Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models.

The essential product of the Duchenne muscular dystrophy (DMD) gene is dystrophin1, a rod-like protein2 that protects striated myocytes from contraction-induced injury3,4. Dystrophin-related protein (or utrophin) retains most of the structural and protein binding elements of dystrophin5. Importantly, normal thymic expression in DMD patients6 should protect utrophin by central immunologic tolerance. We designed a codon-optimized, synthetic transgene encoding a miniaturized utrophin (µUtro), deliverable by adeno-associated virus (AAV) vectors. Here, we show that µUtro is a highly functional, non-immunogenic substitute for dystrophin, preventing the most deleterious histological and physiological aspects of muscular dystrophy in small and large animal models. Following systemic administration of an AAV-µUtro to neonatal dystrophin-deficient mdx mice, histological and biochemical markers of myonecrosis and regeneration are completely suppressed throughout growth to adult weight. In the dystrophin-deficient golden retriever model, µUtro non-toxically prevented myonecrosis, even in the most powerful muscles. In a stringent test of immunogenicity, focal expression of µUtro in the deletional-null German shorthaired pointer model produced no evidence of cell-mediated immunity, in contrast to the robust T cell response against similarly constructed µDystrophin (µDystro). These findings support a model in which utrophin-derived therapies might be used to treat clinical dystrophin deficiency, with a favorable immunologic profile and preserved function in the face of extreme miniaturization.

A robust Pax7EGFP mouse that enables the visualization of dynamic behaviors of muscle stem cells.

BACKGROUND: Pax7 is a transcription factor involved in the specification and maintenance of muscle stem cells (MuSCs). Upon injury, MuSCs leave their quiescent state, downregulate Pax7 and differentiate, contributing to skeletal muscle regeneration. In the majority of regeneration studies, MuSCs are isolated by fluorescence-activated sorting (FACS), based on cell surface markers. It is known that MuSCs are a heterogeneous population and only a small percentage of isolated cells are true stem cells that are able to self-renew. A strong Pax7 reporter line would be valuable to study the in vivo behavior of Pax7-expressing stem cells. METHODS: We generated and characterized the muscle properties of a new transgenic Pax7EGFP mouse. Utilizing traditional immunofluorescence assays, we analyzed whole embryos and muscle sections by fluorescence microscopy, in addition to whole skeletal muscles by 2-photon microscopy, to detect the specificity of EGFP expression. Skeletal muscles from Pax7EGFP mice were also evaluated in steady state and under injury conditions. Finally, MuSCs-derived from Pax7EGFP and control mice were sorted and analyzed by FACS and their myogenic activity was comparatively examined. RESULTS: Our studies provide a new Pax7 reporter line with robust EGFP expression, detectable by both flow cytometry and fluorescence microscopy. Pax7EGFP-derived MuSCs have identical properties to that of wild-type MuSCs, both in vitro and in vivo, excluding any positional effect due to the transgene insertion. Furthermore, we demonstrated high specificity of EGFP to label MuSCs in a temporal manner that recapitulates the reported Pax7 expression pattern. Interestingly, immunofluorescence analysis showed that the robust expression of EGFP marks cells in the satellite cell position of adult muscles in fixed and live tissues. CONCLUSIONS: This mouse could be an invaluable tool for the study of a variety of questions related to MuSC biology, including but not limited to population heterogeneity, polarity, aging, regeneration, and motility, either by itself or in combination with mice harboring additional genetic alterations.

Genetic Structure of the Rice Blast Pathogen (Magnaporthe oryzae) over a Decade in North Central California Rice Fields.

Rice blast, caused by the ascomycete Magnaporthe oryzae, is one of the most destructive rice diseases worldwide. Even though the disease has been present in California since 1996, there is no data for the pathogen population biology in the state. Using amplified fragment length polymorphisms and mating-type markers, the M. oryzae population diversity was investigated using isolates collected when the disease was first established in California and isolates collected a decade later. While in the 1990 samples, a single multilocus genotype (MLG) was identified (MLG1), over a decade later, we found 14 additional MLGs in the 2000 isolates. Some of these MLGs were found to infect the only rice blast-resistant cultivar (M-208) available for commercial production in California. The same samples also had a significant decrease of MLG1. MLG1 was found infecting the resistant rice cultivar M-208 on one occasion whereas MLG7 was the most common genotype infecting the M-208. MLG7 was identified in the 2000 samples, and it was not present in the M. oryzae population a decade earlier. Our results demonstrate a significant increase in genotypic diversity over time with no evidence of sexual reproduction and suggest a recent introduction of new virulent race(s) of the pathogen. In addition, our data could provide information regarding the durability of the Pi-z resistance gene of the M-208. This information will be critical to plant breeders in developing strategies for deployment of other rice blast resistance genes/cultivars in the future.

Suite of clinically relevant functional assays to address therapeutic efficacy and disease mechanism in the dystrophic mdx mouse.

Duchenne muscular dystrophy (DMD) is a progressive primary myodegenerative disease caused by a genetic deficiency of the 427-kDa cytoskeletal protein dystrophin. Despite its single-gene etiology, DMD's complex pathogenesis remains poorly understood, complicating the extrapolation from results of preclinical studies in genetic homologs to the design of informative clinical trials. Here we describe novel phenotypic assays which when applied to the mdx mouse resemble recently used primary end points for DMD clinical trials. By coupling force transduction, high-precision motion tracking, and respiratory measurements, we have achieved a suite of integrative physiological tests that provide novel insights regarding normal and pathological responses to muscular exertion. A common feature of these physiological assays is the precise tracking and analysis of volitional movement, thereby optimizing the relevance to clinical tests. Unexpectedly, the measurable biological distinction between dystrophic and control mice at early time points in the disease process is better resolved with these tests than with the majority of previously used, labor-intensive studies of individual muscle function performed ex vivo. For example, the dramatic loss of volitional movement following a novel, standardized grip test distinguishes control mice from mdx mice by a 17.4-fold difference of the means (3.5 ± 2.2 vs. 60.9 ± 12.1 units of activity, respectively; effect size 1.99). The findings have both mechanistic and translational implications of potential significance to the fields of basic myology and neuromuscular therapeutics.NEW & NOTEWORTHY This study uses novel phenotypic assays which when applied to the mdx mouse resemble recently used primary end points for DMD clinical trials. A measurable distinction between dystrophic and control mice was seen at early time points in vivo compared with invasive muscle studies performed ex vivo. These assays shed light on normal and pathological responses to muscular exertion and have significant mechanistic and translational implications for the fields of basic myology and neuromuscular therapeutics.

Statin Medication Use and Nosocomial Infection Risk in the Acute Phase of Stroke.

GOAL: Statins have immunomodulatory and peripheral anti-inflammatory properties that are independent of their lipid-lowering action. Whether these properties reduce the risk for developing poststroke infection is debated in clinical literature. We estimated the risk for developing nosocomial poststroke infection based on statin exposure in patients aged 18 or older hospitalized for ischemic stroke. MATERIALS AND METHODS: A consecutive sample of acute care hospital electronic medical records was retrospectively analyzed. Patients were assigned to the exposed cohort either when statin use preceded infection or statin medication was used, but no infection developed. The unexposed cohort included patients not on statins or initiating statins after infection developed. The association of statin exposure with infection was examined with conditional logistic regression adjusted for poststroke infection risk factors. Cochran-Mantel-Haenszel analyses examined the association of statin exposure and infection status within strata of binary predictor variables that increased infection risk. FINDINGS: Up to 1612 records were analyzed: 1151 in the exposed cohort and 461 in the unexposed cohort. Infection developed in 20% of the statin-exposed patients and in 41% of the statin-unexposed patients (P < .001). Exposure to statins reduced odds for developing nosocomial infection by 58% over no exposure (adjusted odds ratio = .418, P < .001). Statins lowered the infection risk for both sexes, patients with a nasogastric tube, and patients with dysphagia (P < .05). Statins did not change infection risk for patients with endotracheal intubation. CONCLUSIONS: In patients with ischemic stroke and without endotracheal intubation, statin medications were associated with reduced risk of nosocomial infections.

Global analyses of endonucleolytic cleavage in mammals reveal expanded repertoires of cleavage-inducing small RNAs and their targets.

In mammals, small RNAs are important players in post-transcriptional gene regulation. While their roles in mRNA destabilization and translational repression are well appreciated, their involvement in endonucleolytic cleavage of target RNAs is poorly understood. Very few microRNAs are known to guide RNA cleavage. Endogenous small interfering RNAs are expected to induce target cleavage, but their target genes remain largely unknown. We report a systematic study of small RNA-mediated endonucleolytic cleavage in mouse through integrative analysis of small RNA and degradome sequencing data without imposing any bias toward known small RNAs. Hundreds of small cleavage-inducing RNAs and their cognate target genes were identified, significantly expanding the repertoire of known small RNA-guided cleavage events. Strikingly, both small RNAs and their target sites demonstrated significant overlap with retrotransposons, providing evidence for the long-standing speculation that retrotransposable elements in mRNAs are leveraged as signals for gene targeting. Furthermore, our analysis showed that the RNA cleavage pathway is also present in human cells but affecting a different repertoire of retrotransposons. These results show that small RNA-guided cleavage is more widespread than previously appreciated. Their impact on retrotransposons in non-coding regions shed light on important aspects of mammalian gene regulation.

Systemic attenuation of the TGF-ß pathway by a single drug simultaneously rejuvenates hippocampal neurogenesis and myogenesis in the same old mammal.

Stem cell function declines with age largely due to the biochemical imbalances in their tissue niches, and this work demonstrates that aging imposes an elevation in transforming growth factor ß (TGF-ß) signaling in the neurogenic niche of the hippocampus, analogous to the previously demonstrated changes in the myogenic niche of skeletal muscle with age. Exploring the hypothesis that youthful calibration of key signaling pathways may enhance regeneration of multiple old tissues, we found that systemically attenuating TGF-ß signaling with a single drug simultaneously enhanced neurogenesis and muscle regeneration in the same old mice, findings further substantiated via genetic perturbations. At the levels of cellular mechanism, our results establish that the age-specific increase in TGF-ß1 in the stem cell niches of aged hippocampus involves microglia and that such an increase is pro-inflammatory both in brain and muscle, as assayed by the elevated expression of ß2 microglobulin (B2M), a component of MHC class I molecules. These findings suggest that at high levels typical of aged tissues, TGF-ß1 promotes inflammation instead of its canonical role in attenuating immune responses. In agreement with this conclusion, inhibition of TGF-ß1 signaling normalized B2M to young levels in both studied tissues.

Side effects of commonly prescribed analgesic medications.

Analgesics, including opioids, steroidal and nonsteroidal anti-inflammatory drugs, aspirin, acetaminophen, antiepileptics, and serotonin-norepinephrine reuptake inhibitors, are medications commonly used to treat many forms of pain. However, all of these agents may have significant adverse side effects. Adverse effects may occasionally be inseparable from desired effects. Side effects are often dose dependent and time dependent. It is critical that the prescribing practitioner and the dispensing pharmacist provide a thorough, understandable review of the potential side effects to all patients before these drugs are administered. Proper monitoring and follow-up during therapy are crucial.

Medication regimen complexity and hospital readmission for an adverse drug event.

BACKGROUND: Adverse drug events (ADEs) are costly, dangerous, and often preventable. Little is known about the link between medication regimen complexity and rehospitalization as a result of an ADE. OBJECTIVE: The objective of this study was to compare admission and discharge medication regimen complexity in 2 cohorts: patients readmitted for an ADE within 30 days and patients not readmitted for an ADE. METHODS: The study used a retrospective parallel-group case-control design. Participants from 4 urban acute care hospitals were included in the revisit cohort if they were rehospitalized within 30 days as a result of an adverse event coded as accidental poisoning. The no-revisit cohort was formed by randomly sampling patients with the same disease classification codes as the revisit group but without history of a readmission within 30 days. Complexity of medication regimens at the initial admission and discharge was quantified with the medication regimen complexity index (MRCI). RESULTS: The revisit group comprised 92 individuals and the no-revisit group, 228. The revisit group had a significantly higher MRCI score at admission and discharge than the no-revisit group (all P < .005). Receiver operating characteristic curves, used to determine a potential MRCI cutoff score for risk of an ADE, revealed MRCI scores of 8 or greater to optimally predict increased risk for readmission caused by an ADE. CONCLUSIONS: Complex medication regimens at hospital admission are predictive of rehospitalizations for ADEs. This finding suggests that medication regimen complexity be considered as a target for interventions to decrease the risk for readmission.

Accurate identification of A-to-I RNA editing in human by transcriptome sequencing.

RNA editing enhances the diversity of gene products at the post-transcriptional level. Approaches for genome-wide identification of RNA editing face two main challenges: separating true editing sites from false discoveries and accurate estimation of editing levels. We developed an approach to analyze transcriptome sequencing data (RNA-seq) for global identification of RNA editing in cells for which whole-genome sequencing data are available. We applied the method to analyze RNA-seq data of a human glioblastoma cell line, U87MG. Around 10,000 DNA-RNA differences were identified, the majority being putative A-to-I editing sites. These predicted A-to-I events were associated with a low false-discovery rate (∼5%). Moreover, the estimated editing levels from RNA-seq correlated well with those based on traditional clonal sequencing. Our results further facilitated unbiased characterization of the sequence and evolutionary features flanking predicted A-to-I editing sites and discovery of a conserved RNA structural motif that may be functionally relevant to editing. Genes with predicted A-to-I editing were significantly enriched with those known to be involved in cancer, supporting the potential importance of cancer-specific RNA editing. A similar profile of DNA-RNA differences as in U87MG was predicted for another RNA-seq data set obtained from primary breast cancer samples. Remarkably, significant overlap exists between the putative editing sites of the two transcriptomes despite their difference in cell type, cancer type, and genomic backgrounds. Our approach enabled de novo identification of the RNA editome, which sets the stage for further mechanistic studies of this important step of post-transcriptional regulation.

Analysis of transcriptome complexity through RNA sequencing in normal and failing murine hearts.

RATIONALE: Accurate and comprehensive de novo transcriptome profiling in heart is a central issue to better understand cardiac physiology and diseases. Although significant progress has been made in genome-wide profiling for quantitative changes in cardiac gene expression, current knowledge offers limited insights to the total complexity in cardiac transcriptome at individual exon level. OBJECTIVE: To develop more robust bioinformatic approaches to analyze high-throughput RNA sequencing (RNA-Seq) data, with the focus on the investigation of transcriptome complexity at individual exon and transcript levels. METHODS AND RESULTS: In addition to overall gene expression analysis, the methods developed in this study were used to analyze RNA-Seq data with respect to individual transcript isoforms, novel spliced exons, novel alternative terminal exons, novel transcript clusters (ie, novel genes), and long noncoding RNA genes. We applied these approaches to RNA-Seq data obtained from mouse hearts after pressure-overload-induced by transaortic constriction. Based on experimental validations, analyses of the features of the identified exons/transcripts, and expression analyses including previously published RNA-Seq data, we demonstrate that the methods are highly effective in detecting and quantifying individual exons and transcripts. Novel insights inferred from the examined aspects of the cardiac transcriptome open ways to further experimental investigations. CONCLUSIONS: Our work provided a comprehensive set of methods to analyze mouse cardiac transcriptome complexity at individual exon and transcript levels. Applications of the methods may infer important new insights to gene regulation in normal and disease hearts in terms of exon utilization and potential involvement of novel components of cardiac transcriptome.

Association of antidepressant medication therapy with inpatient rehabilitation outcomes for stroke, traumatic brain injury, or traumatic spinal cord injury.

OBJECTIVE: To study whether outcomes in patients who have undergone inpatient rehabilitation for stroke, traumatic brain injury (TBI), or traumatic spinal cord injury (TSCI) differ based on antidepressant medication (ADM) use. DESIGN: Retrospective cohort study of 867 electronic medical records of patients receiving inpatient rehabilitation for stroke, TBI, or TSCI. Four cohorts were formed within each rehabilitation condition: patients with no history of ADM use and no indication of history of depression; patients with no history of ADM use but with a secondary diagnostic code for a depressive illness; patients with a history of ADM use prior to and during inpatient rehabilitation; and patients who began ADM therapy in inpatient rehabilitation. SETTING: Freestanding inpatient rehabilitation facility (IRF). PARTICIPANTS: Patients diagnosed with stroke (n=625), TBI (n=175), and TSCI (n=67). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: FIM, rehabilitation length of stay (LOS), deviation between actual LOS and expected LOS, and functional gain per day. RESULTS: In each impairment condition, patients initiating ADM therapy in inpatient rehabilitation had longer LOS than patients in the same impairment condition on ADM at IRF admission, and had significantly longer LOS than patients with no history of ADM use and no diagnosis of depression (P<.05). LOS for patients initiating ADM therapy as inpatients even exceeded LOS for patients without ADM history, but who had a diagnosis for a depressive disorder. Deviation in LOS was significantly larger in the stroke and TBI groups initiating ADM in IRF than their counterparts with no history of ADM use, illustrating that the group initiating ADM therapy in rehabilitation significantly exceeded expected LOS. Increased LOS did not translate into functional gains, and in fact, functional gain per day was lower in the group initiating ADM therapy in IRF. CONCLUSIONS: Explanations for unexpectedly long LOS in patients initiating ADM in inpatient rehabilitation focus on the potential for ADM to inhibit therapy-driven remodeling of the nervous system when initiated close in time to nervous system injury, or the possibility that untreated sequelae (eg, depressive symptoms or fatigue) were limiting progress in therapy, which triggered ADM treatment.

Contrasting genetic structure between Magnaporthe grisea populations associated with the golf course turfgrasses Lolium perenne (perennial ryegrass) and Pennisetum clandestinum (kikuyugrass).

Gray leaf spot (GLS) disease of perennial ryegrass (Lolium perenne) and kikuyugrass (Pennisetum clandestinum) in golf courses in California was first noted in 2001 and 2003, respectively, and within 5 years had become well established. The causal agent of the disease is the fungus Magnaporthe grisea, which is known to consist primarily of clonal lineages that are highly host specific. Therefore, our objective was to investigate host specificity and population dynamics among isolates associated primarily from perennial ryegrass and kikuyugrass since the disease emerged at similar times in California. We also obtained isolates from additional hosts (tall fescue, St. Augustinegrass, weeping lovegrass, and rice) and from the eastern United States for comparative purposes. A total of 38 polymorphic amplified fragment length polymorphism makers were scored from 450 isolates which clustered by host with high bootstrap support (71 to 100%). Genetic structure between kikuyugrass and perennial ryegrass isolates differed significantly. Isolates from kikuyugrass were genotypically diverse (n = 34), possessed both mating types, and some tests for random mating could not be rejected, whereas isolates from perennial ryegrass were less genotypically diverse (n = 10) and only consisted of a single mating type. Low genotypic diversity was also found among the other host specific isolates which also only consisted of a single mating type. This is the first study to document evidence for the potential of sexual reproduction to occur in M. grisea isolates not associated with rice (Oryza sativa). Moreover, given the significant host specificity and contrasting genetic structures between turfgrass-associated isolates, the recent emergence of GLS on various grass hosts in California suggests that potential cultural practices or environmental changes have become conducive for the disease and that the primary inoculum may have already been present in the state, despite the fact that two genotypes associated with perennial ryegrass and St. Augustinegrass in California were the same as isolates collected from the eastern United States.

Population structure of Rhizoctonia oryzae-sativae in California rice fields.

Six pairs of single-locus microsatellite primers were developed to study the population structure of Rhizoctonia oryzae-sativae, the cause of aggregate sheath spot disease of rice, among and within three rice-growing areas in California over a 3-year period. A high level of gene flow among growing areas was indicated by low population subdivision according to analysis of molecular variance and moderate to no population differentiation between pairs of populations based on the fixation index (F(ST)). Gametic equilibrium of most pairs of microsatellite loci, high numbers of unique multilocus genotypes, and high genotypic diversity indicated extensive sexual recombination within growing areas. Because there was little differentiation among populations in all hierarchical levels, including among growing areas within sampling years, fields within growing areas, and corners within individual fields, a high level of gene flow was revealed in all levels. Basidiospores were likely the main vehicle of gene flow among populations, including short and long distances. Asexual inocula (sclerotia and mycelia) probably overwinter because a few clones were detected over a 2-year period within the same field. A few clones were shared among fields but were not commonly shared among growing areas.

Controller-area-network bus control and monitor system for a radio astronomy interferometer.

We describe the design and implementation of a controller-area-network bus (CANbus) monitor and control system for a millimeter wave interferometer. The Combined Array for Research in Millimeter-wave Astronomy (CARMA) is a 15-antenna connected-element interferometer for astronomical imaging, created by the merger of two university observatories. Its new control system relies on a central computer supervising a variety of subsystem computers, many of which control distributed intelligent nodes over CANbus. Subsystems are located in the control building and in individual antennas and communicate with the central computer via Ethernet. Each of the CAN modules has a very specific function, such as reading an antenna encoder or tuning an oscillator. Hardware for the modules was based on a core design including a commercial CANbus-enabled single-board computer and some standard circuitry for interfacing to peripherals. Hardware elements were added or changed as necessary for the specific module types. Similarly, a base set of embedded code was implemented for essential common functions such as CAN message handling and time keeping and extended to implement the required functionality for the different hardware. Using a standard CAN messaging protocol designed to fit the requirements of CARMA and a well-defined interface to the high-level software allowed separate development of high-level code and embedded code with minimal integration problems. Over 30 module types have been implemented and successfully deployed in CARMA, which is now delivering excellent new science data.