Correction to: Comprehensive genetic analyses using targeted next-generation sequencing and genotype-phenotype correlations in 53 Japanese patients with osteogenesis imperfecta.

The original article has been corrected.

Comprehensive genetic analyses using targeted next-generation sequencing and genotype-phenotype correlations in 53 Japanese patients with osteogenesis imperfecta.

To elucidate mutation spectrum and genotype-phenotype correlations in Japanese patients with OI, we conducted comprehensive genetic analyses using NGS, as this had not been analyzed comprehensively in this patient population. Most mutations were located on COL1A1 and COL1A2. Glycine substitutions in COL1A1 resulted in the severe phenotype. INTRODUCTION: Most cases of osteogenesis imperfecta (OI) are caused by mutations in COL1A1 or COL1A2, which encode α chains of type I collagen. However, mutations in at least 16 other genes also cause OI. The mutation spectrum in Japanese patients with OI has not been comprehensively analyzed, as it is difficult to identify using classical Sanger sequencing. In this study, we aimed to reveal the mutation spectrum and genotype-phenotype correlations in Japanese patients with OI using next-generation sequencing (NGS). METHODS: We designed a capture panel for sequencing 15 candidate OI genes and 19 candidate genes that are associated with bone fragility or Wnt signaling. Using NGS, we examined 53 Japanese patients with OI from unrelated families. RESULTS: Pathogenic mutations were detected in 43 out of 53 individuals. All mutations were heterozygous. Among the 43 individuals, 40 variants were identified including 15 novel mutations. We found these mutations in COL1A1 (n = 30, 69.8%), COL1A2 (n = 12, 27.9%), and IFITM5 (n = 1, 2.3%). Patients with glycine substitution on COL1A1 had a higher frequency of fractures and were more severely short-statured. Although no significant genotype-phenotype correlation was observed for bone mineral density, the trabecular bone score was significantly lower in patients with glycine substitutions. CONCLUSION: We identified pathogenic mutations in 81% of our Japanese patients with OI. Most mutations were located on COL1A1 and COL1A2. This study revealed that glycine substitutions on COL1A1 resulted in the severe phenotype among Japanese patients with OI.

MIB-1 index is unlikely to predict relapse-free survival in patients who underwent R0-esophagectomy for esophageal squamous cell carcinoma.

MIB-1 is a cell proliferation marker and has previously been investigated as a diagnostic or prognostic indicator of malignancy. Previous studies have investigated MIB-1 index and clinicopathological factors in relation to prognosis of patients with esophageal cancer, with conflicting results. The aim of this study is to assess the prognostic significance of MIB-1 index in patients with thoracic esophageal squamous cell carcinoma. A total of 78 patients who underwent R0-esophagectomy for thoracic esophageal squamous cell carcinoma were enrolled in this study. Preoperatively, 29 patients underwent chemotherapy, six underwent chemoradiotherapy, and the remaining did not undergo any preoperative therapy. The MIB-1 labeling index was reported by counting 500 tumor cells in the hot spots of nuclear labeling. Correlations between MIB-1 index, clinicopathological factors, and relapse-free survival (RFS) were investigated. The mean MIB-1 index was 39.3 ± 21.0 (range: 0-91.3). There was no significant correlation between clinicopathological factors and MIB-1 index in the study patients, irrespective of whether they underwent preoperative therapy. Univariate analysis revealed no significant association between MIB-1 index and RFS. However, depth of tumor invasion, lymph node metastasis and stage, all showed a significant correlation to RFS. Multivariate analysis of RFS revealed that stage was the only significant factor. Conversely, MIB-1 index was not significantly related to RFS (p = 0.41). In conclusion, MIB-1 index is unlikely to be a significant prognostic indicator for esophageal cancer.

Angiopoietin-like protein 2 promotes chondrogenic differentiation during bone growth as a cartilage matrix factor.

OBJECTIVE: Chondrocyte differentiation is crucial for long bone growth. Many cartilage extracellular matrix (ECM) proteins reportedly contribute to chondrocyte differentiation, indicating that mechanisms underlying chondrocyte differentiation are likely more complex than previously appreciated. Angiopoietin-like protein 2 (ANGPTL2) is a secreted factor normally abundantly produced in mesenchymal lineage cells such as adipocytes and fibroblasts, but its loss contributes to the pathogenesis of lifestyle- or aging-related diseases. However, the function of ANGPTL2 in chondrocytes, which are also differentiated from mesenchymal stem cells, remains unclear. Here, we investigate whether ANGPTL2 is expressed in or functions in chondrocytes. METHODS: First, we evaluated Angptl2 expression during chondrocyte differentiation using chondrogenic ATDC5 cells and wild-type epiphyseal cartilage of newborn mice. We next assessed ANGPTL2 function in chondrogenic differentiation and associated signaling using Angptl2 knockdown ATDC5 cells and Angptl2 knockout mice. RESULTS: ANGPTL2 is expressed in chondrocytes, particularly those located in resting and proliferative zones, and accumulates in ECM surrounding chondrocytes. Interestingly, long bone growth was retarded in Angptl2 knockout mice from neonatal to adult stages via attenuation of chondrocyte differentiation. Both in vivo and in vitro experiments show that changes in ANGPTL2 expression can also alter p38 mitogen-activated protein kinase (MAPK) activity mediated by integrin α5ß1. CONCLUSION: ANGPTL2 contributes to chondrocyte differentiation and subsequent endochondral ossification through α5ß1 integrin and p38 MAPK signaling during bone growth. Our findings provide insight into molecular mechanisms governing communication between chondrocytes and surrounding ECM components in bone growth activities.

Glycaemic control boosts glucosylated nanocarrier crossing the BBB into the brain.

Recently, nanocarriers that transport bioactive substances to a target site in the body have attracted considerable attention and undergone rapid progression in terms of the state of the art. However, few nanocarriers can enter the brain via a systemic route through the blood-brain barrier (BBB) to efficiently reach neurons. Here we prepare a self-assembled supramolecular nanocarrier with a surface featuring properly configured glucose. The BBB crossing and brain accumulation of this nanocarrier are boosted by the rapid glycaemic increase after fasting and by the putative phenomenon of the highly expressed glucose transporter-1 (GLUT1) in brain capillary endothelial cells migrating from the luminal to the abluminal plasma membrane. The precisely controlled glucose density on the surface of the nanocarrier enables the regulation of its distribution within the brain, and thus is successfully optimized to increase the number of nanocarriers accumulating in neurons.There are only a few examples of nanocarriers that can transport bioactive substances across the blood-brain barrier. Here the authors show that by rapid glycaemic increase the accumulation of a glucosylated nanocarrier in the brain can be controlled.

Cytological markers for predicting ALK-positive pulmonary adenocarcinoma.

BACKGROUND: ALK gene rearrangement is an important class of gene mutations in pulmonary adenocarcinoma. ALK-positive pulmonary adenocarcinoma exhibits characteristic histological features, such as signet ring cell carcinoma (SRCC) and a mucinous cribriform structure. However, when insufficient histological specimens are obtained, ALK-positivity must be predicted based on cytological features. The purpose of this study was to clarify the cytological characteristics of ALK-positive pulmonary adenocarcinoma. METHODS: We compared the cytological findings of 16 ALK-positive cases with 40 ALK-negative cases. We examined various cytoplasmic features of SRCC, including the presence of pink, yellow, or orange mucin; green, vacuolar, or vesicular cytoplasm; and green globular cytoplasmic secretions. We also examined whether the SRCC cells exhibited a pattern of individually scattered cells, the formation of cell clusters, and formation of a mucinous cribriform pattern. RESULTS: A univariate analysis showed that significantly frequent cytological findings included pink mucin, green cytoplasm, vacuolar cytoplasm, vesicular cytoplasm, green globular cytoplasmic secretions, an individually scattered pattern, cluster formation, and a mucinous cribriform structure (all, P < .05). A stepwise multivariate logistic regression analysis identified three significant contributing factors: pink mucin (P = .03), vesicular cytoplasm (P = .06), and an individually scattered pattern (P = .01) of SRCC. If the specimens showed two or three of these features, the sensitivity and specificity were both 88% for the prediction of ALK-positive cancers. CONCLUSION: Three cytological features of SRCC (pink mucin, vesicular cytoplasm, and an individually scattered pattern) could be useful cytological markers for the prediction of ALK-positive pulmonary adenocarcinoma.

Hydroxychloroquine-conjugated gold nanoparticles for improved siRNA activity.

Current technology of siRNA delivery relies on pharmaceutical dosage forms to route maximal doses of siRNA to the tumor. However, this rationale does not address intracellular bottlenecks governing silencing activity. Here, we tested the impact of hydroxychloroquine conjugation on the intracellular fate and silencing activity of siRNA conjugated PEGylated gold nanoparticles. Addition of hydroxychloroquine improved endosomal escape and increased siRNA guide strand distribution to the RNA induced silencing complex (RISC), both crucial obstacles to the potency of siRNA. This modification significantly improved gene downregulation in cellulo. Altogether, our data suggest the benefit of this modification for the design of improved siRNA delivery systems.

A Pharmacokinetic/Pharmacodynamic Drug-Drug Interaction Study of Tofogliflozin (a New SGLT2 Inhibitor) and Selected Anti-Type 2 Diabetes Mellitus Drugs.

OBJECTIVE: Tofogliflozin is an oral hypoglycemic agent with a novel mechanism of action that reduces blood glucose levels by promoting glucose excretion in urine, achieved by selectively inhibiting sodium-glucose co-transporter 2 (SGLT2). We evaluated the effects of several selected anti-type 2 diabetes mellitus (T2DM) drugs-glimepiride, metformin, sitagliptin, pioglitazone, miglitol, nateglinide, and voglibose-on the pharmacokinetics and pharmacodynamics of tofogliflozin, and the effects of tofogliflozin on the pharmacokinetics of these anti-T2DM drugs in healthy male volunteers. METHODS: A single dose of either tofogliflozin alone, one of the anti-T2DM drugs alone, or co-administration of tofogliflozin and the anti-T2DM drug was administered to 108 healthy men. Cmax, AUCinf, and cumulative urine glucose excretion after co-administration of tofogliflozin and each of the anti-T2DM drugs was evaluated relative to the values of those parameters after administration of each drug alone. RESULTS: None of the anti-T2DM drugs had any effect on tofogliflozin exposure. Tofogliflozin had no or little effect on the exposure of any anti-T2DM drug. No anti-T2DM drug had any major effect on the cumulative urine glucose excretion induced by tofogliflozin. There were no safety concerns evident after administration of any drug alone or in co-administration. CONCLUSIONS: Neither the pharmacokinetics nor the pharmacodynamics of tofogliflozin was affected by any of the anti-T2DM drugs evaluated in this study, nor was the pharmacokinetics of any of the anti-T2DM drugs affected by tofogliflozin in healthy male volunteers.

Music before Dental Surgery Suppresses Sympathetic Activity Derived from Preoperative Anxiety: A Randomized Controlled Trial.

The aim of this study was to estimate the relieving effect of music intervention on preoperative anxiety by using heart rate variability (HRV) analysis. In this randomized controlled trial, 86 adult patients were scheduled to undergo impacted tooth extraction under intravenous sedation and local anesthesia and were classified as either fearful or nonfearful based on a questionnaire. Thereafter, the patients were subdivided into 2 groups: those who listened to music from the time that they arrived at the outpatient clinic until immediately before entering the operating room and those who did not listen to music. The effect of music intervention was evaluated by assessing 1) the low-frequency/high-frequency ratio of HRV, in which positive changes indicate increased sympathetic nervous activity, and 2) the coefficient of component variance for high frequency, in which positive changes indicate increased parasympathetic nervous activity, assessed by means of HRV analysis. Subjective preoperative anxiety was evaluated on a visual analog scale. For fearful patients, the mean magnitude of low-frequency/high frequency changes from baseline among those who listened to music was significantly lower as compared with those who did not listen to music (in the private room: -1.45 ± 1.88 vs. 1.05 ± 1.88, P = 0.0096, 95% confidence interval of effect size = -4.52 to -0.48, Cohen's d = -0.75; in the operating waiting room: -2.18 ± 2.39 vs. -0.10 ± 3.37, P = 0.011, 95% confidence interval of effect size = -3.94 to -0.22, Cohen's d = -0.71, respectively). Visual analog scale scores were also significantly different. Coefficient of component variance for high frequency and heart rate did not differ significantly between the 2 groups. From the perspective of autonomic nervous activity, music intervention is useful for relieving anxiety in patients with dental fear before they enter a dental outpatient operating room. Music intervention may relieve anxiety by reducing sympathetic nervous activity, while parasympathetic nervous activity is not involved (UMIN000016882). Knowledge Transfer Statement: The results of this study revealed that music intervention is useful for clinicians when planning preoperative anxiety management of patients with dental fear who undergo impacted tooth extraction under intravenous sedation and local anesthesia. As a bridging intervention, music intervention enables stress management to continue uninterrupted from the patient's arrival at the dental outpatient clinic to intravenous sedation until completion of the dental surgery. With consideration of cost-effectiveness, absence of adverse physical effects, immediate effect, safety in terms of not using drugs, and lack of concerns about recovery, this information could lead to more appropriate decisions regarding anxiety management in dentistry.

3-T MRI safety assessments of magnetic dental attachments and castable magnetic alloys.

OBJECTIVES: To assess the safety of different magnetic dental attachments during 3-T MRI according to the American Society for Testing and Materials F2182-09 and F2052-06e1 standard testing methods and to develop a method to determine MRI compatibility by measuring magnetically induced torque. METHODS: The temperature elevations, magnetically induced forces and torques of a ferromagnetic stainless steel keeper, a coping comprising a keeper and a cast magnetic alloy coping were measured on MRI systems. RESULTS: The coping comprising a keeper demonstrated the maximum temperature increase (1.42 °C) for the whole-body-averaged specific absorption rate and was calculated as 2.1 W kg⁻¹ with the saline phantom. All deflection angles exceeded 45°. The cast magnetic alloy coping had the greatest deflection force (0.33 N) during 3-T MRI and torque (1.015 mN m) during 0.3-T MRI. CONCLUSIONS: The tested devices showed minimal radiofrequency (RF)-induced heating in a 3-T MR environment, but the cast magnetic alloy coping showed a magnetically induced deflection force and torque approximately eight times that of the keepers. For safety, magnetic dental attachments should be inspected before and after MRI and large prostheses containing cast magnetic alloy should be removed. Although magnetic dental attachments may pose no great risk of RF-induced heating or magnetically induced torque during 3-T MRI, their magnetically induced deflection forces tended to exceed acceptable limits. Therefore, the inspection of such devices before and after MRI is important for patient safety.

Incidence of deepening of the upper eyelid sulcus in prostaglandin-associated periorbitopathy with a latanoprost ophthalmic solution.

PURPOSE: Among some local side effects of prostaglandin-associated periorbitopathy (PAP), deepening of the upper eyelid sulcus (DUES) is the most prominent clinical feature, and is one of the most significant adverse cosmetic events. Here, we prospectively investigated the incidence of DUES in Japanese open-angle glaucoma patients initially treated with latanoprost (Xalatan 0.005%) ophthalmic solution. METHODS: This was an open-label prospective study. Facial photographs and subjective reports of the recognition of DUES were obtained at the beginning of latanoprost treatment and at 2, 4, and 6 months thereafter. Intraocular pressure (IOP) was measured at three consecutive visits before and after treatment with latanoprost. The incidence of DUES was evaluated objectively by three blinded investigators who compared the series of photographs. RESULTS: A total of 52 eyes of 52 newly diagnosed open-angle glaucoma Japanese patients (28 males, 24 females) were evaluated. The objective rate of DUES was 1/52 (2%; 95% CI 0.05 to 10.7%) at 2 months, 2/52 (4%; 95% CI 0.5 to 13.9%) at 4 months, and 3/52 (6%; 95% CI 1.2 to 16.9%) at 6 months. During this period, no patient self-reported an occurrence of DUES. Mean IOPs before and after treatment were 16.5±2.9 and 13.8±3.0 mm Hg, respectively. Latanoprost reduced the IOP significantly (P<0.0001, paired t-test). CONCLUSIONS: Latanoprost caused DUES rarely and had a robust IOP-lowering effect in Japanese glaucoma patients.

Effects of the nanotopographic surface structure of commercially pure titanium following anodization-hydrothermal treatment on gene expression and adhesion in gingival epithelial cells.

The long-term stability and maintenance of endosseous implants with anodized-hydrothermally treated commercially pure titanium surfaces and a nanotopographic structure (SA-treated c.p.Ti) depend on the barrier function provided by the interface between the transmucosal portion of the implant surface and the peri-implant epithelium. This study investigated the effects of extracellular and intracellular gene expression in adherent gingival epithelial cells cultured for 1-7 days on SA-treated c.p.Ti implant surfaces compared to anodic oxide (AO) c.p.Ti and c.p.Ti disks. Scanning electron microscopy (SEM) showed filopodium-like extensions bound closely to the nanotopographic structure of SA-treated c.p.Ti at day 7 of culture. Gene expressions of focal adhesion kinase, integrin-α6ß4, and laminin-5 (α3, ß3, γ2) were significantly higher on SA-treated c.p.Ti than on c.p.Ti or AO c.p.Ti after 7 days (P<0.05). Our results confirmed that gingival epithelial cells adhere to SA-treated c.p.Ti as the transmucosal portion of an implant, and that this interaction markedly improves expression of focal adhesion molecules and enhances the epithelial cell phenotype. The cellular gene expression responses driving extracellular and intracellular molecular interactions thus play an important role in maintenance at the interface between SA-treated c.p.Ti implant surfaces and the gingival epithelial cells.

Influence of cycloplegia with topical cyclopentolate on higher-order aberrations in myopic children.

PURPOSE: To investigate the influence of cycloplegia with topical cyclopentolate on wavefront aberrations in myopic children. DESIGN: This is a prospective, comparative study. METHODS: Twenty-eight myopic children with a mean age of 7.25 ± 2.55 were enrolled in this study. We evaluated refraction and wavefront aberrations before and after cycloplegia with 1% cyclopentolate hydrochloride. Ocular and corneal aberrations were simultaneously measured and compared with each other. Individual Zernike components were also analyzed up to the sixth order. All these parameters were compared before and after cycloplegia. RESULTS: Ocular higher-order aberrations (HOAs) significantly increased after cycloplegia (P=0.012 for spherical-like and P=0.015 for total HOAs). Corneal HOAs did not change after cycloplegia. When corneal and ocular HOAs were compared, the ocular HOAs were significantly smaller than the corneal HOAs in spherical-like aberrations (P<0.001) and total HOAs (P=0.006). As for individual Zernike components, ocular aberration generally showed smaller or equivalent values in comparison with corneal aberration. In addition, each Zernike component showed a large standard deviation. CONCLUSIONS: Internal optics compensates for corneal HOAs in myopic children, and paralysis of tonic accommodation with cyclopentolate considerably affects ocular HOAs. However, inter-individual variation in each Zernike component is quite large in myopic children.

Radiofrequency heating of metallic dental devices during 3.0 T MRI.

OBJECTIVES: To estimate the risk of injury from radiofrequency (RF) heating of metallic dental devices in use during 3.0 T MRI. METHODS: The whole-body specific absorption rate (WB-SAR) was calculated on the basis of saline temperature elevation under the maximum RF irradiation for 15 min to determine the operation parameters for the heating test. The temperature changes of three types of three-unit bridges, a full-arch fixed dental prosthesis and an orthodontic appliance in use during MRI with a 3.0 T MR system (Magnetom(®) Verio; Siemens AG, Erlangen, Germany) were then tested in accordance with the American Society for Testing and Materials F2182-09 standardized procedure under the maximum RF heating during 15 min RF irradiation. RESULTS: The system console-predicted WB-SAR was approximately 1.4 W kg(-1) and that measured with a saline phantom was 2.1 W kg(-1). In the assessment of RF heating, the highest temperature increase was +1.80 °C in the bridges, +1.59 °C in the full-arch fixed dental prosthesis and +2.61 °C in the orthodontic appliance. CONCLUSIONS: The relatively minor RF heating of dental casting material-based prostheses in Magnetom Verio systems in the normal operating mode should not pose a risk to patients. However, orthodontic appliances may exhibit RF heating above the industrial standard (CENELEC standard prEN45502-2-3); therefore, the wire should be removed from the bracket or a spacer should be used between the appliance and the oral mucosa during MRI.

Supercooling and vitrification of aqueous glycerol solutions at normal and high pressures.

The supercooling and vitrification of aqueous glycerol solutions was studied at high pressures. Homogeneous ice nucleation temperatures (T(H)) were obtained for aqueous glycerol solutions of R=50, 30, 20, 12, and 10 (R: moles of water/moles of glycerol) up to 300MPa. The R=20 glycerol solution formed a glass above 200MPa at a cooling rate of 200°C/min, indicating that pressure enhances glass-formation of aqueous glycerol solutions. The (dT(g)/dP) values were obtained for vitrified aqueous glycerol solutions of R=3, 5, 10, and 20. These data can be used for the development of cryo-preservation liquids for living cells at high pressures.

Radiofrequency heating and magnetically induced displacement of dental magnetic attachments during 3.0 T MRI.

OBJECTIVE: The aim of this study was to estimate the risk of injury from dental magnetic attachments due to their radiofrequency (RF) heating and magnetically induced displacement during 3.0 T MRI. METHODS: To examine the magnetic attachments, we adopted the American Society for Testing and Materials F2182-02a and F2052-06 standards in two MRI systems (Achieva 3.0 T Nova Dual; Philips, Tokyo, Japan, and Signa HDxt 3.0 T; GE Healthcare, Milwaukee, WI). The temperature change was measured in a cylindrical keeper (GIGAUSS D600; GC, Tokyo, Japan) with coping of the casting alloy and a keeper with a dental implant at the maximum specific absorption rate (SAR) for 20 min. To measure the magnetically induced displacement force, three sizes of keepers (GIGAUSS D400, D600 and D1000) were used in deflection angle tests conducted at the point of the maximum magnetic field strength. RESULTS: Temperature elevations of both coping and implant were higher in the Signa system than in the Achieva system. The highest temperature changes in the keeper with implant and keeper with coping were 0.6 °C and 0.8 °C in the Signa system, respectively. The temperature increase did not exceed 1.0 °C at any location. The deflection angle (α) was not measurable because it exceeded 90°. GIGAUSS D400 required an extra 3.0 g load to constrain the deflection angle to less than 45°; GIGAUSS D600 and D1000 required 5.0 and 9.0 g loads, respectively. CONCLUSIONS: Dental magnetic attachments pose no risk due to RF heating and magnetically induced displacement at 3.0 T MRI. However, it is necessary to confirm that these keepers are securely attached to the prosthesis before imaging.

MicroRNA-494 suppresses cell proliferation and induces senescence in A549 lung cancer cells.

OBJECTIVES: MicroRNAs (miRNAs) are small functional RNAs that regulate mRNAs for degradation or translational suppression. In the present study, we aimed to reveal functional importance of miRNA-494 (miR-494) in A549 human lung cancer cells. MATERIALS AND METHODS: We established A549 cells that constitutively expressed miR-494. Next, we sought to investigate insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) mRNA as an miR-494 target. For this, we constructed a reporter plasmid bearing potential miR-494 binding sequences derived from the 3'-untranslated region (3'-UTR) of IGF2BP1 mRNA in the 3'-UTR of the luciferase gene. RESULTS: Through comparison between miR-494 expressing cells and control cells, we revealed that miR-494 suppressed cell proliferation and colony forming activity, and induced senescence. Reporter activity was inhibited by miR-494. In addition, IGF2BP1 mRNA levels were down-regulated in A549 cells that constitutively expressed miR-494. IGF2BP1 has been shown to bind and suppress IGF2 mRNA, and this could be a reason why IGF2BP1 can regulate cell function. Therefore, we analysed IGF2 mRNA levels and revealed that IGF2 was up-regulated in A549 cells that constitutively expressed miR-494. Finally, elevated IGF2 mRNA levels in A549 cells that constitutively expressed miR-494 were suppressed to basal level by an miR-494 inhibitor. CONCLUSIONS: Taken together, IGF2BP1 and its downstream target IGF2 could be a crucial axis for miR-494 in regulation of the destiny of A549 cells.

Effect of integrin targeting and PEG shielding on polyplex micelle internalization studied by live-cell imaging.

α(v)ß(3) and α(v)ß(5) integrins are attractive target structures for cancer therapy as they are upregulated in tumor and tumor associated host cells and play a pivotal role for tumor growth and metastasis. Gene vectors such as polyplex micelles consisting of thiolated PEG-block-poly(lysine) copolymers complexed with plasmid DNA can be targeted to these specific integrins by equipment with a cyclic RGD peptide. In this study, we analyzed the effect of the RGD ligand on micelle endocytosis by comparing fluorescently labeled, targeted and untargeted micelles in live-cell imaging experiments with highly sensitive fluorescence microscopy and flow cytometry. Two micelle types with 12 kDa (PEG12) and 17 kDa (PEG17) PEG shell layers were examined to evaluate the influence of surface shielding on the internalization characteristics. Our results reveal three major effects: First, the RGD ligand accelerates the internalization of micelles into integrin expressing HeLa cells without changing the uptake pathway of the micelles. Both targeted as well as untargeted micelles are predominantly internalized via clathrin mediated endocytosis. Second, the PEG shielding of micelles has an important effect on their targeting specificity. At high PEG shielding selective endocytosis of integrin targeted micelles occurs, whereas at low PEG shielding targeted and untargeted micelles show comparable internalization. In addition, PEG17 RGD(+) micelles induce the highest reporter gene expression. Third, our data demonstrate a clear influence of the applied micelle dose on the internalization of integrin targeted micelles. We propose that PEG17 shielded micelles equipped with a cyclic RGD ligand are the favored system of choice for clinical therapy as they exhibit higher transgene expression, a higher specificity for integrin-dependent endocytosis compared to PEG12 shielded micelles, and are functional at low doses as well.

Serum level of pepsinogen significantly associated with gastric distress induced by amino-bisphosphonates.

UNLABELLED: To elucidate whether serum levels of pepsinogens are associated with the occurrence of gastrointestinal adverse events induced by amino-bisphosphonates (amino-BP), the serum levels of pepsinogen were measured in amino-BP users. Our results indicate that measurement of pepsinogen I is useful in predicting gastric distress induced by amino-BP in osteoporosis. INTRODUCTION: To elucidate whether serum levels of pepsinogens are associated with the occurrence of gastrointestinal adverse events induced by amino-BP, the serum levels of pepsinogen I and II were measured in amino-BP users. METHODS: When the patients complained of gastric distress symptoms during the first 6 months after amino-BP use resulting in discontinuation of the drug, endoscopical examinations were performed to assess whether gastric lesions were present. A total of 223 amino-BP users were enrolled in the study, of which 47 patients refused to take the drug due to gastric distress symptoms. The remaining 176 patients did not complain of any gastric distress. RESULTS: Among 47 patients, eight patients showed obvious gastric lesions such as gastric or duodenal ulcers and acute gastric mucosal lesions in the endoscopical examination. The remaining 39 patients did not show any gastric lesions. The possible confounding factors, such as a Helicobactor pylori infection or concurrent use of ulcerogenic agents, did cause not affect gastric distress in amino-BP users. The serum pepsinogen I level was significantly associated with severity of the gastric lesion 46.8 ± 27.7, 60.8 ± 32.4, and 103.4 ± 49.2 ng/ml for patients without any gastric distress, with gastric distress accompanied no gastric lesions, and with gastric distress accompanied gastric lesions, respectively. CONCLUSIONS: ROC analysis revealed that the cutoff value of pepsinogen I for expectation of gastric regions was 76.8 ng/ml. The results clearly indicate that measurement of pepsinogen I may be useful in predicting gastric distress induced by amino-BP in osteoporosis.

Supercooling of aqueous dimethylsulfoxide solution at normal and high pressures: Evidence for the coexistence of phase-separated aqueous dimethylsulfoxide solutions of different water structures.

Supercooling behavior of aqueous dimethylsulfoxide (DMSO) solution was investigated as a function of DMSO concentration and at high pressures. A linear relationship was observed for T(H) (homogeneous ice nucleation temperature) and T(m) (melting temperature) for the supercooling of aqueous DMSO solution at normal pressure. Analysis of the DTA (differential thermal analysis) traces for homogeneous ice crystallization in the bottom region of the T(H) curve for a DMSO solution of R=20 (R: moles of water/moles of DMSO) at high pressures supported the contention that the second critical point (SCP) of liquid water should exist at P(c2)= approximately 200 MPa and at T(c2)<-100 degrees C (P(c2): pressure of SCP, T(c2): temperature of SCP). The presence of two T(H) peaks for DMSO solutions (R=15, 12, and 10) suggests that phase separation occurs in aqueous DMSO solution (R