CRISPR interference to evaluate modifiers of C9ORF72-mediated toxicity in FTD.

Treatments for neurodegenerative disease, including Frontotemporal dementia (FTD) and Amyotrophic lateral sclerosis (ALS), remain rather limited, underscoring the need for greater mechanistic insight and disease-relevant models. Our ability to develop novel disease models of genetic risk factors, disease modifiers, and other FTD/ALS-relevant targets is impeded by the significant amount of time and capital required to develop conventional knockout and transgenic mice. To overcome these limitations, we have generated a novel CRISPRi interference (CRISPRi) knockin mouse. CRISPRi uses a catalytically dead form of Cas9, fused to a transcriptional repressor to knockdown protein expression, following the introduction of single guide RNA against the gene of interest. To validate the utility of this model we have selected the TAR DNA binding protein (TDP-43) splicing target, stathmin-2 (STMN2). STMN2 RNA is downregulated in FTD/ALS due to loss of TDP-43 activity and STMN2 loss is suggested to play a role in ALS pathogenesis. The involvement of STMN2 loss of function in FTD has yet to be determined. We find that STMN2 protein levels in familial FTD cases are significantly reduced compared to controls, supporting that STMN2 depletion may be involved in the pathogenesis of FTD. Here, we provide proof-of-concept that we can simultaneously knock down Stmn2 and express the expanded repeat in the Chromosome 9 open reading frame 72 (C9ORF72) gene, successfully replicating features of C9-associated pathology. Of interest, depletion of Stmn2 had no effect on expression or deposition of dipeptide repeat proteins (DPRs), but significantly decreased the number of phosphorylated Tdp-43 (pTdp-43) inclusions. We submit that our novel CRISPRi mouse provides a versatile and rapid method to silence gene expression in vivo and propose this model will be useful to understand gene function in isolation or in the context of other neurodegenerative disease models.

Inhibitory effect of lanosterol on cataractous lens of cynomolgus monkeys using a subconjunctival drug release system.

Background: To evaluate the effect of lanosterol on cataractous lens of cynomolgus monkeys using a subconjunctival drug release system. Methods: Nine elder cynomolgus monkeys were used, consisting of three monkeys without cataract as controls, three monkeys with naturally occurring cortical cataract, and three monkeys with nuclear cataract as intervention groups. Nanoparticulated thermogel with lanosterol and fluorescein was administered by subconjunctival injection in the monkeys with cataract. Fluorescence changes of injected thermogel and cataract progression were observed. Lanosterol concentration in aqueous humor, solubility changes in lens proteins, and oxidative stress levels were analyzed in the lenses of the control and intervention groups. Results: Injected thermogel showed decreased fluorescence during follow up. Lanosterol concentration in aqueous humor increased in the first 2 weeks and then gradually decreased, which was in accordance with the changes in cortical lens clarity. However, lenses with nuclear opacification showed little change. In the cortical region of lenses with cortical cataract, solubility of α-crystallin was significantly increased after administration of lanosterol, as well as the reduction of oxidative stress. Conclusions: We demonstrated the effect of lanosterol on cataract progression based on in vivo models of primates. Lanosterol showed a short-term and reliable reversal effect on reducing cataract severity in cortical cataract in the early stages, possibly due to the increase in the solubility of lens proteins and changes in the oxidative stress status. Lanosterol administration using subconjunctival drug release system could be a promising nonsurgical approach for future clinical studies of cataract prevention and treatment.

Development and multi-center clinical trials of an up-converting phosphor technology-based point-of-care (UPT-POCT) assay for rapid COVID-19 diagnosis and prediction of protective effects.

BACKGROUND: Quantitative point-of-care testing assay for detecting antibodies is critical to COVID-19 control. In this study, we established an up-conversion phosphor technology-based point-of-care testing (UPT-POCT), a lateral flow assay, for rapid COVID-19 diagnosis, as well as prediction of seral neutralizing antibody (NAb) activity and protective effects. METHODS: UPT-POCT was developed targeting total antibodies against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. Using ELISA as a contrast method, we evaluated the quantitation accuracy with NAb and serum samples. Cutoff for serum samples was determined through 70 healthy and 140 COVID-19 patients. We evaluated the cross-reactions with antibodies against other viruses. Then, we performed multi-center clinical trials of UPT-POCT, including 782 patients with 387 clinically confirmed COVID-19 cases. Furthermore, RBD-specific antibody levels were detected using UPT-POCT and microneutralization assay for samples from both patients and vaccinees. Specifically, the antibodies of recovered patients with recurrent positive (RP) reverse transcriptase-polymerase chain reaction test results were discussed. RESULTS: The ratios of signal intensities between the test and control bands on the lateral flow strip, namely, T/C ratios, was defined as the results of UPT-POCT. T/C ratios had excellent correlations with concentrations of NAb, as well as OD values of ELISA for serum samples. The sensitivity and specificity of UPT-POCT were 89.15% and 99.75% for 782 cases in seven hospitals in China, respectively. We evaluated RBD-specific antibodies for 528 seral samples from 213 recovered and 99 RP COVID-19 patients, along with 35 seral samples from inactivated SARS-CoV-2 vaccinees, and we discovered that the total RBD-specific antibody level indicated by T/C ratios of UPT-POCT was significantly related to the NAb titers in both COVID-19 patients (r = 0.9404, n = 527; ρ = 0.6836, n = 528) and the vaccinees (r = 0.9063, ρ = 0.7642, n = 35), and it was highly relevant to the protection rate against RP (r = 0.9886, n = 312). CONCLUSION: This study reveals that the UPT-POCT for quantitative detection of total RBD-specific antibody could be employed as a surrogate method for rapid COVID-19 diagnosis and prediction of protective effects.

Safety and efficacy of grazoprevir/elbasvir in the treatment of acute hepatitis C in hemodialysis patients.

Treatment of hepatitis C virus (HCV) infection with direct-acting antiviral agents (DAAs) in hemodialysis patients requires extensive consideration. At present, studies regarding DAAs for acute HCV infection in such patients are limited. The present study aims to evaluate the efficacy and safety of grazoprevir (GZR) plus elbasvir (EBR) treatment in acute hepatitis C (AHC) patients undergoing hemodialysis. Patients undergoing hemodialysis who had a nosocomial acute HCV infection were enrolled. All patients received GZR 100 mg/EBR 50 mg once daily for 12 weeks and were followed up for 12 weeks. Serum alanine transaminase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), and HCV RNA levels were monitored during treatment and follow-up periods. Sustained virologic response at 12 weeks after treatment cessation and treatment-emergent adverse events (AEs) were assessed. A total of 68 AHC patients were enrolled. All patients were infected with HCV genotype 1b and achieved SVR12. Decreasing ALT, AST, and TBIL were observed over time in the first 4 weeks and became steady thereafter. Forty-eight (70.59%) patients reported at least one AEs. The most common AEs were fatigue, headache, and nausea. Two AHC patients discontinued treatment due to serious but drug-unrelated AEs. In conclusion, GZR/EBR has a high efficacy and safety profile in hemodialysis-dependent patients with genotype 1b AHC.

Cell-based fluorescent microsphere incorporated with carbon dots as a sensitive immunosensor for the rapid detection of Escherichia coli O157 in milk.

The rapid and early detection of foodborne pathogens in contaminated food is important for ensuring food safety and quality. In this study, a highly sensitive fluorescent immunosensor was developed to detect Escherichia coli O157:H7 in milk, by using microspheres labeled with carbon dots (CDs). The CDs-microspheres were prepared with Staphylococcus aureus cells as the carrier to incorporate CDs particles. Characterization of the microsphere revealed strong intensity, good stability and high uniformity in fluorescence. With Staphylococcal Protein A (SPA) on the surface of S. aureus cells, the microsphere could be easily coupled with various antibodies (e.g., immunoglobulin G). In combination with the immunomagnetic beads technique, a CDs-microsphere immunosensor was established for the specific detection of E. coli O157:H7 in milk. The limit of detection for E. coli O157:H7 is 2.4 × 102 colony-forming unit (CFU)/mL, comparable to that of real-time PCR methods. Milk samples spiked with E. coli O157:H7 at concentrations from 2.4 × 102 to 2.4 × 107 CFU/mL could be detected within 30 min. The coefficients of variation of the intra-assay tests were less than 10%, indicating a good repeatability. Moreover, the method was able to detect trace amounts of E. coli O157:H7 (<10 CFU) in real milk samples, with a 100% (10/10) accuracy after bacterial enrichment. This CDs-microsphere immunosensor shows considerable potential as a rapid and sensitive tool to detect pathogens in milk and other foods.

Early Corneal Wound Healing Response After Small Incision Lenticule Extraction.

PURPOSE: To evaluate the corneal wound healing response after small incision lenticule extraction surgery. METHODS: Small incision lenticule extraction was performed in both eyes of 12 New Zealand White rabbits. The refractive spherical correction was set at -6.00 D. Two animals were analyzed at each time point (1 hour, 4 hours, 1 day, 3 days, 7 days, and 28 days). The corneas were evaluated using slit-lamp and in vivo confocal microscopy. After euthanatization, the corneal tissues were subjected to light microscopy, transferase 2'-Deoxyuridine 5'-Triphosphate (dUTP) nick end labeling assay, and immunofluorescence microscopy (CD11b, fibronectin, tenascin, alpha-smooth muscle actin [α-SMA]). RESULTS: The corneas did not show any opacity at any time point except at the side-cut incision. By contrast, there was obvious scar tissue at the side-cut incision. Scattered, hyperreflective spots were seen by confocal microscopy from 1 hour postoperatively. Transferase dUTP nick end labeling-positive keratocytes were abundant near the femtosecond laser incision area at 1 hour and reached a peak at 4 hours postoperatively and then decreased. Inflammatory cells migrated from the incision into the central cornea, and this process began 1 hour after surgery and peaked at 7 days. Extracellular matrix components were deposited at the beginning of day 1 postoperatively, and the distribution pattern differed between the central cornea and the incision site. α-SMA-positive myofibroblasts were only detected at the side-cut incision. CONCLUSIONS: The scar tissue response in the peripheral cornea is related to the epithelium debridement. Inflammatory cells begin to be recruited by 1 hour after surgery. Therefore, it is necessary to implement antiinflammation interventions at a very early stage.

In vivo confocal laser microscopy of morphologic changes after small incision lenticule extraction with accelerated cross-linking (SMILE Xtra) in patients with thin corneas and high myopia.

PURPOSE: To evaluate the microstructural modifications and safety of small incision lenticule extraction combined with accelerated cross-linking (SMILE Xtra) in high myopia and thin corneas by means of in vivo confocal microscopy (IVCM) and 3D-OCT after a 6-month follow-up. METHODS: Forty-three eyes with high myopia and thin corneas were enrolled. All eyes underwent SMILE procedure. After the lenticule was extracted, 0.25% riboflavin was injected into the interface and allowed to diffuse for 60 s. The eye was irradiated with UVA radiation of 30 mW/cm2 for 90 s through the cap. The total energy delivered was 2.7 J/cm2. Morphologic modifications of corneal architecture were evaluated prior to SMILE Xtra and 7 days, 1, 3, and 6 months after SMILE by in vivo confocal microscopy (IVCM) and 3D-OCT. RESULTS: The corneal epithelial cells showed slight damage until 3 months postoperatively. The subepithelial nerve plexus decreased but no absence within the treatment zone at the first week after treatment, recolonized at 3 months postoperatively, and had mostly recovered at the 6 months postoperative but remained less than its normal baseline state. Keratocytes were absent in the surgical interface area, and the presence of strong reflective particles and cicatricial reaction in the anterior stroma were observed during the entire 6-month examination period. Increased hyperreflectivity was observed from the cap side at a depth of 60 µm to stroma bed at a depth of 388 µm through 6 months. The depth of the demarcation line in 40 eyes (93.0%) was at a mean depth of 296.12 ± 47.86 µm (range, 211-388 µm). No particular change between preoperative and postoperative corneal endothelium was observed. CONCLUSIONS: Confocal microscopy showed increased hyperreflectivity in the SMILE Xtra eyes, and no changes in corneal endothelium. We confirmed the safety of the SMILE Xtra but recognize that larger and longer-term studies of SMILE Xtra are necessary.

Evaluation of Human Corneal Lenticule Quality After SMILE With Different Cap Thicknesses Using Scanning Electron Microscopy.

PURPOSE: To evaluate the surface characteristics of lenticules created by small-incision lenticule extraction (SMILE) with different cap thicknesses. METHODS: This prospective study included 20 consecutive patients who underwent bilateral SMILE. Surface regularity of the extracted corneal lenticule was analyzed using scanning electron microscopy (SEM) combined with 2 methods: qualitative and quantitative regularity. Qualitative regularity of SEM images was graded by masked observers using an established scoring system. Quantitative regularity of SEM images was assessed by counting the total number and areas of tissue bridges using Image-Pro Plus software. Four different cap thickness of 120, 130, 140, and 150 µm were compared. Refractive outcomes of patients were measured at baseline and 1 month after surgery. RESULTS: As 10 specimens were not analyzable, only 30 eyes were included. Postoperatively, all eyes had postoperative uncorrected distance visual acuity of 20/20 or better; 43% had an unchanged corrected distance visual acuity; 43% gained 1 line; 10% lost 1 line. Ultrastructurally, surface irregularity was primarily caused by tissue bridges. The average surface regularity score obtained was 10.87 ± 2.40 for 120 µm, 10.78 ± 2.60 for 130 µm, 8.76 ± 2.16 for 140 µm, and 8.70 ± 2.66 for 150 µm (P < 0.001). The total number and areas of tissue bridges of 120 to 130 µm were significantly less than 140 to 150 µm (P < 0.05). Surface regularity decreased as cap thickness increased (P < 0.05). CONCLUSIONS: There is smoother appearance of the lenticular surface as seen through SEM when a thin cap is created compared with a thick cap qualitatively and quantitatively.

Aspheric Micro-monovision LASIK in Correction of Presbyopia and Myopic Astigmatism: Early Clinical Outcomes in a Chinese Population.

PURPOSE: To analyze early clinical outcomes of aspheric micro-monovision LASIK for correction of presbyopia and myopia with or without astigmatism. METHODS: Prospective, non-comparative case series of 80 eyes of 40 patients with a mean age of 43.4 ± 4.9 years (range: 38 to 63 years) treated bilaterally using an aspheric micro-monovision protocol. The target refraction was plano for the distance eye and between -0.75 and -2.25 diopters (D) for the near eye. Visual acuity, ocular aberrations, contrast sensitivity, corneal topography, amplitude of accommodation, binocular sensorial function, and satisfaction score questionnaires were evaluated at 3 months after surgery. RESULTS: Three months after surgery, the mean spherical equivalent (SE) refraction in the distance eye was -0.08 ± 0.27 D, whereas the attempted and achieved SE in the near eye were -1.41 ± 0.28 and -1.32 ± 0.35 D, respectively. Ninety-three percent of eyes were within ±0.50 D of target correction of SE. The uncorrected distance visual acuity (UDVA) after surgery was -0.10 ± 0.06 logMAR (20/15.5), 0.22 ± 0.12 logMAR (20/34), and -0.11 ± 0.06 logMAR (20/15), for distance eyes, near eyes, and binocularly, respectively. Ninety-five percent of patients achieved simultaneously uncorrected distance visual acuity 0.0 logMAR (20/20) or better and uncorrected near visual acuity J2 (20/25) or better. Stability was achieved from 1 week of follow-up. The overall satisfaction score for surgery was 92 ± 6. CONCLUSIONS: The aspheric micro-monovision protocol provided a well-tolerated and effective means for treating myopic astigmatism and alleviating presbyopic symptoms simultaneously. [J Refract Surg. 2016;32(10):680-685.].

Impact of Suction Loss During Small Incision Lenticule Extraction (SMILE).

PURPOSE: To evaluate the clinical results of suction loss in eyes during small incision lenticule extraction (SMILE). METHODS: This prospective paired-eye case study enrolled 8,490 eyes of 4,296 patients, of which 35 eyes experienced suction loss during the SMILE procedure while being treated for myopia or myopic astigmatism. The eyes with suction loss were re-treated with SMILE, femtosecond laser-assisted LASIK (FS-LASIK), or pseudo SMILE, and the fellow eyes were treated with SMILE. Patients were examined before surgery and at 1 day, 1 week, and 1 and 3 months postoperatively. Corrected distance visual acuity (CDVA) and uncorrected distance visual acuity (UDVA), manifest refraction, higher order aberrations (HOAs), and morphologic modifications of corneal architecture were evaluated. At 3 months, patients were asked if they preferred the vision in either eye. RESULTS: High-contrast CDVA was worse in the eyes that suffered loss of suction than in the fellow eyes at 1 week postoperatively (two-tailed paired t tests, P = .04), but not at 1 and 3 months. There was no statistical significance between the two treatments in the safety and efficacy indices or the 3rd and 4th order aberrations at 3 months postoperatively (two-tailed paired t tests, all P > .05). No apparent abnormalities were observed in the corneas by frequency-domain optical coherence tomography. CONCLUSIONS: Re-treatment with femtosecond laser for incomplete SMILE was safe, predictable, and effective, and the patients did not perceive a difference in vision. [J Refract Surg. 2016;32(10):686-692.].

Reversible Femtosecond Laser-Assisted Endokeratophakia Using Cryopreserved Allogeneic Corneal Lenticule.

PURPOSE: To evaluate outcomes, reversibility, and wound healing response after the femtosecond laser-assisted endokeratophakia procedure in a rabbit model. METHODS: Allogeneic rabbit corneal lenticules were cryopreserved in liquid nitrogen for 3 months. Twenty rabbits underwent the monocular endokeratophakia procedure and were divided into four groups according to the follow-up periods. The first three groups were killed at 3 days, 2 weeks, and 6 months after endokeratophakia, respectively. The rabbits in the fourth group received re-extraction of implanted lenticules at 6 months after endokeratophakia and were killed at 1 month after re-extraction. The rabbits were monitored by slit-lamp microscopy, ultrasonic pachymetry, in vivo confocal microscopy, optical coherence tomography (OCT), Corvis ST tonometry (Oculus Optikgeräte, Wetzlar, Germany), and Ocular Response Analyzer (Reichert Ophthalmic Instruments, Depew, NY). The tissue responses were analyzed by immunohistochemistry and transmission electron microscopy. RESULTS: After endokeratophakia, corneal clarity improved continually with time. The changes in the refraction and corneal thickness were stable after implantation and could be reversed after re-extracting the lenticules. The interfaces were clearly visible on confocal microscopy and transmission electron microscopy images over the entire follow-up period. There were significant numbers of TUNEL-positive keratocytes in lenticules after endokeratophakia. CD11b-positive cells and deposition of fibronectin and tenascin were observed at earlier follow-up times. No alpha-smooth muscle actin-positive fibroblasts could be detected. In addition, the corneal biomechanics parameters were not significantly increased after endokeratophakia. CONCLUSIONS: The endokeratophakia procedure using allogeneic cryopreserved lenticules was clinically stable and could be reversed. The wound healing response was mild, limited, and produced no scars. [J Refract Surg. 2016;32(8):569-576.].

Detection of clarithromycin-resistant Helicobacter pylori in clinical specimens by molecular methods: A review.

Various molecular methods have been developed to rapidly detect clarithromycin (CLR) resistance in Helicobacter pylori isolates in clinical specimens. All of these assays for detecting CLR resistance in H. pylori are based on detection of mutations in the 23S rRNA gene. In this article, we summarise current knowledge regarding the detection of H. pylori CLR resistance in clinical specimens by molecular tests. The available data showed that restriction fragment length polymorphism (RFLP), 3'-mismatch PCR, DNA sequencing, the PCR line probe assay (PCR-LiPA) and fluorescence in situ hybridisation assay (FISH) are able to detect CLR-resistant H. pylori in clinical specimens with excellent specificity and sensitivity. However, several factors limit their clinical application, including fastidious, time-consuming preparation and low-throughput as well as carrying a risk of contamination. Furthermore, as an invasive method, FISH is not suitable for children or the elderly. Among the molecular methods, one that is most promising for the future is real-time PCR probe hybridisation technology using fluorescence resonance energy transfer (FRET) probes, which can rapidly detect CLR resistance with high sensitivity and specificity in biopsies and stool specimens, even though mixed infections are present in clinical specimens. Moreover, due to the advantages that this method is simple, rapid and economical, real-time PCR is technically feasible for clinical application in small- and medium-sized hospitals in developing countries. Second, with high sensitivity, specificity and throughput, DNA chips will also be a valuable tool for detecting resistant H. pylori isolates from cultures and clinical specimens.

Comparison of 120- and 140-µm SMILE Cap Thickness Results in Eyes With Thick Corneas.

PURPOSE: To evaluate clinical outcomes after small incision lenticule extraction (SMILE) with different cap thicknesses in thick corneas. METHODS: Forty patients with central corneal thickness of more than 560 µm were recruited in this prospective, randomized, masked, paired-eye study. Patients were randomized to receive SMILE with a 120-µm cap thickness in 1 eye and 140-µm cap thickness in the other. Uncorrected and corrected distance visual acuity (CDVA), contrast sensitivity (CS), higher-order aberrations (HOAs), and morphologic modifications of corneal architecture were measured during the 3-month follow-up period. RESULTS: Postoperative refractive outcomes, visual outcomes, CS, and the changes in HOAs were similar between both groups. The persistence of brightly reflective particles in the corneal interface layer was 1388.6 ± 219.5/mm in eyes with 120-µm cap thickness and 54.7 ± 8.6/mm in eyes with 140-µm cap thickness (P < 0.001). The hyperreflectivity line at the interface layer almost disappeared in all eyes with 140-µm cap thickness, and it still persisted in 43% of the fellow eyes at 3 months postoperatively. The anterior surfaces of lenticules in the 140-µm cap thickness group exhibited more smoothness than in the 120-µm cap thickness group. CONCLUSIONS: There was a lower level corneal wound-healing response after SMILE with a 140-µm cap thickness than with a 120-µm cap thickness, although the thickness of cap creation did not affect visual outcomes by 3 months postoperatively.

Clinical Outcomes After SMILE and Femtosecond Laser-Assisted LASIK for Myopia and Myopic Astigmatism: A Prospective Randomized Comparative Study.

PURPOSE: To compare the clinical outcomes of small-incision lenticule extraction (SMILE) and femtosecond laser-assisted LASIK (FS-LASIK) for the correction of myopia and myopic astigmatism. METHODS: In this prospective, randomized, comparative study, SMILE surgery was performed in 113 eyes with a mean spherical equivalent (MSE) of -5.22 ± 1.70 diopters (D) and FS-LASIK was performed in 84 eyes with an MSE of -5.18 ± 1.93 D. Contrast sensitivity and uncorrected and corrected distance visual acuities were measured preoperatively and 1 day, 1 week, 1, 3, and 6 months postoperatively. Higher-order aberrations were evaluated preoperatively and 6 months postoperatively. RESULTS: At postoperative day 1, the uncorrected distance visual acuity in the FS-LASIK group was better (P < 0.05), whereas no statistically significant difference was found at 6 months postoperatively (P = 0.9). There was no significant difference in terms of logMAR corrected distance visual acuity and MSE during the 6 months of follow-up between the SMILE and FS-LASIK groups (all P > 0.05). The contrast sensitivity in the SMILE group was lower at 1 day and 1 week postoperatively (all P < 0.05). The spherical aberration at 6 mm diameter at 6 months postoperatively was lower (P < 0.05) in the SMILE group (0.12 ± 0.22 µm) than in the FS-LASIK group (0.28 ± 0.26 µm). CONCLUSIONS: Both FS-LASIK and SMILE procedures achieved good visual outcomes in the correction of myopia and myopic astigmatism. SMILE had a lower induction rate of spherical aberration at 6 months postoperatively in the analysis of 6 mm diameter than that of FS-LASIK.

Femtosecond Laser-assisted Endokeratophakia Using Allogeneic Corneal Lenticule in a Rabbit Model.

PURPOSE: To investigate the feasibility of allogeneic corneal lenticule implantation using the femtosecond laser as a method for cornea remodeling. METHODS: Small incision lenticule extraction was performed on the right eyes of 15 New Zealand white rabbits. Corneal intrastromal pockets were created with a femtosecond laser on the left eyes. Allogeneic corneal lenticule implantation was performed on the left eye immediately after the lenticule was extracted from the right eye. All animals had preoperative and postoperative slit-lamp photography, ultrasonic pachymetry, corneal endothelial cell count, anterior segment optical coherence tomography, in vivo confocal microscopy, and retinoscopy refraction during the observation period of 8 weeks. The rabbits were killed 8 weeks after surgery. Corneal wound healing response was analyzed by transmission electron microscopy, hematoxylin-eosin staining, TUNEL assay, and immunofluorescent staining. RESULTS: Mild corneal edema and decreased clarity were noted the first few days after implantation, improving to normal 8 weeks after surgery. The corneal thickness and retinoscopy refraction were stable during the observation period. Viable keratocytes could be detected within the lenticule lamellae by ultrastructural analysis 8 weeks after surgery. The anterior and posterior border of the lenticule showed acellular layers with highly irregular collagen arrangement on transmission electron microscope images. Proliferating Ki-67 positive cells were present only in the epithelium layer. CONCLUSION: Femtosecond laser-assisted endokeratophakia using allogeneic corneal lenticule may be feasible for reshaping cornea, providing a new possibility in refractive surgery and keratoconus treatment.

Decentration of optical zone center and its impact on visual outcomes following SMILE.

PURPOSE: To evaluate decentration from pupil center and corneal vertex normal following femtosecond laser small incision lenticule extraction (SMILE) for the treatment of myopia and myopic astigmatism. METHODS: This study evaluated 101 eyes of 101 patients with a spherical equivalent (SE) of -5.30 ± 1.72 diopters (D). Decentration was measured in the pupil-centered group (PC group) by video capture images and in the vertex normal-centered group (VNC group) using the WaveLight Oculyzer II (Alcon). The PC and VNC groups were further divided into 4 subgroups (1 PC, 2 PC and 1 VNC, 2 VNC) based on the displacement of the lenticule center. Uncorrected and corrected distance visual acuities, manifest refraction, and wavefront errors were measured preoperatively, and at 1 day, 1 week, 1 month, and 3 and 6 months postoperatively. RESULTS: Regarding efficacy and safety, at 6 months postoperatively, 65, 71, 83, and 53% of eyes had unchanged corrected distance visual acuity in 1 PC, 1 VNC, 2 PC and 2 VNC groups, respectively. Also, 97, 98, 100, and 97% of treated eyes had a postoperative uncorrected distance visual acuity 20/20 or better in the subgroups, respectively. In the groups mentioned above, 96, 100, 100, and 95% of eyes, respectively, were within ±0.5 D of the targeted SE. In terms of total higher-order aberrations, the 1 PC group was significantly higher than the 1 VNC group (P = 0.04), whereas the 2 VNC group was significantly higher than the 2 PC group (P = 0.02). CONCLUSIONS: Our results demonstrate that better refractive outcomes will be achieved when the lenticule center is closer to the corneal vertex normal.

Corneal regeneration after femtosecond laser small-incision lenticule extraction: a prospective study.

PURPOSE: To evaluate the morphologic changes of corneal after small incision lenticule extraction (SMILE) using the Heidelberg Retina Tomograph III (HRT III)/Rostock Cornea Module confocal microscope and three-dimensional optical coherence tomography (3D-OCT). METHODS: Thirty eyes of 30 patients who underwent SMILE for the correction of myopia and myopic astigmatism in this study. Morphologic modifications of corneal architecture and the density of corneal subbasal nerve were evaluated prior to SMILE and 7 days, 1, 3, and 6 months after SMILE by in-vivo confocal microscopy (IVCM) and 3D-OCT. RESULTS: The corneal epithelium morphology showed no particular changes during the entire 6-month examination period. Subbasal nerve density recovered to preoperative levels within 3 months after SMILE (P > 0.05). On the interface layer, intense keratocyte activity was noted in all eyes after SMILE. The corneal fibrotic tissues on the interface layer diminished with time, but the persistence of brightly reflective particles still existed. No particular change between preoperative and postoperative corneal endothelium. The incision margin of SMILE had the appearance of a less clearly identified fibrotic scar with epithelial plug. CONCLUSIONS: IVCM and 3D-OCT analysis showed that there is a low-level corneal wound-healing response after SMILE procedure in the early postoperative period. Subbasal nerve regeneration is recovered at the third month, but the reflective particles elevated up to the sixth month.

Overexpression of integrin αv in the human nasopharyngeal carcinoma associated with metastasis and progression.

BACKGROUND: Integrins are cell-surface adhesion molecules, regulate normal cellular interactions, and which are consisting of α and ß subunits and facilitate signal transduction in a bidirectional manner. Tumor cells have been found to express a wide variety of integrins, overexpression of integrin αv has been detected in a growing number of human malignancy types, However, the reports obout expression of integrin αv in nasopharyngeal carcinoma (NPC) are very rare. OBJECTIVE: This study aims to detect the expression of integrin αv in NPC, and to evaluate the correlation between integrin αv expression and clinicopathological factors. METHODS: Real-time polymerase chain reaction (real-time PCR) assay and immunohistochemical staining were performed to detect the expression of integrin αv in NPC tissue samples. The correlation between the integrin αv expression and clinicopathological factors was evaluated. RESULTS: In NPC tissues, the expression levels of integrin αv mRNA was significantly higher than those in the nasopharyngeal inflammation tissues (P< 0.05), and the expression level were significantly correlated with T, N and clinical stage (all P < 0.05). Moreover, the expression rates of integrin αv protein in NPC tissues was 76.92%, significantly higher than that of nasopharyngeal inflammation tissues (6.25%, P< 0.05). We also observed that the protein expression of integrin αv was significantly related to T, N and clinical stage (all P< 0.05). CONCLUSIONS: All these findings suggest that overexpression of integrin αv is closely associated with metastasis and progression of NPC. Therefore, we can speculate that integrin αv could be effective prognostic markers in the future for individualized treatment of patients with NPC.

Enzymic colorimetry-based DNA chip: a rapid and accurate assay for detecting mutations for clarithromycin resistance in the 23S rRNA gene of Helicobacter pylori.

Macrolide drugs, such as clarithromycin (CAM), are a key component of many combination therapies used to eradicate Helicobacter pylori. However, resistance to CAM is increasing in H. pylori and is becoming a serious problem in H. pylori eradication therapy. CAM resistance in H. pylori is mostly due to point mutations (A2142G/C, A2143G) in the peptidyltransferase-encoding region of the 23S rRNA gene. In this study an enzymic colorimetry-based DNA chip was developed to analyse single-nucleotide polymorphisms of the 23S rRNA gene to determine the prevalence of mutations in CAM-related resistance in H. pylori-positive patients. The results of the colorimetric DNA chip were confirmed by direct DNA sequencing. In 63 samples, the incidence of the A2143G mutation was 17.46 % (11/63). The results of the colorimetric DNA chip were concordant with DNA sequencing in 96.83 % of results (61/63). The colorimetric DNA chip could detect wild-type and mutant signals at every site, even at a DNA concentration of 1.53 x 10(2) copies microl(-1). Thus, the colorimetric DNA chip is a reliable assay for rapid and accurate detection of mutations in the 23S rRNA gene of H. pylori that lead to CAM-related resistance, directly from gastric tissues.

Bifurcations of an epidemic model with non-monotonic incidence rate of saturated mass action.

In this paper, we study the bifurcations of an epidemic model with non-monotonic incidence rate of saturated mass action, which describes the psychological effects of the community on certain serious diseases when the number of infective is getting larger. By carrying out the bifurcation analysis of the model, we show that there exist some values of the model parameters such that numerous kinds of bifurcation occur for the model, such as Hopf bifurcation, Bogdanov-Takens bifurcation.