Multi-center safety and efficacy study of a negative-pressure intraoral device in obstructive sleep apnea.

BACKGROUND: Intraoral devices, with or without negative oral pressure, can stabilize the oropharynx and reduce obstructive sleep apneas. We tested the hypothesis that treatment with the iNAP® Sleep Therapy System, which applies negative oral pressure through an intra-oral appliance, would reduce the severity of obstructive sleep apnea in a multi-center, prospective, first-night-randomized-order cross-over study. METHODS/PATIENTS: 130 patients fulfilled the entry criteria (age <75, AHI 15-55, BMI <33), and 63 entered the primary endpoint cohort (Total Sleep Time ≥4 h/night on the baseline polysomnogram and an oral negative vacuum time maintained by iNAP® ≥ 4 h/night and total sleep time ≥4 h/night during the first treatment study). 54 patients completed a second treatment sleep study at least 28 days after the first sleep study. RESULTS: Among the primary endpoint cohort (n = 63, age = 53.2 ± 11.3, BMI = 27.1 ± 2.8), 33 patients (52 %; 95 % confidence interval = 40%-64 %, p < 0.001) responded to iNAP treatment according to the Sher criteria (>50 % reduction in AHI and an AHI ≤20 events/hr). The average oxy-hemoglobin saturation increased by 1-2%, and the average percent oxygen desaturation decreased (was less severe) by 1 % while using the iNAP device. The incidence of adverse events, all self-limited, was low. The reduction in the apnea-hypopnea index was durable over the 28-day study. Patients used iNAP on average 5.6 h per night during the study period. CONCLUSION: The iNAP® Sleep Therapy System achieved a durable benefit in more than half the patients with moderate to severe obstructive sleep apnea and may be considered in patients who object to or failed continuous positive airway pressure. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02698059.

Pre-clinical evaluation of APrevent® VOIS for unilateral vocal fold paralysis medialization.

Objective: To evaluate the concept and efficacy of an adjustable implant (Prototype SH30: porcine implant and APrevent® VOIS: human concept) for treatment of unilateral vocal fold paralysis (UVFP) via in vivo mini-pig studies, human computed tomographic (CT) and magnetic resonance (MR) image analysis, ex-vivo aerodynamic and acoustic analysis. Methods: Feasibility testing and prototype implantation were performed using in-vivo UVFP porcine model (n = 8), followed by a dimensional finding study using CT and MR scans of larynges (n = 75) for modification of the implant prototypes. Acoustic and aerodynamic measurements were recorded on excised canine (n = 7) larynges with simulated UVFP before and after medialization with VOIS-Implant. Results: The prototype showed in the in-vivo UVFP porcine model an improved glottic closure from grade 6 incomplete closure to complete closure (n = 5), to grade 2 incomplete closure (n = 2) and grade 3 incomplete closure (n = 1). On human CT/MR scans the identification of the correct size was successful in 97.3% using the thyroid cartilage alar "distance S" as the only parameter, which is an important step towards procedure standardization and implant design. Results were confirmed with implantation in human laryngeal cadavers (n = 44). Measurements of the acoustic and aerodynamic effects after implantation showed a significant decreased phonation threshold pressure (p = .0187), phonation threshold flow (p = .0001) and phonation threshold power (p = .0046) on excised canine larynges with simulated UVFP. Percent jitter and percent shimmer decreased (p = .2976; p = .1771) but not significant. Conclusions: Based on the preclinical results four sizes, differing in medial length, implant width and expansion direction of silicone cushions, seem to be enough to satisfy laryngeal size variations. This concept is significantly effective in medializing UVFP and improving the aerodynamic and acoustic qualities of phonation as reported in a preliminary clinical outcome study with long-term implantation. Level of Evidence: N/A.

Mitochondrial replication, transcription, and function in obstructive sleep apnea.

We assessed mitochondrial replication, transcription, and function in the upper airways of obstructive sleep apnea (OSA) patients and the effects of uvulopalatopharyngoplasty. Twenty subjects with mild and 40 with moderate to severe OSA requiring uvulopalatopharyngoplasty were included. Mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in uvula specimens were assessed by immunohistochemical staining, and their mRNA and protein expression was examined using reverse-transcription polymerase chain reaction and western blotting, respectively. The mitochondrial to nuclear DNA (Mt/N) ratio in the blood, exhaled breath condensate (EBC), and uvula was measured using quantitative reverse-transcription polymerase chain reaction. TFAM and PGC-1α protein concentrations in the plasma and EBC were determined using enzyme-linked immunosorbent assay. All tested parameters were higher in the OSA group than in the control. Three months later, 21 uvulopalatopharyngoplasty-responsive patients with OSA showed decreased TFAM and PGC-1α concentrations and EBC Mt/N ratio while these remained high in 19 uvulopalatopharyngoplasty-unresponsive patients. The OSA group showed severe inflammation, increased mitochondrial replication and transcription-related signaling, and mitochondrial dysfunction in the uvula. Successful OSA treatment using uvulopalatopharyngoplasty restored the TFAM and PGC-1α levels and EBC Mt/N ratio.

Nasal Extranodal Natural Killer/T-cell Lymphoma Imitating Squamous Cell Carcinoma: A Case Report.

Nasal extranodal NK/T-cell lymphoma (NNKTL) is a lethal disease due to poor prognosis with rapid progress. A 56-year-old man complained of left nasal obstruction and blood-stained nasal drip for two months. Incisional biopsies were performed at the outpatient department three times, and the diagnosis of SCC was made. The patient underwent wide excision of the entire lesion via endoscopic sinus surgery with navigation. Final pathologic report revealed NNKTL. Pathological examination of the tumor revealed overlying epithelium presenting as pseudoepitheliomatous hyperplasia (PEH), which mimicked SCC invasion, with infiltration of atypical lymphocytes in the deeper sections. Immunohistochemistry supported the diagnosis of NNKTL. Chemoradiotherapy was administered, and a complete response was achieved at the two-year follow-up. The correct diagnosis of NNKTL is essential for prompt treatment and prevention of superfluous surgery. Although the link between PEH and NNKTL may lead to a misdiagnosis of SCC, multiple large and deep biopsies can prevent this dilemma. A biopsy showing ulceration or necrosis can indicate PEH and imply potential malignancy. Repeated biopsies and complete immunohistochemical studies are important for diagnosing NNKTL.

Increased nasality of connected speech in patients underwent modified Lothrop procedure for refractory chronic rhinosinusitis: Cross-sectional and prospective study.

BACKGROUND: The endoscopic modified Lothrop procedure (EMLP) is performed to create a large frontal neostium in patients who had failed previous frontal sinus surgeries. EMLP causes obvious changes in the volume and anatomy of the sinuses, which may cause a more significant change in nasality. This study aimed to evaluate the changes in nasalance in patients who underwent EMLP by comparing their preoperative nasalance to the established normative values and postoperative nasalance. METHODS: This was a prospective study. Twenty-one patients diagnosed with refractory frontal sinusitis who were indicated to undergo EMLP were enrolled. One hundred one healthy participants were enrolled as norm references. The Nasometer II Model 6400 (KayPENTAX) was used to analyze the nasalance scores. Nasalance scores were tested before surgery and 1 and 3 months after the surgery. RESULTS: The normative references of nasalance were (mean ± SD) 14.6% ± 6.7%, 39.4% ± 8.4%, and 55.8% ± 8.3% for the oral passage, oral-nasal passage, and nasal sentences, respectively. The mean nasalance scores pre-EMLP and 1 and 3 months post-EMLP were 23.2% ± 9.6%, 29.0% ± 9.3%, and 29.9% ± 0.4% for the oral passage; 48.7% ± 10.7%, 54.7% ± 7.7%, and 56.4% ± 7.2% for the oral-nasal passage; and 62.7% ± 10.9%, 69.8% ± 6.7%, and 70.7% ± 6.4% for the nasal sentences, respectively. Compared with the normative references, pre-EMLP nasalance was higher for all the three speech stimuli (t-test, p < 0.05). Post-EMLP nasalance also significantly increased for all the three stimuli at the 1- and 3-month follow-up visits (Paired t-test, p < 0.05). CONCLUSION: EMLP has a short-term impact on resonance; however, long-term follow-up is required for further study.

Factors influencing surgeon decision-making by measuring waiting time for pediatric ventilation tube insertions.

BACKGROUND: The surgeon and physician's decision-making may be influenced by many factors. The clinical practice guideline suggested that watchful waiting for 3 months should be the initial management for pediatric otitis media with effusion. The waiting time of ventilation tube insertion for pediatric patients is a proper measurement for physician decision-making. This study investigated factors influencing the waiting time for pediatric ventilation tube insertion and to explore factors influencing physician decision-making. METHODS: Information associated with all patients under 18 years of age who received ventilation tube insertions from July 1, 2000 to December 31, 2009 were retrieved and analyzed from a nationwide, population-based administrative database. The waiting time before ventilation tube insertions from the time of diagnosis of otitis media with effusion was recorded. Certain factors that would influence the waiting time were identified. At the same time, how these factors influenced clinical decision-making were also identified. RESULTS: The waiting time decreased as patient age increased (p < 0.001), and increased as the recent frequency of upper respiratory tract infection diagnosis increased (p < 0.001). Patients who received simultaneously bilateral ventilation tube insertions had shorter waiting time than those who had unilateral surgery (p < 0.01) and patients who had undergone ventilation tube insertions in a tertiary referral center generally had longer waiting times (p < 0.001). CONCLUSION: The waiting time of ventilation tube insertions for pediatric otitis media with effusion can be influenced by many factors. Patients with older age and undergone simultaneously bilateral ventilation tube insertion had shorter waiting time. Patients who had more upper respiratory tract infection episodes and who received ventilation tube insertions in a tertiary referral center setting were subject to longer waiting times.

A Giant Schwannoma Extending from Medial Portion of Middle Cranial Fossa to Parapharyngeal Space and Deep Parotid Space.

Trigeminal schwannomas are rare tumours comprising 0.2% of all intracranial tumours and 0.5% of all head and neck tumours. Patients with trigeminal schwannomas presented with facial hypoesthesia and pain. We presented a case with left bulging oropharynx. The CT scan showed a 3.8x2.6x4.9cm left parapharyngeal tumour compressed to the oropharynx and middle cranial fossa. We performed 3 ways in two times of operation to excise the whole tumour. We chose the transoral approach for parapharyngeal space, trans-parotid approach for deep parotid part and the endoscopic endonasal trans-pterygoid approach and trans-maxillary with Canine fossa trephination for intracranial lesions. The pathology showed schwannoma. A huge schwannoma extended from intracranial to several spaces is difficult to resect just by one approach. We should separate the tumour to several parts by clinical image before the operation and design a plan to remove the whole tumour in different approach. The different space of tumour involvement had several ways to access. We needed to choose the less harm but with better surgical field.

Olfactory Outcomes After Middle Turbinate Resection in Endoscopic Transsphenoidal Surgery: A Prospective Randomized Study.

OBJECTIVE: Endoscopic endonasal transsphenoidal surgery is safe and effective for sellar and parasellar tumor removal. Partial middle turbinate (MT) resection is sometimes performed to optimize the surgical field and facilitate postoperative care. Disturbances in olfaction are concerning because of the lack of randomized studies in this field. STUDY DESIGN: Prospective randomized trial. SETTING: Single academic medical center. METHODS: We resected the lower halves of bilateral MTs in the resected group and laterally fractured bilateral MTs in the preserved group. Olfactory outcomes and sinonasal conditions were assessed by using the validated Taiwan Smell Identification Test and Lund-Kennedy Endoscopy Score, respectively. Forty-nine patients were enrolled in the final analysis, of whom 23 underwent partial MT resection. RESULTS: The average Taiwan Smell Identification Test result was 36.9 one month after surgery, with a significant change of -4.4 ± 3.1 (mean ± SD; P < .01) from baseline. The impact was not significant at 3 months (-2.1 ± 2.6, P = .13) or 6 months (0.3 ± 2.0, P = .79). Between the MT resection and preservation groups, there were no significant differences at postoperative 1 month (P = .60), 3 months (P = .86), and 6 months (P > .99). Lund-Kennedy Endoscopy Score was still higher at 3 months (P = .006) after surgery but returned to the preoperative level at 6 months (P = .63). CONCLUSIONS: Endoscopic endonasal transsphenoidal surgery may affect olfaction at 1 month after surgery, and olfactory function is expected to return after 3 months. Partial MT resection did not result in additional olfactory loss. It is safe to perform partial MT resection during surgery without compromising the olfactory outcomes.

Glaucocalyxin A attenuates allergic responses by inhibiting mast cell degranulation through HMGB1/TLR4/NF-κB signaling pathways / 药学学报

The study is to investigate the effect of glaucocalyxin A (GLA) on mast cell-mediated anaphylaxis. The animal welfare and experimental process of this experiment followed the regulations of the Animal Ethics Committee of Yanbian University. BALB/c mice were used in the animal experiment and randomly divided into five groups, control group, model group, and GLA low, medium, and high dose groups (10, 20, and 40 mg·kg-1). Mice were sensitized by intradermal injection of anti-dinitrophenyl-immunoglobulin E (DNP-IgE) into the ears and challenged with a mixture of DNP-human serum albumin (HSA) and 4% evans blue into the tail veins to prepare an animal skin passive cutaneous anaphylaxis (PCA) model, which was collected from both ears for measurement of dye staining and histology. Rat peritoneal mast cells (RPMCs) were used in the cell experiment and divided into control, IgE + antigen (Ag), and IgE + Ag + GLA groups to determine histamine release as well as calcium influx levels. High-affinity IgE receptor (FcεRI)-mediated signaling pathway proteins and HMGB1/TLR4/NF-κB (high mobility group box 1/toll like receptor 4/nuclear transcription factor kappa B) signaling proteins were detected by Western blot. The results of animal experiments suggest that GLA inhibits PCA, reduces evans blue dye exudation, and reduces ear inflammation and ear thickness in mice. The results of cellular experiments suggested that GLA could reduce histamine release and calcium influx, and inhibit tumor necrosis factor-α (TNF-α), interleukin (IL)-4, IL-13, and IL-1β production; Western blot results showed that GLA inhibited FcεRI-mediated phosphorylation levels of spleen tyrosine kinase (Syk), Lck/Yes novel tyrosine kinase (Lyn), tyrosine kinase Fyn (Fyn), growth-factor receptor-bound protein 2 (Gab2), and phospholipase C (PLC) γ1, while GLA inhibited HMGB1/TLR4 signaling pathway to limit NF-κB p65 nuclear metastasis. The results indicate that GLA inhibits mast cell degranulation and attenuates allergic inflammation through the HMGB1/TLR4/NF-κB signaling pathway.

Building a model to precisely target the responders of a novel intermittent negative air pressure device-with mechanism definition.

OBJECTIVE: Due to the complexity of obstructive sleep apnea syndrome (OSAS), engaging patients in the right treatment poses a constant challenge. A novel oral pressure therapy device, the intermittent negative air pressure Sleep Therapy System (iNAP), has proven to ameliorate respiratory events for OSAS patients. However, the mode of action and the characteristics of its responders are not yet fully understood. Therefore, we have first disclosed the mechanism and provided systemic models to predict the treatment response. METHODS: Series of imaging studies were carried out to differentiate the anatomical features of iNAP responders versus non-responders. Compatible electroencephalography was used to evaluate sleep status during magnetic resonance imaging (MRI) assessments. RESULTS: The upper airway volume was statistically widened under the iNAP treatment while patients were naturally asleep (p < 0.05). Negative predictors included several parameters related to oral-tissue redundancy, enlarged middle pharyngeal space, and longer distance of hyoidale to mandibular plane. Positive predictors included larger angulation of sella-articulate-gonion, longer distance of anterior nasal spine to posterior nasal spine, and elongated tongue, which could correspond to the fact that the iNAP had a greater ability to widen the retropalatal region. Furthermore, algorithms developed by these predictors were built to predict treatment response. CONCLUSIONS: We were able to confirm the effect of the iNAP in widening the upper airway. Anatomic features that can be visually observed or obtained through X-ray films, accompanied with the resulting algorithms, were provided to facilitate physicians' ability to predict patients' treatment response to the iNAP with greater sensitivity and efficiency.

Dysregulation of Dual-Specificity Phosphatases by Epstein-Barr Virus LMP1 and Its Impact on Lymphoblastoid Cell Line Survival.

The strongest evidence of the oncogenicity of Epstein-Barr virus (EBV) in vitro is its ability to immortalize human primary B lymphocytes into lymphoblastoid cell lines (LCLs). Yet the underlying mechanisms explaining how the virus tempers the growth program of the host cells have not been fully elucidated. The mitogen-activated protein kinases (MAPKs) are implicated in many cellular processes and are constitutively activated in LCLs. We questioned the expression and regulation of the dual-specificity phosphatases (DUSPs), the main negative regulator of MAPKs, during EBV infection and immortalization. Thirteen DUSPs, including 10 typical and 3 atypical types of DUSPs, were tested. Most of them were downregulated after EBV infection. Here, a role of viral oncogene latent membrane protein 1 (LMP1) in limiting DUSP6 and DUSP8 expression was identified. Using MAPK inhibitors, we found that LMP1 activates extracellular signal-regulated kinase (ERK) or p38 to repress the expression of DUSP6 and DUSP8, with corresponding substrate specificity. Morphologically, overexpression of DUSP6 and DUSP8 attenuates the ability of EBV-immortalized LCL cells to clump together. Mechanistically, apoptosis induced by restoring DUSP6 and DUSP8 in LCLs indicated a novel mechanism for LMP1 to provide a survival signal during EBV immortalization. Collectively, this report provides the first description of the interplay between EBV genes and DUSPs and contributes considerably to the interpretation of MAPK regulation in EBV immortalization.IMPORTANCE Infections by the ubiquitous Epstein-Barr virus (EBV) are associated with a wide spectrum of lymphomas and carcinomas. It has been well documented that activation levels of MAPKs are found in cancer cells to translate various external or intrinsic stimuli into cellular responses. Physiologically, the dual-specificity phosphates (DUSPs) exhibit great ability in regulating MAPK activities with respect to their capability of dephosphorylating MAPKs. In this study, we found that DUSPs were generally downregulated after EBV infection. EBV oncogenic latent membrane protein 1 (LMP1) suppressed DUSP6 and DUSP8 expression via MAPK pathway. In this way, LMP1-mediated MAPK activation was a continuous process. Furthermore, DUSP downregulation was found to contribute greatly to prevent apoptosis of EBV-infected cells. To sum up, this report sheds light on a novel molecular mechanism explaining how EBV maintains the unlimited proliferation status of the immortalized cells and provides a new link to understand EBV-induced B cell survival.

Free mucosal grafts and anterior pedicled flaps to prevent ostium restenosis after endoscopic modified Lothrop (frontal drillout) procedure: a randomized, controlled study.

BACKGROUND: The endoscopic modified Lothrop procedure (EMLP) is used to manage ongoing refractory frontal sinusitis after failed previous endoscopic sinus surgery (ESS), but this approach has a significant restenosis rate. We evaluated the potential benefits of mucosal grafts and pedicled flaps on the opening of the newly formed frontal ostium. METHODS: Fifty patients with refractory frontal sinusitis or mucoceles after ESS were randomized to undergo EMLP, either with (n = 27) or without (n = 23) mucosal grafts and pedicled flap reconstruction of the neo-ostium. The frontal neo-ostium was measured with Lindholm distending forceps, and anteroposterior (A-P) and lateral dimensions were measured intraoperatively, and then again at 6 weeks, 6 months, and 12 months postoperatively. Olfaction outcomes were assessed using the Taiwan Smell Identification Test (TWSIT) and a smell visual analog scale (VAS) score preoperatively and at 6 months postsurgery. RESULTS: The initial intraoperative mean lateral and A-P dimensions were 23.2 ± 2.7 mm and 14.8 ± 2.3 mm and were significantly decreased at all time-points postoperatively. The mucosal grafts and pedicled flaps had greater lateral and A-P dimensions, and a greater percentage of intraoperative frontal neo-ostium area at all time-points postoperatively (all p < 0.05). At 6 months postoperatively, TWSIT (p = 0.027), but not the smell VAS score (p = 0.063), was significantly improved compared with baseline. TWSIT and smell VAS score changes did not differ between groups (p = 0.92 and p = 0.85, respectively). CONCLUSION: The use of mucosal grafts and pedicled flaps reduces stenosis of the frontal neo-ostium postsurgery and should be considered after EMLP.

Epidemiology of common otologic surgical procedures in pediatric patients: A population-based birth cohort study.

OBJECTIVE: It is generally recognized the most common pediatric otologic surgical procedure is ventilation tube insertion (VTI). Tympanoplasty and mastoidectomy are more frequently performed on adults. In this study we examined the incidence and age distribution of these procedures by use of a population-based birth cohort design, in order to provide an overall view of the role of these procedures in the pediatric population. MATERIALS AND METHODS: We used the national health insurance research database in Taiwan. We retrieved data on all patients born in the years 2000 and 2001, subsequently underwent VTI, tympanoplasty or mastoidectomy from 2000 to 2013. The incidence and age distribution of these procedures were analyzed. RESULTS: The cumulative incidence of VTI, tympanoplasty, and mastoidectomy was 0.41%, 0.02% and 0.025%, respectively. VTI were more often performed on children 4 or 5 years of age. Tympanoplasties are frequently done on children older than 5, and 30.7% of them had earlier VTI. The time interval from VTI to tympanoplasty was 5.18 ±â€¯2.27 years (mean ±â€¯SD). Mastoidectomies are more often performed on children from 2 to 9 years of age. CONCLUSIONS: VTI was the most frequent otologic surgery for the pediatric population, and was more often performed on children 4-5 years old. Also, tympanoplasty is more frequently performed on children older than 5, and a third of them had prior VTI. Overall, the time interval from VTI to tympanoplasty was 5.18 years. Furthermore, children with cleft palate and congenital metabolic disorder were more prone to otologic surgical procedures.

Differential effects of postoperative oral corticosteroid on eosinophilic vs. non-eosinophilic CRSwNP subtypes.

PURPOSE: The efficacy of postoperative oral corticosteroids on surgical outcomes in chronic rhinosinusitis with nasal polyps (CRSwNP) patients following endoscopic sinus surgery (ESS) remains controversial. This study evaluated the potential benefits of postoperative oral corticosteroids on surgical outcomes in CRSwNP patients and investigated the differential effects on eosinophilic CRSwNP (ECRSwNP) and noneosinophilic CRSwNP (NECRSwNP). MATERIALS AND METHODS: Patients with bilateral CRSwNP who underwent ESS were enrolled and randomized to receive either oral prednisolone (30 mg/day) or placebo for 2 weeks after surgery. Visual analog scale (VAS) and Sino-Nasal Outcome Test 22 (SNOT-22) scores were chosen as the subjective outcomes, evaluated at preoperative baseline and 1, 3, and 6 months postoperatively. Lund-Kennedy Endoscopic Scores (LKESs) were used as the objective outcome, evaluated at preoperative baseline and at 2 weeks and 2, 3, and 6 months postoperatively. RESULTS: In total, 100 patients with bilateral CRSwNP were enrolled, of whom only 82 completed the 6-month follow-up. The subjective outcomes showed no significant difference at each follow-up points. Of the objective outcomes, the corticosteroid group reporting a trend of improvement in LKESs at 6 months postoperatively (p = 0.05). After stratification by tissue eosinophils, only patients with NECRSwNP (<10 eosinophils/HPF) demonstrated a significant improvement in LKESs at 3 months postoperatively (p = 0.03). CONCLUSIONS: Postoperative oral corticosteroids did not provide additional improvements in VAS and SNOT-22 scores; nevertheless, a trend of LKES improvement was noted at 6 months postoperatively. After stratification by tissue eosinophils, this effect was significant only among NECRSwNP patients at 3 months follow-up.

The Mechanism of Cathepsins S Inhibitor Affected Ischemia-induced Neovascularization in Experimental Mice / 中国循环杂志

Objectives: To investigate the potential mechanism of cathepsins S inhibitor (CatS-I) affected ischemia-induced neovascularization in experimental mice. Methods: 8 week-old wild type (C57/BL6) mice were randomly divided into 2 groups: Control group, the mice received basic diet with intraperitoneal injection of 5% carboxymethylcellulose sodium and CatS-I group, the mice received basic diet with intraperitoneal injection of CatS-I (1mg/kg·d); all animals were treated for 17 days. n=20 in each group. Hind limb ischemia model was established at 3 days after injection in both groups. Blood flow was measured by laser Doppler blood flow analyzer, the ratio of ischemic area to non-ischemic area was calculated; protein expressions of peroxidase proliferation activated receptors-γ (PPAR-γ), p-Akt, p-eNOS and VEGF were examined by Western blot analysis at day 4 after the operation; frozen section of ischemic skeletal muscle was taken at 7 days after operation to measure capillary density by immunohistochemistry.Results: ① CatS-I inhibited blood flow recovery. Compared with Control group, CatS-I group had slower blood flow recovery in ischemic hind limb, P<0.05. ② CatS-I inhibited capillary formation. At 14 days after operation, capillary formation in non-ischemic skeletal muscles was similar between 2 groups, P>0.05; while in ischemic skeletal muscles, capillary density was lower in CatS-I group than Control group, P<0.01. ③ Compared with Control group, CatS-I group showed decreased protein expressions of PPAR-γ, p-Akt, p-Enos and VEGF, P<0.05. Conclusions: CatS-I regulated ischemia-induced neovascularization might be related to PPAR-γ activation and PI3K/Akt/eNOS signaling pathway in experimental mice.

miR-24-mediated knockdown of H2AX damages mitochondria and the insulin signaling pathway

Mitochondrial deficits or altered expressions of microRNAs are associated with the pathogenesis of various diseases, and microRNA-operated control of mitochondrial activity has been reported. Using a retrovirus-mediated short-hairpin RNA (shRNA) system, we observed that miR-24-mediated H2AX knockdown (H2AX-KD) impaired both mitochondria and the insulin signaling pathway. The overexpression of miR-24 decreased mitochondrial H2AX and disrupted mitochondrial function, as indicated by the ATP content, membrane potential and oxygen consumption. Similar mitochondrial damage was observed in shH2AX-mediated specific H2AX-KD cells. The H2AX-KD reduced the expression levels of mitochondrial transcription factor A (TFAM) and mitochondrial DNA-dependent transcripts. H2AX-KD mitochondria were swollen, and their cristae were destroyed. H2AX-KD also blocked the import of precursor proteins into mitochondria and the insulin-stimulated phosphorylation of IRS-1 (Y632) and Akt (S473 and T308). The rescue of H2AX, but not the nuclear form of ΔC24-H2AX, restored all features of miR-24- or shH2AX-mediated impairment of mitochondria. Hepatic miR-24 levels were significantly increased in db/db and ob/ob mice. A strong feedback loop may be present among miR-24, H2AX, mitochondria and the insulin signaling pathway. Our findings suggest that H2AX-targeting miR-24 may be a novel negative regulator of mitochondrial function and is implicated in the pathogenesis of insulin resistance.

Influenza M1 Virus-Like Particles Consisting of Toxoplasma gondii Rhoptry Protein 4

Toxoplasma gondii infections occur throughout the world, and efforts are needed to develop various vaccine candidates expressing recombinant protein antigens. In this study, influenza matrix protein (M1) virus-like particles (VLPs) consisting of T. gondii rhoptry antigen 4 (ROP4 protein) were generated using baculovirus (rBV) expression system. Recombinant ROP4 protein with influenza M1 were cloned and expressed in rBV. SF9 insect cells were coinfected with recombinant rBVs expressing T. gondii ROP4 and influenza M1. As the results, influenza M1 VLPs showed spherical shapes, and T. gondii ROP4 protein exhibited as spikes on VLP surface under transmission electron microscopy (TEM). The M1 VLPs resemble virions in morphology and size. We found that M1 VLPs reacted with antibody from T. gondii-infected mice by western blot and ELISA. This study demonstrated that T. gondii ROP4 protein can be expressed on the surface of influenza M1 VLPs and the M1 VLPs containing T. gondii ROP4 reacted with T. gondii-infected sera, indicating the possibility that M1 VLPs could be used as a coating antigen for diagnostic and/or vaccine candidate against T. gondii infection.