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Pathomechanistic studies of neurodegenerative diseases have documented the toxic effects of mutant protein expression, misfolding, and aggregation. However, alterations in the expression of the corresponding wild-type (WT) gene, due to either variations in copy number or transcriptional regulation, have also been linked to Alzheimer's and Parkinson's diseases. Another striking example of this mutant and WT duality is spinocerebellar ataxia type 1 (SCA1) caused by an ATXN1 polyglutamine protein, although subtle variations in WT AXTN1 levels also lead to ataxia. In this issue of Genes & Development, Nitschke and colleagues (pp. 1147–1160) delve into posttranscriptional events that fine-tune ATXN1 expression and uncover a key role for 5′ untranslated region (5′ UTR)–miR760 interactions. Thus, this study not only provides significant insights into the complexities of modulating the expression of a dosage-sensitive gene but also highlights the critical importance of identifying noncoding polymorphisms as disease risk factors.

Short tandem repeats (STRs) are prone to expansion mutations that cause multiple hereditary neurological and neuromuscular diseases. To study pathomechanisms using mouse models that recapitulate the tissue specificity and developmental timing of an STR expansion gene, we used rolling circle amplification and CRISPR/Cas9-mediated genome editing to generate Dmpk CTG expansion (CTGexp) knockin models of myotonic dystrophy type 1 (DM1). We demonstrate that skeletal muscle myoblasts and brain choroid plexus epithelial cells are particularly susceptible to Dmpk CTGexp mutations and RNA missplicing. Our results implicate dysregulation of muscle regeneration and cerebrospinal fluid homeostasis as early pathogenic events in DM1.

Purpose: The purpose of this article is to review and analyze reported cases of ocular venous air embolism (OVAE) to develop a reliable clinical definition of OVAE and effective prevention strategies. Methods: We reviewed all reports of suspected air embolism during vitrectomy published in PubMed since the introduction of pars plana vitrectomy, and 5 cases found elsewhere and separately reported concurrent with this review. Results: OVAE is a precipitous drop in end-tidal CO2, a choroidal detachment, or a choroidal wound, followed by signs of impending or actual cardiovascular collapse, during vitrectomy air infusion. In each case meeting the above clinical definition, entrained air was found whenever it was sought (8/8, 100%), either by antemortem imaging or postmortem forensic investigations. Most OVAE cases were fatal (9/13, 69%), with 8 of 9 deaths (89%) occurring the day of surgery. Conclusions: OVEA is a rare but usually fatal complication of air infusion into the eye during vitrectomy. Although received with skepticism when first reported (2005), OVAE may be the most lethal type of surgical air embolization because of its high entrainment pressure and proximity to the heart. Because the effective response time to avoid a fatal OVAE outcome can be less than 1 minute, use of preventive measures is critical—most notably a “time out” before air infusion to confirm infusion cannula positioning, and immediate cessation of air infusion if choroidal detachment is detected.

Background Mosquito feeding assays using venous blood are commonly used for evaluating the transmission potential of malaria infected individuals. To improve the accuracy of these assays, care must be taken to prevent premature activation or inactivation of gametocytes before they are fed to mosquitoes. This can be challenging in the field where infected individuals and insectary facilities are sometimes very far apart. In this study, a simple, reliable, field applicable method is presented for storage and transport of gametocyte infected blood using a thermos flask. Methods The optimal storage conditions for maintaining the transmissibility of gametocytes were determined initially using cultured Plasmodium falciparum gametocytes in standard membrane feeding assays (SMFAs). The impact of both the internal thermos water temperature (35.5 to 37.8 °C), and the external environmental temperature (room temperature to 42 °C) during long-term (4 h) storage, and the impact of short-term (15 min) temperature changes (room temp to 40 °C) during membrane feeding assays was assessed. The optimal conditions were then evaluated in direct membrane feeding assays (DMFAs) in Burkina Faso and The Gambia where blood from naturally-infected gametocyte carriers was offered to mosquitoes immediately and after storage in thermos flasks. Results Using cultured gametocytes in SMFAs it was determined that an internal thermos water temperature of 35.5 °C and storage of the thermos flask between RT (~ 21.3 °C) and 32 °C was optimal for maintaining transmissibility of gametocytes for 4 h. Short-term storage of the gametocyte infected blood for 15 min at temperatures up to 40 °C (range: RT, 30 °C, 38 °C and 40 °C) did not negatively affect gametocyte infectivity. Using samples from natural gametocyte carriers (47 from Burkina Faso and 16 from The Gambia), the prevalence of infected mosquitoes and the intensity of oocyst infection was maintained when gametocyte infected blood was stored in a thermos flask in water at 35.5 °C for up to 4 h. Conclusions This study determines the optimal long-term (4 h) storage temperature for gametocyte infected blood and the external environment temperature range within which gametocyte infectivity is unaffected. This will improve the accuracy, reproducibility, and utility of DMFAs in the field, and permit reliable comparative assessments of malaria transmission epidemiology in different settings.

Myotonic dystrophy type I (DM1) is a multisystemic autosomal-dominant inherited human disorder that is caused by CTG microsatellite repeat expansions (MREs) in the 3' untranslated region of DMPK. Toxic RNAs expressed from such repetitive sequences can be eliminated using CRISPR-mediated RNA targeting, yet evidence of its in vivo efficacy and durability is lacking. Here, using adult and neonatal mouse models of DM1, we show that intramuscular or systemic injections of adeno-associated virus (AAV) vectors encoding nuclease-dead Cas9 and a single-guide RNA targeting CUG repeats results in the expression of the RNA-targeting Cas9 for up to three months, redistribution of the RNA-splicing protein muscleblind-like splicing regulator 1, elimination of foci of toxic RNA, reversal of splicing biomarkers and amelioration of myotonia. The sustained reversal of DM1 phenotypes provides further support that RNA-targeting Cas9 is a viable strategy for treating DM1 and other MRE-associated diseases.

Purpose: The purpose of this case series is to report 5 new cases of ocular venous air embolism (OVAE). We define OVAE as a precipitous drop in end-tidal carbon dioxide, a choroidal detachment, or a choroidal wound, followed by signs of impending or actual cardiovascular collapse, during vitrectomy air infusion. Methods: A case report series was retrospectively reviewed. Results: Four of the 5 OVAE cases (80%) were fatal, occurring in conjunction with repair of rhegmatogenous retinal detachment (2 cases); a large surgical wound of the choroid (1 case); and vitrectomy repair of an injured eye (1 case). One patient survived OVAE during choroidal melanoma endoresection after prompt discontinuance of air infusion by anesthesia personnel previously alerted to the OVAE risk. Conclusions: OVAE is a rare but usually fatal complication of air infusion into the eye during vitrectomy. Because the effective response time to avoid a fatal OVAE outcome can be less than 1 minute, use of preventive measures is critical.

Significance Myotonic dystrophy type 1 (DM1) is a model for RNA-mediated disease in microsatellite expansion disorders. DM1 is caused by CTG expansions (CTGexp) and expression of CUGexp RNAs that sequester muscleblind-like (MBNL) proteins, while also triggering hyperphosphorylation of CUGBP1/ETR3-like factor 1 (CELF1). These proteins regulate developmental transitions in RNA processing, so DM1 is characterized by retention of fetal RNA processing patterns in adults. Although current evidence indicates that CELF1 is a specific antagonist of MBNL activity, this study reveals that another protein, HNRNPA1, is also downregulated during normal development but upregulated in DM1, where it also induces fetal splicing shifts. Thus, DM1 disease results from an imbalance in the expression of multiple RNA processing factors important for both proliferation and differentiation., Studies on myotonic dystrophy type 1 (DM1) have led to the RNA-mediated disease model for hereditary disorders caused by noncoding microsatellite expansions. This model proposes that DM1 disease manifestations are caused by a reversion to fetal RNA processing patterns in adult tissues due to the expression of toxic CUG RNA expansions (CUGexp) leading to decreased muscleblind-like, but increased CUGBP1/ETR3-like factor 1 (CELF1), alternative splicing activities. Here, we test this model in vivo, using the mouse HSALR poly(CUG) model for DM1 and recombinant adeno-associated virus (rAAV)-mediated transduction of specific splicing factors. Surprisingly, systemic overexpression of HNRNPA1, not previously linked to DM1, also shifted DM1-relevant splicing targets to fetal isoforms, resulting in more severe muscle weakness/myopathy as early as 4 to 6 wk posttransduction, whereas rAAV controls were unaffected. Overexpression of HNRNPA1 promotes fetal exon inclusion of representative DM1-relevant splicing targets in differentiated myoblasts, and HITS-CLIP of rAAV-mycHnrnpa1-injected muscle revealed direct interactions of HNRNPA1 with these targets in vivo. Similar to CELF1, HNRNPA1 protein levels decrease during postnatal development, but are elevated in both regenerating mouse muscle and DM1 skeletal muscle. Our studies suggest that CUGexp RNA triggers abnormal expression of multiple nuclear RNA binding proteins, including CELF1 and HNRNPA1, that antagonize MBNL activity to promote fetal splicing patterns.

Myotonic dystrophy (DM) is a multisystemic disorder caused by microsatellite expansion mutations in two unrelated genes leading to similar, yet distinct, diseases. DM disease presentation is highly variable and distinguished by differences in age-of-onset and symptom severity. In the most severe form, DM presents with congenital onset and profound developmental defects. At the molecular level, DM pathogenesis is characterized by a toxic RNA gain-of-function mechanism that involves the transcription of noncoding microsatellite expansions. These mutant RNAs disrupt key cellular pathways, including RNA processing, localization, and translation. In DM, these toxic RNA effects are predominantly mediated through the modulation of the muscleblind-like and CUGBP and ETR-3-like factor families of RNA binding proteins (RBPs). Dysfunction of these RBPs results in widespread RNA processing defects culminating in the expression of developmentally inappropriate protein isoforms in adult tissues. The tissue that is the focus of this review, skeletal muscle, is particularly sensitive to mutant RNA-responsive perturbations, as patients display a variety of developmental, structural, and functional defects in muscle. Here, we provide a comprehensive overview of DM1 and DM2 clinical presentation and pathology as well as the underlying cellular and molecular defects associated with DM disease onset and progression. Additionally, fundamental aspects of skeletal muscle development altered in DM are highlighted together with ongoing and potential therapeutic avenues to treat this muscular dystrophy. © 2018 American Physiological Society. Compr Physiol 8:509-553, 2018.

Summary: Myotonic dystrophy types 1 (DM1) and 2 (DM2) are dominantly inherited neuromuscular disorders caused by a toxic gain of function of expanded CUG and CCUG repeats, respectively. Although both disorders are clinically similar, congenital myotonic dystrophy (CDM), a severe DM form, is found only in DM1. CDM is also characterized by muscle fiber immaturity not observed in adult DM, suggesting specific pathological mechanisms. Here, we revealed upregulation of the interleukin-6 (IL-6) myokine signaling pathway in CDM muscles. We also found a correlation between muscle immaturity and not only IL-6 expression but also expanded CTG repeat length and CpG methylation status upstream of the repeats. Aberrant CpG methylation was associated with transcriptional dysregulation at the repeat locus, increasing the toxic RNA burden that upregulates IL-6. Because the IL-6 pathway is involved in myocyte maturation and muscle atrophy, our results indicate that enhanced RNA toxicity contributes to severe CDM phenotypes through aberrant IL-6 signaling. : Congenital myotonic dystrophy (CDM) manifests characteristic genetic (very large CTG repeat expansions), epigenetic (CpG hypermethylation upstream of the repeat), and phenotypic (muscle immaturity) features not seen in adult DM. Nakamori et al. find phenotype-genotype and epigenotype correlation in CDM muscle and reveal involvement of the IL-6 myokine signaling pathway in the disease process. Keywords: CTCF, ER stress, IL-6, muscular dystrophy, NF-κB, trinucleotide, cytokine, splicing

In this issue of Molecular Cell, Yap et al. (2018) identify a novel lncRNA (PNCTR) that contains short tandem repeats that trap the RNA splicing factor PTBP1 in the perinucleolar compartment and link this sequestration activity to cancer cell development.

IntroductionA large proportion of malaria-infected individuals in endemic areas do not experience symptoms that prompt treatment-seeking. These asymptomatically infected individuals may retain their infections for many months during which sexual-stage parasites (gametocytes) are produced that may be transmissible to mosquitoes. Reductions in malaria transmission could be achieved by detecting and treating these infections early. This study assesses the impact of enhanced community case management (CCM) and monthly screening and treatment (MSAT) on the prevalence and transmissibility of malaria infections.Methods and analysisThis cluster-randomised trial will take place in Sapone, an area of intense, highly seasonal malaria in Burkina Faso. In total, 180 compounds will be randomised to one of three interventions: arm 1 - current standard of care with passively monitored malaria infections; arm 2 - standard of care plus enhanced CCM, comprising active weekly screening for fever, and detection and treatment of infections in fever positive individuals using conventional rapid diagnostic tests (RDTs); or arm 3 - standard of care and enhanced CCM, plus MSAT using RDTs. The study will be conducted over approximately 18 months covering two high-transmission seasons and the intervening dry season. The recruitment strategy aims to ensure that overall transmission and force of infection is not affected so we are able to continuously evaluate the impact of interventions in the context of ongoing intense malaria transmission. The main objectives of the study are to determine the impact of enhanced CCM and MSAT on the prevalence and density of parasitaemia and gametocytaemia and the transmissibility of infections. This will be achieved by molecular detection of infections in all study participants during start and end season cross-sectional surveys and routine sampling of malaria-positive individuals to assess their infectiousness to mosquitoes.Ethics and disseminationThe study has been reviewed and approved by the London School of Hygiene and Tropical Medicine (LSHTM) (Review number: 14724) and The Centre National de Recherche et de Formation sur le Paludisme institutional review board (IRB) (Deliberation N° 2018/000002/MS/SG/CNRFP/CIB) and Burkina Faso national medical ethics committees (Deliberation N° 2018-01-010).Findings of the study will be shared with the community via local opinion leaders and community meetings. Results may also be shared through conferences, seminars, reports, theses and peer-reviewed publications; disease occurrence data and study outcomes will be shared with the Ministry of Health. Data will be published in an online digital repository.Trial registration numberNCT03705624.

Short tandem repeat (STR) or microsatellite, expansions underlie more than 50 hereditary neurological, neuromuscular and other diseases, including myotonic dystrophy types 1 (DM1) and 2 (DM2). Current disease models for DM1 and DM2 propose a common pathomechanism, whereby the transcription of mutant DMPK (DM1) and CNBP (DM2) genes results in the synthesis of CUG and CCUG repeat expansion (CUGexp, CCUGexp) RNAs, respectively. These CUGexp and CCUGexp RNAs are toxic since they promote the assembly of ribonucleoprotein (RNP) complexes or RNA foci, leading to sequestration of Muscleblind-like (MBNL) proteins in the nucleus and global dysregulation of the processing, localization and stability of MBNL target RNAs. STR expansion RNAs also form phase-separated gel-like droplets both in vitro and in transiently transfected cells, implicating RNA-RNA multivalent interactions as drivers of RNA foci formation. Importantly, the nucleation and growth of these nuclear foci and transcript misprocessing are reversible processes and thus amenable to therapeutic intervention. In this review, we provide an overview of potential DM1 and DM2 pathomechanisms, followed by a discussion of MBNL functions in RNA processing and how multivalent interactions between expanded STR RNAs and RNA-binding proteins (RBPs) promote RNA foci assembly.

BACKGROUND Juvenile osteochondritis dissecans (OCD) of the medial femoral condyle (MFC) is one of the most common causes of knee pain in adolescents. Wilson sign reproduces knee pain with internal rotation of the tibia during extension of the knee from 90 to 30 degrees due to impingement of the tibial eminence on the MFC. This impingement may result in microtrauma and contribute to lesion formation. The purpose of this study was to evaluate anatomic factors that may increase the likelihood of impingement by using magnetic resonance imaging scans of patients with MFC OCD lesions to measure tibial eminence height and femoral notch width. METHODS A retrospective, case-control study was performed using the radiology database at our institution between July 2009 and February 2014. Magnetic resonance imagings of patients with MFC OCD lesions and matched controls were identified. For each patient, tibial eminence height and femoral notch width were measured and then normalized for patient size [creating the tibial eminence height normalized, and the notch width index (NWI), respectively]. Values for OCD and control knees were compared using Student t test. Interrater and intrarater reliability were calculated using intraclass correlation coefficients. RESULTS Thirty-five MFC OCD patients and matched controls were identified. Comparison of the groups showed a significantly smaller NWI in MFC OCD knees than in the matched controls (0.2620±0.0248 vs. 0.2886 ±0.0323, P=0.0003). There was no difference in tibial eminence height normalized between groups (0.1387±0.0161 vs. 0.1428±0.0108, P=0.21). Interrater and intrarater reliability of all measurements was good to excellent (0.81 to 1.00) when measurements were made using bony margins. CONCLUSIONS Knees with MFC OCD lesions have significantly smaller NWIs than matched controls. This anatomic factor may increase the likelihood of tibial eminence impingement and contribute to OCD lesion formation. LEVEL OF EVIDENCE Level III-case-control study.

Significance A number of hereditary neurological and neuromuscular diseases are caused by the abnormal expansion of short tandem repeats, or microsatellites, resulting in the expression of repeat expansion RNAs and proteins with pathological properties. Although these microsatellite expansions may occur in either the coding or noncoding regions of the genome, trinucleotide CNG repeats predominate in exonic coding and untranslated regions while intron mutations vary from trinucleotide to hexanucleotide GC-rich, and A/AT-rich, repeats. Here, we use transcriptome analysis combined with complementary experimental approaches to demonstrate that GC-rich intronic expansions are selectively associated with host intron retention. Since these intron retention events are detectable in both affected tissues and peripheral blood, they provide a sensitive and disease-specific diagnostic biomarker.

Matrix-induced autologous chondrocyte implantation (MACI) is an established technique to treat articular cartilage defects in the knee. Traditionally, the chondral graft is harvested arthroscopically and then implanted via an open or mini-open technique. However, an arthroscopic implantation technique carries with it the potential for reduced pain, improved rehabilitation, and reduced arthrofibrosis. The arthroscopic implantation first involves a standard arthroscopic biopsy followed by cell culture and seeding onto a collagen membrane. The implantation procedure itself is performed 6–8 weeks later. A standard arthroscopy is performed and the defect is debrided to stable vertical walls using curettes and arthroscopic shavers. All irrigation fluid is subsequently evacuated such that the rest of the procedure is performed as a dry arthroscopy. The defect is sized using a graduated probe. If required, a template may be cut out of the supplied membrane. A wide-bore valveless cannula, inserted through the working portal, permits the repeated atraumatic passage of the prepared membrane. The probe is used to position the graft and check for size. A definitive graft is then cut and placed in the knee. Fibrin glue is applied to the base of the defect, the membrane is positioned, and an embolectomy or indwelling catheter balloon is inflated to apply even pressure to the graft while the glue sets. A validated, accelerated rehabilitation program is recommended, with full weight-bearing at 8-week post-procedure. Results using this technique have shown this to be a safe, reliable, and reproducible procedure with good clinical and radiological outcomes at the 5-year follow-up.

Myotonic dystrophy (DM) is a progressive, multisystem disorder affecting skeletal muscle, heart, and central nervous system. In both DM1 and DM2, microsatellite expansions of CUG and CCUG RNA repeats, respectively, accumulate and disrupt functions of alternative splicing factors, including muscleblind (MBNL) proteins. Grey matter loss and white matter changes, including the corpus callosum, likely underlie cognitive and executive function deficits in DM patients. However, little is known how cerebral cortical circuitry changes in DM. Here, flavoprotein optical imaging was used to assess local and contralateral responses to intracortical motor cortex stimulation in DM-related mouse models. In control mice, brief train stimulation generated ipsilateral and contralateral homotopic fluorescence increases, the latter mediated by the corpus callosum. Single pulse stimulation produced an excitatory response with an inhibitory-like surround response mediated by GABAA receptors. In a mouse model of DM2 (Mbnl2 KO), we observed prolonged and increased responsiveness to train stimulation and loss of the inhibition from single pulse stimulation. Conversely, mice overexpressing human MBNL1 (MBNL1-OE) exhibited decreased contralateral response to train stimulation and reduction of inhibitory-like surround to single pulse stimulation. Therefore, altering levels of two key DM-associated splicing factors modifies functions of local cortical circuits and contralateral responses mediated through the corpus callosum.

The dominantly inherited, multi-systemic disease myotonic dystrophy type I (DM1) is caused by triplet repeat CTG expansions in the DMPK gene and is the most common form of adult-onset muscular dystrophy. Elimination of the toxic, repetitive CUG RNA constitutes a therapeutic for this disease. We report an RNA-targeting Cas9 (RCas9) system that supports efficient reversal of DM1 phenotypes via delivery to adult poly(CUG) DM1 mouse muscle using adeno-associated virus (AAV). We observe elimination of CUG RNA, restoration of CUG foci-associated Mbnl1 protein to wild-type subcellular localization, correction of DM1-type alternative splicing patterns in candidate genes including the voltage-gated chloride channel 1 (Clcn1) responsible for characteristic myotonia, recovery of Clcn1 staining, and reduction in centralized myonuclei. Our results establish RCas9 as a potential long-term in vivo therapeutic for DM1.One Sentence SummaryA repurposed CRISPR system termed RNA-targeting Cas9 reverses the molecular pathology associated with the most common type of adult onset muscular dystrophy in adult mouse muscle.

Myotonic dystrophy type 1 (DM1) is a CTG microsatellite expansion (CTGexp) disorder caused by expression of CUGexp RNAs. These mutant RNAs alter the activities of RNA processing factors, including MBNL proteins, leading to re-expression of fetal isoforms in adult tissues and DM1 pathology. While this pathogenesis model accounts for adult-onset disease, the molecular basis of congenital DM (CDM) is unknown. Here, we test the hypothesis that disruption of developmentally regulated RNA alternative processing pathways contributes to CDM disease. We identify prominent alternative splicing and polyadenylation abnormalities in infant CDM muscle, and, although most are also misregulated in adult-onset DM1, dysregulation is significantly more severe in CDM. Furthermore, analysis of alternative splicing during human myogenesis reveals that CDM-relevant exons undergo prenatal RNA isoform transitions and are predicted to be disrupted by CUGexp-associated mechanisms in utero. To test this possibility and the contribution of MBNLs to CDM pathogenesis, we generated mouse Mbnl double (Mbnl1; Mbnl2) and triple (Mbnl1; Mbnl2; Mbnl3) muscle-specific knockout models that recapitulate the congenital myopathy, gene expression, and spliceopathy defects characteristic of CDM. This study demonstrates that RNA misprocessing is a major pathogenic factor in CDM and provides novel mouse models to further examine roles for cotranscriptional/post-transcriptional gene regulation during development.

Summary Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington's disease, and C9orf72 -linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes. Here, we describe the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs. We observe specific targeting and efficient elimination of microsatellite repeat expansion RNAs both when exogenously expressed and in patient cells. Importantly, RNA-targeting Cas9 (RCas9) reverses hallmark features of disease including elimination of RNA foci among all conditions studied (DM1, DM2, C9-ALS, polyglutamine diseases), reduction of polyglutamine protein products, relocalization of repeat-bound proteins to resemble healthy controls, and efficient reversal of DM1-associated splicing abnormalities in patient myotubes. Finally, we report a truncated RCas9 system compatible with adeno-associated viral packaging. This effort highlights the potential of RCas9 for human therapeutics.

SummaryTranscription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs’ endothelial corneal dystrophy, andC9orf72-ALS/FTD. Eliminating or reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that a deactivated form of the Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction in the abundance of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences. Aberrant splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of DM1, DM2, andC9orf72-ALS/FTD cells was drastically decreased. Systemic delivery of dCas9/gRNA by adeno-associated virus led to reductions in pathological RNA foci, rescue of chloride channel 1 protein expression, and decreased myotonia. These observations suggest that transcription of microsatellite repeat-containing RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention.

To compare life expectancy (LE) losses attributable to three imaging strategies for appendicitis in adults-computed tomography (CT), ultrasonography (US) followed by CT for negative or indeterminate US results, and magnetic resonance (MR) imaging-by using a decision-analytic model.In this model, for each imaging strategy, LE losses for 20-, 40-, and 65-year-old men and women were computed as a function of five key variables: baseline cohort LE, test performance, surgical mortality, risk of death from delayed diagnosis (missed appendicitis), and LE loss attributable to radiation-induced cancer death. Appendicitis prevalence, test performance, mortality rates from surgery and missed appendicitis, and radiation doses from CT were elicited from the published literature and institutional data. LE loss attributable to radiation exposure was projected by using a separate organ-specific model that accounted for anatomic coverage during a typical abdominopelvic CT examination. One- and two-way sensitivity analyses were performed to evaluate effects of model input variability on results.Outcomes across imaging strategies differed minimally-for example, for 20-year-old men, corresponding LE losses were 5.8 days (MR imaging), 6.8 days (combined US and CT), and 8.2 days (CT). This order was sensitive to differences in test performance but was insensitive to variation in radiation-induced cancer deaths. For example, in the same cohort, MR imaging sensitivity had to be 91% at minimum (if specificity were 100%), and MR imaging specificity had to be 62% at minimum (if sensitivity were 100%) to incur the least LE loss. Conversely, LE loss attributable to radiation exposure would need to decrease by 74-fold for combined US and CT, instead of MR imaging, to incur the least LE loss.The specific imaging strategy used to diagnose appendicitis minimally affects outcomes. Paradigm shifts to MR imaging owing to concerns over radiation should be considered only if MR imaging test performance is very high.

BackgroundMalaria elimination and its ultimate eradication will require drugs targeting all stages of the parasite’s life cycle. Yet, very few drugs are known to be effective on the sexual stages (gametocytes) of Plasmodium falciparum. Artemisinin-based combination therapy (ACT) has been shown to have some early-stage gametocytocidal effects on in vitro and in feeding experiments. However, field studies showed that artesunate reduces but does not prevent post-treatment transmission of P. falciparum to mosquitos.Methods763 children and adult patients with acute uncomplicated Plasmodium sp. malaria were included in a phase IIIb/IV comparative, randomised, multi-centre, open label, parallel 3-arm clinical trial to assess safety and efficacy of repeated administration of pyronaridine-artesunate, dihydroartemisinin-piperaquine or artemether-lumefantrine or artesunate-amodiaquine over a two-year period. Drugs were given based on the body weight and volunteers were followed up for 42 days. Clinical signs and symptoms were recorded and filter paper and blood smears collected during each visit. Malaria parasites were assessed and parasite density development stages determined by light microscopy.ResultsP. falciparum gametocyte was 1.9%, during the two years of follow-up. From the three treatment arms, artesunate-amodiaquine was the arm bearing more P. falciparum gametocyte with 68.7%, dihydroartemisinin-piperaquine accounted for 6.3% and pyronaridine-artesunate for 25%. P. falciparum gametocyte was more pronounced in populations having parasite density ≤1 00 000 parasites/µl compared to above parasitaemia.ConclusionRepeated ACTs treatment didn’t clear P. falciparum gametocyte in a population infected with uncomplicated malaria.

Objective To develop RNA splicing biomarkers of disease severity and therapeutic response in myotonic dystrophy type 1 (DM1) and type 2 (DM2).

Background Bohler's historical tuber-joint angle of the calcaneus has been used since 1931. Surprisingly, there is a paucity of literature on its use. Objectives To confirm the normal range for Bohler's angle and determine the angle with the highest accuracy in the diagnosis of calcaneal fractures. Methods A retrospective cohort study was performed. The study cohort comprised 424 patients spanning a 5-year period from April 2005 to March 2010. Bohler's angle was measured by two independent observers on lateral x-ray study using the digital angle tool from the Picture Archival and Communication System. Data were analyzed using Stata 8 statistical software package. Results The mean Bohler's angle in patients without calcaneal fracture was 29.4°. In this group there was no difference in Bohler's angle between male and female patients, left and right feet, or across age. In those patients with calcaneal fractures, a Bohler's angle below 25° was moderately predictive of calcaneal fracture (sensitivity = 100%, specificity = 82%), an angle below 23° was highly predictive of calcaneal fracture (sensitivity = 100%, specificity = 89%), and an angle below 21° was strongly suggestive of calcaneal fracture (sensitivity = 99%, specificity = 99%). A Bohler's angle of ≤ 20° had the highest diagnostic accuracy. Conclusion A Bohler's angle of 20° or less is highly accurate in determining the presence or absence of calcaneal fracture. Bohler’s angle serves as a useful screening tool in fracture diagnosis.

Myotonic dystrophy (DM) is a multi‐systemic disease that impacts cardiac and skeletal muscle as well as the central nervous system (CNS). DM is unusual because it is an RNA‐mediated disorder due to the expression of toxic microsatellite expansion RNAs that alter the activities of RNA processing factors, including the muscleblind‐like (MBNL) proteins. While these mutant RNAs inhibit MBNL1 splicing activity in heart and skeletal muscles, Mbnl1 knockout mice fail to recapitulate the full‐range of DM symptoms in these tissues. Here, we generate mouse Mbnl compound knockouts to test the hypothesis that Mbnl2 functionally compensates for Mbnl1 loss. Although Mbnl1 −/− ; Mbnl2 −/− double knockouts (DKOs) are embryonic lethal, Mbnl1 −/− ; Mbnl2 +/− mice are viable but develop cardinal features of DM muscle disease including reduced lifespan, heart conduction block, severe myotonia and progressive skeletal muscle weakness. Mbnl2 protein levels are elevated in Mbnl1 −/− knockouts where Mbnl2 targets Mbnl1‐regulated exons. These findings support the hypothesis that compound loss of MBNL function is a critical event in DM pathogenesis and provide novel mouse models to investigate additional pathways disrupted in this RNA‐mediated disease.

Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A. We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy., Patients with myotonic dystrophy (MD) suffer from severe cardiac issues of unknown aetiology. Freyermuth et al. show that fatal changes in cardiac electrophysiological properties in humans and mice with MD may arise from misregulation of the alternative splicing of the cardiac Na+ channel SCN5A transcript, resulting in expression of its fetal form.

Clinical Considerations Concerning Detection of Venous Air Embolism. By Maurice S. Albin, Robert G. Carroll, Joseph C. Maroon. Neurosurgery 1978; 3:380-84. Abstract used with permission from the Congress of Neurological Surgeons, copyright 1978. Venous air embolism during neurosurgical procedures (detected by Doppler ultrasound and aspiration via a right atrial catheter) was noted in 100 of 400 patients in the sitting position, 5 of 60 patients in the lateral position, 7 of 48 patients in the supine position, and 1 of 10 patients in the monitored prone position. We confirmed venous air embolism in many of these patients by using serial technetium-microaggregated albumin lung scans. Gravitational gradients from the venous portal of entrance to the right side of the heart were as small as 5.0 cm, with aspiration of 200 ml of air occurring. Doppler ultrasonic air bubble detection and aspiration through a previously inserted right atrial catheter are critical factors in the diagnosis and treatment of this condition.

Temozolomide (TMZ) is an oral alkylating agent that has been widely used in the treatment of refractory glioma, although inherent and acquired resistance to this drug is common. The clinical use of valproic acid (VPA) as an anticonvulsant and mood-stabilizing drug has been reported primarily for the treatment of epilepsy and bipolar disorder and less commonly for major depression. VPA is also used in the treatment of glioma-associated seizures with or without intracranial operation. In this study, we evaluated the potential synergistic effect of TMZ and VPA in human glioma cell lines. Compared with the use of TMZ or VPA alone, concurrent treatment with both drugs synergistically induced apoptosis in U87MG cells as evidenced by p53 and Bax expression, mitochondrial transmembrane potential loss, reactive oxygen species production, and glutathione depletion. This synergistic effect correlated with a decrease in nuclear translocation of the nuclear factor-erythroid 2 p45-related factor and corresponded with reduced heme oxygenase-1 and γ-glutamylcysteine synthetase expression. Pretreatment with N-acetylcysteine partially recovered the apoptotic effect of the TMZ/VPA combination treatment. The same degree of synergism is also seen in p53-mutant Hs683 cells, which indicates that p53 may not play a major role in the increased proapoptotic effect of the TMZ/VPA combination. In conclusion, VPA enhanced the apoptotic effect of TMZ, possibly through a redox regulation mechanism. The TMZ/VPA combination may be effective for treating glioma cancer and may be a powerful agent against malignant glioma. This drug combination should be further explored in the clinical setting.

Nearly two decades have passed since the discovery that the expansion of microsatellite trinucleotide repeats is responsible for a prominent class of neurological disorders, including Huntington disease and fragile X syndrome. These hereditary diseases are characterized by genetic anticipation or the intergenerational increase in disease severity accompanied by a decrease in age-of-onset. The revelation that the variable expansion of simple sequence repeats accounted for anticipation spawned a number of pathogenesis models and a flurry of studies designed to reveal the molecular events affected by these expansions. This work led to our current understanding that expansions in protein-coding regions result in extended homopolymeric amino acid tracts, often polyglutamine or polyQ, and deleterious protein gain-of-function effects. In contrast, expansions in noncoding regions cause RNA-mediated toxicity. However, the realization that the transcriptome is considerably more complex than previously imagined, as well as the emerging regulatory importance of antisense RNAs, has blurred this distinction. In this review, we summarize evidence for bidirectional transcription of microsatellite disease genes and discuss recent suggestions that some repeat expansions produce variable levels of both toxic RNAs and proteins that influence cell viability, disease penetrance and pathological severity.

To determine how physicians' diagnoses, diagnostic uncertainty, and management decisions are affected by the results of computed tomography (CT) in emergency department settings.This study was approved by the institutional review board and compliant with HIPAA. Data were collected between July 12, 2012, and January 13, 2014. The requirement to obtain patient consent was waived. In this prospective, four-center study, patients presenting to the emergency department who were referred for CT with abdominal pain, chest pain and/or dyspnea, or headache were identified. Physicians were surveyed before and after CT to determine the leading diagnosis, diagnostic confidence (on a scale of 0% to 100%), alternative "rule out" diagnosis, and management decisions. Primary measures were the proportion of patients for whom the leading diagnosis or admission decision changed and median changes in diagnostic confidence. Secondary measures addressed alternative diagnoses and return-to-care visits (eg, to emergency department) at 1-month follow-up. Regression analysis was used to identify associations between primary measures and site and participant characteristics.Both surveys were completed for 1280 patients by 245 physicians. The leading diagnosis changed in 235 of 460 patients with abdominal pain (51%), 163 of 387 with chest pain and/or dyspnea (42%), and 103 of 433 with headache (24%). Pre-CT diagnostic confidence was inversely associated with the likelihood of a diagnostic change (P.0001). Median changes in confidence were substantial (increases of 25%, 20%, and 13%, respectively, for patients with abdominal pain, chest pain and/or dyspnea, and headache; P.0001); median post-CT confidence was high (95% for all three groups). CT helped confirm or exclude at least 95% of alternative diagnoses. Admission decisions changed in 116 of 457 patients with abdominal pain (25%), 72 of 387 with chest pain and/or dyspnea (19%), and 81 of 426 with headache (19%). During follow-up, 70 of 450 patients with abdominal pain (15%), 53 of 387 with chest pain and/or dyspnea (14%), and 49 of 433 with headache (11%) returned for the same indication. In general, changes in leading diagnosis, diagnostic confidence, and admission decisions were not well explained with site or participant characteristics.Physicians' diagnoses and admission decisions changed frequently after CT, and diagnostic uncertainty was alleviated.

The differentiation of fibroblasts into myofibroblasts mediates tissue wound healing and fibrotic remodelling, although the molecular programme underlying this process remains poorly understood. Here we perform a genome-wide screen for genes that control myofibroblast transformation, and identify the RNA-binding protein muscleblind-like1 (MBNL1). MBNL1 overexpression promotes transformation of fibroblasts into myofibroblasts, whereas loss of Mbnl1 abrogates transformation and impairs the fibrotic phase of wound healing in mouse models of myocardial infarction and dermal injury. Mechanistically, MBNL1 directly binds to and regulates a network of differentiation-specific and cytoskeletal/matrix-assembly transcripts to promote myofibroblast differentiation. One of these transcripts is the nodal transcriptional regulator serum response factor (SRF), whereas another is calcineurin Aβ. CRISPR-Cas9-mediated gene-editing of the MBNL1-binding site within the Srf 3′UTR impairs myofibroblast differentiation, whereas in vivo deletion of Srf in fibroblasts impairs wound healing and fibrosis. These data establish a new RNA-dependent paradigm for myofibroblast formation through MBNL1., Fibroblast-to-myofibroblast differentiation is crucial for wound healing and regeneration. Davis et al. describe a new regulatory mechanism underlying myofibroblast differentiation via the RNA-binding protein MBNL1, which promotes the maturation of certain mRNA transcripts that are integral nodes in fibroblast differentiation.

Background Although esophageal strictures caused by epidermolysis bullosa are often treated with balloon dilations, complete obstruction has few effective therapies except esophagectomy with colonic replacement. Objective Resolution of esophageal obstructive lesion without surgical intervention. Design Case study. Setting Interventional radiology. Patient Epidermolysis bullosa with esophageal stricture. Intervention Endoscopic- and guidewire-guided sharp recanalization. Main Outcome Measurement Radiologic evidence of stricture resolution. Results Successful recanalization. Limitations Experience of operators (anesthesiologist, endoscopist, interventional radiologist). Conclusions Sharp recanalization of a complete stricture in patients with epidermolysis bullosa is feasible in a controlled setting.

In myotonic dystrophy (DM), expression of RNA containing expanded CUG or CCUG repeats leads to misregulated alternative splicing of pre-mRNA. The repeat-bearing transcripts accumulate in nuclear foci, together with proteins in the muscleblind family, MBNL1 and MBNL2. In transgenic mice that express expanded CUG repeats, we show that the splicing defect selectively targets a group of exons that share a common temporal pattern of developmental regulation. These exons undergo a synchronized splicing switch between post-natal day 2 and 20 in wild-type mice. During this post-natal interval, MBNL1 protein translocates from a predominantly cytoplasmic to nuclear distribution. In the absence of MBNL1, these physiological splicing transitions do not occur. The splicing defect induced by expanded CUG repeats in mature muscle fibers is closely reproduced by deficiency of MBNL1 but not by deficiency of MBNL2. A parallel situation exists in human DM type 1 and type 2. MBNL1 is depleted from the muscle nucleoplasm because of sequestration in nuclear foci, and the associated splicing defects are remarkably similar to those observed in MBNL1 knockout mice. These results indicate that MBNL1 participates in the post-natal remodeling of skeletal muscle by controlling a key set of developmentally regulated splicing switches. Sequestration of MBNL1, and failure to maintain these splicing transitions, has a pivotal role in the pathogenesis of muscle disease in DM.

Myotonic dystrophy type I (DM1), which is caused by a non-coding CTG-repeat expansion in the dystrophia myotonica-protein kinase (DMPK) gene, is an RNA-mediated disease. Expanded CUG repeats in transcripts of mutant DMPK form nuclear foci that recruit muscleblind-like (MBNL) proteins, a family of alternative splicing factors. Although transcripts of mutant DMPK and MBNL proteins accumulate in nuclear RNA foci, it is not clear whether foci formation is required for splicing mis-regulation. Here, we use a co-transfection strategy to show that both CUG and CAG repeats form RNA foci that colocalize with green fluorescent protein (GFP)-MBNL1 and endogenous MBNL1. However, only CUG repeats alter splicing of the two tested pre-mRNAs, cardiac troponin T (cTNT) and insulin receptor (IR). Using FRAP, we demonstrate that GFP-MBNL1 in CUG and CAG foci have similar half-times of recovery and fractions of immobile molecules, suggesting that GFP-MBNL1 is bound by both CUG and CAG repeats. We also find an immobile fraction of GFP-MBNL1 in DM1 fibroblasts and a similar rapid exchange in endogenous CUG RNA foci. Therefore, formation of RNA foci and disruption of MBNL1-regulated splicing are separable events.

We examined the effects of thyroid hormones (THs) on left ventricular (LV) function and myocyte remodeling in rats with spontaneously hypertensive heart failure (SHHF). SHHF rats were treated with three different TH doses from 20–21 mo of age. In terminal experiments, LV function (as determined by echocardiography and catheterization) and isolated myocyte shape were examined in SHHF rat groups and age-matched Wistar-Furth control animals. Compared with Wistar-Furth rats, the ratio of α- to β-myosin was reduced in untreated SHHF rats. The α-to-β-myosin ratio increased in all TH groups, which suggests a reversal of the fetal gene program. Low-dose TH produced no changes in LV myocyte size or function, but high-dose TH produced signs of hyperthyroidism (e.g., increased heart weight, tachycardia). The chamber diameter-to-wall thickness ratio declined with increasing dose due to reduced chamber diameter and increased wall thickness. This resulted in a 38% reduction in LV systolic wall stress in the middle- and high-dose groups despite sustained hypertension. Isolated myocyte data indicated that chamber remodeling and reduced wall stress were due to a unique alteration in myocyte transverse shape (e.g., reduced major diameter and increased minor diameter). Based on our present understanding of ventricular remodeling and wall stress, we believe these changes are likely beneficial. Results suggest that TH may be an important regulator of myocyte transverse shape in heart disease.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are caused by genomic expansions of CTG or CCTG repeats. When transcribed, these mutations give rise to repeat expansion RNAs that form nuclear inclusions and compromise the function of myonuclei. Here, we have used in situ hybridization and immunofluorescence to compare DM1 and DM2 and search for proteins that associate with the RNA nuclear (ribonuclear) inclusions. Although muscle disease is generally more severe in DM1, the ribonuclear inclusions were 8- to 13-fold more intense in DM2, implying greater amounts of repeat expansion RNA. Expression of repeat expansion RNA in myoblasts has been implicated in the pathogenesis of congenital DM1. However, we found that repeat expansion RNA is also expressed in myoblasts in DM2, a disorder that has not been associated with a congenital phenotype. Of 10 putative CUG binding proteins tested for colocalization with mutant RNA, only proteins in the muscleblind family were recruited into ribonuclear inclusions. Previous studies have shown activation of the protein kinase, PKR, by expanded CUG repeats in vitro. However, breeding experiments utilizing PKR knockout mice indicate that this kinase is not required for disease pathogenesis in a transgenic mouse model of DM1. We conclude that ribonuclear inclusions are a key feature of the muscle pathology in DM and that sequestration of muscleblind proteins may have a direct role in the disease process.

Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1., The splicing of the penultimate exon of the dystrophin gene is developmentally regulated. Here the authors show that the dysregulation of this exon's splicing leads to the expression of an embryonic dystrophin form with a C-terminus distinct from the adult isoform, which leads to muscle wasting in zebrafish and mice.

Background According to the guidelines of the Burkina Faso National Malaria Control Programme, artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) are the first-line drugs for uncomplicated malaria treatments. However, in some contexts where individuals will experience more than 1 episode of clinical malaria per year, it is unknown to what extent giving any of these ACTs repeatedly is safe. In the framework of the activities of the West African Network for Antimalarial Drugs (WANECAM) network, we have compared the efficacy and tolerability of repeated use of dihydroartemisinin-piperaquine (DHA-PQ) or artesunate-pyronaridine (PYR) with artesunate-amodiaquine (ASAQ). Methods A randomised open-label parallel 3 arms trial was conducted to compare the efficacy of a three-day regimen of DHA-PQ and PYR with ASAQ. The trial involved children and adults with uncomplicated falciparum malaria. Participants were randomly assigned to one of the three treatment arms at the first clinical episode. During the subsequent clinical episodes, the same drug was administered. Follow-up duration was 42 days for each episode. Study duration was two years for each participant. Primary endpoints were the incidence rate of uncomplicated malaria over a period of 2 years and PCR corrected/uncorrected ACPR at day 28 and day 42. Safety parameters were also assessed. Results Of the 763 patients enrolled, the incidence rate of clinical malaria was 1.4, 1.2, and 1.5 episodes / person-year at risk in the ASAQ, DHA-PQ and PYRAMAX arms, respectively. The PCR-uncorrected efficacy at day 28 versus day 42 was: ASAQ 93.4% vs 79.5%; PYR 98.1% vs 74.8%; and DHA-PQ 99.5% vs 95.2%. Bronchitis, rhinitis, abdominal pain, cough, QTc prolongation, headache, and vomiting were registered as the main adverse events in each of the three groups. Conclusions The findings from our study support the current recommendations for using artemisinin-based combinations in the treatment of uncomplicated malaria in areas of high malaria transmission such as Burkina Faso.

Myotonic dystrophy (DM) is associated with a (CTG) (n) triplet repeat expansion in the 3'-untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. Using electron microscopy, we visualized large RNAs containing up to 130 CUG repeats and studied the binding of purified CUG-binding protein (CUG-BP) to these RNAs. Electron microscopic examination revealed perfect double-stranded (ds)RNA segments whose lengths were that expected for duplex RNA. The RNA dominant mutation model for DM pathogenesis predicts that the expansion mutation acts at the RNA level by forming long dsRNAs that sequester certain RNA-binding proteins. To test this model, we examined the subcellular distribution and RNA-binding properties of CUG-BP. While previous studies have demonstrated that mutant DMPK transcripts accumu-late in nuclear foci, the localization pattern of CUG-BP in both normal and DM cells was similar. Although CUG-BP in nuclear extracts preferentially photocrosslinked to DMPK transcripts, this binding was not proportional to (CUG) (n) repeat size. Moreover, CUG-BP localized to the base of the RNA hairpin and not along the stem, as visualized by electron micro-scopy. These results provide the first visual evidence that the DM expansion forms an RNA hairpin structure and suggest that CUG-BP is unlikely to be a sequestered factor.

The role of inhaled nitric oxide in the immediate post-bypass period after surgical repair of congenital heart disease is uncertain. In a controlled, randomized, double-blind study, we tested the hypothesis that inhaled nitric oxide (NO) would reduce pulmonary hypertension immediately after surgical repair of congenital heart disease in 40 patients with preoperative evidence of pulmonary hypertension (mean pulmonary arterial pressure [MPAP] exceeding 50% of mean systemic arterial pressure [MSAP]). Patients were then followed in the intensive care unit (ICU) to document the incidence of severe pulmonary hypertension. Of the patients, 36% (n = 13) emerged from bypass with MPAP >50% MSAP. In these patients, inhaled NO reduced MPAP by 19% (P = 0.008) versus an increase of 9% in the placebo group. No effect on MPAP was observed in patients emerging from bypass without pulmonary hypertension (n = 23). Inhaled NO was required five times in the ICU, always in the patients who had emerged from cardiopulmonary bypass with pulmonary hypertension (5 of 13 [38%] versus 0 of 23). We conclude that, in infants and children undergoing congenital heart surgery, inhaled NO selectively reduces MPAP in patients who emerge from cardiopulmonary bypass with pulmonary hypertension and has no effect on those who emerge without it. Implications: In a randomized double-blind study, inhaled nitric oxide selectively reduced pulmonary artery pressures in pediatric patients who developed pulmonary hypertension (high blood pressure in the lungs) immediately after cardiopulmonary bypass and surgical repair.

RNA-binding proteins (RBPs) play pivotal roles in multiple cellular pathways from transcription to RNA turnover by interacting with RNA sequence and/or structural elements to form distinct RNA-protein complexes. Since these complexes are required for the normal regulation of gene expression, mutations that alter RBP functions may result in a cascade of deleterious events that lead to severe disease. Here, we focus on a group of hereditary disorders, the microsatellite expansion diseases, which alter RBP activities and result in abnormal neurological and neuromuscular phenotypes. While many of these diseases are classified as adult-onset disorders, mounting evidence indicates that disruption of normal RNA-protein interaction networks during embryogenesis modifies developmental pathways, which ultimately leads to disease manifestations later in life. Efforts to understand the molecular basis of these disorders has already uncovered novel pathogenic mechanisms, including RNA toxicity and repeat-associated non-ATG (RAN) translation, and current studies suggest that additional surprising insights into cellular regulatory pathways will emerge in the future.

Background Persistent pulmonary hypertension of the newborn causes systemic arterial hypoxemia because of increased pulmonary vascular resistance and right-to-left shunting of deoxygenated blood. Inhaled nitric oxide decreases pulmonary vascular resistance in newborns. We studied whether inhaled nitric oxide decreases severe hypoxemia in infants with persistent pulmonary hypertension. Methods In a prospective, multicenter study, 58 full-term infants with severe hypoxemia and persistent pulmonary hypertension were randomly assigned to breathe either a control gas (nitrogen) or nitric oxide (80 parts per million), mixed with oxygen from a ventilator. If oxygenation increased after 20 minutes and systemic blood pressure did not decrease, the treatment was considered successful and was continued at lower concentrations. Otherwise, it was discontinued and alternative therapies, including extracorporeal membrane oxygenation, were used. Results Inhaled nitric oxide successfully doubled systemic oxygenation in 16 of 30 infants (53 percent), whereas conventional therapy without inhaled nitric oxide increased oxygenation in only 2 of 28 infants (7 percent). Long-term therapy with inhaled nitric oxide sustained systemic oxygenation in 75 percent of the infants who had initial improvement. Extracorporeal membrane oxygenation was required in 71 percent of the control group and 40 percent of the nitric oxide group (P=0.02). The number of deaths was similar in the two groups. Inhaled nitric oxide did not cause systemic hypotension or increase methemoglobin levels. Conclusions Inhaled nitric oxide improves systemic oxygenation in infants with persistent pulmonary hypertension and may reduce the need for more invasive treatments.

The muscleblind-like (MBNL) genes encode alternative splicing factors that are essential for the postnatal development of multiple tissues, and the inhibition of MBNL activity by toxic C(C)UG repeat RNAs is a major pathogenic feature of the neuromuscular disease myotonic dystrophy. While MBNL1 controls fetal-to-adult splicing transitions in muscle and MBNL2 serves a similar role in the brain, the function of MBNL3 in vivo is unknown. Here, we report that mouse Mbnl3, which encodes protein isoforms that differ in the number of tandem zinc-finger RNA-binding motifs and subcellular localization, is expressed primarily during embryonic development but also transiently during injury-induced adult skeletal muscle regeneration. Mbnl3 expression is required for normal C2C12 myogenic differentiation and high-throughput sequencing combined with cross-linking/immunoprecipitation analysis indicates that Mbnl3 binds preferentially to the 3' untranslated regions of genes implicated in cell growth and proliferation. In addition, Mbnl3ΔE2 isoform knockout mice, which fail to express the major Mbnl3 nuclear isoform, show age-dependent delays in injury-induced muscle regeneration and impaired muscle function. These results suggest that Mbnl3 inhibition by toxic RNA expression may be a contributing factor to the progressive skeletal muscle weakness and wasting characteristic of myotonic dystrophy.

International audience; Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology.

There is ongoing confusion within the clinical literature about which group model (i.e., a disease-and-recovery model or a cognitive-behavioral model) is effective for persons who have a comorbid disorder including substance abuse and mental problems. Both the disease-and-recovery model and the cognitive-behavioral model hold clinical promise in terms of providing comprehensive and integrated group therapy treatment for persons with substance abuse and personality disorders. The purpose of this article is to describe and suggest two integrated group therapy protocols developed and empirically tested specifically for the integrated treatment of persons having both a substance abuse disorder and a personality disorder. Practical guidelines are also offered based on the author's clinical group therapy practice experience with this challenging population.

The neuromuscular disease myotonic dystrophy (DM) is caused by microsatellite repeat expansions at two different genomic loci. Mutant DM transcripts are retained in the nucleus together with the muscleblind (Mbnl) proteins, and these abnormal RNAs somehow interfere with pre-mRNA splicing regulation. Here, we show that disruption of the mouse Mbnl1 gene leads to muscle, eye, and RNAsplicing abnormalities that are characteristic of DM disease. Our results support the hypothesis that manifestations of DM can result from sequestration of specific RNAbinding proteins by a repetitive element expansion in a mutant RNA.

Background— Cardiac hypertrophy is a common response to circulatory or neurohumoral stressors as a mechanism to augment contractility. When the heart is under sustained stress, the hypertrophic response can evolve into decompensated heart failure, although the mechanism(s) underlying this transition remain largely unknown. Because phosphorylation of cardiac myosin light chain 2 (MLC2v), bound to myosin at the head-rod junction, facilitates actin-myosin interactions and enhances contractility, we hypothesized that phosphorylation of MLC2v plays a role in the adaptation of the heart to stress. We previously identified an enzyme that predominantly phosphorylates MLC2v in cardiomyocytes, cardiac myosin light-chain kinase (cMLCK), yet the role(s) played by cMLCK in regulating cardiac function in health and disease remain to be determined. Methods and Results— We found that pressure overload induced by transaortic constriction in wild-type mice reduced phosphorylated MLC2v levels by ≈40% and cMLCK levels by ≈85%. To examine how a reduction in cMLCK and the corresponding reduction in phosphorylated MLC2v affect function, we generated Mylk3 gene-targeted mice and transgenic mice overexpressing cMLCK specifically in cardiomyocytes. Pressure overload led to severe heart failure in cMLCK knockout mice but not in mice with cMLCK overexpression in which cMLCK protein synthesis exceeded degradation. The reduction in cMLCK protein during pressure overload was attenuated by inhibition of ubiquitin-proteasome protein degradation systems. Conclusions— Our results suggest the novel idea that accelerated cMLCK protein turnover by the ubiquitin-proteasome system underlies the transition from compensated hypertrophy to decompensated heart failure as a result of reduced phosphorylation of MLC2v.

Fluoride in drinking water above permissible levels is responsible for dental and skeletal fluorosis. In this study, removal of fluoride ions from water using phosphoric acid treated lime was investigated in continuous and point-of-use system opera tions. In the continuous column operations, fluoride removal performance was investigated as a function of the fluoride concentration, flow rate and amount of adsorbent mass. Early saturation and lower fluoride removal were observed at higher flow rate and initial concentration, and at lower mass. Two domestic defluoridation point-of-use systems operated inter mittently to process 20 l∙day −1 of water were used. High fluoride uptake capacity (FUC) from groundwater was observed depicting the suitability of the new media in defluoridation. However, further research is required to optimise the point-ofuse systems performances.