Computational Analysis of miR-140 and miR-135 as Potential Targets to Develop Combinatorial Therapeutics for Degenerative Tendinopathy.

Background: Degenerative tendinopathy, a condition causing movement restriction due to high pain, highly impacts productivity and quality of life. The healing process is a complex phenomenon and involves a series of intra-cellular and inter-cellular processes. Proliferation and differentiation of the tenocyte is a major and essential process to heal degenerative tendinopathy. The recent development in microRNA (miRNA)-mediated reprogramming of the cellular function through specific pathways opened door for the development of new regenerative therapeutics. Based on information about gene expression and regulation of tendon injury and healing, we attempted to evaluate the combinatorial effect of selected miRNAs for better healing of degenerative tendinopathy. Methods: The present study was designed to evaluate the combinatorial effect of two miRNAs (has-miR-140 and has-miR-135) in the healing process of the tendon. Publicly available information/data were retrieved from appropriate platforms such as PubMed. Only molecular data, directly associated with tendinopathies, including genes/proteins and miRNAs, were used in this study. The miRNAs involved in tendinopathy were analyzed by a Bioinformatics tools (e.g., TargetScan, miRDB, and the RNA22v2). Interactive involvement of the miRNAs with key proteins involved in tendinopathy was predicted by the Insilco approach. Results: Based on information available in the public domain, tendon healing-associated miRNAs were predicted to explore their therapeutic potentials. Based on computation analysis, focusing on the potential regulatory effect on tendon healing, the miR-135 and miR-140 were selected for this study. These miRNAs were found as key players in tendon healing through Rho-associated coiled-coil containing protein kinase 1 (ROCK1), IGF-1/PI3K/Akt, PIN, and Wnt signaling pathways. It was also predicted that these miRNAs may reprogram the cells to induce proliferation and differentiation activity. Many miRNAs are likely to regulate genes important for the tendinopathy healing process, and the result of this study allows an approach for miRNA-mediated regeneration of the tenocyte for tendon healing. Based on computational analysis, the role of these miRNAs in different pathways was established, and the results provided insights into the combinatorial approach of miRNA-mediated cell reprogramming. Conclusions: In this study, the association between miRNAs and the disease was evaluated to correlate the tendinopathy genes and the relevant role of different miRNAs in their regulation. Through this study, it was established that the synergistic effect of more than one miRNA on directed reprogramming of the cell could be helpful in the regeneration of damaged tissue. It is anticipated that this study will be helpful for the design of miRNA cocktails for the orchestration of cellular reprogramming events.

Impact of COVID-19 outbreak on acute gallbladder disease in the emergency department.

OBJECTIVE: Acute gallbladder disease (AGD) is frequent in the emergency department (ED) and usually requires emergency surgery. However, only a few studies have reported the impact of COVID-19 on AGD. The goal of this study was to evaluate the time between symptom onset and surgery and the perioperative severity of AGD during the COVID-19 pandemic compared to before the era of COVID-19. METHODS: This retrospective, single-center cohort study included patients who presented to the ED with suspected AGD and who underwent emergency cholecystectomy. We designed a before-after comparative study, and the intervention was the COVID-19 outbreak. The 6-month period after the COVID-19 outbreak was defined as the post-COVID group, whereas the pre-COVID group consisted of the same period in the previous year. The primary outcome was the time from symptoms to surgery. We evaluated the time intervals between symptom onset and ED arrival and between ED arrival and surgery. The secondary outcomes were preoperative and postoperative severity indexes. RESULTS: A total of 316 patients was analyzed. The post-COVID group showed longer duration from symptom onset to ED arrival (34.0 hours vs. 15.0 hours, P<0.001) and longer time interval from ED arrival to surgery (16.2 hours vs. 10.2 hours, P<0.001) than the pre-COVID group. The overall time interval between symptom onset to surgery was longer in the post-COVID group than the pre-COVID group (71.5 hours vs. 33.5 hours, P<0.001). The post-COVID group showed higher preoperative Simplified Acute Physiology Score II scores than the pre-COVID group (20.1 vs. 18.2, P=0.045). The proportion of moderate or severe disease increased in the post-COVID group (78% vs. 65%, P=0.017). The durations of hospital stay (7.0 days vs. 5.0 days, P<0.001) and intensive care unit stay (27.1 hours vs. 10.8 hours, P=0.008) were longer in the post-COVID group than in the pre-COVID group. CONCLUSION: During the pandemic, the time interval between symptom onset to surgery was significantly increased among patients with AGD. Concomitantly, higher preoperative severity indexes and longer hospital stay were reported with a delay in emergency surgery.

Comparison of Primary Versus Revision Lumbar Discectomy Using a Biportal Endoscopic Technique.

STUDY DESIGN: Retrospective study. OBJECTIVE: To compare the clinical outcomes of the biportal endoscopic technique for primary lumbar discectomy (BE-LD) and revision lumbar discectomy (BE-RLD). METHODS: Eighty-one consecutive patients who underwent BE-LD or BE-RLD, and could be followed up for at least 12 months were divided into two groups: Group A (BE-LD; n = 59) and Group B (BE-RLD; n = 22). Clinical outcomes included the visual analog scale (VAS), Oswestry Disability Index (ODI), and modified MacNab's criteria. Perioperative results included operation time (OT), length of hospital stay (LOS), amount of surgical drain, and kinetics of serum creatine phosphokinase (CPK) and C-reactive protein (CRP). Clinical and perioperative outcomes were assessed preoperatively and postoperatively at 2 days and at 3, 6, and 12 months. Postoperative complications were noted. RESULTS: Both groups showed significant improvement in pain (VAS) and disability (ODI) compared to baseline values at postoperative day 2, which lasted until the final follow-up. There were no significant differences in the improvement of the VAS and ODI scores between the groups. According to the modified MacNab's criteria, 88.1 and 90.9% of the patients were excellent or good in groups A and B, respectively. OT, LOS, amount of surgical drain, and kinetics in serum CRP and CPK levels were comparable. Complications in Group A included incidental durotomy (n = 2), epidural hematoma (n = 1), and local recurrence (n = 1) and in Group B incidental durotomy (n = 1) and epidural hematoma (n = 1). CONCLUSION: BE-RLD showed favorable clinical outcomes, less postoperative pain, and early laboratory recovery equivalent to BE-LD.

Comparison of Extracorporeal Shock Wave Therapy and Ultrasound-Guided Shoulder Injection Therapy in Patients with Supraspinatus Tendinitis.

Background: The present study compared the clinical effect of extracorporeal shock wave therapy (ESWT) with that of ultrasound (US)-guided shoulder steroid injection therapy in patients with supraspinatus tendinitis. We hypothesized that the two treatments would show comparable results. Methods: The inclusion criteria were age over 20 years and diagnosis of supraspinatus tendinitis using US. Ultimately, 26 patients were assigned using blocked randomization: 13 in the US-guided shoulder injection group and 13 in the ESWT group. Treatment outcomes were evaluated using the pain visual analog scale (pVAS), the American Shoulder and Elbow Society (ASES) score, and the Constant score at baseline and at 1 and 3 months after the procedure. Results: At 1 month after the intervention, pVAS, ASES, and constant score were significantly higher in the US-guided shoulder injection group than in the ESWT group, but not at 3 months after the intervention. Both groups showed clinically significant treatment effects at 3 months after the intervention compared to baseline. No significance was shown using equivalence testing. Conclusions: US-guided shoulder injection therapy was not superior to ESWT therapy. Considering the complications and rebound phenomenon of steroid injections, interventions using ESWT may be a good alternative to treat patients with supraspinatus tendinitis.

Exposure to an accidental trichlorosilane spill: three case reports.

Chlorosilane is a hazardous chemical compound which is used as a raw material in the production of silicone. Despite strict restrictions, accidental spillage of chlorosilane is often reported. However, human exposure was rarely reported in the past decades and the effect on humans is barely known. We report cases of human exposure to an accidental trichlorosilane spill. Three middle aged male industrial workers visited our emergency department after exposure to trichlorosilane. They presented with shortness of breath and burns on multiple sites. Chest radiograph and laboratory studies were performed. None of the reports showed serious results and were discharged after conservative management.

Evaluation of the efficacy and safety of conventional and biportal endoscopic decompressive laminectomy in patients with lumbar spinal stenosis (ENDO-B trial): a protocol for a prospective, randomized, assessor-blind, multicenter trial.

BACKGROUND: Recent studies on biportal endoscopic spine surgery in patients with lumbar spinal stenosis have reported good clinical results. However, these studies have been limited by the small sample sizes and use of a retrospective study design. Therefore, we aim to compare the efficacy and safety of biportal endoscopic decompressive laminectomy with those of conventional decompressive laminectomy in a multicenter, prospective, randomized controlled trial. METHODS: This study will include 120 patients (60 per group, aged 20-80 years) with 1- or 2-level lumbar spinal stenosis, who will be recruited from six hospitals. The study will be conducted from July 2021 to December 2024. The primary outcome (Oswestry Disability Index at 12 months after surgery) will be evaluated through a modified intention-to-treat method. The secondary outcomes will include the following: visual analog scale score for low back and lower extremity radiating pain, EuroQol 5-dimensions score, surgery satisfaction, walking time, postoperative return to daily life period, postoperative surgical scars, and some surgery-related variables. Radiographic outcomes will be analyzed using magnetic resonance imaging or computed tomography. All outcomes will be evaluated before the surgery and at 2 weeks, 3 months, 6 months, and 12 months postoperatively. This protocol adheres to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines for reporting of clinical trial protocols. DISCUSSION: It is hypothesized that the efficacy and safety of biportal endoscopic and conventional decompressive laminectomy will be comparable in patients with lumbar spinal stenosis. The results of this trial will provide a high level of evidence for the efficacy and safety of the biportal endoscopic technique in patients with lumbar spinal stenosis and facilitate the development of clinical practice guidelines. Furthermore, the results of this study may indicate the feasibility of the biportal endoscopic technique for other types of spinal surgery. TRIAL REGISTRATION: The ENDO-B trial is registered at Clinical Research Information Service (CRIS, cris.nih.go.kr ) (KCT0006057; April 52,021).

Graphene collage on Ni-rich layered oxide cathodes for advanced lithium-ion batteries.

The energy storage performance of lithium-ion batteries (LIBs) depends on the electrode capacity and electrode/cell design parameters, which have previously been addressed separately, leading to a failure in practical implementation. Here, we show how conformal graphene (Gr) coating on Ni-rich oxides enables the fabrication of highly packed cathodes containing a high content of active material (~99 wt%) without conventional conducting agents. With 99 wt% LiNi0.8Co0.15Al0.05O2 (NCA) and electrode density of ~4.3 g cm-3, the Gr-coated NCA cathode delivers a high areal capacity, ~5.4 mAh cm-2 (~38% increase) and high volumetric capacity, ~863 mAh cm-3 (~34% increase) at a current rate of 0.2 C (~1.1 mA cm-2); this surpasses the bare electrode approaching a commercial level of electrode setting (96 wt% NCA; ~3.3 g cm-3). Our findings offer a combinatorial avenue for materials engineering and electrode design toward advanced LIB cathodes.

Graphene/PVDF Composites for Ni-rich Oxide Cathodes Toward High-Energy Density Li-ion Batteries.

Li-ion batteries (LIBs) employ porous, composite-type electrodes, where few weight percentages of carbonaceous conducting agents and polymeric binders are required to bestow electrodes with electrical conductivity and mechanical robustness. However, the use of such inactive materials has limited enhancements of battery performance in terms of energy density and safety. In this study, we introduced graphene/polyvinylidene fluoride (Gr/PVdF) composites in Ni-rich oxide cathodes for LIBs, replacing conventional conducting agents, carbon black (CB) nanoparticles. By using Gr/PVdF suspensions, we fabricated highly dense LiNi0.85Co0.15Al0.05O2 (NCA) cathodes having a uniform distribution of conductive Gr sheets without CB nanoparticles, which was confirmed by scanning spreading resistance microscopy mode using atomic force microscopy. At a high content of 99 wt.% NCA, good cycling stability was shown with significantly improved areal capacity (Qareal) and volumetric capacity (Qvol), relative to the CB/PVdF-containing NCA electrode with a commercial-level of electrode parameters. The NCA electrodes using 1 wt.% Gr/PVdF (0.9:0.1) delivered a high Qareal of ~3.7 mAh cm-2 (~19% increment) and a high Qvol of ~774 mAh cm-3 (~18% increment) at a current rate of 0.2 C, as compared to the conventional NCA electrode. Our results suggest a viable strategy for superseding conventional conducting agents (CB) and improving the electrochemical performance of Ni-rich cathodes for advanced LIBs.

Assessment of the abuse potential of methamnetamine in rodents: a behavioral pharmacology study.

RATIONALE: Methamnetamine (MNA; PAL-1046) is a new psychoactive substance that acts as a full biogenic amine transporter (BAT) substrate. BAT substrates promote neurotransmitter release from the nerve terminal and can be abused as stimulants. However, scientific information on the abuse potential of methamnetamine is lacking. OBJECTIVE: We evaluated the abuse liability of methamnetamine. METHODS: The effective dose range of methamnetamine was determined using a climbing behavior test. The rewarding effect and reinforcing effect of the test compound were evaluated in mice by conditioned place preference (CPP) testing and self-administration (SA) testing at the selected doses. Dopamine level changes were analyzed using synaptosomes and in vivo microdialysis to investigate the effects of methamnetamine on the central nervous system. Drug discrimination experiments were used to examine the potential similarity of the interoceptive effects of methamnetamine and cocaine. RESULTS: A significant response was observed in the climbing behavior test with 10 and 40 mg/kg intraperitoneally administered methamnetamine. In the CPP test, mice intraperitoneally administered methamnetamine (10 and 20 mg/kg) showed a significant preference for the drug-paired compartment. In the SA test, mice that intravenously received 1 mg/kg/infusion showed significant active-lever responses. Dopamine was significantly increased in synaptosomes and in in vivo microdialysis tests. Furthermore, methamnetamine showed cross-generalization with cocaine in a dose-dependent manner. CONCLUSIONS: Methamnetamine exhibits interceptive stimulus properties similar to those of cocaine and induces rewarding and reinforcing effects, suggesting its dependence liability potential.

Tendon Regeneration After Partial-Thickness Peroneus Longus Tendon Harvesting: Magnetic Resonance Imaging Evaluation and In Vivo Animal Study.

BACKGROUND: In recent years, the use of the anterior half of the peroneus longus tendon (AHPLT) as an autograft source for ligament reconstruction has gained popularity. However, no reports are available regarding tendon regeneration after harvesting of the AHPLT. HYPOTHESIS: When half of the tendon is preserved during tendon harvesting, the quality of the regenerated tendon is better than that of the regenerated tendon after full-thickness harvesting. STUDY DESIGN: Case series; Level of evidence, 4; controlled laboratory study. METHODS: A total of 21 patients who underwent AHPLT harvesting for lower extremity ligament reconstruction participated in the magnetic resonance imaging (MRI) study to evaluate tendon regeneration 1 year after the harvesting. An in vivo animal study was performed to compare the quality of the regenerated tendon after partial-thickness and full-thickness tendon harvesting. A total of 30 adult female Sprague-Dawley rats were allocated to 2 groups-15 rats underwent partial-thickness Achilles tendon harvesting (partial-thickness harvesting [PTH] group), and 15 rats underwent full-thickness Achilles tendon harvesting (full-thickness harvesting [FTH] group). The quality of the regenerated tendons was compared 180 days after tendon harvesting. RESULTS: All 21 patients showed regeneration of the peroneus longus tendon (PLT) (homogeneously dark on both T1- and T2-weighted sequences). The cross-sectional area of the regenerated tendon divided by that of the preoperative tendon was 92.6% and 84.5% at 4 cm and 9 cm proximal to the tip of the distal fibula, respectively. In the animal study, the mean histologic score was better for the PTH group compared with the FTH group (9.17 ± 1.35 vs 14.72 ± 0.74; P < .001). The ultimate strength and the stiffness of the regenerated Achilles tendon were significantly higher for the PTH group compared with the FTH group (35.5 ± 8.3 vs 22.4 ± 8.3 N, P = .004; and 31.6 ± 7.7 vs 23.5 ± 4.8 N/mm, P = .016). CONCLUSION: The PLT was found to regenerate after partial-thickness harvesting on MRI. In the animal study, the quality of the regenerated tendon when half of the tendon was preserved during tendon harvesting was better than that after full-thickness tendon harvesting.

Detection of somatic variants and EGFR mutations in cell-free DNA from non-small cell lung cancer patients by ultra-deep sequencing using the ion ampliseq cancer hotspot panel and droplet digital polymerase chain reaction.

Highly sensitive genotyping assays can detect mutations in cell-free DNA (cfDNA) from cancer patients, reflecting the biology of each patient's cancer. Because circulating tumor DNA comprises a small, variable fraction of DNA circulating in the blood, sensitive parallel multiplexing tests are required to determine mutation profiles. We prospectively examined the clinical utility of ultra-deep sequencing analysis of cfDNA from 126 non-small cell lung cancer (NSCLC) patients using the Ion AmpliSeq Cancer Hotspot Panel v2 (ICP) and validated these findings with droplet digital polymerase chain reaction (ddPCR). ICP results were compared with tumor tissue genotyping (TTG) results and clinical outcomes. A total of 853 variants were detected, with a median of four variants per patient. Overall concordance of ICP and TTG analyses was 90% for EGFR exon 19 deletion and 88% for the L858R mutation. Of 34 patients with a well-defined EGFR activating mutation defined based on the results of ICP and TTG, 31 (81.6%) showed long-term disease control with EGFR TKI treatment. Of 56 patients treated with an EGFR tyrosine kinase inhibitor (TKI), the presence of the de novo T790M mutation was confirmed in 28 (50%). Presence of this de novo mutation did not have a negative effect on EGFR TKI treatment. Ultra-deep sequencing analysis of cfDNA using ICP combined with confirmatory ddPCR was effective at defining driver genetic changes in NSCLC patients. Comprehensive analysis of tumor DNA and cfDNA can increase the specificity of molecular diagnosis, which could translate into tailored treatment.

Assessment of Optical Quality at Different Contrast Levels in Pseudophakic Eyes.

Purpose. To assess visual function using Optical Quality Analysis System (OQAS) at varying levels of contrast in pseudophakic eyes. Methods. The study included patients admitted to Seoul St. Mary's Hospital between January and February 2012: 143 pseudophakic eyes with one of five intraocular lens types, examined 2-6 months after cataract surgery, and 93 normal eyes (enhanced visual acuity (VA) < 0.1 logMAR) in age-matched controls. Subjects were examined at three contrast levels using the OQAS. Results. At 100%, 20%, and 9% contrast, simulated mean VA was 0.16 ± 0.18 logMAR, 0.30 ± 0.18 logMAR, and 0.52 ± 0.17 logMAR, in normal eyes, and 0.16 ± 0.12 logMAR, 0.33 ± 0.20 logMAR, and 0.56 ± 0.21 logMAR, respectively, in pseudophakic eyes. Simulated VA decreased significantly when contrast was reduced, regardless of ocular status, age group, and lens type (p < 0.05). There were no significant differences between normal and pseudophakic eyes among subjects aged 50-69 (p > 0.05). Among subjects aged 70-79, pseudophakic eyes showed improved simulated VA (p = 0.000) and objective scattering index values (p = 0.008). Conclusions. Patients with intraocular lenses have similar or superior visual function when compared to those with normal eyes at 2-6 months after cataract surgery, even under low-contrast conditions.

Rab3a promotes brain tumor initiation and progression.

The Rab protein family is composed of small GTP-binding proteins involved in intracellular vesicle trafficking. In particular, Rab3a which is one of four Rab3 proteins (a, b, c, and d isoforms) is associated with synaptic vesicle trafficking in normal brain. However, despite the elevated level of Rab3a in tumors, its role remains unclear. Here we report a tumorigenic role of Rab3a in brain tumors. Elevated level of Rab3a expression in human was confirmed in both glioma cell lines and glioblastoma multiforme patient specimens. Ectopic Rab3a expression in glioma cell lines and primary astrocytes promoted cell proliferation by increasing cyclin D1 expression, induced resistance to anti-cancer drug and irradiation, and accelerated foci formation in soft agar and tumor formation in nude mice. The overexpression of Rab3a augmented the tumorsphere-forming ability of glioma cells and p53(-/-) astrocytes and increased expression levels of various stem cell markers. Taken together, our results indicate that Rab3a is a novel oncogene involved in glioma initiation and progression.

Mass spectrometric screening of ovarian cancer with serum glycans.

Changes of glycosylation pattern in serum proteins have been linked to various diseases including cancer, suggesting possible development of novel biomarkers based on the glycomic analysis. In this study, N-linked glycans from human serum were quantitatively profiled by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and compared between healthy controls and ovarian cancer patients. A training set consisting of 40 healthy controls and 40 ovarian cancer cases demonstrated an inverse correlation between P value of ANOVA and area under the curve (AUC) of each candidate biomarker peak from MALDI-TOF MS, providing standards for the classification. A multibiomarker panel composed of 15 MALDI-TOF MS peaks resulted in AUC of 0.89, 80~90% sensitivity, and 70~83% specificity in the training set. The performance of the biomarker panel was validated in a separate blind test set composed of 23 healthy controls and 37 ovarian cancer patients, leading to 81~84% sensitivity and 83% specificity with cut-off values determined by the training set. Sensitivity of CA-125, the most widely used ovarian cancer marker, was 74% in the training set and 78% in the test set, respectively. These results indicate that MALDI-TOF MS-mediated serum N-glycan analysis could provide critical information for the screening of ovarian cancer.

Enhancement of ovarian tumor classification by improved reproducibility in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of serum glycans.

The serum N-glycome is a promising source of biomarker discovery. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) profiling of serum N-glycans was attempted for differentiating borderline ovarian tumor from benign cases, for which a low data spread is essential. An experimental protocol using matrix-prespotted MALDI plates and fast vacuum drying of the loaded N-glycan samples was developed, thereby minimizing the intensity variations in the replicates to an average relative standard deviation (RSD) of 3.96% for the highest N-glycan peak (m/z 1485.53) of the Sigma-Aldrich serum standard. When applied to sera of ovarian tumors, this procedure exhibited an average RSD of 5.74% for m/z 1485.53 and of 7.28% for all MS peaks. This improved reproducibility combined with the OVA-Beyond(®) screening software resulted in 75.1% and 79.4% correct classification for benign and borderline tumor samples, respectively, while the classification rates by the conventional ovarian tumor marker CA-125 were 54.4% and 53.1%, respectively. Both true positive rate and true negative rate fluctuated with small numbers of markers and converged as the number of markers increased. Cross-validations were performed in comparison with CA-125. These results suggest that our optimized process for MALDI-TOF MS of the serum glycome has a great potential for the screening of early stage ovarian cancer.

Changes in anterior and posterior corneal parameters in patients with keratoconus after intrastromal corneal-ring segment implantation.

PURPOSE: To compare changes in the anterior and the posterior corneal parameters and the anterior chamber depth (ACD) measured by a rotating Scheimpflug camera and scanning-slit topography before and after intrastromal corneal ring segment (ICRS) implantation. METHODS: Twenty-five eyes of 21 keratoconus patients who had Intacs (Addition Technology, Sunnyvale, CA) ICRS implantation were included. The anterior segment parameters of the participants were measured before and 3 months after Intacs ICRS implantation with a rotating Scheimpflug camera and scanning-slit topography. Outcome measures included uncorrected (UDVA) and corrected (CDVA) distance visual acuities, refraction and corneal topography. RESULTS: The UDVA improved in 21 eyes (84%) and CDVA in 18 eyes (72%). The anterior average keratometry decreased from 51.80 D preoperatively to 49.59 D postoperatively (p < 0.001) using the rotating Scheimpflug camera and Sim K decreased from 51.21 to 47.44 D using the scanning-slit topography. The posterior average keratometry decreased from -8.03 D preoperatively to -8.42 D postoperatively (p < 0.001) using the rotating Scheimpflug camera. ACD decreased from 3.44 to 3.32 mm (p < 0.001) and from 3.28 to 3.19 mm (p < 0.001) using the rotating Scheimpflug camera and scanning-slit topography, respectively. CONCLUSIONS: The implantation of Intacs segments induced corneal flattening of the anterior surface and corneal steepening of the posterior surface. Both anterior corneal flattening and posterior corneal steepening contributed to the decrease in the keratometric power.

Novel noncontact meibography with anterior segment optical coherence tomography: Hosik meibography.

PURPOSE: To present a novel noncontact meibography system with anterior segment optical coherence tomography (OCT), which is widely used in clinics for the anterior segment (cornea, anterior chamber angle, etc.), and compare the results with preexisting infrared meibography. METHODS: This research was carried out at the Seoul St Mary's Hospital with 2 volunteers. Preexisting infrared meibography was performed on the subjects, and photographs of the meibomian gland were taken again with the anterior segment OCT. With the anterior segment OCT, a tomogram of the meibomian gland could be taken and a picture of the whole meibomian gland could be taken from the infrared images for monitoring. RESULTS: The resolution of the preexisting infrared meibography was a pixel size of 640 × 480. In the anterior segment OCT, the meibomian glands were clearly identified just beneath the palpebral conjunctiva. There was no problem in grading the meibomian gland with the infrared images for monitoring. The resolution was 239 × 178 or 129 × 95 pixels, depending on the save option of the photograph. CONCLUSIONS: This novel meibography using anterior OCT yielded meibomian gland tomograms without any additional equipment, and overall infrared meibography was possible for grading. We believe this new technique will be useful in clinics for meibomian gland dysfunction and the like.

Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition.

Heightened interest in relevant models for human disease increases the need for improved methods for germline transgenesis. We describe a significant improvement in the creation of transgenic laboratory mice and rats by chemical modification of Sleeping Beauty transposons. Germline transgenesis in mice and rats was significantly enhanced by in vitro cytosine-phosphodiester-guanine methylation of transposons prior to injection. Heritability of transgene alleles was also greater from founder mice generated with methylated versus non-methylated transposon. The artificial methylation was reprogrammed in the early embryo, leading to founders that express the transgenes. We also noted differences in transgene insertion number and structure (single-insert versus concatemer) based on the influence of methylation and plasmid conformation (linear versus supercoiled), with supercoiled substrate resulting in efficient transpositional transgenesis (TnT) with near elimination of concatemer insertion. Combined, these substrate modifications resulted in increases in both the frequency of transgenic founders and the number of transgenes per founder, significantly elevating the number of potential transgenic lines. Given its simplicity, versatility and high efficiency, TnT with enhanced Sleeping Beauty components represents a compelling non-viral approach to modifying the mammalian germline.

Erythroid-specific expression of ß-globin from Sleeping Beauty-transduced human hematopoietic progenitor cells.

Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34(+) cells with DsRed and a hybrid IHK-ß-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased ß-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p=0.05), indicating expression of ß-globin from the integrated SB transgene. IHK-ß-globin mRNA was found in non-erythroid cell types, similar to native ß-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK-ß-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK-ß-globin transgene for gene therapy of sickle cell disease.