contribute to adaptive change have been identified in nature; one exception is the genetic basis of camouflaging pigmentation of oldfield mice (Peromyscus polionotus) that have colonized the sandy dunes of Florida's Gulf Coast. To quantify the genomic impact of colonization as well as the signature of selection, we resequenced 5000 1.5-kb noncoding loci as well as a 160-kb genomic region surrounding the melanocortin-1 receptor (Mc1r), a gene that contributes to pigmentation differences, in beach and mainland populations. Using a genome-wide phylogenetic approach, we recovered a single monophyletic group comprised of beach mice, consistent with a single colonization event of the Gulf Coast. We also found evidence of a severe founder event, estimated to have occurred less than 3000 years ago. In this demographic context, we show that all beach subspecies share a single derived light Mc1r allele, which was likely selected from standing genetic variation that originated in the mainland. Surprisingly, we were unable to identify a clear signature of selection in the Mc1r region, despite independent evidence that this locus contributes to adaptive coloration. Nonetheless, these data allow us to reconstruct and compare the evolutionary history of populations and alleles to better understand how adaptive evolution, following the colonization of a novel habitat, proceeds in nature., (© 2012 The Author(s). Evolution© 2012 The Society for the Study of Evolution.)

The ability to efficiently and accurately determine genotypes is a keystone technology in modern genetics, crucial to studies ranging from clinical diagnostics, to genotype-phenotype association, to reconstruction of ancestry and the detection of selection. To date, high capacity, low cost genotyping has been largely achieved via "SNP chip" microarray-based platforms which require substantial prior knowledge of both genome sequence and variability, and once designed are suitable only for those targeted variable nucleotide sites. This method introduces substantial ascertainment bias and inherently precludes detection of rare or population-specific variants, a major source of information for both population history and genotype-phenotype association. Recent developments in reduced-representation genome sequencing experiments on massively parallel sequencers (commonly referred to as RAD-tag or RADseq) have brought direct sequencing to the problem of population genotyping, but increased cost and procedural and analytical complexity have limited their widespread adoption. Here, we describe a complete laboratory protocol, including a custom combinatorial indexing method, and accompanying software tools to facilitate genotyping across large numbers (hundreds or more) of individuals for a range of markers (hundreds to hundreds of thousands). Our method requires no prior genomic knowledge and achieves per-site and per-individual costs below that of current SNP chip technology, while requiring similar hands-on time investment, comparable amounts of input DNA, and downstream analysis times on the order of hours. Finally, we provide empirical results from the application of this method to both genotyping in a laboratory cross and in wild populations. Because of its flexibility, this modified RADseq approach promises to be applicable to a diversity of biological questions in a wide range of organisms.

The identification of regulatory sequences in animal genomes remains a significant challenge. Comparative genomic methods that use patterns of evolutionary conservation to identify non-coding sequences with regulatory function have yielded many new vertebrate enhancers. However, these methods have not contributed significantly to the identification of regulatory sequences in sequenced invertebrate taxa. We demonstrate here that this differential success, which is often attributed to fundamental differences in the nature of vertebrate and invertebrate regulatory sequences, is instead primarily a product of the relatively small size of sequenced invertebrate genomes. We sequenced and compared loci involved in early embryonic patterning from four species of true fruit flies (family Tephritidae) that have genomes four to six times larger than those of Drosophila melanogaster. Unlike in Drosophila, where virtually all non-coding DNA is highly conserved, blocks of conserved non-coding sequence in tephritids are flanked by large stretches of poorly conserved sequence, similar to what is observed in vertebrate genomes. We tested the activities of nine conserved non-coding sequences flanking the even-skipped gene of the teprhitid Ceratis capitata in transgenic D. melanogaster embryos, six of which drove patterns that recapitulate those of known D. melanogaster enhancers. In contrast, none of the three non-conserved tephritid non-coding sequences that we tested drove expression in D. melanogaster embryos. Based on the landscape of non-coding conservation in tephritids, and our initial success in using conservation in tephritids to identify D. melanogaster regulatory sequences, we suggest that comparison of tephritid genomes may provide a systematic means to annotate the non-coding portion of the D. melanogaster genome. We also propose that large genomes be given more consideration in the selection of species for comparative genomics projects, to provide increased power to detect functional non-coding DNAs and to provide a less biased view of the evolution and function of animal genomes.

The gene expression pattern specified by an animal regulatory sequence is generally viewed as arising from the particular arrangement of transcription factor binding sites it contains. However, we demonstrate here that regulatory sequences whose binding sites have been almost completely rearranged can still produce identical outputs. We sequenced the even-skipped locus from six species of scavenger flies (Sepsidae) that are highly diverged from the model species Drosophila melanogaster, but share its basic patterns of developmental gene expression. Although there is little sequence similarity between the sepsid eve enhancers and their well-characterized D. melanogaster counterparts, the sepsid and Drosophila enhancers drive nearly identical expression patterns in transgenic D. melanogaster embryos. We conclude that the molecular machinery that connects regulatory sequences to the transcription apparatus is more flexible than previously appreciated. In exploring this diverse collection of sequences to identify the shared features that account for their similar functions, we found a small number of short (20-30 bp) sequences nearly perfectly conserved among the species. These highly conserved sequences are strongly enriched for pairs of overlapping or adjacent binding sites. Together, these observations suggest that the local arrangement of binding sites relative to each other is more important than their overall arrangement into larger units of cis-regulatory function., Competing Interests: The authors have declared that no competing interests exist.

Background: Inhibition of N-methyl-D-aspartate (NMDA) receptors by anesthetic gases and vapors may play an important role in anesthesia and neuroprotection. However, the site of action of these agents on the NMDA receptor is unknown. The authors show that xenon and isoflurane compete for the binding of the coagonist glycine on the NMDA receptor NR1 subunit., Methods: Using a novel application of grand canonical Monte Carlo simulations, the authors predict the binding site of xenon on NMDA receptors. They test this prediction using electrophysiology on recombinant NMDA receptors., Results: The authors' modeling predicts that xenon binds at the glycine site of the NMDA receptor. The authors show that inhibition of NMDA receptors by xenon and isoflurane increases as glycine concentration is decreased, consistent with the prediction of competitive inhibition at the glycine site. Lineweaver-Burk analysis shows that isoflurane inhibition seems purely competitive with glycine, but for xenon, there is an additional component of noncompetitive inhibition. The loss of inhibitory effect of xenon and isoflurane in mutant NR1(F639A)/NR2A receptors is explained by increased glycine affinity of the mutant receptors, and inhibition is restored at low glycine concentrations., Conclusions: Xenon and isoflurane inhibit NMDA receptors by binding at the same site as the coagonist glycine. This finding may have important implications for general anesthesia and neuroprotection. Neuroprotectants that act at the glycine site of the NMDA receptor antagonists are well tolerated in patients, being devoid of psychotomimetic side effects, and the mechanism of inhibition may play a role in their clinical profile.

In addition to contractile function, muscle provides a metabolic buffer by degrading protein in times of organismal need. Protein is also degraded during adaptive muscle remodeling upon exercise, but extreme degradation in diverse clinical conditions can compromise function(s) and threaten life. Here, we show how two independent signals interact to control protein degradation. In striated muscles of Caenorhabditis elegans, reduction of insulin-like signaling via DAF-2 insulin/IGF receptor or its intramuscular effector PtdIns-3-kinase (PI3K) causes unexpected activation of MAP kinase (MAPK), consequent activation of pre-existing systems for protein degradation, and progressive impairment of mobility. Degradation is prevented by mutations that increase signal downstream of PI3K or by disruption of autocrine signal from fibroblast growth factor (FGF) via the FGF receptor and its effectors in the Ras-MAPK pathway. Thus, the activity of constitutive protein degradation systems in normal muscle is minimized by a balance between directly interacting signaling pathways, implying that physiological, pathological, or therapeutic alteration of this balance may contribute to muscle remodeling or wasting.

Gene expression in metazoans is regulated not only at the level of individual genes but also in a coordinated manner across large chromosomal domains (for example centromeres, telomeres and imprinted gene clusters) and along entire chromosomes (for example X-chromosome dosage compensation). The primary DNA sequence usually specifies the regulation of individual genes, but the nature of cis-acting information that controls genes over large regions has been elusive: higher-order DNA structure, specific histone modifications, subnuclear compartmentalization and primary DNA sequence are possibilities. One paradigm of chromosome-wide gene regulation is Caenorhabditis elegans dosage compensation in which a large dosage compensation complex (DCC) is targeted to both X chromosomes of hermaphrodites to repress transcript levels by half. This essential process equalizes X-linked gene expression between the sexes (XO males and XX hermaphrodites). Here we report the discovery and dissection of cis-acting sites that mark nematode X chromosomes as targets for gene repression by the DCC. These rex (recruitment element on X) sites are widely dispersed along X and reside in promoters, exons and intergenic regions. rex sites share at least two distinct motifs that act in combination to recruit the DCC. Mutating these motifs severely reduces or abolishes DCC binding in vivo, demonstrating the importance of primary DNA sequence in chromosome-wide regulation. Unexpectedly, the motifs are not enriched on X, but altering motif numbers within rex sites demonstrates that motif co-occurrence in unusually high densities is essential for optimal DCC recruitment. Thus, X-specific repression is established through sequences not specific to X. The distribution of common motifs provides the foundation for repression along an entire chromosome.

To discover and study intracellular signals that regulate proteolysis in muscle, we have employed transgenic strains of Caenorhabditis elegans that produce a soluble LacZ reporter protein limited to body-wall and vulval muscles. This reporter protein is stable in well-fed wild-type animals, but its degradation is triggered upon a shift to 25 degrees C in a strain carrying a temperature-sensitive activating mutation in the Ras oncogene homologue let-60. These mutants are not physiologically starved, inasmuch as growth rates are normal at 25 degrees C. Ras-induced degradation is not prevented by the presence of cycloheximide added at or before the temperature shift and thus uses preexisting proteolytic systems and signaling components. Furthermore, degradation is triggered when adult animals are shifted to conditions of 25 degrees C, confirming that Ras acutely promotes protein degradation in muscles whose developmental history is normal. Reduction-of-function mutations in the downstream protein kinase Raf (lin-45), MEK (mek-2), or mitogen-activated protein kinase (MAPK) (mpk-1) prevent Ras-induced protein degradation, whereas activated MPK-1 is sufficient to trigger degradation, indicating that this kinase cascade is the principal route by which Ras signaling triggers protein degradation in muscle. This pathway is activated in hypodermal cells by the LET-23 epidermal growth factor receptor homologue, but an activating mutation in let-23 does not promote proteolysis in muscle. Starvation-induced LacZ reporter degradation is unaffected by reduction-of-function mutations in Ras, Raf, MEK, or MAPK, implying that Ras activation and starvation trigger proteolysis by mechanisms that are at least partially independent. This is the first evidence that Ras-Raf-MEK-MAPK signaling activates protein degradation in differentiated muscle.