Vorinostat Improves Myotonic Dystrophy Type 1 Splicing Abnormalities in DM1 Muscle Cell Lines and Skeletal Muscle from a DM1 Mouse Model.

Myotonic dystrophy type 1 (DM1), the most common form of adult muscular dystrophy, is caused by an abnormal expansion of CTG repeats in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The expanded repeats of the DMPK mRNA form hairpin structures in vitro, which cause misregulation and/or sequestration of proteins including the splicing regulator muscleblind-like 1 (MBNL1). In turn, misregulation and sequestration of such proteins result in the aberrant alternative splicing of diverse mRNAs and underlie, at least in part, DM1 pathogenesis. It has been previously shown that disaggregating RNA foci repletes free MBNL1, rescues DM1 spliceopathy, and alleviates associated symptoms such as myotonia. Using an FDA-approved drug library, we have screened for a reduction of CUG foci in patient muscle cells and identified the HDAC inhibitor, vorinostat, as an inhibitor of foci formation; SERCA1 (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) spliceopathy was also improved by vorinostat treatment. Vorinostat treatment in a mouse model of DM1 (human skeletal actin-long repeat; HSALR) improved several spliceopathies, reduced muscle central nucleation, and restored chloride channel levels at the sarcolemma. Our in vitro and in vivo evidence showing amelioration of several DM1 disease markers marks vorinostat as a promising novel DM1 therapy.

Advanced Trauma Life Support Course Delivery: Comparison of Outcomes From Modifications During Covid-19.

Introduction The Advanced Trauma Life Support (ATLS) Course is a two-day long medical training course developed by the American College of Surgeons (ACS) to help train and prepare healthcare providers to care for severely injured patients. The coronavirus disease 2019 (COVID-19) pandemic has resulted in the modification or cancellation of many education programs across the world. At the University of South Florida's Center for Advanced Medical Learning and Simulation (CAMLS) two different models of ATLS were delivered in response to the COVID-19 pandemic with both models utilizing the ACS's online mobile ATLS (mATLS). In this study three different models of ATLS delivered by USF CAMLS between 2019 and 2020 were compared to determine if there were any impacts on the education and functionality of the ATLS course between the three different models of ATLS: a baseline ATLS course, an augmented ATLS course that used mATLS, and an ATLS course that used mATLS as a replacement for in-person lectures. Material and methods To compare the three models of ATLS delivery, a total of six ATLS courses were studied: a baseline face-to-face ATLS course from June 2019, two Mobile ATLS (mATLS) courses from September 2020, and three augmented ATLS courses that contained both face-to-face and mATLS delivery from October, November and December 2020. The only differences between the traditional ATLS courses from 2020 and the pre-COVID ATLS course from 2019 were that the courses from 2020 utilized mATLS and that the course days were longer due to cleaning time. These courses were selected to have a non-significant difference in the number of learners in each model of ATLS course. The data that were collected from these courses included: post-test results from learners, learner feedback surveys, and interviews with the ATLS Course Director, ATLS Course Coordinator, and the Educational Coordinator. Results The only courses with significant differences in the post-test mean scores were for the baseline ATLS course compared to the mATLS courses. The augmented courses showed similar post-test performance to the mATLS courses. Students viewed the courses favorably with the only major complaint between the 2019 and the 2020 courses being a high amount of downtime for the 2020 courses due to time required to disinfect skill stations and equipment. The main difficulties for the ATLS Course Director, ATLS Course Coordinator, and the Educational Coordinator with the ATLS courses in 2020 were concerned with challenges from COVID-19, like social distancing, and not with mATLS or the shortened instruction time with the hybrid model. Discussion This preliminary study analyzed three delivery models of ATLS. The mATLS may be able to replace in-person lectures of ATLS courses as courses using alternative delivery formats showed post-test scores as good or better than the baseline face-to-face course.

Combined Pathology-Driven Algorithmic Testing and Integrated Reporting for Bone Marrow Examination.

CONTEXT.­: The College of American Pathologists published guideline recommending bone marrow synoptic reporting for hematologic neoplasms. OBJECTIVE.­: To evaluate the impact of pathology-driven algorithmic testing (PDAT) with integrated reporting for bone marrow examination on test utilization, ability to render a specific World Health Organization diagnosis, and clinician satisfaction 1 year after implementation. DESIGN.­: We reviewed the hematopathology reports, integrated synoptic reports, and ancillary test results generated during a 12-month period. The initial diagnosis from the hematopathology report was compared with the final diagnosis on the integrated synoptic reports. Test utilization data were compared with a previous year in which ancillary testing was ordered at clinician discretion. Clinicians were anonymously surveyed to assess their satisfaction with PDAT and integrated reporting. RESULTS.­: Integrated reporting resulted in a World Health Organization diagnosis for 80 of 85 cases (94%) compared with 54 (64%) for the hematopathology report alone. Unnecessary testing decreased from 45% pre-PDAT (124 of 274 cases) to 0.7% PDAT (2 of 268 cases), and PDAT resulted in fewer omissions of necessary tests. Clinicians preferred PDAT and valued integrated reporting for a variety of reasons, including the ease of finding relevant prognostic information. CONCLUSIONS.­: Pathology-driven algorithmic testing with integrated reporting improves the pathologist's ability to render a specific World Health Organization diagnosis and improves test utilization. Clinicians prefer PDAT to clinician-ordered testing. This is the first study to examine how synoptic reporting can modify hematologic diagnoses.

Evidence-based review of trauma center care and routine palliative care processes for geriatric trauma patients; A collaboration from the American Association for the Surgery of Trauma Patient Assessment Committee, the American Association for the Surgery of Trauma Geriatric Trauma Committee, and the Eastern Association for the Surgery of Trauma Guidelines Committee.

BACKGROUND: Despite an aging population and increasing number of geriatric trauma patients annually, gaps in our understanding of best practices for geriatric trauma patients persist. We know that trauma center care improves outcomes for injured patients generally, and palliative care processes can improve outcomes for disease-specific conditions, and our goal was to determine effectiveness of these interventions on outcomes for geriatric trauma patients. METHODS: A priori questions were created regarding outcomes for patients 65 years or older with respect to care at trauma centers versus nontrauma centers and use of routine palliative care processes. A query of MEDLINE, PubMed, Cochrane Library, and EMBASE was performed. Letters to the editor, case reports, book chapters, and review articles were excluded. GRADE (Grading of Recommendations Assessment, Development and Evaluation) methodology was used to perform a systematic review and create recommendations. RESULTS: We reviewed seven articles relevant to trauma center care and nine articles reporting results on palliative care processes as they related to geriatric trauma patients. Given data quality and limitations, we conditionally recommend trauma center care for the severely injured geriatric trauma patients but are unable to make a recommendation on the question of routine palliative care processes for geriatric trauma patients. CONCLUSIONS: As our older adult population increases, injured geriatric patients will continue to pose challenges for care, such as comorbidities or frailty. We found that trauma center care was associated with improved outcomes for geriatric trauma patients in most studies and that utilization of early palliative care consultations was generally associated with improved secondary outcomes, such as length of stay; however, inconsistency and imprecision prevented us from making a clear recommendation for this question. As caregivers, we should ensure adequate support for trauma systems and palliative care processes in our institutions and communities and continue to support robust research to study these and other aspects of geriatric trauma. LEVEL OF EVIDENCE: Systematic review/guideline, level III.

Muscle-specific microRNA-206 targets multiple components in dystrophic skeletal muscle representing beneficial adaptations.

Over the last several years, converging lines of evidence have indicated that miR-206 plays a pivotal role in promoting muscle differentiation and regeneration, thereby potentially impacting positively on the progression of neuromuscular disorders, including Duchenne muscular dystrophy (DMD). Despite several studies showing the regulatory function of miR-206 on target mRNAs in skeletal muscle cells, the effects of overexpression of miR-206 in dystrophic muscles remain to be established. Here, we found that miR-206 overexpression in mdx mouse muscles simultaneously targets multiple mRNAs and proteins implicated in satellite cell differentiation, muscle regeneration, and at the neuromuscular junction. Overexpression of miR-206 also increased the levels of several muscle-specific mRNAs/proteins, while enhancing utrophin A expression at the sarcolemma. Finally, we also observed that the increased expression of miR-206 in dystrophin-deficient mouse muscle decreased the production of proinflammatory cytokines and infiltration of macrophages. Taken together, our results show that miR-206 acts as a pleiotropic regulator that targets multiple key mRNAs and proteins expected to provide beneficial adaptations in dystrophic muscle, thus highlighting its therapeutic potential for DMD.

HuR Mediates Changes in the Stability of AChR ß-Subunit mRNAs after Skeletal Muscle Denervation.

Acetylcholine receptors (AChRs) are heteromeric membrane proteins essential for neurotransmission at the neuromuscular junction. Previous work showed that muscle denervation increases expression of AChR mRNAs due to transcriptional activation of AChR subunit genes. However, it remains possible that post-transcriptional mechanisms are also involved in controlling the levels of AChR mRNAs following denervation. We examined whether post-transcriptional events indeed regulate AChR ß-subunit mRNAs in response to denervation. First, in vitro stability assays revealed that the half-life of AChR ß-subunit mRNAs was increased in the presence of denervated muscle protein extracts. A bioinformatics analysis revealed the existence of a conserved AU-rich element (ARE) in the 3'-untranslated region (UTR) of AChR ß-subunit mRNA. Furthermore, denervation of mouse muscle injected with a luciferase reporter construct containing the AChR ß-subunit 3'UTR, caused an increase in luciferase activity. By contrast, mutation of this ARE prevented this increase. We also observed that denervation increased expression of the RNA-binding protein human antigen R (HuR) and induced its translocation to the cytoplasm. Importantly, HuR binds to endogenous AChR ß-subunit transcripts in cultured myotubes and in vivo, and this binding is increased in denervated versus innervated muscles. Finally, p38 MAPK, a pathway known to activate HuR, was induced following denervation as a result of MKK3/6 activation and a decrease in MKP-1 expression, thereby leading to an increase in the stability of AChR ß-subunit transcripts. Together, these results demonstrate the important contribution of post-transcriptional events in regulating AChR ß-subunit mRNAs and point toward a central role for HuR in mediating synaptic gene expression. SIGNIFICANCE STATEMENT: Muscle denervation is a convenient model to examine expression of genes encoding proteins of the neuromuscular junction, especially acetylcholine receptors (AChRs). Despite the accepted model of AChR regulation, which implicates transcriptional mechanisms, it remains plausible that such events cannot fully account for changes in AChR expression following denervation. We show that denervation increases expression of the RNA-binding protein HuR, which in turn, causes an increase in the stability of AChR ß-subunit mRNAs in denervated muscle. Our findings demonstrate for the first time the contribution of post-transcriptional events in controlling AChR expression in skeletal muscle, and points toward a central role for HuR in mediating synaptic development while also paving the way for developing RNA-based therapeutics for neuromuscular diseases.

A reduction in the human adenovirus virion size through use of a shortened fibre protein does not enhance muscle transduction following systemic or localised delivery in mice.

We have investigated whether reducing the overall size of adenovirus (Ad), through use of a vector containing a shortened fibre, leads to enhanced distribution and dissemination of the vector. Intravenous or intraperitoneal injection of Ad5SlacZ (12 nm fibre versus the normal Ad5 37 nm fibre) or Ad5SpKlacZ (shortened fibre with polylysine motif in the H-I loop of fibre knob domain) led to similar levels of lacZ expression compared to Ad5LlacZ (native Ad5 fibre) in the liver of treated animals, but did not enhance extravasation into the tibialis anterior muscle. Direct injection of the short-fibre vectors into the tibialis anterior muscle did not result in enhanced spread of the vector through muscle tissue, and led to only sporadic transgene expression in the spinal cord, suggesting that modifying the fibre length or redirecting viral infection to a more common cell surface receptor does not enhance motor neuron uptake or retrograde transport.

Resveratrol induces expression of the slow, oxidative phenotype in mdx mouse muscle together with enhanced activity of the SIRT1-PGC-1α axis.

Slower, more oxidative muscle fibers are more resistant to the dystrophic pathology in Duchenne muscular dystrophy (DMD) patients as well as in the preclinical mdx mouse model of DMD. Therefore, one therapeutic strategy for DMD focuses on promoting expression of the slow, oxidative myogenic program. In the current study, we explored the therapeutic potential of stimulating the slow, oxidative phenotype in mdx mice by feeding 6-wk-old animals with the natural phenol resveratrol (RSV; ~100 mg·kg(-1)·day(-1)) for 6 wk. Sirtuin 1 (SIRT1) activity and protein levels increased significantly, as well as peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) activity, in the absence of alterations in AMPK signaling. These adaptations occurred concomitant with evidence of a fast, glycolytic, to slower, more oxidative fiber type conversion, including mitochondrial biogenesis and increased expression of slower myosin heavy chain isoforms. These positive findings raised the question of whether increased exposure to RSV would result in greater therapeutic benefits. We discovered that an elevated RSV dose of ~500 mg·kg(-1)·day(-1) across a duration of 12 wk was clearly less effective at muscle remodeling in mdx mice. This treatment protocol failed to influence SIRT1 or AMPK signaling and did not result in a shift towards a slower, more oxidative phenotype. Taken together, this study demonstrates that RSV can stimulate SIRT1 and PGC-1α activation, which in turn may promote expression of the slow, oxidative myogenic program in mdx mouse muscle. The data also highlight the importance of selecting an appropriate dosage regimen of RSV to maximize its potential therapeutic effectiveness for future application in DMD patients.

New onset pancytopenia in adults: a review of underlying pathologies and their associated clinical and laboratory findings.

Abstract Pancytopenia is regularly encountered in hematology practice, yet there exist few published assessments of the frequencies of various etiologies, and these frequencies exhibit substantial geographic variation. We reviewed bone marrow specimens from pancytopenic adults to determine the most common etiologies and to identify associations with clinical and laboratory findings. Of 132 patients with no history of hematolymphoid neoplasia, no prior bone marrow study for pancytopenia and no recent cytotoxic chemotherapy, 64% had clonal hematopoietic disorders. Most common were myeloid processes: 26% of patients had acute myeloid leukemia, and 17% had myelodysplasia. Less common were lymphoid neoplasms such as non-Hodgkin lymphoma (6%), hairy cell leukemia (5%) and precursor B acute lymphoblastic leukemia (4%). Among non-clonal cases, the most common specific diagnoses were aplastic anemia (5%), megaloblastic anemia (2%) and human immunodeficiency virus (HIV)-related changes (2%). Clonal diagnoses were associated with more severe cytopenias than non-clonal cases. Circulating nucleated erythroid precursors, immature granulocytes and blasts were seen more frequently in clonal cases. Nearly two-thirds of cases of new onset pancytopenia in adults in our North American practice setting have a clonal etiology, with myeloid neoplasms being most common. Blood counts and peripheral smear findings can provide insights into the likelihood of a clonal etiology.

Converging pathways involving microRNA-206 and the RNA-binding protein KSRP control post-transcriptionally utrophin A expression in skeletal muscle.

Several reports have previously highlighted the potential role of miR-206 in the post-transcriptional downregulation of utrophin A in cultured cells. Along those lines, we recently identified K-homology splicing regulator protein (KSRP) as an important negative regulator in the post-transcriptional control of utrophin A in skeletal muscle. We sought to determine whether these two pathways act together to downregulate utrophin A expression in skeletal muscle. Surprisingly, we discovered that miR-206 overexpression in cultured cells and dystrophic muscle fibers causes upregulation of endogenous utrophin A levels. We further show that this upregulation of utrophin A results from the binding of miR-206 to conserved sites located in the 3'-UTR (untranslated region) of KSRP, thus causing the subsequent inhibition of KSRP expression. This miR-206-mediated decrease in KSRP levels leads, in turn, to an increase in the expression of utrophin A due to a reduction in the activity of this destabilizing RNA-binding protein. Our work shows that miR-206 can oscillate between direct repression of utrophin A expression via its 3'-UTR and activation of its expression through decreased availability of KSRP and interactions with AU-rich elements located within the 3'-UTR of utrophin A. Our study thus reveals that two apparent negative post-transcriptional pathways can act distinctively as molecular switches causing repression or activation of utrophin A expression.

Activation of p38 signaling increases utrophin A expression in skeletal muscle via the RNA-binding protein KSRP and inhibition of AU-rich element-mediated mRNA decay: implications for novel DMD therapeutics.

Several therapeutic approaches are currently being developed for Duchenne muscular dystrophy (DMD) including upregulating the levels of endogenous utrophin A in dystrophic fibers. Here, we examined the role of post-transcriptional mechanisms in controlling utrophin A expression in skeletal muscle. We show that activation of p38 leads to an increase in utrophin A independently of a transcriptional induction. Rather, p38 controls the levels of utrophin A mRNA by extending the half-life of transcripts via AU-rich elements (AREs). This mechanism critically depends on a decrease in the functional availability of KSRP, an RNA-binding protein known to promote decay of ARE-containing transcripts. In vitro and in vivo binding studies revealed that KSRP interacts with specific AREs located within the utrophin A 3' UTR. Electroporation experiments to knockdown KSRP led to an increase in utrophin A in wild-type and mdx mouse muscles. In pre-clinical studies, treatment of mdx mice with heparin, an activator of p38, causes a pronounced increase in utrophin A in diaphragm muscle fibers. Together, these studies identify a pathway that culminates in the post-transcriptional regulation of utrophin A through increases in mRNA stability. Furthermore, our results constitute proof-of-principle showing that pharmacological activation of p38 may prove beneficial as a novel therapeutic approach for DMD.

Chronic AMPK activation evokes the slow, oxidative myogenic program and triggers beneficial adaptations in mdx mouse skeletal muscle.

A therapeutic approach for Duchenne muscular dystrophy (DMD) is to up-regulate utrophin in skeletal muscle in an effort to compensate for the lack of dystrophin. We previously hypothesized that promotion of the slow, oxidative myogenic program, which triggers utrophin up-regulation, can attenuate the dystrophic pathology in mdx animals. Since treatment of healthy mice with the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) enhances oxidative capacity and elicits a fast-to-slow fiber-type transition, we evaluated the effects of chronic AMPK stimulation on skeletal muscle phenotype and utrophin expression in mdx mice. Daily AICAR administration (500 mg/kg/day, 30 days) of 5-7-week-old mdx animals induced an elevation in mitochondrial cytochrome c oxidase enzyme activity, an increase in myosin heavy-chain type IIa-positive fibers and slower twitch contraction kinetics in the fast, glycolytic extensor digitorum longus muscle. Utrophin expression was significantly enhanced in response to AICAR, which occurred coincident with an elevated ß-dystroglycan expression along the sarcolemma. These adaptations were associated with an increase in sarcolemmal structural integrity under basal conditions, as well as during damaging eccentric contractions ex vivo. Notably, peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) and silent information regulator two ortholog 1 protein contents were significantly higher in muscle from mdx mice compared with wild-type littermates and AICAR further increased PGC-1α expression. Our data show that AICAR-evoked muscle plasticity results in beneficial phenotypic adaptations in mdx mice and suggest that the contextually novel application of this compound for muscular dystrophy warrants further study.

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

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

FISH detection of t(14;18) in follicular lymphoma on Papanicolaou-stained archival cytology slides.

BACKGROUND: The t(14;18)(q32;q21) translocation is present in about 85% of follicular lymphomas (FL) and can be identified using fluorescence in situ hybridization (FISH). In the diagnostic laboratory setting, the cytologic archival material consists of stained slides, and only rarely is material saved for molecular testing. The authors proposed FISH for FL using Papanicolaou-stained archival cytology material as a practical ancillary technique for diagnosing FL. METHODS: Cases included 35 FL, 6 small lymphocytic lymphomas/chronic lymphocytic leukemias (SLL/CLL), 4 mantle cell lymphomas (MCL), 4 marginal zone lymphomas (MZL), 1 lymphoplasmacytic lymphoma (LPL), and 10 reactive lymphoid tissues (RLT). FISH was performed on Papanicolaou-stained archival cytology slides using probes for immunoglobulin heavy chain (IGH) on chromosome 14 and BCL2 on chromosome 18. RESULTS: In all, 25 of 32 (81%) FL cases exhibited the t(14;18) translocation, whereas 7 of 32 (19%) lacked the translocation. No cases of non-FL were positive for t(14;18). This series shows a sensitivity of 81% and specificity of 100% for detecting the t(14;18) translocation as a diagnostic tool in FL. CONCLUSIONS: When performed on Papanicolaou-stained cytology slides, FISH for t(14;18) is relatively sensitive and quite specific for FL. These findings are similar to those reported on other specimens, such as paraffin-embedded tissue and unstained cytology slides. The authors proposed that their technique would allow the pathologist and clinician the flexibility to utilize previously stained fine-needle aspiration slides for FISH evaluation.

The RNA-binding protein HuR binds to acetylcholinesterase transcripts and regulates their expression in differentiating skeletal muscle cells.

During myogenic differentiation, acetylcholinesterase (AChE) transcript levels are known to increase dramatically. Although this increase can be attributed in part to increased transcriptional activity, posttranscriptional mechanisms have also been implicated in the high levels of AChE mRNA in myotubes. In this study, we observed that transfection of a luciferase reporter construct containing the full-length AChE 3'-untranslated region (UTR) resulted in significantly higher (5-fold) luciferase activity in differentiated myotubes versus myoblasts. RNA-electrophoretic mobility shift assays (REMSAs) performed with a full-length AChE 3'-UTR probe and the AU-rich element revealed that the intensity of RNA-binding protein complexes increased as myogenic differentiation proceeded. Using several complementary approaches including supershift REMSA, mRNA-binding protein pull-down assays, and immunoprecipitation followed by reverse transcription-PCR, we found that the mRNA-stabilizing protein HuR interacts directly with AChE transcripts. Stable overexpression of HuR in C2C12 cells increased the expression of endogenous AChE transcripts as well as that of the luciferase reporter construct containing the AChE 3'-UTR. In vitro stability assays performed with protein extracts from these cells versus controls resulted in a slower rate of AChE mRNA decay. The down-regulation of HuR expression mediated through small interfering RNA further confirmed the role of HuR in the regulation of AChE mRNA levels. Taken together, these studies demonstrate that HuR interacts with the AChE 3'-UTR to regulate posttranscriptionally the expression of AChE mRNA during myogenic differentiation.

Use of adenovirus protein IX (pIX) to display large polypeptides on the virion--generation of fluorescent virus through the incorporation of pIX-GFP.

The adenovirus (Ad) protein IX (pIX) is a minor component of the Ad capsid and associates with the hexons that make up the facets of the icosahedron. In this study, we investigated whether a large protein tag could be fused to pIX without compromising the Ad vector itself. As proof-of-principle, we generated a pIX-green fluorescent protein (GFP) fusion protein. We show that a virus encoding the pIX-GFP can be generated and that pIX-GFP fusion protein was incorporated into the Ad capsid as efficiently as native pIX. In tissue culture, translocation of Ad/pIX-GFP from the outside of the cell to the nucleus could be followed using fluorescence microscopy, and the timing of migration to the nucleus was similar to that previously reported for Ad. We also could track the virus after injection into the tibialis anterior muscle of mice. Shortly after injection, the majority of the Ad/pIX-GFP accumulated in pockets adjacent to the muscle fibers, with some migration of the virus between fibers. Our ability to attach GFP to the Ad virion, through fusion to pIX, provides a valuable tool for virus tracking in vitro and in vivo. Moreover, our data indicate that pIX can be used as a platform to anchor proteins to the Ad capsid, such as large ligands for cell-type-specific targeting of the vector.

Ste20-like kinase SLK displays myofiber type specificity and is involved in C2C12 myoblast differentiation.

Cell growth and terminal differentiation are controlled by complex signaling cascades that regulate the expression of specific subsets of genes implicated in cell fate and morphogenic processes. We have recently cloned and characterized a novel Ste20-like kinase termed SLK that is associated with adhesion structures during cell adhesion and spreading. However, the specific function of SLK is poorly understood. To gain further insight into the role of SLK, we have characterized its activity, expression, and distribution in skeletal muscle and during the in vitro differentiation of C2C12 myoblasts. Although SLK is expressed ubiquitously in adult tissues, our results show that it is predominantly expressed in muscle masses during development. Furthermore, SLK activity is upregulated during the differentiation of C2C12 myoblasts. In addition, we have found that SLK localizes presynaptically at neuromuscular junctions and that it is preferentially expressed in types I and IIA myofibers at major myofibrillar striations. Supporting a role in myoblast function and differentiation, SLK expression is induced in Myf5- and Pax7-positive activated satellite cells during regeneration and expression of dominant negative SLK in C2C12 cultures impairs myoblast fusion, suggesting a role for SLK in muscle cell differentiation.