Using global remote camera data of a solitary species complex to evaluate the drivers of group formation.

The social system of animals involves a complex interplay between physiology, natural history, and the environment. Long relied upon discrete categorizations of "social" and "solitary" inhibit our capacity to understand species and their interactions with the world around them. Here, we use a globally distributed camera trapping dataset to test the drivers of aggregating into groups in a species complex (martens and relatives, family Mustelidae, Order Carnivora) assumed to be obligately solitary. We use a simple quantification, the probability of being detected in a group, that was applied across our globally derived camera trap dataset. Using a series of binomial generalized mixed-effects models applied to a dataset of 16,483 independent detections across 17 countries on four continents we test explicit hypotheses about potential drivers of group formation. We observe a wide range of probabilities of being detected in groups within the solitary model system, with the probability of aggregating in groups varying by more than an order of magnitude. We demonstrate that a species' context-dependent proclivity toward aggregating in groups is underpinned by a range of resource-related factors, primarily the distribution of resources, with increasing patchiness of resources facilitating group formation, as well as interactions between environmental conditions (resource constancy/winter severity) and physiology (energy storage capabilities). The wide variation in propensities to aggregate with conspecifics observed here highlights how continued failure to recognize complexities in the social behaviors of apparently solitary species limits our understanding not only of the individual species but also the causes and consequences of group formation.

Chemical Proteomics and Phenotypic Profiling Identifies the Aryl Hydrocarbon Receptor as a Molecular Target of the Utrophin Modulator Ezutromid.

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease arising from mutations in the dystrophin gene. Upregulation of utrophin to compensate for the missing dystrophin offers a potential therapy independent of patient genotype. The first-in-class utrophin modulator ezutromid/SMT C1100 was developed from a phenotypic screen through to a Phase 2 clinical trial. Promising efficacy and evidence of target engagement was observed in DMD patients after 24 weeks of treatment, however trial endpoints were not met after 48 weeks. The objective of this study was to understand the mechanism of action of ezutromid which could explain the lack of sustained efficacy and help development of new generations of utrophin modulators. Using chemical proteomics and phenotypic profiling we show that the aryl hydrocarbon receptor (AhR) is a target of ezutromid. Several lines of evidence demonstrate that ezutromid binds AhR with an apparent KD of 50 nm and behaves as an AhR antagonist. Furthermore, other reported AhR antagonists also upregulate utrophin, showing that this pathway, which is currently being explored in other clinical applications including oncology and rheumatoid arthritis, could also be exploited in future DMD therapies.

GATA1-mutant clones are frequent and often unsuspected in babies with Down syndrome: identification of a population at risk of leukemia.

Transient abnormal myelopoiesis (TAM), a preleukemic disorder unique to neonates with Down syndrome (DS), may transform to childhood acute myeloid leukemia (ML-DS). Acquired GATA1 mutations are present in both TAM and ML-DS. Current definitions of TAM specify neither the percentage of blasts nor the role of GATA1 mutation analysis. To define TAM, we prospectively analyzed clinical findings, blood counts and smears, and GATA1 mutation status in 200 DS neonates. All DS neonates had multiple blood count and smear abnormalities. Surprisingly, 195 of 200 (97.5%) had circulating blasts. GATA1 mutations were detected by Sanger sequencing/denaturing high performance liquid chromatography (Ss/DHPLC) in 17 of 200 (8.5%), all with blasts >10%. Furthermore low-abundance GATA1 mutant clones were detected by targeted next-generation resequencing (NGS) in 18 of 88 (20.4%; sensitivity ∼0.3%) DS neonates without Ss/DHPLC-detectable GATA1 mutations. No clinical or hematologic features distinguished these 18 neonates. We suggest the term "silent TAM" for neonates with DS with GATA1 mutations detectable only by NGS. To identify all babies at risk of ML-DS, we suggest GATA1 mutation and blood count and smear analyses should be performed in DS neonates. Ss/DPHLC can be used for initial screening, but where GATA1 mutations are undetectable by Ss/DHPLC, NGS-based methods can identify neonates with small GATA1 mutant clones.

Ets-2 repressor factor silences extrasynaptic utrophin by N-box mediated repression in skeletal muscle.

Utrophin is the autosomal homologue of dystrophin, the protein product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin expression is temporally and spatially regulated being developmentally down-regulated perinatally and enriched at neuromuscular junctions (NMJs) in adult muscle. Synaptic localization of utrophin occurs in part by heregulin-mediated extracellular signal-regulated kinase (ERK)-phosphorylation, leading to binding of GABPalpha/beta to the N-box/EBS and activation of the major utrophin promoter-A expressed in myofibers. However, molecular mechanisms contributing to concurrent extrasynaptic silencing that must occur to achieve NMJ localization are unknown. We demonstrate that the Ets-2 repressor factor (ERF) represses extrasynaptic utrophin-A in muscle. Gel shift and chromatin immunoprecipitation studies demonstrated physical association of ERF with the utrophin-A promoter N-box/EBS site. ERF overexpression repressed utrophin-A promoter activity; conversely, small interfering RNA-mediated ERF knockdown enhanced promoter activity as well as endogenous utrophin mRNA levels in cultured muscle cells in vitro. Laser-capture microscopy of tibialis anterior NMJ and extrasynaptic transcriptomes and gene transfer studies provide spatial and direct evidence, respectively, for ERF-mediated utrophin repression in vivo. Together, these studies suggest "repressing repressors" as a potential strategy for achieving utrophin up-regulation in DMD, and they provide a model for utrophin-A regulation in muscle.

Mechanisms of Progression of Myeloid Preleukemia to Transformed Myeloid Leukemia in Children with Down Syndrome.

Myeloid leukemia in Down syndrome (ML-DS) clonally evolves from transient abnormal myelopoiesis (TAM), a preleukemic condition in DS newborns. To define mechanisms of leukemic transformation, we combined exome and targeted resequencing of 111 TAM and 141 ML-DS samples with functional analyses. TAM requires trisomy 21 and truncating mutations in GATA1; additional TAM variants are usually not pathogenic. By contrast, in ML-DS, clonal and subclonal variants are functionally required. We identified a recurrent and oncogenic hotspot gain-of-function mutation in myeloid cytokine receptor CSF2RB. By a multiplex CRISPR/Cas9 screen in an in vivo murine TAM model, we tested loss-of-function of 22 recurrently mutated ML-DS genes. Loss of 18 different genes produced leukemias that phenotypically, genetically, and transcriptionally mirrored ML-DS.

Alternative utrophin mRNAs contribute to phenotypic differences between dystrophin-deficient mice and Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal disorder caused by absence of functional dystrophin protein. Compensation in dystrophin-deficient (mdx) mice may be achieved by overexpression of its fetal paralogue, utrophin. Strategies to increase utrophin levels by stimulating promoter activity using small compounds are therefore a promising pharmacological approach. Here, we characterise similarities and differences existing within the mouse and human utrophin locus to assist in high-throughput screening for potential utrophin modulator drugs. We identified five novel 5'-utrophin isoforms (A',B',C,D and F) in adult and embryonic tissue. As the more efficient utrophin-based response in mdx skeletal muscle appears to involve independent transcriptional activation of conserved, myogenic isoforms (A' and F), elevating their paralogues in DMD patients is an encouraging therapeutic strategy.

Myostatin propeptide-mediated amelioration of dystrophic pathophysiology.

Mutations in myostatin (GDF8) cause marked increases in muscle mass, suggesting that this transforming growth factor-beta (TGF-beta) superfamily member negatively regulates muscle growth. Myostatin blockade therefore offers a strategy for reversing muscle wasting in Duchenne's muscular dystrophy (DMD) without resorting to genetic manipulation. Here, we demonstrate that pharmacological blockade using a myostatin propeptide stabilized by fusion to IgG-Fc improved pathophysiology of the mdx mouse model of DMD. Functional benefits evidenced by specific force improvement, exceeded those reported previously using myostatin antibody-mediated blockade. More importantly, use of a propeptide blockade strategy obviates possibilities of anti-idiotypic responses that could potentially limit the effectiveness of antibody-mediated myostatin blockade strategies over time. This study provides a novel pharmacological strategy for treatment of diseases associated with muscle wasting such as DMD and since it uses an endogenous inhibitor of myostatin should help circumvent technical hurdles and toxicity associated with conventional gene or cell based therapies.

Therapeutics for Duchenne muscular dystrophy: current approaches and future directions.

Duchenne muscular dystrophy (DMD) is the most common X-linked neuromuscular disorder. The devastating nature of DMD has led to an intense effort toward finding a cure for this disease, dating back to the time when Duchenne first initiated clinical trials using faradic stimulation for DMD patients. Unfortunately despite the passage of some 150 years the disease remains incurable, and its medical management is largely supportive. However, the discovery of the DMD gene about 20 years ago has allowed a change in the focus of therapeutic strategy dramatically toward delivery of the missing gene/protein. Indeed, some degree of success has been achieved in preclinical animal studies using such strategies, and gene therapy trials are currently underway in humans. Pharmacological approaches for DMD are also being developed since they can circumvent some of the technical problems associated with gene and cell based therapy. This review explores developments in therapeutic approaches for DMD.

Ets, Ap-1 and GATA factor families regulate the utrophin B promoter: potential regulatory mechanisms for endothelial-specific expression.

Duchenne muscular dystrophy is caused by dystrophin deficiency, which can be prevented in the mdx mouse model by over-expression of an autosomal homologue, utrophin. Utrophin has two characterised full-length promoters, A and B. No data are available on the transcriptional regulation of B utrophin, which has been recently localised to the endothelium. Similar to characterised endothelial promoters, Ets and Ap-1 individually trans-activate the human B core promoter. Synergistic activation by GATA-2 and c-jun to the order of 20-fold was observed.

The role of utrophin in the potential therapy of Duchenne muscular dystrophy.

Duchenne muscular dystrophy is an X-linked recessive muscle wasting disease caused by the absence of the muscle cytoskeletal protein, dystrophin. Dystrophin is a member of the spectrin superfamily of proteins and is closely related in sequence similarity and functional motifs to three proteins that constitute the dystrophin related protein family, including the autosomal homologue, utrophin. An alternative strategy circumventing many problems associated with somatic gene therapies for Duchenne muscular dystrophy has arisen from the demonstration that utrophin can functionally substitute for dystrophin and its over-expression in muscles of dystrophin-null transgenic mice completely prevents the phenotype arising from dystrophin deficiency. One potential approach to increase utrophin levels in muscle for possible therapeutic purpose in humans is to increase expression of the utrophin gene at a transcriptional level via promoter activation. This has lead to an interest in the identification and manipulation of important regulatory regions and/or molecules that increase the expression of utrophin and their delivery to dystrophin-deficient tissue. As pre-existing cellular mechanisms are utilized, this approach would avoid many problems associated with conventional gene therapies.

Emulating natural disturbances for declining late-successional species: a case study of the consequences for cerulean warblers (Setophaga cerulea).

Forest cover in the eastern United States has increased over the past century and while some late-successional species have benefited from this process as expected, others have experienced population declines. These declines may be in part related to contemporary reductions in small-scale forest interior disturbances such as fire, windthrow, and treefalls. To mitigate the negative impacts of disturbance alteration and suppression on some late-successional species, strategies that emulate natural disturbance regimes are often advocated, but large-scale evaluations of these practices are rare. Here, we assessed the consequences of experimental disturbance (using partial timber harvest) on a severely declining late-successional species, the cerulean warbler (Setophaga cerulea), across the core of its breeding range in the Appalachian Mountains. We measured numerical (density), physiological (body condition), and demographic (age structure and reproduction) responses to three levels of disturbance and explored the potential impacts of disturbance on source-sink dynamics. Breeding densities of warblers increased one to four years after all canopy disturbances (vs. controls) and males occupying territories on treatment plots were in better condition than those on control plots. However, these beneficial effects of disturbance did not correspond to improvements in reproduction; nest success was lower on all treatment plots than on control plots in the southern region and marginally lower on light disturbance plots in the northern region. Our data suggest that only habitats in the southern region acted as sources, and interior disturbances in this region have the potential to create ecological traps at a local scale, but sources when viewed at broader scales. Thus, cerulean warblers would likely benefit from management that strikes a landscape-level balance between emulating natural disturbances in order to attract individuals into areas where current structure is inappropriate, and limiting anthropogenic disturbance in forests that already possess appropriate structural attributes in order to maintain maximum productivity.

Recent advances in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD), an allelic X-linked progressive muscle-wasting disease, is one of the most common single-gene disorders in the developed world. Despite knowledge of the underlying genetic causation and resultant pathophysiology from lack of dystrophin protein at the muscle sarcolemma, clinical intervention is currently restricted to symptom management. In recent years, however, unprecedented advances in strategies devised to correct the primary defect through gene- and cell-based therapeutics hold particular promise for treating dystrophic muscle. Conventional gene replacement and endogenous modification strategies have greatly benefited from continued improvements in encapsidation capacity, transduction efficiency, and systemic delivery. In particular, RNA-based modifying approaches such as exon skipping enable expression of a shorter but functional dystrophin protein and rapid progress toward clinical application. Emerging combined gene- and cell-therapy strategies also illustrate particular promise in enabling ex vivo genetic correction and autologous transplantation to circumvent a number of immune challenges. These approaches are complemented by a vast array of pharmacological approaches, in particular the successful identification of molecules that enable functional replacement or ameliorate secondary DMD pathology. Animal models have been instrumental in providing proof of principle for many of these strategies, leading to several recent trials that have investigated their efficacy in DMD patients. Although none has reached the point of clinical use, rapid improvements in experimental technology and design draw this goal ever closer. Here, we review therapeutic approaches to DMD, with particular emphasis on recent progress in strategic development, preclinical evaluation and establishment of clinical efficacy. Further, we discuss the numerous challenges faced and synergistic approaches being devised to combat dystrophic pathology effectively.

Pharmacologically targeting the primary defect and downstream pathology in Duchenne muscular dystrophy.

DMD is a devastatingly progressive muscle wasting disorder of childhood that significantly shortens life expectancy. Despite efforts to develop an effective therapy that dates back over a century, clinical interventions are still restricted to management of symptoms rather than a cure. The rationale to develop effective therapies changed in 1986 with the discovery of the dystrophin gene. Since then extensive research into both the molecular basis and pathophysiology of DMD has paved the way not only for development of strategies which aim to correct the primary defect, but also towards the identification of countless therapeutic targets with the potential to alleviate the downstream pathology. In addition to gene and cell-based therapies, which aim to deliver the missing gene and/or protein, an exciting spectrum of pharmacological approaches aimed at modulating therapeutic targets within DMD muscle cells through the use of small drugs are also being developed. This review presents promising pharmacological approaches aimed at targeting the primary defect, including suppression of nonsense mutations and functional compensation by upregulation of the dystrophin homologue, utrophin. Downstream of the primary membrane fragility, inflammation and fibrosis are reduced by blocking NF-κB, TGF-α and TGF-ß, and free radical damage has been targeted using antioxidants and dietary/nutritional supplements. There are new hopes that ACE and PDE5 inhibitors can protect against skeletal as well as cardiac pathology, and modulating Ca2+ influx, NO, BMP, protein degradation and the mitochondrial permeability pore hold further promise in tackling the complex pathogenesis of this multifaceted disorder.

Plasma-Lyte 148 vs 0.9% saline for fluid resuscitation in diabetic ketoacidosis.

PURPOSE: The purpose of the study was to determine the effects of Plasma-Lyte 148 (PL) vs 0.9% saline (NS) fluid resuscitation in diabetic ketoacidosis (DKA). METHODS: A multicenter retrospective analysis of adults admitted for DKA to the intensive care unit, who received almost exclusively PL or NS infusion up until 12 hours, was performed. RESULTS: Nine patients with PL and 14 patients with NS were studied. Median serum bicarbonate correction was higher in the PL vs NS groups at 4 to 6 hours (8.4 vs 1.7 mEq/L) and 6 to 12 hours (12.8 vs 6.2 mEq/L) from baseline (P < .05). Median standard base excess improved by 10.5 vs 4.2 mEq/L at 4 to 6 hours and by 16.0 vs 9.1 mEq/L at 6 to 12 hours in the PL and NS groups, respectively (P < .05). Chloride levels increased significantly in the NS vs PL groups over 24 hours. Potassium levels were lower at 6 to 12 hours in the PL group. Mean arterial blood pressure was higher at 2 to 4 hours in the PL group, whereas cumulative urine output was lower at 4 to 6 hours in the NS group. There were no differences in glycemic control or duration of intensive care unit stay. CONCLUSION: Patients with DKA resuscitated with PL instead of NS had faster initial resolution of metabolic acidosis and less hyperchloremia, with a transiently improved blood pressure profile and urine output.

An ungracious host for an unwelcome guest.

Persistence of latently infected CD4(+) T cell "reservoirs" presents a major obstacle for current HIV-1 antiretroviral therapies. In Cell Host & Microbe, two separate studies use model systems of latent infection to dissect the influence of host genes on HIV-1 transcription, yielding intriguing, though partially contradictory, conclusions.

Transcription-dependent gene looping of the HIV-1 provirus is dictated by recognition of pre-mRNA processing signals.

HIV-1 provirus, either as a chromosomal integrant or as an episomal plasmid in HeLa cells, forms a transcription-dependent gene loop structure between the 5'LTR promoter and 3'LTR poly(A) signal. Flavopiridol-mediated inhibition of RNA polymerase II elongation blocks 5' to 3'LTR juxtaposition, indicating that this structure is maintained during transcription. Analysis of mutant or hybrid HIV-1 plasmids demonstrates that replacement of the 5'LTR promoter with CMV or the 3'LTR poly(A) signal with a synthetic element (SPA) permits gene loop formation, suggesting that these interactions are not retroviral specific. In addition, activation of the 5'LTR poly(A) signal or inactivation of the 3'LTR poly(A) signal abolishes gene loop formation. Overall, we demonstrate that both ongoing transcription and pre-mRNA processing are essential for gene loop formation, and predict that these structures represent a defining feature of active gene transcription.

Late-Onset visceral presentation with cardiomyopathy and without neurological symptoms of adult Sanfilippo A syndrome.

Sanfilippo A syndrome, mucopolysaccharidosis type IIIA, is caused by a deficiency of heparan sulphamidase activity, and usually presents in childhood with neurodegeneration leading to death in teenage years. Visceral symptoms are limited to coarsening and diarrhea. We now describe an adult patient who presented with cardiomyopathy. At age 45 years she had hypertension, and the next year she developed a progressively worsening cardiomyopathy with prominent apical hypertrophy and atrial fibrillation. At age 53, she had severe concentric hypertrophic nonobstructive cardiomyopathy in both ventricles. There was no coarsening of features. Neurologic function, skeleton, cornea, liver, and spleen were normal. Percutaneous endomyocardial biopsy showed ballooned cardiomyocytes with storage vacuoles, containing acid mucopolysaccharides. Leucocytes, uterus, and brain biopsy did not show this storage material. There was a slight increase in total urine mucopolysaccharides, with an increased proportion of heparan sulfates. Heparan sulphamidase activity was deficient in leukocytes and heparan sulphamidase protein and activity were reduced in cultured fibroblasts. No mutations were identified after sequencing of the heparan sulphamidase gene at the cDNA and the genomic level. This new clinical presentation expands the clinical spectrum of Sanfilippo A syndrome to include a primary visceral presentation of cardiomyopathy without neurologic symptoms in the adult. The late onset may be related to the residual heparan sulphamidase activity. The genetic basis of this new variant is still unclear. Physicians evaluating adults must remain aware of possible new adult presentations of storage conditions.