Potential limitations of microdystrophin gene therapy for Duchenne muscular dystrophy.

Clinical trials delivering high doses of adeno-associated viruses (AAVs) expressing truncated dystrophin molecules (microdystrophins) are underway for Duchenne muscular dystrophy (DMD). We examined the efficiency and efficacy of this strategy with 4 microdystrophin constructs (3 in clinical trials and a variant of the largest clinical construct), in a severe mouse model of DMD, using AAV doses comparable with those in clinical trials. We achieved high levels of microdystrophin expression in striated muscles with cardiac expression approximately 10-fold higher than that observed in skeletal muscle. Significant, albeit incomplete, correction of skeletal muscle disease was observed. Surprisingly, a lethal acceleration of cardiac disease occurred with 2 of the microdystrophins. The detrimental cardiac effect appears to be caused by variable competition (dependent on microdystrophin design and expression level) between microdystrophin and utrophin at the cardiomyocyte membrane. There may also be a contribution from an overloading of protein degradation. The significance of these observations for patients currently being treated with AAV-microdystrophin therapies is unclear since the levels of expression being achieved in the DMD hearts are unknown. However, these findings suggest that microdystrophin treatments need to avoid excessively high levels of expression in the heart and that cardiac function should be carefully monitored in these patients.

Myospreader improves gene editing in skeletal muscle by myonuclear propagation.

Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader," a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development.

Myo10 tail is crucial for promoting long filopodia.

Filopodia are slender cellular protrusions containing parallel actin bundles involved in environmental sensing and signaling, cell adhesion and migration, and growth cone guidance and extension. Myosin 10 (Myo10), an unconventional actin-based motor protein, was reported to induce filopodial initiation with its motor domain. However, the roles of the multifunctional tail domain of Myo10 in filopodial formation and elongation remain elusive. Herein, we generated several constructs of Myo10-full-length Myo10, Myo10 with a truncated tail (Myo10 HMM), and Myo10 containing four mutations to disrupt its coiled-coil domain (Myo10 CC mutant). We found that the truncation of the tail domain decreased filopodial formation and filopodial length, while four mutations in the coiled-coil domain disrupted the motion of Myo10 toward filopodial tips and the elongation of filopodia. Furthermore, we found that filopodia elongated through multiple elongation cycles, which was supported by the Myo10 tail. These findings suggest that Myo10 tail is crucial for promoting long filopodia.

Direct electrical stimulation impacts on neuromuscular junction morphology on both stimulated and unstimulated contralateral soleus.

BACKGROUND: There is increasing evidence of crosstalk between organs. The neuromuscular junction (NMJ) is a peripheral chemical synapse whose function and morphology are sensitive to acetylcholine (ACh) release and muscle depolarization. In an attempt to improve our understanding of NMJ plasticity and muscle crosstalk, the effects of unilateral direct electrical stimulation of a hindlimb muscle on the NMJ were investigated in rats exposed long-term post-synaptic neuromuscular blockade. METHODS: Sprague Dawley rats were subjected to post-synaptic blockade of neuromuscular transmission by systemic administration of α-cobrotoxin and mechanically ventilated for up to 8 days and compared with untreated sham operated controls and animals exposed to unilateral chronic electrical stimulation 12 h/day for 5 or 8 days. RESULTS: NMJs produced axonal and glial sprouts (growth of processes that extend beyond the confines of the synapse defined by high-density aggregates of acetylcholine receptors [AChRs]) in response to post-synaptic neuromuscular blockade, but less than reported after peripheral denervation or pre-synaptic blockade. Direct electrical soleus muscle stimulation reduced the terminal Schwann cell (tSC) and axonal sprouting in both stimulated and non-stimulated contralateral soleus. Eight days chronic stimulation reduced (P < 0.001) the number of tSC sprouts on stimulated and non-stimulated soleus from 6.7 ± 0.5 and 6.9 ± 0.5 sprouts per NMJ, respectively, compared with 10.3 ± 0.9 tSC per NMJ (P < 0.001) in non-stimulated soleus from rats immobilized for 8 days. A similar reduction of axonal sprouts (P < 0.001) was observed in stimulated and non-stimulated contralateral soleus in response to chronic electrical stimulation. RNAseq-based gene expression analyses confirmed a restoring effect on both stimulated and unstimulated contralateral muscle. The cross-over effect was paralleled by increased cytokine/chemokine levels in stimulated and contralateral unstimulated muscle as well as in plasma. CONCLUSIONS: Motor axon terminals and terminal Schwann cells at NMJs of rats subjected to post-synaptic neuromuscular blockade exhibited sprouting responses. These axonal and glial responses were likely dampened by a muscle-derived myokines released in an activity-dependent manner with both local and systemic effects.

Filopodia powered by class x myosin promote fusion of mammalian myoblasts.

Skeletal muscle fibers are multinucleated cellular giants formed by the fusion of mononuclear myoblasts. Several molecules involved in myoblast fusion have been discovered, and finger-like projections coincident with myoblast fusion have also been implicated in the fusion process. The role of these cellular projections in muscle cell fusion was investigated herein. We demonstrate that these projections are filopodia generated by class X myosin (Myo10), an unconventional myosin motor protein specialized for filopodia. We further show that Myo10 is highly expressed by differentiating myoblasts, and Myo10 ablation inhibits both filopodia formation and myoblast fusion in vitro. In vivo, Myo10 labels regenerating muscle fibers associated with Duchenne muscular dystrophy and acute muscle injury. In mice, conditional loss of Myo10 from muscle-resident stem cells, known as satellite cells, severely impairs postnatal muscle regeneration. Furthermore, the muscle fusion proteins Myomaker and Myomixer are detected in myoblast filopodia. These data demonstrate that Myo10-driven filopodia facilitate multinucleated mammalian muscle formation.

Infections of Soil-Transmitted Helminth in Refugees from North Korea.

Soil-transmitted helminthiases (STH) are now no longer public health problems in the Republic of Korea (South Korea), but their status are unavailable in the residents of North Korea (NK) despite the expectation of large scale traffic and future reunification of the Korean Peninsula. A total of 20 female refugees from NK who had been admitted to the Division of Gastroenterology, Dankook University Hospital, were subjected in this study. Among them, 15 refugees were examined by the colonoscopy and 10 ones were examined with the stool examination (formalin-ether sedimentation). Both diagnostic methods were commonly adopted in 5 patients. Eggs of Trichuris trichiura were detected in 7 out of 10 refugees in the stool examination. In the colonoscopy, T. trichiura worms were found in 6 (40.0%) out of 15 refugees. Total 9 (45.0%) peoples were confirmed to be infected with human whipworms. Additionally, 1 case of clonorchiasis was diagnosed in the stool examination and a worm of Ascaris lumbricoides was discovered from a trichuriasis case. These findings suggested that STH is highly prevalent in NO, in which living conditions are not so good in the aspect of general hygiene and medical care.

Obstructive sleep apnea in postmenopausal women: a comparative study using drug induced sleep endoscopy / Apneia obstrutiva do sono em mulheres na pós-menopausa: estudo comparativo usando endoscopia do sono induzido por fármaco

Abstract Introduction: The key to successful treatment of OSAS is to individually tailor such treatment. Thus, it is very important to determine the severity of OSAS, its pattern, and the extent of collapse, by gender, age, and BMI. Objective: The objective of the study was to understand the characteristics of obstructive sleep apnea in postmenopausal women by comparing postmenopausal and premenopausal subjects, and men, using DISE. We hope that our work will help the medical community to consult on, diagnose, and treat OSAS more effectively. Methods: A total of 273 patients (195 males and 78 females) diagnosed with OSAS were enrolled. Female patients were divided into pre-menopausal (n = 41) and post-menopausal patients (n = 37). The group of post-menopausal female patients was matched with a group of male patients with similar age and body mass index (BMI). DISE findings were compared between pre-menopausal female patients and post-menopausal female patients, and also between post-menopausal female patients and male patients matched for age and BMI. Results: Upon PSG examination, post-menopausal patients (who had a significantly higher BMI than did pre-menopausal patients; 25.6 kg/m2 vs. 23.5 kg/m2; p = 0.019) tended to have a higher AHI and a lower lowest SaO2, but the differences did not attain statistical significance. With DISE analysis, post-menopausal female patients showed higher values in all obstruction sites, with significantly higher value in lateral diameter of retropalatal (1.49 vs. 0.90; p = 0.001) and retrolingual levels (1.14 vs. 0.61; p = 0.003) compared to pre-menopausal females patients. Post-menopausal female patients showed significantly more retrolingual collapse (antero-posterior, AP, p ≤ 0.0001, and lateral diameter, p = 0.042) in the lower BMI group (BMI < 25) and more concentric retropalatal collapse (lateral diameter, p = 0.017 and tonsillar obstruction, p = 0.003) in higher BMI group (BMI ≥ 25) than BMI and age matched male patients. Conclusion: Post-menopausal female patients showed a different pattern of airway obstruction compared to pre-menopausal female patients and male patients matched for age and BMI based on DISE findings.

Resumo Introdução: A chave para o sucesso do tratamento da síndrome da apneia obstrutiva do sono (SAOS) é adaptar individualmente esse tratamento. Assim, é muito importante determinar a gravidade da SAOS, seu padrão e a medida do colapso, por sexo, idade e IMC. Objetivo: O objetivo do estudo foi compreender as características da apneia obstrutiva do sono em mulheres na pós-menopausa, comparando estas características entre mulheres na pós-menopausa e pré-menopausa, e homens, utilizando endoscopia do sono induzido por fármacos (DISE). Esperamos que o nosso estudo ajude a comunidade médica a diagnosticar e tratar a SAOS de maneira mais eficaz. Método: Foram recrutados 273 pacientes (195 do sexo masculino e 78 do feminino) com diagnóstico de SAOS. As pacientes do sexo feminino foram divididas em pacientes na pré-menopausa (n = 41) e na pós-menopausa (n = 37). O grupo de pacientes do sexo feminino na pós-menopausa foi pareada com um grupo de pacientes do sexo masculino com idade e Índice de Massa Corporal (IMC) semelhantes. Os achados da DISE foram comparados entre as pacientes do sexo feminino na pré-menopausa e as pacientes do sexo feminino pós-menopausa e também entre pacientes do sexo feminino na pós-menopausa e pacientes do sexo masculino pareados por idade e IMC. Resultados: Ao exame de PSG, as pacientes na pós-menopausa (que tinham um IMC significativamente maior do que as pacientes na pré-menopausa; 25,6 vs. 23,5 kg/m2; p = 0,019) tenderam a ter um IAH superior e uma saturação arterial de oxigênio (SaO2) mínima menor, mas as diferenças não atingiram significância estatística. Na análise do DISE, pacientes do sexo feminino pós-menopausa apresentaram valores mais elevados em todos os locais de obstrução, com um valor significativamente maior de diâmetro lateral dos níveis retropalatal (1,49 vs. 0,90; p = 0,001) e retrolingual (1,14 vs. 0,61; p = 0,003) em comparação com pacientes do sexo feminino na pré-menopausa. As pacientes do sexo feminino na pós-menopausa apresentaram colapso significativamente mais retrolingual (anteroposterior, AP, p ≤ 0,0001 e diâmetro lateral, p = 0,042) no grupo de IMC menor (IMC < 25) e colapso retropalatal mais concêntrico (diâmetro lateral, p = 0,017 e obstrução tonsilar, p = 0,003) no grupo de maior IMC (IMC ≥ 25) do que pacientes do sexo masculino pareados por IMC e idade. Conclusão: Com base nos achados do DISE, as pacientes do sexo feminino na pós-menopausa apresentaram um padrão diferente de obstrução das vias respiratórias em comparação com pacientes do sexo feminino na pré-menopausa e com os pacientes do sexo masculino pareados por idade e IMC.

The Sihler staining study of the infraorbital nerve and its clinical complication.

The infraorbital nerve (ION) is a cardinal cutaneous nerve that provides general sensation to the mid face. Its twigs are vulnerable to iatrogenic damage during medical and dental manipulations. The aims of this study were to elucidate the distribution pattern of the ION and thus help to prevent nerve damage during medical procedures and to enable accurate prognostic evaluation where complications do occur. This was achieved by treating 7 human hemifaces with the Sihler modified staining protocol, which enables clear visualization of the course and distribution of nerves without the accidental displacement of these structures that can occur during classic dissection. The twigs of the ION can be classified into the usual 5 groups: inferior palpebral, innervating the lower eyelid in a fan-shaped area; external and internal nasal, reaching the nosewing and philtrum including the septal area between the nostrils, respectively; as well as medial and lateral superior labial, supplying the superior labial area from the midline to the mouth corner. Of particular note, the superior labial twigs fully innervated the infraorbital triangle formed by the infraorbital foramen, the most lateral point of the nosewing, and the mouth corner. In the superior 3-quarter area, the ION twigs made anastomoses with the buccal branches of the facial nerve, forming an infraorbital nervous plexus. The infraorbital triangle may be considered a dangerous zone with respect to the risk for iatrogenic complications associated with the various medical interventions such as implant placement.

Work-relatedness of lung cancer by smoking and histologic type in Korea.

OBJECTIVES: This study investigated the distribution of causative agents related to occupational lung cancer, their relationships with work, and associations between work-relatedness and the histologic type of lung cancer. METHODS: We used data from the occupational surveillance system in Korea in 2013. In addition, data from 1,404 participants diagnosed with lung cancer were collected through interviews. We included the patients' longest-held job in the analysis. Work-relatedness was categorized as "definite," "probable," "possible," "suspicious," "none," or "undetermined." RESULTS: Among the subjects, 69.3% were men and 30.7% were women. Regarding smoking status, current smokers were the most prevalent (35.5%), followed by non-smokers (32.3%), ex-smokers (32.2%). Regarding the causative agents of lung cancer, asbestos (1.0%) and crystalline silica (0.9%) were the most common in definite work-related cases, while non-arsenical insecticide (2.8%) was the most common in probable cases followed by diesel engine exhaust (1.9%) and asbestos (1.0%). Regarding histologic type, adenocarcinoma was the most common (41.7%), followed by squamous cell carcinoma (21.2%). Among current smokers, squamous cell carcinoma was the most common among definite and probable cases (13.4%), while non-small cell lung cancer was the least common (7.1%). Among non-smokers, squamous cell carcinoma was the most common (21.4%), while the least common was adenocarcinoma (1.6%). CONCLUSIONS: Approximately, 9.5% of all lung cancer cases in Korea are occupational-related lung cancer. Well-known substances associated with lung cancer, such as crystalline silica, asbestos, and diesel engine exhaust, are of particular concern. However, the histologic types of lung cancer related to smoking were inconsistent with previous studies when work-relatedness was taken into account. Future studies are required to clarify the incidence of occupational lung cancer in agricultural workers exposed to non-arsenical insecticides and the associations between work-relatedness and the histologic type of lung cancer.

Treadmill exercise represses neuronal cell death and inflammation during Aß-induced ER stress by regulating unfolded protein response in aged presenilin 2 mutant mice.

Alzheimer's disease (AD) is characterized by the deposition of aggregated amyloid-beta (Aß), which triggers a cellular stress response called the unfolded protein response (UPR). The UPR signaling pathway is a cellular defense system for dealing with the accumulation of misfolded proteins but switches to apoptosis when endoplasmic reticulum (ER) stress is prolonged. ER stress is involved in neurodegenerative diseases including AD, but the molecular mechanisms of neuronal apoptosis and inflammation by Aß-induced ER stress to exercise training are not fully understood. Here, we demonstrated that treadmill exercise (TE) prevented PS2 mutation-induced memory impairment and reduced Aß-42 deposition through the inhibition of ß-secretase (BACE-1) and its product, C-99 in cortex and/or hippocampus of aged PS2 mutant mice. We also found that TE down-regulated the expression of GRP78/Bip and PDI proteins and inhibited activation of PERK, eIF2α, ATF6α, sXBP1 and JNK-p38 MAPK as well as activation of CHOP, caspase-12 and caspase-3. Moreover, TE up-regulated the expression of Bcl-2 and down-regulated the expressions of Bax in the hippocampus of aged PS2 mutant mice. Finally, the generation of TNFα and IL-1α and the number of TUNEL-positive cells in the hippocampus of aged PS2 mutant mice was also prevented or decreased by TE. These results showed that TE suppressed the activation of UPR signaling pathways as well as inhibited the apoptotic pathways of the UPR and inflammatory response following Aß-induced ER stress. Thus, therapeutic strategies that modulate Aß-induced ER stress through TE could represent a promising approach for the prevention or treatment of AD.

A comparative study on the accuracy of the devices for measuring the implant stability.

STATEMENT OF PROBLEM: How the ISQ values measured by Osstell™ and Osstell™ Mentor are related, and whether the ISQ values acquired from the two machines changes in accordance with changes in implant stability are not yet fully understood. PURPOSE: The aim of this study was to find out correlation between the ISQ values acquired from Osstell™ and Osstell™ Mentor, and to evaluate the clinical effectiveness and accuracy of two devices. MATERIAL AND METHODS: Sixty two implants were inserted into 47 patients, and their ISQ values were measured using Osstell™ and Osstell™ Mentor. In the first stage surgery, the ISQ values of forty four implants inserted into thirty five patients were measured. In the second stage surgery, the values of fifty implants inserted into thirty seven patients were measured. The values were analyzed to determine the difference between the mean ISQ values of Osstell™ and Osstell™ Mentor. In addition, the correlation between implants used in the first and second stage of surgery with regard to their types and areas of insertion were analyzed. The difference between the ISQ values of 32 implants in each patient during the first and second stage was analyzed. The statistical assessment was carried out using SPSS V. 12.0 for Win. (SPSS Inc., Chicago, USA). The Pearson correlation coefficient was used to examine the correlation between Osstell™ and Osstell™ Mentor in the first and second stages of surgery, whereas the difference between their ISQ values was evaluated using a paired t-test. RESULTS: In the first stage, the mean ISQ value for Osstell™ and Osstell™ Mentor was 70.84 and 75.09, respectively, showing a significant difference (P < .01). In the second stage, the mean ISQ value of Osstell™ and Osstell™ Mentor was 71.76 and 75.94, respectively, also showing a significant difference (P < .01). The difference between the ISQ values in patients in the first and the second stages was significant with both instruments. CONCLUSION: The significant difference in the values obtained using the Osstell™ and Osstell™ Mentor between the first and second stages of implant surgery indicates that these values can be a convenient and precise way for evaluating the implant stability in clinical practice.

An anatomic study on the overlap patterns of structural components in the keystone area in noses of koreans.

OBJECTIVES: The keystone area has critical importance in maintaining the stability of the nasal dorsum. However, overlap patterns between structural components in the keystone area have rarely been studied, especially in the noses of Koreans. METHODS: Dissections were performed on 18 cadaveric noses. The length and width of the structural components in the keystone area were measured. The shape of the caudal margin of the nasal bone and the overlap patterns between the upper lateral cartilage and the nasal bone were classified. RESULTS: THE SHAPE OF THE CAUDAL MARGIN OF THE NASAL BONE WERE CLASSIFIED AS FOLLOWS: type A, curvilinear margin; type B, paramedian retractions or "M" shape; type C, elongated midline; and type D, retracted midline. The overlap area between the upper lateral cartilage and the nasal bone was classified as follows: type Ao, crescent shape; type Bo, short midline extension; and type Co, long midline extension. The cephalocaudal length of the overlap area between the upper lateral cartilage and the nasal bone was 4-10 mm (mean, 7 mm) in the midline, and 0-7 mm (mean, 3.1 mm) in the paramedian area. The overlap width of the upper lateral cartilage with the nasal bone was 8-14 mm (mean, 9.7 mm). CONCLUSION: The overlap pattern of the structural components in the keystone area is variable. Therefore, a thorough understanding and a cautious evaluation of the relationships of these components before and during surgery is important in performing safe and effective nasal procedures.

Expression of CD95 and CD95L on astrocytes in the CA1 area of the immature rat hippocampus after hypoxia-ischemia injury.

The immature brain is affected profoundly by hypoxia-ischemia (HI) injury, which can lead to permanent neurologic sequelae in survivors. Neuronal degeneration after HI injury usually is achieved through apoptosis. Both CD95 and its natural ligand, CD95L, which are key molecules in the regulation of apoptosis, are constitutively expressed by neurons and astrocytes during embryonic and early postnatal stages. Further, CD95 or CD95L may have a functional relationship in glial cells and lead to apoptosis of these cells. The hippocampus, especially the CA1 area, is particularly susceptible to HI injury. We therefore investigated the temporal and spatial alterations in CD95 and CD95L expression in the CA1 area of 7-d-old rats after unilateral ligation of the carotid artery. Using immunohistochemistry and Western blotting, we showed that expression of CD95 and CD95L in the hippocampus peaked at 12 h and then decreased. In addition, we used terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick end-labeling to demonstrate apoptosis among CD95- and CD95L-reactive cells. Our findings show that increases in the expression of CD95 and CD95L after HI injury may involve astrocytic apoptosis in the 7-d-old rat hippocampus, and these molecules may act as targets or inducers of cell death.

p21 as a prognostic factor in non-small cell lung carcinomas.

Few studies have focused on the correlation between p21 expression and survival for patients with non-small cell lung carcinoma (NSCLC), and the results are not consistent. We investigated the expression of p21 in 90 cases of NSCLC to evaluate the correlation between the p21 expression level and the clinicopathologic characteristics with patient survival. p21 was expressed in the nuclei of all the NSCLCs. The percentage of immunoreactive cells varied from 1% to 70%. All the patients were subdivided into a high and a low p21 expression group on the basis of the median percentage (17.05). There was no significant correlation between the p21 expression level and age, gender, histologic type, histologic grade, or stage. Using uni- and multivariate analyses, survival was significantly associated with gender, stage, and the p21 expression level. The survival rate for the high p21 expression group was higher than that for the low p21 expression group in the entire patient group, and especially for stage II and III patients, males, adenocarcinomas, or p53-positive tumors. Our findings showed that high p21 expression was an independent prognostic factor for NSCLC. p21 may be useful for determining the candidates for adjuvant therapies and also for selecting the appropriate chemotherapeutic agents.

Perfusion imaging of the brain using Z-score and dynamic images obtained by subtracting images from before and after contrast injection.

OBJECTIVE: The aim of this study was to examine the feasibility of perfusion imaging of the brain using the Z-score and subtraction dynamic images obtained from susceptibility contrast MR images. MATERIALS AND METHODS: Five patients, each with a normal MRI, Moya-moya, a middle cerebral artery occlusion, post-trauma syndrome, and a metastatic brain tumor, were selected for a presentation. A susceptibility-contrast echo-planar image after a routine MRI was taken as the source image with a rapid manual injection of 0.1 mmol/kg of Gd-DTPA. The inflow and washout patterns were observed from the time-signal intensity curve of the serial scans using the standard program of an MRI machine. The repeated Z-score images of the peak and late phases were made using the threshold Z-score values between 1.4 and 2.0 in four to five studies of the pre-contrast, peak, and late phases. Dynamic subtraction images were produced by subtracting sequential post-contrast images from a pre-contrast image and coloring these images using a pseudocolor mapping method. RESULTS: In the diseases with perfusion abnormalities, the Z-score images revealed information about the degree of perfusion during the peak and late phases. However, the quality varied with the Z-score threshold and the studies selected in a group. The dynamic subtraction images were of sufficient quality with no background noise and more clearly illustrated the temporal changes in perfusion and delayed perfusion. CONCLUSION: The Z-scores and dynamic subtraction images illustrated the degree of perfusion and sequential changes in the pattern of perfusion, respectively. These images can be used as a new complimentary method for observing the perfusion patterns in brain diseases.