Injectable in situ cross-linking hyaluronan hydrogel for easier removal of posterior vitreous cortex in vitrectomy.

PURPOSE: Removing transparent vitreous tissues, such as a residual vitreous cortex (VC) or proliferative membrane, without damaging the retina is often problematic in vitrectomy. We examined the feasibility of an injectable in situ cross-linking hyaluronan hydrogel (XL-HA) for vitrectomy. STUDY DESIGN: Experiments using ex vivo and in vivo animal models. METHODS: HA-dibenzocyclooctyne and HA-azidoethylamine solutions were mixed to form XL-HA, which then gradually formed a hydrogel. We tested the function of XL-HA in ex vivo porcine eyes. We then examined the performance of XL-HA in in vivo rabbit models of posterior vitreous detachment, posterior VC removal, and proliferative vitreoretinopathy. RESULTS: The ex vivo study showed that XL-HA rapidly embedded triamcinolone acetonide, mimicking VC attached to the retina, and became hard enough to be pinched with tweezers within 3 minutes, allowing us to remove only the triamcinolone acetonide without impairing the internal limiting membrane. In the in vivo rabbit models, XL-HA injection improved posterior vitreous detachment, and the thin and fragile posterior VC or fibrous proliferative membrane was readily peeled off without any damage to the underlying retina as compared with untreated controls. A short-term intraocular biocompatibility test demonstrated that the intraocular pressure remained normal with XL-HA injected into the eye. In addition, transmission electron microscopy showed no obvious abnormalities in the cornea or in the inner and outer retina. CONCLUSION: The results indicate that XL-HA is a potential adjunctive device to help make vitrectomy safe, effective, and successful.

Development of in situ crosslinked hyaluronan as an adjunct to vitrectomy surgery.

Ophthalmologists have used hyaluronan (HA) products as adjuncts to ocular surgery since the 1970s. However, HA products are not always functional in surgeries of the posterior eye segment due to their lack of biomechanical strength. In this study, we developed an in situ crosslinked HA (XL-HA) and evaluated its potential as an adjunct to vitrectomy surgery in an in vitro model with a triamcinolone acetonide (TA) layer used as a pseudo residual vitreous cortex (RVC). Within a few minutes at concentrations over 0.9%, XL-HA, generated by the click chemistry of HA-dibenzocyclooctyne and HA-azidoethylamine, formed a hydrogel with the appropriate hardness for tweezers peeling. XL-HA (concentration, 0.76-1.73%) without dispersion successfully entered the TA layer and removed more than 45% of the total TA. Dynamic viscoelasticity helps to explain the rheological behavior of hydrogels, and the assessment results for XL-HA indicated that suitable concentrations were between 0.97% and 1.30%. For example, 1.30% XL-HA hydrogel reached sufficient hardness at 3 min for tweezers peeling, and the TA removal ability exceeded 70%. These results demonstrated that XL-HA was a potential adjunct to successful vitrectomy.

Simultaneous production of N-acetylheparosan and recombinant chondroitin using gene-engineered Escherichia coli K5.

N-Acetylheparosan and chondroitin are increasingly needed as alternative sources of animal-derived sulfated glycosaminoglycans (GAGs) and as inert substances in medical devices and pharmaceuticals. The N-acetylheparosan productivity of E. coli K5 has achieved levels of industrial applications, whereas E.coli K4 produces a relatively lower amount of fructosylated chondroitin. In this study, the K5 strain was gene-engineered to co-express K4-derived, chondroitin-synthetic genes, namely kfoA and kfoC. The productivities of total GAG and chondroitin in batch culture were 1.2 g/L and 1.0 g/L respectively, which were comparable to the productivity of N-acetylheparosan in the wild K5 strain (0.6-1.2 g/L). The total GAG of the recombinant K5 was partially purified by DEAE-cellulose chromatography and was subjected to degradation tests with specific GAG-degrading enzymes combined with HPLC and 1H NMR analyses. The results indicated that the recombinant K5 simultaneously produced both 100-kDa chondroitin and 45-kDa N-acetylheparosan at a weight ratio of approximately 4:1. The content of chondroitin in total GAG partially purified was 73.2%. The molecular weight of recombinant chondroitin (100 kDa) was 5-10 times higher than that of commercially available chondroitin sulfate. These results indicated that the recombinant K5 strain acquired the chondroitin-producing ability without altering the total GAG productivity of the host.

Biochemical and crystallographic assessments of the effect of 4,6-O-disulfated disaccharide moieties in chondroitin sulfate E on chondroitinase ABC I activity.

O-sulfated N-acetyl-d-galactosamine (GalNAc) residues in chondroitin sulfate (CS) play a crucial role in chondroitinase ABC I (cABC-I) activity. CSA containing mainly 4-O-monosulfated GalNAc was a good substrate for the enzyme, but not CSE containing mainly 4,6-O-disulfated GalNAc [GalNAc(4S,6S)]. Each CS isomer exhibits structural heterogeneity; CSE has di-sulfated disaccharide units and mono-sulfated disaccharide units. Disaccharide composition analysis of digested products revealed that mono-sulfated disaccharide units in CSE contributed to the enzyme reactivity. Although enough substrate (CSA) was present in mixtures of CSA and CSE for reaction, the reactivity was reduced depending on the amount of CSE in the mixture. These results suggested that CSE is not only resistant to enzyme digestion but also attenuates enzyme activity. To understand the mechanism of action, crystallography of cABC-I in complex with unsaturated CSE-disaccharide, ΔDi-(4,6)S, was performed. Both 4-O- and 6-O-sulfate groups in ΔDi-(4,6)S interact with Arg500, suggesting that there was a greater interaction between ΔDi-(4,6)S and Arg500 than between mono-sulfated disaccharides and Arg500. Besides, this interaction attenuated enzyme activity by interfering with a function of Arg500, which is the charge neutralization of the carboxy group of D-glucuronic acid (GlcA) residues in CS. When interacting with the CSE-disaccharide unit [GlcAß1-3GalNAc(4S,6S)] in CS, cABC-I cannot interact with other CS-disaccharide units until it has digested the CSE-disaccharide unit. The low reactivity of cABC-I with CSE is attributable to two suggested factors: (a) resistance of E-units in CSE molecules to digestion by cABC-I, and (b) tendency of E-units in CSE molecules to attenuate cABC-I activity.

Highly sensitive quantification of bacterial chondroitin in a culture based on ELISA techniques.

Some bacteria produce non-sulfated chondroitin (CH). Accurate, rapid, and high throughput methods to quantify CH in fermented cultures helps to improve microbial breeding and fermentation conditions efficiently. In this study, highly sensitive methods to quantify bacterial CH were developed based on ELISA techniques. An assay using an anti-K4 antiserum successfully determined the concentration of fructosylated CH in the range from 9 to 800 ng/mL. The method also enabled the determination of CH concentration exceeding 9 µg/mL. To improve the assay sensitivity for CH, hyaluronan (HA) binding protein (HABP) was applied instead of a capture antibody. HABP was bound to CH, but not to chemically desulfated chondroitin sulfate or fructosylated CH. The quantification limit of CH was 18 µg/mL in the HA assay using HABP. Replacing the HA-coated microplate with a CH-coated microplate increased the sensitivity >1000 times (assay range = 14 to 1000 ng/mL). Pretreatment with hyaluronidase enabled us to accurately quantify CH in samples mixed with HA.

[Clinical Functionality of Dispersive OVDs: Improvement of One of the Properties of 3% Hyaluronic Acid and 4% Chondroitin Sulfate Combination].

Ophthalmic viscosurgical devices (OVDs) are mainly divided into two general categories: cohesive and dispersive. Dispersive OVDs such as the 3% hyaluronic acid and 4% chondroitin sulfate (HA/CS) combination have excellent adhesion to ocular tissues and protect the corneal endothelium to a greater extent than cohesive OVDs. Herein, we summarize our recent findings regarding one of the properties of the HA/CS combination related to clinical performance. (i) The room temperature stability of OVDs and needle clogging by OVDs remain clinical issues. We demonstrated that adding d-sorbitol to the HA/CS combination preserved its viscosity, which was equivalent after 2 year-storage at room temperature to the viscosity of HA/CS combination stored under refrigeration for 2 years without d-sorbitol. Besides, the HA/CS combination with d-sorbitol could be used repeatedly without cleaning or replacing the needle, suggesting that the addition of d-sorbitol prevents drying and solidification of the OVD on the needle. (ii) Although it can be inferred from numerous studies that the tissue adhesion of OVDs influences their retention by the eye, little is known about the physical properties of OVDs that contribute to intraocular retention. To address this issue, we compared two types of adhesive forces, detachment force and repulsive force, for each OVD. Compared with other dispersive OVDs, the HA/CS combination showed higher values for both adhesive forces. These results suggest that adhesive forces may be used as an index of dispersive OVD retention in the eye.

Advances in Understanding the Mechanism of Ophthalmic Viscosurgical Device Retention in the Anterior Chamber or on the Corneal Surface during Ocular Surgery.

Retention durability, especially in the eye, is one of the most important properties of ophthalmic viscosurgical devices (OVDs) during ocular surgery. However, the information on the physical properties of OVDs is insufficient to explain their retention durability. The purpose of this study is to clarify the mechanism of OVD retention to improve understanding of the behavior of OVDs during ocular surgery. To elucidate the mechanism of OVD retention, we have developed a new test method for measuring repulsive force. As a result, the maximum repulsive force of OVDs was positively and well correlated with the retention durability of investigated OVDs. Consequently, we demonstrated that the repulsive force could be used as an index of retention durability on the ocular surface and in the eye. We directly compared the intraocular retention durability of three OVDs (Shellgan, Viscoat, and Opegan-Hi) in ex vivo porcine eyes. Opegan-Hi was immediately removed from the anterior chamber, but Shellgan and Viscoat remained largely in the anterior chamber as determined by fluorescence imaging. These results showed that the intraocular retention behavior of OVDs was similar to their ocular surface behavior in our previous report, suggesting that retention durability is dependent on the OVD itself. The retention durability of Shellgan seemed to be higher than that of Viscoat, and the maximum repulsive force of Shellgan was 1.35-fold higher than that of Viscoat. Therefore, the repulsive force might be a useful index for assessing the difference in the retention durability between OVDs such as Shellgan and Viscoat.

Quantitative Assessment of Ophthalmic Viscosurgical Devices on Visibility, Spreadability, and Durability as Corneal Wetting Agents for the Wet Shell Technique.

PURPOSE: To evaluate ophthalmic viscosurgical devices (OVDs) as corneal wetting agents for the wet shell technique, a common procedure in Japan to maintain the wettability of corneal surfaces. METHODS: We surveyed Japanese ophthalmologists to determine the current state of the wet shell technique. After developing three ex vivo testing methods, we evaluated the corneal wetting properties of OVDs including 3% hyaluronic acid (HA) solution and OVD products, Opegan, Opelead, Viscoat, Shellgan, Discovisc, and Opegan-Hi. RESULTS: Overall, 214 ophthalmologists (70%) had performed the wet shell technique, and 91% of ophthalmologists who performed vitreous surgery had performed this technique. Using a questionnaire, we evaluated the performance of OVD as corneal wetting agents as follows: (i) visibility, smoothness of OVD surface; (ii) spreadability, coverage of the cornea; and (iii) retention durability, residual ratio of OVD on the corneal surface. The smoothness and spreadability of Opegan, Opelead, and 3% HA were superior to other OVDs. Adding an appropriate amount of balanced salt solution to the other OVDs improved smoothness and spreadability similar to that of Opelead or 3% HA. Shellgan and Viscoat, combination OVDs consisting of 3% HA and 4% chondroitin sulfate, showed high retention durability, resulting in remaining longer on the cornea compared with other OVDs. CONCLUSIONS: Physical properties of OVDs tested in this study may provide useful information for ophthalmologists to select a suitable OVD when performing the wet shell technique.

International Cross-Sectional Study on the Effectiveness of Okada Purifying Therapy, a Biofield Therapy, for the Relief of Various Symptoms.

Objective: To investigate whether differences exist in the effectiveness/safety of a single session of Okada Purifying Therapy (OPT), a type of biofield therapy, among those from different ethnicity/cultures, and to analyze factors associated with the outcomes in a real-world setting. Design: Pre-post test design using convenience sampling methods. Setting: Home setting. Subjects: A total of 11,303 individuals aged 16 years or older from 14 different countries (>1000 individuals each from Japan, the United States, Thailand, Chile/Peru, and <200 individuals each from Portugal, Spain, Argentina, Mexico, Brazil, South Korea, Taiwan, Belgium, and France). More than 50% of the subjects were themselves OPT practitioners, and more than 50% of the treatments were administered in an environment where the practice of OPT was promoted. Intervention: Participants received a single session of OPT lasting 30 min or longer from the volunteer practitioners. They self-reported the changes in overall symptoms, physical pain, anxiety/depression, and dizziness/palpitation. Outcome measures: Improvement/exacerbation rates of each symptom and factors associated with symptom improvement were analyzed. Results: Of the participants, 77.5%, 75.6%, 78.4%, and 73.8% reported an improvement of overall symptoms, physical pain, anxiety/depression, and dizziness/palpitation, respectively. The improvement rates were consistently higher among participants from Chile/Peru than those from Japan, the United States, and Thailand (p < 0.001), and among those who had received a longer therapy (p < 0.001). Spanish/Portuguese speaking countries almost always showed high improvement rates; conversely, Japan showed a lower rate in each symptom. Participants' gender, reasons for participation, previous experience, and location of the session were also associated with the improvement of different symptoms. These findings occurred regardless of the participants' age or presence/absence of illness. In terms of safety, the exacerbation rates of symptoms were 2.8%, 2.5%, 0.8%, and 1.7%, respectively. Of those who expressed symptoms exacerbation, 71.6% recovered in a few hours. None of them needed emergency medical treatment. Conclusions: In those who were often sympathetic to OPT and/or in an authorized location, OPT was effective and safe across countries with ethnic/cultural differences; however, participants' country of residence and duration of the session were independently associated with the changes in various symptoms. (ClinicalTrials.gov NCT03994809).

D-sorbitol can keep the viscosity of dispersive ophthalmic viscosurgical device at room temperature for long term.

The combination of 3% sodium hyaluronate (HA) and 4% sodium chondroitin sulfate (CS) is used as a dispersive ophthalmic viscosurgical device (OVD) during cataract surgery. For most OVDs containing HA, storage at 2-8 °C is recommended to preserve product characteristics. In order to develop a dispersive OVD that can be stored at room temperature, in this study, we searched additives which can stably maintain the viscosity, a key parameter of OVD, under preservation stability testing at 60 °C. The addition of D-sorbitol to a combination OVD, 3% HA and 4% CS, suppressed the reduction in viscosity compared with other OVDs with or without additives. The addition of D-sorbitol was also effective in improving the residual viscosity of the combination OVD after thermal treatment and light irradiation. Moreover, the OVD containing D-sorbitol can be stored at 25 °C with stably maintaining the initial viscosity for at least 24 months. In conclusion, the new dispersive OVD, 3% HA, 4% CS, and 0.5% D-sorbitol, can be stored at room temperature instead of under cold conditions and may represent an attractive option for clinical use because it is not necessary to bring the product to room temperature prior to use.

[Evaluation of Rheological Properties of Cohesive Ophthalmic Viscosurgical Devices Composed of Sodium Hyaluronate with High Molecular Weight-2019].

Ophthalmic viscosurgical devices (OVDs), mainly containing sodium hyaluronate (HA), are used in cataract surgeries to protect the cornea endothelium. In this study, the rheological properties of 9 launched products (containing 1% HA) were evaluated. The molecular weights (MWs) of HA estimated based on intrinsic viscosity varied widely, between 1100-2500 kDa, and showed a particular value for each product. Of the 9 products, 6 are classified as cohesive OVDs and their product specifications show the same value for intrinsic viscosity (25-45 dL/g), with high MW HA (>2000 kDa); however, the MW of each HA showed a particular value (2200-2500 kDa) within the range of the product specification. As with the MW of HA, apparent viscosity and dynamic rheological parameters showed particular values for each OVD. The product Opegan-Hi exhibited the highest value of apparent viscosity at low shear rate, and a solid-like behavior among the OVDs. In a questionnaire survey among 198 cataract surgeons, 42% of surgeons had experienced a difference in ability to maintain the depth of anterior chamber during surgery among the different cohesive OVDs used. This suggested that surgeons select OVD properties based on surgical procedure and patient cases. In conclusion, we demonstrated that each OVD has particular rheological properties within the range of the product specification defined by the intrinsic viscosity. The results might provide useful information for surgeons in their selection of OVDs based on their experience.

Rheological and Adhesive Properties to Identify Cohesive and Dispersive Ophthalmic Viscosurgical Devices.

The purpose of this study is to evaluate the usefulness of the rheological properties and adhesive force of ophthalmic viscosurgical devices (OVDs) as parameters for understanding and identifying the surgical behavior of cohesive and dispersive OVDs. The apparent viscosity, and the storage and loss moduli (dynamic rheological parameters) of 50% chondroitin sulfate (CS), 3% sodium hyaluronate (HA), Shellgan (the combination of 3% HA and 4% CS), Opegan (1% HA with a low molecular mass) and Opegan-Hi (1% HA with a high molecular mass) were obtained with a rheometer. The adhesive force of each sample was measured by using a texture analyzer. Opegan-Hi showed a solid-like behavior, while 50% CS showed a fluid-like behavior from their apparent viscosity and dynamic rheological parameters. Shellgan, 3% HA, and Opegan exhibited similar rheological properties and intermediate characteristics between Opegan-Hi and 50% CS, although their respective values were slightly different. Among these OVD samples, the adhesive force was higher in the order of 50% CS > Shellgan, 3% HA > Opegan > Opegan-Hi. The adhesive force of dispersive OVDs tended to be higher than that of cohesive OVDs, which correlated well with the removal times of OVDs from the eye that have previously been reported. In conclusion, we demonstrated that cohesive OVDs and dispersive OVDs have particular rheological and adhesive properties that can be applied to identify both types. These parameters obtained in this study provide useful information for a greater understanding and prediction of the behavior of OVDs in the eye during surgery.

The Influence of a Multimodal Health Program with Diet, Art, and Biofield Therapy on the Quality of Life of People in Japan.

OBJECTIVE: To investigate whether the frequency of the practice of each of diet, art, and biofield therapy influences improvement in quality of life (QOL), and to examine whether the simultaneous practice of all three components increasingly improves QOL in a real-world setting. DESIGN: Pre-post-test design using convenience sampling methods. SETTING: Home setting. SUBJECTS: A total of 4681 individuals aged 16 years or older who answered the questionnaire appropriately. INTERVENTION: Participants agreed to practice the three components daily and self-evaluated the frequency of their weekly practice for three consecutive months. At the beginning and end of the study, they completed the MOA quality-of-life questionnaire (10-item MOA quality-of-life questionnaire [MQL-10]). OUTCOME MEASURES: Factors associated with the increase in MQL-10 scores for each component, and the relationship between the simultaneous practice of multiple components and the changes in MQL-10 scores were analyzed. RESULTS: Frequent practice of the diet and/or art components was associated with an increase in the term-end MQL-10 score (p < 0.001); however, receiving biofield therapy frequently was not. Participants' age, gender, and qualification as a practitioner of biofield therapy had no relationship with changes in scores, but the reasons for participation had a significant influence on changes in scores (p < 0.001). Participants who initially did not practice any components frequently but who subsequently increased the number of components and frequency of each practice had a higher likelihood of exhibiting an increase in the term-end score (p < 0.01). Participants who initially practiced all three components frequently but later decreased the number of components practiced frequently had a lower chance of increase and a higher risk of decrease in scores (p < 0.01). CONCLUSIONS: The data suggest that the frequent practice of the diet and art components is associated with improvement in QOL. Simultaneous practice of diet, art, and biofield therapy is more likely to improve QOL. ( ClinicalTrials.gov NCT01927250).

Effectiveness of Biofield Therapy for Patients Diagnosed With Fibromyalgia.

Context • Fibromyalgia (FM) is a disorder with an unknown etiology; symptoms include physical and psychological stress, widespread chronic pain, insomnia, and depression. Mind-body medicine and aerobic exercise have shown positive effects for symptom control. Several studies have reported positive effects for biofield therapy for FM, but when other studies have compared the treatment with a sham control, they have not found those beneficial effects. Objective • The study intended to examine the effects of a biofield therapy called Okada purifying therapy on patients' FM symptoms. Design • An open-label, self-controlled study was conducted with 2 groups of FM patients. Setting • The study was conducted at an integrative medicine clinic in Portugal. Participants • Twelve patients, aged 25 to 59 y, with symptoms of FM for the 3 mo before the study, participated. Participants agreed not to receive any new treatment other than the intervention throughout the study as a condition for participation. Intervention • Participants received 50 min of biofield therapy 2 ×/wk for 3 mo, either during the first half of the 6-mo study for group A or the second half for group B. The second half of the study was designated as a sham control for group A, and the first half of the study was designated as a sham control for group B. Outcome Measures • Measures included the Beck depression inventory (BDI), the fibromyalgia impact questionnaire (FIQ), the tender point index (TPI), and changes in the dosage of prescribed medication. Results • A significant change was observed for scores in the FIQ (P = .027), BDI (P = .027), and TPI (P = .027) in the second group of patients who received the intervention in the second half of the 6-mo study. Seven of 11 participants taking prescribed medications reduced their dosage. Conclusion • Biofield therapy may help reduce symptoms of depression, chronic widespread pain, and tenderness among patients suffering from FM. Larger studies with rigorous designs are required for further accurate evaluation.

Isolation and characterization of monoclonal antibodies specific for chondroitin sulfate E.

Chondroitin sulfate E (CSE) is a polysaccharide containing mainly disaccharide units of D-glucuronic acid (GlcA) and 4,6-O-disulfated N-acetyl-D-galactosamine (GalNAc) residues (E-unit) in the amount of ∼ 60%. CSE is involved in many biological and pathological processes. In this study, we established new monoclonal antibodies, termed E-12C and E-18H, by using CSE that contained more than 70% of E-units as an immunogen. These antibodies recognized CSE but not other CSs isomers or dermatan sulfate (DS). We evaluated the reactivities of the antibodies to 6-O-sulfated CSA (6S-CSA) and DS (6S-DS) that possessed ∼ 60% of GalNAc (4S, 6S) moieties in their structures. Neither of the antibodies reacted with 6S-DS. The antibodies strictly distinguished the structural difference of GlcA and L-iduronic acid in the polysaccharide. Binding affinities of the antibodies were determined by a surface plasmon resonance assay using CSE and 6S-CSA. The binding affinities were strongly associated with the molecular weight of CSE and the E-unit content of 6S-CSA. Moreover, we demonstrated that the antibodies are applicable to histochemical analysis. In conclusion, the new anti-CSE monoclonal antibodies specifically recognize the E-unit of CSE. The antibodies will become useful tools for the investigation of the biological and pathological significance of CSE.

The association between impaired proinsulin processing and type 2 diabetes mellitus in non-obese Japanese individuals.

We aimed to examine the association between impaired proinsulin processing in pancreatic beta cells and type 2 diabetes mellitus in non-obese Japanese patients. Participants were divided into groups for normal glucose tolerance, prediabetes, and type 2 diabetes based on the oral glucose tolerance test (OGTT). Activities of prohormone convertase (PC) 1/3 and PC2 in fasting states were estimated. Multiple regression analysis was undertaken to ascertain if alteration of the activities of these enzymes contributes to the development of impaired glucose tolerance by comparison with HOMA-ß and the oral disposition index (DI(O)). Overall, 452 subjects were included. PC1/3 activity tended to decrease in type 2 diabetes compared with normal glucose tolerance. PC2 activity showed no difference among the three groups. Decreased estimated PC1/3 activity was significantly associated with type 2 diabetes after adjustment for sex, age, creatinine, triglycerides, HOMA-ß and DI(O). Odds ratios (95% CI) of PC1/3, HOMA-ß, and DI(O) were 2.16 (1.12-4.19), 3.44 (1.82-6.52) and 14.60 (7.87-27.11), respectively. Furthermore, decreased PC1/3(≤1.7) combined with decreased HOMA-ß (≤30) had a sensitivity of 73% and specificity of 62%. Decreased PC1/3 activity may be a useful measurement of beta-cell function alongside decreased HOMA-ß or DI(O). A combined decrease in estimated fasting PC1/3 activity and HOMA-ß measurement led to suspicion of type 2 diabetes in the non-obese Japanese population studied.

Heparosan-glucuronate 5-epimerase: Molecular cloning and characterization of a novel enzyme.

Iduronic acid (IdoA) is a critical component of heparan sulfate in its interaction with functional proteins. Heparosan-N-sulfate-glucuronate 5-epimerase (HNSG-5epi) converts d-glucuronic acid (GlcA) residues in N-sulfated heparosan (NS-heparosan), as an intermediate in heparan sulfate biosynthesis, to IdoA. In the present study, the authors discovered a different 5-epimerase, designated HG-5epi (heparosan-glucuronate 5-epimerase), that is involved in acharan sulfate biosynthesis and possesses novel substrate specificity. A candidate cDNA of HG-5epi was cloned from the cDNA library of Achatina fulica. The cloned cDNA contained a whole coding region that predicts a type II transmembrane protein composed of 601 amino acid residues. The amino acid sequence of HG-5epi is homologous to that of HNSG-5epi. Recombinant HG-5epi was expressed in insect cells and its enzymatic properties characterized. As expected, HG-5epi epimerizes GlcA residues in heparosan, but not in NS-heparosan. Conversion of IdoA to GlcA was also catalyzed by HG-5epi when completely desulfated N-acetylated heparin was used as the substrate, indicating a reversible reaction mechanism. At equilibrium of the epimerization, the proportion of IdoA in the reaction product reached up to 30% of total hexuronic acid. To our knowledge, this is the first report to describe an enzyme that catalyzes the epimerization of non-sulfated heparosan. This new enzyme may be applied to the study of synthetic heparan sulfate-related polysaccharides having certain biological and pharmacological activities. In addition, a new method using anion-exchange HPLC connected to a post-column fluorescent labeling system was developed for analyzing hexuronic acid isomers.

Estimated proinsulin processing activity of prohormone convertase (PC) 1/3 rather than PC2 is decreased in pancreatic ß-cells of type 2 diabetic patients.

Type 2 diabetic (T2D) patients exhibit fasting relative hyperproinsulinemia owing to pancreatic ß-cell dysfunction. To clarify the mechanism underlying this hyperproinsulinemic state, we evaluated the activities of the endopeptidases prohormone convertase (PC) 1/3 and PC2 in T2D patients. Fasting blood levels of intact proinsulin (IPI), total proinsulin (t-PI) and C-peptide were measured simultaneously, and intravenous glucagon loading was performed to investigate the dynamics of circulating proinsulin-related molecules released from pancreatic ß-cells in 12 healthy volunteers and 18 T2D patients. Taking advantage of the 95% cross-reactivity between proinsulin and des-31,32-proinsulin (des-31,32-PI) with the human proinsulin radioimmunoassay kit used in this study, we estimated PC1/3 and PC2 activities using the following formulas: des-31,32-PI = (t-PI-IPI)/0.95; PC1/3 activity = des-31,32-PI/IPI; and PC2 activity = C-peptide/des-31,32-PI. C-peptide responses to glucagon were slightly lower among T2D patients. IPI and the IPI/C-peptide ratio were significantly higher in T2D patients (p<0.05 and p<0.01, respectively). There was no difference in des-31,32-PI levels or PC2 activity between the two groups. However, PC1/3 activity was significantly lower in T2D patients than in the control group (p<0.01). We propose that decreased activity of PC1/3 rather than PC2 in pancreatic ß-cells is involved in the impaired proinsulin processing, resulting in elevated IPI levels in T2D patients.

Effectiveness of biofield therapy for individuals with sickle cell disease in Africa.

CONTEXT: Individuals with sickle cell disease (SCD) in Africa have to manage most of their painful episodes at home, mainly due to insufficient medical services. In the past, the research team had observed that some individuals with SCD had experienced symptomatic improvement after administration of biofield therapy. OBJECTIVE: The research team intended to evaluate the influence of repeated administration of biofield therapy on clinical outcomes for individuals with SCD in Africa. DESIGN: The research team designed a prospective, open label, nonrandomized, case-control study. SETTING: This study was performed at Le Centre de Médecine Mixte d'Anémie SS (CMMASS) in Kinshasa, Democratic Republic of Congo (DRC). PARTICIPANTS: Participants were individuals with SCD who received treatments at CMMASS. INTERVENTION: Twenty participants aged 3-36 y underwent Okada purifying therapy (OPT), a form of biofield therapy, every weekday for 1 y (OPT group). OPT was administered by certified practitioners approved by the MOA International Corporation. The research team also constructed an age- and gender-matched control group (n = 20) from regular patients at the clinic. OUTCOME MEASURES: Participants in both groups received blood tests at baseline, after 6 mo, and at end of the 1-y intervention period. A follow-up survey was also conducted 33 mo after the 1-y administration of OPT. RESULTS: The OPT group showed a significant improvement in its blood data, including hemoglobin, total bilirubin, alanine aminotransferase (ALT), creatinine, and white blood cell (WBC) count (P < .001). The control group had less improvement in hemoglobin (P < .001) and total bilirubin (P < .001) than the OPT group. Its creatinine level increased significantly (P < .001), and ALT and WBC count remained at the same level after 1 y. For the OPT group, SCD-related episodes, such as painful crises, blood transfusions, and hospitalizations, decreased more significantly than for the control group (P < .001). The follow-up survey revealed that those who continued using OPT regularly had remained in better condition for approximately 3 y after the intervention period than those who did not (P = .0014). CONCLUSION: Repeated administration of biofield therapy is considered effective for individuals with SCD who have various symptoms, although the care given by the practitioners may have had some influence on the clinical outcomes.

A large cross-sectional, descriptive study of self-reports after biofield therapy in Japan: demography, symptomology, and circumstances of treatment administration.

BACKGROUND: Practitioners apply biofield therapy in diverse situations; however, most previously published reports investigated its efficacy in a clinical setting with a few expert practitioners administering it. OBJECTIVE: The study intended to determine the factors associated with self-reported symptom change after a single session of Okada Purifying Therapy (OPT), a form of biofield therapy, in various settings and to analyze its optimal therapeutic environments. DESIGN: This cross-sectional study used simple questionnaires. The research team trained 100 instructors of OPT to act as investigators for the study. The team recorded the initial lecture to those instructors on video, and the instructors used it to train other certified OPT practitioners in 222 locations. All investigators were volunteers. SETTINGS: The OPT sessions took place in various environments, including at MOA International Corporation's affiliated institutes, with or without clinics; at investigators' homes; and at participants' homes. PARTICIPANTS: The study involved 44 587 participants in total; all were Japanese, aged 16 or older, and suffering from physical pain, palpitation/dizziness, or anxiety/depression. INTERVENTION: The intervention involved one session of OPT lasting 30 minutes or longer administered by the volunteer practitioners. OUTCOME MEASURES: The research team evaluated the severity of symptoms before and after the intervention. The team examined (1) the self-reported change in each category of symptoms as measured after a session and (2) the adjusted odds ratio of the variables related to the outcomes. RESULTS: Of the analyzed participants, 69.7%, 67.5%, and 71.2% reported an improvement in the severity of physical pain, palpitation/dizziness, and anxiety/depression, respectively. Those rates, however, varied based on the participant's gender, the duration or location of the intervention, and the reason for participation. The improvement rate was consistently highest among female participants whose reason for participation involved a hope that OPT would relieve their symptoms or promote their health and who had received the therapy for more than 30 minutes in settings other than their own homes. This finding occurred regardless of the recipient's knowledge of the therapy or past experience of the intervention. The participant's gender, the duration of the intervention, and its location were also significant factors for marked improvements (a decrease in symptoms of 2 or 3 levels) in self-reported symptoms. In evaluating safety, the research team found that the exacerbation rates of reported symptoms were 2.5%, 1.1%, and 0.7% for physical pain, palpitation/dizziness, and anxiety/depression, respectively. None of those who expressed exacerbation of symptoms needed emergency medical treatment. No factor was associated consistently with the exacerbation. CONCLUSION: After a single session of biofield therapy, the participants' responses varied according to their gender, the duration and location of the intervention, and the reasons for participation.