Engineering Human Circulating Monocytes/Macrophages by Systemic Deliverable Gene Editing.

Delivery of plasmid DNA to transfect human primary macrophages is extremely difficult, especially for genetic engineering. Engineering macrophages is imperative for the treatment of many diseases including infectious diseases, cancer, neurological diseases, and aging. Unfortunately, plasmid does not cross the nuclear membranes of terminally differentiated macrophages to integrate the plasmid DNA (pDNA) into their genome. To address this issue, we have developed a core-shell nanoparticle (NP) using our newly created cationic lipid to deliver the anti-inflammatory cytokine IL-4 pDNA (IL-4pDNA-NPs). Human blood monocyte-derived macrophages (MDM) were effectively transfected with IL-4pDNA-NPs. IL-4pDNA-NPs were internalized in MDM within 30 minutes and delivered into the nucleus within 2 hours. Exogenous IL-4 expression was detected within 1 - 2 days and continued up to 30 days. Functional IL-4 expression led to M2 macrophage polarization in vitro and in an in vivo mouse model of inflammation. These data suggest that these NPs can protect pDNA from degradation by nucleases once inside the cell, and can transport pDNA into the nucleus to enhance gene delivery in macrophages in vitro and in vivo. In this research, we developed a new method to deliver plasmids into the nucleus of monocytes and macrophages for gene-editing. Introducing IL-4 pDNA into macrophages provides a new gene therapy solution for the treatment of various diseases.

Effect of Surface Modification for Carbon Cathode Materials on Charge-Discharge Performance of Li-Air Batteries.

Li-air batteries have attracted considerable attention as rechargeable secondary batteries with a high theoretical energy density of 11,400 kWh/g. However, the commercial application of Li-air batteries is hindered by issues such as low energy efficiency and a short lifetime (cycle numbers). To overcome these issues, it is important to select appropriate cathode materials that facilitate high battery performance. Carbon materials are expected to be ideal materials for cathodes due to their high electrical conductivity and porosity. The physicochemical properties of carbon materials are known to affect the performance of Li-air batteries because the redox reaction of oxygen, which is an important reaction for determining the performance of Li-air batteries, occurs on the carbon materials. In this study, we evaluated the effect of the surface modification of carbon cathode materials on the charge-discharge performance of Li-air batteries using commercial Ketjenblack (KB) and KB subjected to vacuum ultraviolet (VUV) irradiation as cathodes. The surface wettability of KB changed from hydrophobic to hydrophilic as a result of the VUV irradiation. The ratio of COOH and OH groups on the KB surface increased after VUV irradiation. Raman spectra demonstrated that no structural change in the KB before and after VUV irradiation was observed. The charge and discharge capacities of a Li-air battery using VUV-irradiated KB as the cathode decreased compared to original KB, whereas the cycling performance of the Li-air battery improved considerably. The sizes and shapes of the discharge products formed on the cathodes changed considerably due to the VUV irradiation. The difference in the cycling performance of the Li-air battery was discussed from the viewpoint of the chemical properties of KB and VUV-irradiated KB.

Systematic Study of Effective Hydrothermal Synthesis to Fabricate Nb-Incorporated TiO2 for Oxygen Reduction Reaction.

Fuel cells are expected to serve as next-generation energy conversion devices owing to their high energy density, high power, and long life performance. The oxygen reduction reaction (ORR) is important for determining the performance of fuel cells; therefore, using catalysts to promote the ORR is essential for realizing the practical applications of fuel cells. Herein, we propose Nb-incorporated TiO2 as a suitable alternative to conventional Pt-based catalysts, because Nb doping has been reported to improve the conductivity and electron transfer number of TiO2. In addition, Nb-incorporated TiO2 can induce the electrocatalytic activity for the ORR. In this paper, we report the synthesis method for Nb-incorporated TiO2 through a hydrothermal process with and without additional load pressures. The electrocatalytic activity of the synthesized samples for the ORR was also demonstrated. In this process, the samples obtained under various load pressures exceeding the saturated vapor pressure featured a high content of Nb and crystalline TiNb2O7, resulting in an ellipsoidal morphology. X-ray diffraction results also revealed that, on increasing the Nb doping amounts, the diffraction peak of the anatase TiO2 shifted to a lower angle and the full width at half maximum decreased. This implies that the Ti atom is exchanged with the Nb atom during this process, resulting in a decrease in TiO2 crystallinity. At a doping level of 10%, Nb-incorporated TiO2 exhibited the best electrocatalytic activity in terms of the oxygen reduction current (iORR) and onset potential for the ORR (EORR); this suggests that 10% Nb-doped samples have the potential for enhancing electrocatalytic activity.

Cytoplasmic Trafficking of Nanoparticles Delivers Plasmid DNA for Macrophage Gene-editing.

BACKGROUND: Successful delivery of gene-editing tools using nano-carriers is dependent on the ability of nanoparticles to pass through the cellular membrane, move through the cytoplasm, and cross the nuclear envelope to enter the nucleus. It is critical that intracellular nanoparticles interact with the cytoskeletal network to move toward the nucleus, and must escape degradation pathways including lysosomal digestion. Without efficient intracellular transportation and nuclear entry, nanoparticles-based gene-editing cannot be effectively used for targeted genomic modification. OBJECTIVE: We have developed nanoparticles with a low molecular weight branched polyethylenimine lipid shell and a PLGA core that can effectively deliver plasmid DNA to macrophages for gene editing while limiting toxicity. METHODS: Core-shell nanoparticles were synthesized by a modified solvent evaporation method and were loaded with plasmid DNA. Confocal microscopy was used to visualize the internalization, intracellular distribution and cytoplasmic transportation of plasmid DNA loaded nanoparticles (pDNA-NPs) in bone marrow-derived macrophages. RESULTS: Core-shell nanoparticles had a high surface charge of +56 mV and narrow size distribution. When loaded with plasmid DNA for transfection, the nanoparticles increased in size from 150 nm to 200 nm, and the zeta potential decreased to +36 mV, indicating successful encapsulation. Further, fluorescence microscopy revealed that pDNA-NPs crossed the cell membrane and interacted with actin filaments. Intracellular tracking of pDNA-NPs showed successful separation of pDNA- NPs from lysosomes, allowing entry into the nucleus at 2 hours, with further nuclear ingress up to 5 hours. Bone marrow-derived macrophages treated with pDNA/GFP-NPs exhibited high GFP expression with low cytotoxicity. CONCLUSION: Together, this data suggests pDNA-NPs are an effective delivery system for macrophage gene-editing.

In situ fabrication of silver/polyimide composite films with enhanced heat dissipation.

In this study, silver/polyimide (Ag/PI) composite films with enhanced heat dissipation properties were prepared. Ag was formed in situ by reducing AgNO3 at various locations according to the reduction method. Two different types of soluble PIs capable of solution processing were used, namely Matrimid and hydroxy polyimide (HPI). Unlike Matrimid with bulky substituents, HPI with polar hydroxy groups formed ion-dipole interactions with Ag ions to form Ag particles with uniform size distribution. The location and distribution of Ag particles affect the heat emission characteristics of the composite films, resulting in better heat dissipation properties with the thermally and photochemically reduced Ag/HPI films having more Ag particles distributed inside of the films than the chemically reduced films.

Characterization and Photocatalytic Performance of Potassium-Doped Titanium Oxide Nanostructures Prepared via Wet Corrosion of Titanium Microspheres.

Potassium doped titanium oxide (KTiOx) nanowires were prepared by the wet corrosion process (WCP) and their photocatalytic effects were systematically characterized. For the synthesis of KTiOx, the potassium hydroxide concentration of the WCP was varied in order to obtain nanostructures with different surface area and surface charge. Structural and crystalline properties of KTiOx were studied by means of X-ray diffraction, scanning and transmission electron microscopy. Chemical composition was determined by X-ray fluorescence and energy-dispersive X-ray analysis. Photocatalytic performance was investigated as a function of the surface area, pH, and crystalline structures by studying the degradation of methylene blue, cardiogreen, and azorubine red dyes upon UV irradiation. The negatively charged crystalline KTiOx nanostructures with high surface area showed significantly higher photocatalytic degradation compared to their TiOx counterpart. They also showed high efficiency for recovery and re-use. Annealing KTiOx nanostructures improved structural properties leading to well-ordered layered structures and improved photocatalysis. However, annealing at temperatures higher than 600 °C yielded formation of rutile grains at the surface of nanowires, significantly affecting the photocatalytic performance. We believe that KTiOx nanostructures produced by WCP are very promising for photocatalysis, especially due to their high photocatalytic efficiency as well as their potential for re-use and durability.

Facile Gene Delivery Derived from Branched Low Molecular Weight Polyethylenimine by High Efficient Chemistry.

Convenient methods for the preparation of gene delivery platforms based on branched low molecular weight polyethylenimine (PEI) were described. Firstly, PEI lipids, with a low molecular weight PEI headgroup and hexadecyl chain tail group, were prepared through a highly efficient ring-opening reaction of glycidyl hexadecyl ether (EpoxyC16) by amine from PEI. Then, the PEI lipids were used as a component of cationic liposomes and as a surfactant for the preparation of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticle (NP) via solvent extraction/evaporation method. As potential effective gene delivery platforms, their preparation, size, size distribution, toxicities, plasmid DNA loading, in vitro transfection and intracellular trafficking were studied. Both facile platforms showed less toxicity and higher transfection efficacy when compared to high molecular weight PEI in vitro, and may have further versatile applications in the gene delivery field.

Diarylheptanoids suppress proliferation of pancreatic cancer PANC-1 cells through modulating shh-Gli-FoxM1 pathway.

Pancreatic cancer is one of the leading causes of cancer, and it has the lowest 5-year survival rates. It is necessary to develop more potent anti-pancreatic cancer drugs to overcome the fast metastasis and resistance to surgery, radiotherapy, chemotherapy, and combinations of these. We have identified several diarylheptanoids as anti-pancreatic cancer agents from Alpinia officinarum (lesser galangal) and Alnus japonica. These diarylheptanoids suppressed cell proliferation and induced the cell cycle arrest of pancreatic cancer cells (PANC-1). Among them, the most potent compounds 1 and 7 inhibited the shh-Gli-FoxM1 pathway and their target gene expression in PANC-1 cells. Furthermore, they suppressed the expression of the cell cycle associated genes that were rescued by the overexpression of exogenous FoxM1. Taken together, (E)-7-(4-hydroxy-3-methoxyphenyl)-1-phenylhept-4-en-3-one (1) from Alpinia officinarum (lesser galangal) and platyphyllenone (7) from Alnus japonica inhibit PANC-1 cell proliferation by suppressing the shh-Gli-FoxM1 pathway, and they can be potential candidates for anti-pancreatic cancer drug development.

Analysis of Dysphagia Patterns Using a Modified Barium Swallowing Test Following Treatment of Head and Neck Cancer.

PURPOSE: The purposes of this study were to evaluate specific dysphagia patterns and to identify the factors affecting dysphagia, especially aspiration, following treatment of head and neck cancer. MATERIALS AND METHODS: A retrospective analysis of 57 patients was performed. Dysphagia was evaluated using a modified barium swallow (MBS) test. The MBS results were rated on the 8-point penetration-aspiration scale (PAS) and swallowing performance status (SPS) score. RESULTS: Reduced base of the tongue (BOT) retraction (64.9%), reduced laryngeal elevation (57.9%), and cricopharyngeus (CP) dysfunction (47.4%) were found. Reduced BOT retraction was correlated with clinical stage (p=0.011) and treatment modality (p=0.001). Aspiration in 42.1% and penetration in 33.3% of patients were observed. Twenty-four patients had PAS values over 6, implying aspiration. Forty-one patients had a SPS score of more than 3, 25 patients had a score greater than 5, and 13 patients had a SPS score of more than 7. Aspiration was found more often in patients with penetration (p=0.002) and in older patients (p=0.026). In older patients, abnormal swallowing caused aspiration even in those with a SPS score of more than 3, irrespective of stage or treatment, contrary to younger patients. Tube feeders (n=20) exhibited older age (65.0%), dysphagia/aspiration related structures (DARS) primaries (75.0%), higher stage disease (66.7%), and a history of radiotherapy (68.8%). CONCLUSION: Reduced BOT retraction was the most common dysphagia pattern and was correlated with clinical stage and treatment regimens including radiotherapy. Aspiration was more frequent in patients who had penetration and in older patients. In contrast to younger patients, older patients showed greater risk of aspiration even with a single abnormal swallowing irrespective of stage or treatment.

New cytotoxic sesquiterpenoids from Siegesbeckia glabrescens.

Two new sesquiterpenoids, siegenolides A (1) and B (2), and two known sesquiterpenes 3 and 4 were isolated from Siegesbeckia glabrescens. Their structures were elucidated by spectroscopic analyses, and they were further evaluated for their cytotoxic activities against human cancer cells (MCF-7, AsPC-1, SW480, HCT 116, HepG2, HeLa). Compounds 1-4 showed differential cytotoxic effects on the target cancer cells with IC50 values in the range of 0.9-33.3 µM.

Preparation and Photocatalytic Activity of Potassium- Incorporated Titanium Oxide Nanostructures Produced by the Wet Corrosion Process Using Various Titanium Alloys.

Nanostructured potassium-incorporated Ti-based oxides have attracted much attention because the incorporated potassium can influence their structural and physico-chemical properties. With the aim of tuning the structural and physical properties, we have demonstrated the wet corrosion process (WCP) as a simple method for nanostructure fabrication using various Ti-based materials, namely Ti-6Al-4V alloy (TAV), Ti-Ni (TN) alloy and pure Ti, which have 90%, 50% and 100% initial Ti content, respectively. We have systematically investigated the relationship between the Ti content in the initial metal and the precise condition of WCP to control the structural and physical properties of the resulting nanostructures. The WCP treatment involved various concentrations of KOH solutions. The precise conditions for producing K-incorporated nanostructured titanium oxide films (nTOFs) were strongly dependent on the Ti content of the initial metal. Ti and TAV yielded one-dimensional nanowires of K-incorporated nTOFs after treatment with 10 mol/L-KOH solution, whereas TN required a higher concentration (20 mol/L-KOH solution) to produce comparable nanostructures. The obtained nanostructures revealed a blue-shift in UV absorption spectra due to the quantum confinement effects. A significant enhancement of the photocatalytic activity was observed via the chromomeric change and the intermediate formation of methylene blue molecules under UV irradiation. This study demonstrates the WCP as a simple, versatile and scalable method for the production of nanostructured K-incorporated nTOFs to be used as high-performance photocatalysts for environmental and energy applications.

Metachronous adenoid cystic carcinoma in the peripheral lung and at base of the tongue.

Primary lung adenoid cystic carcinoma (ACC) is extremely rare and accounts for approximately 0.1%-0.2% of all lung cancers. ACC of the head and neck has generally been regarded as a slow-growing, low-grade malignancy which has a tendency for local recurrence and frequent distant metastasis. When ACC of the lung is identified, physicians must determine whether it represents distant metastasis or a primary lung cancer. Thyroid transcription factor-1 staining is one of the most useful methods to differentiate primary from metastatic lesions in lung cancer. Herein we report a case of metachronous, not synchronous, ACC at the peripheral lung followed by ACC presentation at the base of the tongue, and review of relevant literatures.

Transoral endoscopic thyroidectomy via the tri-vestibular routes: results of a preclinical cadaver feasibility study.

The concept of natural orifice transluminal endoscopic surgery (NOTES) is an emerging experimental alternative to conventional surgery that eliminates skin incisions using an endoscope passed through a natural orifice (e.g., mouth, urethra, or anus). This study was designed to evaluate the feasibility and safety of thyroid resection via an entirely transoral tri-vestibular route using endoscopy, and to introduce NOTES to the head and neck area of medicine. We performed ten complete endoscopic thyroid lobectomies with central lymph node dissection via a tri-vestibular approach in fresh-frozen cadavers. A 5-mm endoscope with a deflectable tip was used to visualize the surgical field. Three cannulas were inserted through the midline and bilateral incision sites in the vestibule to position the instruments and endoscope. We refined and described the surgical technique in each step using video clips. We identified and preserved neighboring critical structures during surgery. We also confirmed that there were no obvious remnant thyroid tissues and no injury to the neighboring structures after exploration. The transoral tri-vestibular approach seems to provide a good view and surgical field for endoscopic thyroidectomy. However, the transoral approach for thyroidectomy remains experimental, and the detailed surgical technique should be refined via further clinical studies.

Nineteen-year oncologic outcomes and the benefit of elective neck dissection in salivary gland adenoid cystic carcinoma.

BACKGROUND: The purposes of this study were to evaluate the oncologic outcomes of salivary gland adenoid cystic carcinoma (ACC) and to confirm the benefits of elective neck dissection. METHODS: We reviewed the records of 61 consecutive patients with ACC. Surgery was performed in all patients. RESULTS: The occult metastasis rate was 15.38% (4 of 26 patients) and no regional recurrence in the elective neck dissection group was identified. Among 4 clinically node positive (cN+) patients, regional metastasis was identified in 3 through therapeutic neck dissection. Regional recurrence was identified in 4 patients (4 of 31) who had never undergone elective neck treatment of clinically node negative (cN-) status, exclusively. Overall regional metastases (overall N+) were identified in 11 patients. The overall survival rate was 84.99% at 5 years, 81.13% at 10 and 15 years in (overall N-) status, contrary to 56.82% at 5 years and 28.41% at 10 years in overall N+ status (p = .025). CONCLUSION: Careful follow-up of regional status is important, and proper therapeutic and elective neck treatment can achieve regional control in ACC. Elective neck dissection is recommendable and can provide valuable staging and prognostic information.

Robot-assisted versus endoscopic submandibular gland resection via retroauricular approach: a prospective nonrandomized study.

The aim of this study was to compare the short-term clinicopathological results of robot-assisted and endoscopic resection of the submandibular gland by the retroauricular approach. We present a prospective, non-random, study of 35 patients who had endoscope-assisted (n=22), or robot-assisted (n=13), resection of the submandibular gland using a retroauricular approach. Patients selected one of the two methods, and short-term clinical outcomes were then compared, including maximum diameter of the tumour and gland, length of incision, total operating time, amount of operative bleeding, amount and duration of drainage, duration of hospital stay, cosmesis, and complications. The operation was feasible in all patients with no appreciable operative complications or need to convert to a conventional open operation. The operating time of the robot-assisted group (63.4 (6.3)min) was comparable with that of the endoscopic group (66.5 (9.1)min) (p=0.15). There were no differences in the other clinical outcomes between the 2 groups. Both groups of patients were extremely satisfied with their postoperative cosmesis, and did not differ significantly (p=0.89). Robotic and endoscopic resection of the submandibular gland gave comparable early surgical outcomes and excellent cosmetic results. Despite the technical convenience for the surgeon, the robot gave no apparent clinical benefit over the endoscope in this series.

Characteristics, management of the neck, and oncological outcomes of malignant minor salivary gland tumours in the oral and sinonasal regions.

The aim of this study was to evaluate the characteristics of malignant tumours of the minor salivary glands in the oral and sinonasal regions, to make sure that the neck was managed correctly, and to identify oncological outcomes, retrospectively, at a single hospital. A total of 60 patients were reviewed. Forty-nine patients had intraoral lesions and 11 had sinonasal lesions. Of the 60 patients, 28 had stage I to II malignant tumours, and 32 had stage III to IV tumours. Treatment was almost exclusively surgical. One of the 16 patients whose nodes were clinically clear had an elective neck dissection. Adjuvant radiotherapy was given if indicated. The mean follow-up period was 52 months (range 13-190). Sinonasal lesions were all advanced T-stage at diagnosis, had more invaded resection margins, and a higher local recurrence rate than intraoral lesions. There was no regional recurrence in those patients whose nodes were clinically invaded and who had therapeutic neck dissection, or in the patient whose nodes were clinically clear and who had an elective neck dissection. Occult metastases developed exclusively in adenoid cystic carcinomas (ACC), the rate of which was 4/16. Regional recurrence developed in 4 patients who had never had elective treatment to the neck 2 of whom mixed pattern ACC and 2 who had low grade mucoepidermoid carcinomas (MEC). The overall survival was 90% at 2 years, 77% at 5 years, and 74% at 10 years. Sinonasal minor salivary gland tumours require careful follow-up because resection margins are more likely to be invaded by tumour, and they have a higher local recurrence rate than intraoral lesions. Elective neck dissection is needed, particularly for MEC and also to prevent regional recurrence in ACC.

Direct electron transfer with enzymes on nanofiliform titanium oxide films with electron-transport ability.

Direct electron transfer (DET) from biomolecules to electrode is a process without electron-mediators, thus superior selectivity and sensitivity is expected in order to monitor electron transfer between electrode and biomolecules without any mediator interference. However, DET is difficult because a redox center which is an electron active center of proteins such as enzymes is buried deep. So, a unique electrode nanostructure to reach the redox center is a critical factor. Here we have systematically investigated terms for DET using various nanofiliformed electrode morphologies and enzyme concentrations. It is pointed out that the reaction site is below 100 nm, the ration amounts of adsorbed enzyme per surface area are below 1.0 are contributed to the DET. As a great application, we have developed a biosensor monitoring the hydrogen peroxide (H(2)O(2)) detecting capability from peroxidase directly. For the fabricated HRP/nTOF/Ti-electrodes observed the catalytic current value was linear according to the increase in the concentration of H(2)O(2) up to 100 µM, which indicates a good potential for an H(2)O(2) biosensor.

Transoral robotic surgery for hypopharyngeal squamous cell carcinoma: 3-year oncologic and functional analysis.

The recent trend in treatment of hypopharyngeal cancer is organ preservation in order to maintain swallowing and speech function as well as improve quality of life. Transoral robotic surgery (TORS) can remove hypopharyngeal lesions successfully without an external incision, preserving physiologic functions of affected organs. However, studies have yet to assess the oncologic and functional results of TORS for the treatment of hypopharyngeal cancer. This prospective study evaluated the oncologic and functional results of TORS for the treatment of hypopharyngeal cancer obtained at our institution over a period of 3 years and confirmed the validity of TORS as a surgical organ-preserving strategy. Between April 2008 and September 2011, 23 patients who were diagnosed with hypopharyngeal cancer underwent TORS for removal of a primary lesion. The da Vinci Robotic system (Intuitive Surgical Inc., Sunnyvale, California) was used to remove the lesion. The Kaplan-Meier method was used to analyze overall survival and disease-free survival. Videopharyngogram study (VEF) was performed and functional outcome swallowing scale (FOSS) was utilized to measure and evaluate swallowing function. Acoustic wave form analysis was conducted to evaluate voice status. Overall survival at 3 years was 89% and disease-free survival was 84%. On the VEF study, serious aspiration or delay of swallowing was not observed during the pharyngeal stage of the swallowing process. Overall, 96% of the patients showed favorable swallowing abilities with an FOSS score ranging from 0 to 2. The fundamental frequency variation (vF0) and jitter were increased upon acoustic waveform analysis (vF0=2.71 ± 0.063, Jitter=2.01 ± 0.034), but the harmonic-to-noise ratio (HNR) and shimmer were maintained close to the normal range (HNR=1.28 ± 0.001, Shim=1.74 ± 0.036). The oncologic and functional results of TORS were quite acceptable for the treatment of hypopharyngeal cancer. TORS is a valid treatment option as a surgical, organ-preserving strategy for select patients with hypopharyngeal cancer.

Prognosis of patients with recurrent facial palsy.

No studies to date have classified recurrent facial palsy according to the House-Brackmann grade, thus limiting any comparisons between patients with recurrent and primary facial palsy. The aims of the study were to analyze the degree of recovery from recurrent facial palsy and factors affecting the degree of recovery and to compare with patients recovering from primary facial palsy. Seventy-two patients with recurrent facial palsy were treated by the same treatment approach as 1,185 patients with general facial palsy. Underlying disease, palsy side, and extent of recovery were measured. Temporal bone magnetic resonance image (MRI), Electroneurography (ENoG), electromyography (EMG), and initial House-Brackmann grade were assessed and compared by statistical analyses. Recovery rate of facial palsy were compared and related risk factors were analyzed. We found that recovery to better than House-Brackmann grade II was more common in patients with primary (88.4%, 1,047/1,185) than with recurrent (72.2%, 52/72) facial palsy. Recovery was observed in 80.6% (25/31) of patients with recurrence on the ipsilateral side and in 72% (18/25) with recurrence on the contralateral side. The accuracies of ENoG and EMG were 76.4 and 84.7%, respectively. In MRI, 26.4% patients showed enhancement findings. Multivariate logistic regression analysis showed that initiation of treatment within 7 days and unfavorable EMG results were significant risk factors in patients with recurrent facial palsy. The recovery rate was lower in patients with recurrent than with primary facial palsy, but there were no significant differences in recovery rates between recurrences on the ipsilateral and contralateral sides. The prognosis of patients with recurrent facial palsy was associated with initiation of treatment within 7 days, with EMG shown to be the most reliable test.