Limiting shapes of confined lipid vesicles.

We theoretically study the shapes of lipid vesicles confined to a spherical cavity, elaborating a framework based on the so-called limiting shapes constructed from geometrically simple structural elements such as double-membrane walls and edges. Partly inspired by numerical results, the proposed non-compartmentalized and compartmentalized limiting shapes are arranged in the bilayer-couple phase diagram which is then compared to its free-vesicle counterpart. We also compute the area-difference-elasticity phase diagram of the limiting shapes and we use it to interpret shape transitions experimentally observed in vesicles confined within another vesicle. The limiting-shape framework may be generalized to theoretically investigate the structure of certain cell organelles such as the mitochondrion.

Medial and lateral laxity in knees with advanced medial osteoarthritis.

OBJECTIVE: An increase in coronal laxity is recognized as a risk factor for progression of knee osteoarthritis (OA). The purpose of this study was to evaluate coronal laxity, which was defined as the angular motion from the neutral, unloaded (baseline) position to the loaded position, in patients with advanced medial knee OA. METHOD: Preoperative coronal laxity was assessed using radiographs in patients with medial knee OA undergoing total knee arthroplasty by applying a force of 150 N with an arthrometer. A consecutive series of 211 knees with OA and 40 normal control knees were examined. A knee with OA was defined as clinically "balanced" when the difference between medial and lateral laxity was 3° or less. Values are expressed as median [25th, 75th percentile]. RESULTS: The laxity was 4° [3, 5] from the baseline on the medial side and 3° [2, 4] on the lateral side. The distribution of medial and lateral laxity indicated that 90% (189/211) of patients fell within 3°. The equivalence test showed that the medial and lateral laxity was similar, with an equivalence margin of 3° (P < 0.001). In the control knees, the laxity was 3° [2, 4] from the baseline on the medial side and 2° [2, 4] on the lateral side. The differences between the knees with advanced OA and the controls were significant (P = 0.005, medial; P = 0.006, lateral). CONCLUSION: This study showed that a clinically balanced knee was maintained even in patients with advanced medial knee OA.

Use of smartphone attached mobile thermography assessing subclinical inflammation: a pilot study.

OBJECTIVE: To verify the reliability and validity of FLIR ONE, a device connected to a smartphone, for the assessment of inflammation based on relative temperature increase compared with the thermography routinely used in pressure ulcer (PU) and diabetic foot assessment. METHOD: Participants in this pilot cross-sectional observational study were recruited from the patients in the PU team rounds and the diabetic foot outpatient clinic at the university hospital in January 2015. Cohen's kappa coefficient with its 95% confidence intervals was used to evaluate the criterion-related validity and inter- and intra-rater reliability for the thermal imaging assessment. For assessing criterion-related validity, a hand-held high-end infrared thermography device was used to provide reference data. Comparison of thermal images between the smartphone-connected device and the hand-held device was performed with both a 'predetermined range' and an 'automatically-set range.' For assessing inter-rater reliability, two assessors evaluated the thermal images taken by the mobile thermography. For assessing intra-rater reliability, one assessor evaluated the thermal images twice. The thermal images were shown to the assessors at random. RESULTS: Among 16 thermal images obtained from eight patients, kappa coefficients for each value were as follows: for the predetermined range and automatically-set range, respectively, the criterion-related validity was 1.00 (95% confidence interval 1.00-1.00) and 1.00 (95% confidence interval 1.00-1.00); the inter-rater reliability was 1.00 (95% confidence interval 1.00-1.00) and 1.00 (95% confidence interval 1.00-1.00); and the intra-rater reliability was 1.00 (95% confidence interval 1.00-1.00) and 1.00 (95% confidence interval 1.00-1.00). CONCLUSION: This pilot study suggests that FLIR ONE can work as an alternative device for assessing subclinical inflammation in PUs and the diabetic foot in clinical settings. Our results may facilitate clinicians in accepting the routine use of thermal imaging assessment at the patients' bedside.

Phosphate binders prevent phosphate-induced cellular senescence of vascular smooth muscle cells and vascular calcification in a modified, adenine-based uremic rat model.

Clinical and experimental studies have reported that phosphate overload plays a central role in the pathogenesis of vascular calcification in chronic kidney disease. However, it remains undetermined whether phosphate induces cellular senescence during vascular calcification. We established a modified uremic rat model induced by a diet containing 0.3% adenine that showed more slowly progressive kidney failure, more robust vascular calcification, and longer survival than the conventional model (0.75% adenine). To determine the effect of phosphate on senescence of vascular smooth muscle cells (VSMCs) and the protective effect of phosphate binders, rats were divided into four groups: (1) normal control rats; (2) rats fed with the modified adenine-based diet (CKD); (3) CKD rats treated with 6% lanthanum carbonate (CKD-LaC); and (4) CKD rats treated with 6% calcium carbonate (CKD-CaC). After 8 weeks, CKD rats showed circumferential arterial medial calcification, which was inhibited in CKD-LaC and CKD-CaC rats. CKD rats showed increased protein expression of senescence-associated ß-galactosidase, bone-related proteins, p16 and p21, and increased oxidative stress levels in the calcified area, which were inhibited by both phosphate binders. However, serum levels of oxidative stress and inflammatory markers, serum fibroblast growth factor 23, and aortic calcium content in CKD-CaC rats were higher than those in CKD-LaC rats. In conclusion, phosphate induces cellular senescence of VSMCs in the modified uremic rat model, and phosphate binders can prevent both cellular senescence and calcification of VSMCs via phosphate unloading. Our modified adenine-based uremic rat model is useful for evaluating uremia-related complications, including vascular calcification.

Polypeptide N-acetylgalactosaminyl transferase 3 independently predicts high-grade tumours and poor prognosis in patients with renal cell carcinomas.

BACKGROUND: The polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) family of enzymes regulates the initial steps of mucin-type O-glycosylation. N-acetylgalactosaminyltransferases might show novel patterns of GalNAc-T glycosylation on tumour-derived proteins, which could influence cancer biology, but its mechanisms are unclear. We investigated the association of GalNAc-T3 and -T6 expressions with clinicopathological features and prognoses of patients with renal cell carcinomas (RCCs). METHODS: Expressions of GalNAc-T3/6 and cell-adhesion molecules were analysed immunohistochemically in 254 paraffin-embedded tumour samples of patients with RCC. RESULTS: Of 138 GalNAc-T3+ cases, 46 revealed significant co-expression with GalNAc-T6. N-acetylgalactosaminyltransferases-3+ expression showed a close relationship to poor clinical performance and large tumour size, or pathologically high Fuhrman's grading, and presence of vascular invasion and necrosis. The GalNAc-T3-positivity potentially suppressed adhesive effects with a significantly low ß-catenin expression. Univariate and multivariate analyses showed the GalNAc-T3+ group, but not the GalNAc-T6+ group, to have significantly worse survival rates. CONCLUSION: N-acetylgalactosaminyltransferases-3 expression independently predicts high-grade tumour and poor prognosis in patients with RCC, and may offer a therapeutic target against RCC.

Effect of methionine on production of naphthoquinones in Impatiens balsamina root cultures and detection of some secondary metabolites.

CONTEXT: Lawsone, lawsone methyl ether and 3,3'-methylelnebislawsone are the main active compounds of Impatiens balsamina L. (Balsaminaceae). These compounds possess various pharmacological activities that have been shown to assist with the treatment of skin diseases. OBJECTIVE: This work focused on increased naphthoquinone production in I. basamina root cultures using methionine feeding. MATERIALS AND METHODS: I. balsamina root cultures were maintained in liquid Gamborg's B5 medium supplemented with 0.1 mg/L α-naphthalene acetic acid, 0.1 mg/L kinetin, 1.0 mg/L 6-benzyladenine and 20 g/L sucrose. The effect of methionine concentration (50, 100, 300, 500 and 1000 mg/L) on naphthoquinone production of I. basamina root cultures was determined. Isolation of secondary metabolites from I. balsamina root cultures was also carried out. RESULTS AND DISCUSSION: Feeding of 300 mg/L methionine to the root cultures at the beginning of the growth cycle increased the production of 3,3'-methylelnebislawsone almost two-fold (0.63 mg/g dry weight, compared to the control group 0.32 mg/g dry weight). Optimization of the feeding conditions showed that adding 500 mg/L methionine to a 21-day old root cultures increased production of lawsone methyl ether and 3,3'-methylenebislawsone up to 2.6- and 3.1-fold higher, respectively, compared to the controls. In addition, various pharmacologically interesting secondary metabolites were isolated from I. balsamina root cultures, such as a flavonoid, luteolin, a naphthoquinone, 2,3-dihydroxy-1,4-naphthoquinone, and a triterpenoid, echinocystic acid. This is the first report of the occurrence of these compounds in this plant.

Lactobacillus pentosus strain b240 suppresses pneumonia induced by Streptococcus pneumoniae in mice.

AIMS: Oral administration of probiotics has been known to improve inflammatory responses against infectious diseases. Here, we describe the inhibitory effect of oral intake of heat-killed Lactobacillus pentosus strain b240 (b240) on pneumococcal pneumonia in a murine experimental model. METHOD AND RESULTS: The mice treated with oral b240 for 21 days before Streptococcus pneumoniae infection exhibited prolonged survival time and less body weight loss, compared with saline-treated control mice. Mild pneumonia with significantly reduced secretion of inflammatory cytokines/chemokines according to related mitogen-activated protein kinase signalling molecules (phosphorylated c-Jun N-terminal kinase) was found in b240-treated mice, whereas severe pneumonia with hypercytokinemia was evident in control mice. Prominent reduction in the number of pneumococci and elevated expression of Toll-like receptor 2 and 4 in the lung tissues was concomitantly noted in b240-treated mice. CONCLUSIONS: These findings indicate that b240 has inhibitory effects on pneumococcal pneumonia induced by Strep. pneumoniae infection and improves inflammatory tissue responses, resulting in reduced damages to the respiratory tissues. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate that oral administration of b240 might protect host animals from Strep. pneumoniae infection by augmentation of innate immune response.

In vivo non-viral gene delivery of human vascular endothelial growth factor improves revascularisation and restoration of euglycaemia after human islet transplantation into mouse liver.

AIMS/HYPOTHESIS: Delivery of the gene for human vascular endothelial growth factor (VEGF, also known as VEGFA) to both the transplanted islets and the surrounding tissue may promote islet revascularisation and survival. We previously showed the effective delivery of VEGF gene to rat myocardium by an ultrasound-mediated gene-transfer method named ultrasound-targeted microbubble destruction (UTMD). Here we examined the effect of non-viral VEGF delivery using UTMD on transplanted islets in vivo. METHODS: A marginal number of human islets were transplanted into livers of mice which were a model for diabetes. Then, non-viral plasmid vectors encoding VEGF (VEGF group, n = 11) or the gene for green fluorescent protein (GFP) (GFP group, n = 7) were introduced into the host liver by UTMD. Transplantation without gene delivery was performed as a control (no-UTMD group, n = 8). Blood glucose, serum human insulin, C-peptide levels and the revascularisation in graft islets were evaluated. RESULTS: Restoration of euglycaemia occurred in 13% in the no-UTMD group and 14% in the GFP group, whereas 73% mice in the VEGF group became euglycaemic at day 30 (p < 0.05 in no-UTMD vs VEGF). Serum human insulin and C-peptide were significantly higher in the VEGF group at day 32 (insulin: no-UTMD, 17 +/- 8; GFP, 37 +/- 17; VEGF, 109 +/- 26 pmol/l, respectively, p < 0.05; C-peptide: no-UTMD, 68 +/- 38; GFP, 115 +/- 58; VEGF, 791 +/- 230 pmol/l, respectively, p < 0.05). Vessel density in graft islets was significantly higher in the VEGF group (no-UTMD, 169 +/- 36; GFP, 227 +/- 39; VEGF, 649 +/- 51 counts/mm(2), respectively, p < 0.05). CONCLUSIONS/INTERPRETATION: Delivery of VEGF gene to host liver using UTMD promoted islet revascularisation after islet transplantation and improved the restoration of euglycaemia.

Regeneration of pancreatic islets in vivo by ultrasound-targeted gene therapy.

This study uses a novel approach to gene therapy in which plasmid DNA is targeted to the pancreas in vivo using ultrasound-targeted microbubble destruction (UTMD) to achieve islet regeneration. Intravenous microbubbles carrying plasmids are destroyed within the pancreatic microcirculation by ultrasound, achieving local gene expression that is further targeted to ß-cells by a modified rat insulin promoter (RIP3.1). A series of genes implicated in endocrine development were delivered to rats 2 days after streptozotocin-induced diabetes. The genes, PAX4, Nkx2.2, Nkx6.1, Ngn3 and Mafa, produced α-cell hyperplasia, but no significant improvement in ß-cell mass or blood glucose level 30 days after UTMD. In contrast, RIP3.1-NeuroD1 promoted islet regeneration from surviving ß-cells, with normalization of glucose, insulin and C-peptide levels at 30 days. In a longer-term experiment, four of six rats had a return of diabetes at 90 days, accompanied by ß-cell apoptosis on Tunel staining. Pretreatment with the JNK inhibitor SP600125 successfully blocked ß-cell apoptosis and resulted in restoration of ß-cell mass and normalization of blood glucose level for up to 90 days. This technique allows in vivo islet regeneration, restoration of ß-cell mass and normalization of blood sugar, insulin and C-peptide in rats without viruses.

DNA sequence and comparative analysis of chimpanzee chromosome 22.

Human-chimpanzee comparative genome research is essential for narrowing down genetic changes involved in the acquisition of unique human features, such as highly developed cognitive functions, bipedalism or the use of complex language. Here, we report the high-quality DNA sequence of 33.3 megabases of chimpanzee chromosome 22. By comparing the whole sequence with the human counterpart, chromosome 21, we found that 1.44% of the chromosome consists of single-base substitutions in addition to nearly 68,000 insertions or deletions. These differences are sufficient to generate changes in most of the proteins. Indeed, 83% of the 231 coding sequences, including functionally important genes, show differences at the amino acid sequence level. Furthermore, we demonstrate different expansion of particular subfamilies of retrotransposons between the lineages, suggesting different impacts of retrotranspositions on human and chimpanzee evolution. The genomic changes after speciation and their biological consequences seem more complex than originally hypothesized.

Effect of phytic acid on postprandial serum uric acid level in healthy volunteers: a randomized, double-blind, crossover study.

Phytic acid, a constituent of various plants, has been related to health benefits. Phytic acid has been shown to inhibit purine nucleotide metabolism in vitro and suppress elevation of plasma uric acid levels after purine administration in animal models. This study investigated the effect of phytic acid on postprandial serum uric acid (SUA) in humans. This randomized, double-blind, crossover design study included 48 healthy subjects with normal fasting SUA. Subjects consumed a control drink and a phytic acid drink with purine-rich food, and serum and urine uric acid levels were measured for 360 min after purine loading. Phytic acid lowered the incremental area under the curve (0-360 min) and incremental maximum concentration of SUA after purine loading (p < 0.05); tended to lower cumulative urinary uric acid excretion (0-360 min) after purine loading (p < 0.10); and suppressed postprandial SUA in this clinical study. Altogether, our findings suggest that phytic acid may play a beneficial role in controlling postprandial SUA.

Actin: volume change on transformation of G-form to F-form.

The volume change occurring on polymerizing actin was measured by dilatometry. A large positive value of + 391 ml/mole was obtained for the volume change during the transformation of G- to F-actin. This large increase in volume could be interpreted as arising from the local change in the ordered water structure on the protein's surface at polymerizing sites.

Prevention of acute liver failure in rats with reversibly immortalized human hepatocytes.

Because of a critical shortage in suitable organs, many patients with terminal liver disease die each year before liver transplantation can be performed. Transplantation of isolated hepatocytes has been proposed for the temporary metabolic support of patients awaiting liver transplantation or spontaneous reversion of their liver disease. A major limitation of this form of therapy is the present inability to isolate an adequate number of transplantable hepatocytes. A highly differentiated cell line, NKNT-3, was generated by retroviral transfer in normal primary adult human hepatocytes of an immortalizing gene that can be subsequently and completely excised by Cre/Lox site-specific recombination. When transplanted into the spleen of rats under transient immunosuppression, reversibly immortalized NKNT-3 cells provided life-saving metabolic support during acute liver failure induced by 90% hepatectomy.

Spontaneous acute subdural haematoma and intracerebral haemorrhage in patient with HELLP syndrome: case report.

HELLP (haemolysis, elevated liver enzymes, and low platelet count) syndrome can result in a fatal intracranial haemorrhage during the perinatal period. We report treatment of a 32-year-old primigravida who fell into a deep coma during labour with fetal distress, complicated by a spontaneous acute subdural haematoma and intracerebral haemorrhage. Simultaneous emergency operations, evacuation of the acute subdural haematoma and a caesarean section, were performed, during which a diagnosis of HELLP syndrome with disseminated intravascular coagulation was made. Both mother and infant recovered, though hemiparesis persisted in the mother. Patients with HELLP syndrome should be managed as high-risk, which requires an excellent working relationship of the physicians involved. Prompt recognition of intracranial haemorrhagic complications and neurosurgical intervention are particularly important.

Evaluation of engraftment after single islet transplantation from a brain-dead donor by the secretory unit of islet transplant objects (SUITO) index.

BACKGROUND: Evaluation of engraftment is important to assess the success of islet transplantation. Recently, we developed a simple index of islet engraftment, the secretory unit of islet transplant objects (SUITO) index. The formula is: 1500 x fasting C-peptide level [ng/dL]/(fasting blood glucose levels [mg/dL]-63). A SUITO index of more than 26 was associated with insulin independence. MATERIALS AND METHODS: In this study, we compared islet engraftment efficacy using the SUITO index after a single infusion of islets from brain-dead donors into 6 recipients. We calculated the SUITO index from postoperative days 3 to 30. We compared the insulin reduction rate with the SUITO index and islet equivalent per kilogram body weight (IE/Kg). We also measured the level of glycosylated hemoglobin (HbA 1C) at 3 months posttransplantation to assess glycemic control after islet transplantation. RESULTS: In 5 cases, islets were cultured before transplantation and in 1 case they were transplanted without culture. Without culture, the SUITO index and insulin reduction rate were highest. The SUITO index significantly correlated with the insulin reduction rate (P = .031, R2 = .728), but the IE/kg was not significantly correlated (P = .303) with the rate of insulin reduction. All cases showed improved HbA 1C to the normal range. CONCLUSIONS: Immediate transplantation without culture substantially improved the efficacy of engraftment of transplanted islets. The SUITO index was a better predictor than islet mass per body weight for clinical outcomes.

Estimation of donor usability for islet isolation with the modified Ricordi method.

BACKGROUND: The quality of donor pancreata is important for successful islet isolation. However, in some countries like Japan, the number of donor pancreata is low. Therefore, marginal donor pancreata have been used with less restrictive donor criteria. In order to use marginal donor pancreata, we established the modified Ricordi method. According to the United Network for Organ Sharing (UNOS) in 2005, more than 6000 pancreata were not clinically usable in the United States. In this study, we reevaluated donor usability based on the Japanese islet donor criteria. MATERIALS AND METHODS: We reviewed donor charts with well-documented cases in Texas from 2005 to 2006. We counted the number of pancreata for pancreas transplantation or islet transplantation. If not used clinically, the reason was also reviewed. Donors were reevaluated based on the Japanese islet donor criteria. RESULTS: We reviewed 236 donor charts, including 29 pancreata used for whole pancreas transplantations and 13 for islet isolation; therefore, 194 pancreata were not used. Among the 194 cases, we were able to identify the reasons that the pancreata were not used in 186 cases. When we applied the Japanese acceptance criteria, an additional 82 of 186 cases (44%) seemed suitable for islet isolations. CONCLUSIONS: With the modified Ricordi method, more than 2500 donor pancreata might be used for islet isolation in the United States when the Japanese criteria are applied.

Method for isolation of mouse pancreatic stem cells.

Replacement of beta-cell mass offers an alternative to standard insulin treatment for diabetes and may overcome the long-term side effects associated with current therapies. Pancreatic stem/progenitor cells could become a useful target for beta-cell replacement therapy in diabetic patients. We have established a method for isolating mouse pancreatic stem cells. In this study, pancreatic stem cells were isolated from 8-week-old mice. After purification on a density gradient, the density range of 1.062-1.11 contained pancreatic stem cells. The islets from the layers were deleted by dithizone staining and hand-picking under a dissecting microscope. The remnant cells were then cultured, inoculated into 96-well plates, and cloned by limiting dilution. One of the wells contained cells, named HN#5 cells, which expressed ductal cell markers, such as cytokeratin-19. HN#5 cells differentiated into insulin-producing cells and albumin-producing cells by induction medium. The isolation technique described here may be useful for identification and isolation of human pancreatic stem/progenitor cells.

Gene expression profiling of human pancreatic islets undergoing a simulated process of instant blood-mediated inflammatory reaction.

OBJECTIVE: Islet cell transplantation, as a treatment for type 1 diabetes mellitus, has historically required islet infusions from more than one donor organ to achieve insulin independence. Significant islet mass may be destroyed upon infusion due to a yet undefined process known as instant blood-mediated inflammatory reaction (IBMIR). Our objective was to identify gene expression changes in islets undergoing a simulated process of IBMIR. MATERIALS AND METHODS: Human pancreatic islets were isolated from 2 cadaveric donors and divided into 3 groups each for a total of 18 samples. Group one (n = 3) was treated with autologous sera, group two (n = 3) with allogeneic sera, and group three (n = 3) with type 1 diabetic sera (T1DM). Each group was treated for 3 hours at 37 degrees C. Islets were washed, lysed using TRI reagent, and mRNA was isolated using the Total Prep mRNA isolation kit. Isolated cRNA was used for microarray analysis using Illumina Gene Chips Hu6_v2. GeneSpring GX software was used for statistical analysis. Results were significant at P < .05. RESULTS: One-way ANOVA statistical analysis of the microarray data revealed that interleukin-11 (IL-11), interleukin-12A (IL-12A), and Ras related associated with diabetes (RRAD) were overexpressed in islets exposed to diabetic sera when normalized to autologous control (P < .01). Under the same conditions, islet cells exposed to T1DM serum had down-regulation of IL-1 receptor antagonist (IL-1RN). CONCLUSION: These findings suggested that T1DM serum elicited an adaptive and innate immune response to the transplanted islet mass making them more susceptible to cytokine-mediated destruction.

Effect of JNK inhibitor during islet isolation and transplantation.

Although islet transplantation has been remarkably improved by the Edmonton protocol, the insulin independence rate after islet transplantation from one donor pancreas has remained low. The c-Jun NH2-terminal kinases (JNKs) are classic stress-activated protein kinases; many cellular stresses have been shown to stimulate JNK activation. JNK in the pancreas is activated during brain death, pancreas procurement, and organ preservation, and its activity is progressively increased during the isolation procedure. Moreover, JNK activity in the transplanted liver after islet transplantation increases markedly within 24 hours. In this study, we show the effect of a JNK inhibitor during islet isolation and transplantation. Use of the JNK inhibitor in pancreas preservation, islet culture, and/or islet transplantation prevents islet cell apoptosis and improves islet graft function. These findings suggest that inhibition of JNK could prevent the impairment of islet cells and improve outcomes after pancreatic islet transplantation.

Continuous, but not occasional, oral ethanol intake reduces the success of intraportal transplanted islets of Langerhans: an experimental study.

BACKGROUND: Islet transplantation is gradually gaining acceptance for the treatment of type 1 diabetes mellitus. One of the unknown questions is alcohol intake; we have prohibited alcohol intake after islet transplantation although there is no solid evidence to support this. MATERIALS AND METHODS: In this study, we employed a mouse model to determine the effect of oral ethanol intake on transplanted islets. Either 500 or 150 islets were infused selectively into the right liver lobe of chemically induced diabetic mice. After transplantation, mice were orally administered either water (as a control) or various concentrations of ethanol for 14 consecutive days occasionally (once per day) or continuously (all intake was alcohol). Blood glucose levels were monitored and oral glucose tolerance tests (OGTT) performed. RESULTS: After 500 islets had been transplanted, all mice were cured from diabetes, but the continuous alcohol intake group showed significantly prolonged time to diabetes reversal and significantly lower glucose clearance rates by OGTT compared with the control group. After 150 islet transplantations, the diabetes cure rate in the continuous alcohol intake group was significantly lower than the control group (continuous alcohol vs control: 3/8 vs 11/12, P < .05). However, the occasional alcohol intake group showed no difference from the control group, even with as few as 150 islets transplanted per mouse. CONCLUSION: The present results demonstrated that continuous but not occasional alcohol intake reduced the success of intraportal islet transplantation.