Weight-dependent Manner Research Articles

Pentosan polysulfate sodium promotes redifferentiation to the original phenotype in micromass-cultured canine articular chondrocytes and exerts molecular weight-dependent effects.

Pentosan polysulfate sodium (PPS) is a heparin-like polysaccharide that is applied as a therapeutic treatment for osteoarthritis (OA) in animals. This study investigated the efficacy of different molecular weights PPS (1,500-7,000 Da) on the phenotype regulatory and chondrogenic properties of canine articular chondrocytes. The cytotoxicity of PPS on chondrocytes was assessed using flow cytometry and 3-(4,5-dimehylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. After 72 hr of exposure, PPS did not induce chondrocyte apoptosis, regardless of molecular weight. In addition, chondrogenic properties were determined according to the mRNA and protein levels in micromass-cultured chondrocytes. Quantitative polymerase chain reaction analysis confirmed that PPS promotes a chondrogenic phenotype in chondrocytes in a molecular weight-dependent manner, with significant upregulation of collagen type II alpha 1 chain, aggrecan, and SRY-box transcription factor 9 (SOX9) mRNA levels relative to those in the control. However, the collagen type I alpha 2 chain mRNA level simultaneously increased after 7,000 Da PPS treatment. PPS exposure also increased collagen type II and SOX9 protein production in a molecular weight-dependent manner and inhibited Akt phosphorylation in chondrocytes. Alcian blue staining indicated that PPS treatment enhanced proteoglycan deposition in micromass cultures, with stronger effects observed in 5,000 and 7,000 Da groups. Overall, these results indicate that PPS exerts protective effects on the chondrocyte phenotype and may represent a potential therapeutic target for OA treatment. Increasing the molecular weight of PPS could enhance these anabolic effects.

In Vivo Anticoagulant and Antithrombic Activity of Depolymerized Glycosaminoglycan from Apostichopus japonicus and Dynamic Effect-Exposure Relationship in Rat Plasma.

Glycosaminoglycan from Apostichopus japonicus (AHG) and its depolymerized fragments (DAHGs) are anticoagulant fucosylated chondroitin sulfate. The aim of this study was to further evaluate the anticoagulant and antithrombic activity of AHG and DAHGs, as well as reveal the dynamic relationship between exposure and effect in vivo. The results demonstrated that AHG100 (Mw~100 kDa), DAHG50 (Mw~50 kDa), and DAHG10 (Mw~10 kDa) exhibited potent anticoagulant activity by inhibiting intrinsic factor Xase complex (FXase) as well as antithrombin-dependent factor IIa (FIIa) and factor Xa (FXa). These glycosaminoglycans markedly prevented thrombosis formation and thrombin-induced platelet aggregation in a dose- and molecular weight-dependent manner in vitro and in vivo. The further bleeding time measurement indicated that DAHG10 exhibited obviously lower hemorrhage risks than native AHG100. Following oral administration, DAHG10 could be absorbed into blood, further dose-dependently prolonging activated partial thromboplastin time (APTT) and thrombin time (TT) as well as inhibiting FXa and FIIa partially through FXase. Anticoagulant activity was positively associated with plasma concentration following oral administration of DAHG10. Our study proposed a new point of view to understand the correlation between effects and exposure of fucosylated chondroitin sulfate as an effective and safe oral antithrombotic agent.

Design of biotin decorated enterocyte targeting muco-inert nanocomplexes for enhanced oral insulin delivery

The natural mucus cover has been a major obstacle to prevent enterocyte targeting particles from contact with the receptors. Thus, mucus penetration and intestinal targeting should be designed into one system. Based on the concept that biotin specifically recognizes epithelium receptors, enterocyte targeting muco-inert nanocomplexes were designed. Firstly, biotinylated chitosan (CS-Biotin) copolymers with different degree of substitution were synthesized and characterized. The nanocomplexes between CS-Biotin and insulin were prepared via self-assembly method. Thereafter, the nanocomplexes were fabricated by coating with various molecular weight hyaluronic acid (HA), which improved penetration efficiency in the mucus layer and small intestine in a HA molecular weight dependent manner. In vivo study indicated that hypoglycemic effect of the nanocomplexes was biotin modification degree and HA molecular weight dependent, with HA (200)-coated CS-Biotin21.8%/Insulin polyelectrolyte complex presenting the best performance. In conclusion, biotin decorated muco-inert nanocomplexes with HA coating are a promising platform for oral insulin delivery.

Water strider females use individual experience to adjust jumping behaviour to their weight within physical constraints of water surface tension

Different species of water striders match leg speeds to their body sizes to maximize their jump take off velocity without breaking the water surface, which might have aided evolution of leg structures optimized for exploitation of the water surface tension. It is not understood how water striders achieve this match. Can individuals modify their leg movements based on their body mass and locomotor experience? Here we tested if water striders, Gerris latiabdominis, adjust jumping behaviour based on their personal experience and how an experimentally added body weight affects this process. Females, but not males, modified their jumping behaviour in weight-dependent manner, but only when they experienced frequent jumping. They did so within the environmental constraint set by the physics of water surface tension. Females’ ability to adjust jumping may represent their adaptation to frequent increases or decreases of the weight that they support as mating bouts, during which males ride on top of females, start or end, respectively. This suggests that natural selection for optimized biomechanics combined with sexual selection for mating adaptations shapes this ability to optimally exploit water surface tension, which might have aided adaptive radiation of Gerromorpha into a diversity of semiaquatic niches.

Abstract 4002: Adipose tissue supernatant of overweight ER positive breast cancer patients promotes migration of MCF-7 breast cancer cells

Abstract Introduction: Adipose tissue is a major component of the breast cancer microenvironment, and preclinical studies have suggested that adipose tissue might play a pivotal role in breast cancer progression. In this study we investigated the effect of adipose tissue from patients with ER positive breast cancer on migration of ER positive MCF-7 breast cancer cells. Materials and Methods: Adipose tissue samples were collected from patients with ER positive breast cancer undergoing a mastectomy. In total, adipose tissue samples from 3 normal weight (BMI: 18.5-24.9) and 7 overweight breast cancer patients (BMI: 25-29.9) were collected. Samples from the mastectomy specimens were minced in small pieces, incubated in RPMI-1640 and after 48 hours (h) the supernatant was harvested. The effect of adipose tissue supernatant on the migration of MCF-7 cells was monitored in real-time for 48 h with the xCELLigence cell invasion and migration plates expressed in delta cell index (DCI). In addition, effect of adipose tissue supernatant on migration was assessed by quantification of F-actin positive lamellipodia of MCF-7 cells in a scratch assay after 24 h. In all experiments, RPMI-1640 with 1% or 20% FCS was used as a negative or positive control respectively. Results: Supernatant of overweight patients significantly induced MCF-7 migration compared to supernatant of normal weight patients after 24 h (xCELLigence DCI: 1.22 ± 0.15 vs. 0.95 ± 0.19 (P=0.038)), 36 h (DCI 1.40 ± 0.21 vs. 0.91 ± 0.31 (P=0.019)) and 48 h (DCI 1.72 ± 0.23 vs. 0.98 ± 0.43 (P=0.007)). In addition, there was a significant difference between the overweight group and negative control at 36 h, and 48 h (DCI 1.40 ± 0.21 vs. 1.06 ± 0.12, P=0.038 and DCI 1.72 ± 0.23 vs. 1.11 ± 0.21, P=0.004 respectively). No significant difference between normal weight group and negative control was found at any of the time points. After 24 h MCF-7 cells incubated with supernatant of overweight patients developed significantly more F-actin positive lamellipodia compared to MCF-7 cells incubated with supernatant of normal weight patients (44.26 ± 3.26% vs. 25.47 ± 1.88% P<0.001). Conclusion: In this study, we showed that adipose tissue supernatant of patients with ER positive breast cancer can promote migration of MCF-7 breast cancer cells in a weight dependent manner. Whether this is the case in other breast cancer subtypes as well, and the potential clinical implication, is of interest for future studies. Citation Format: Jie Ma, Hetty Timmer-Bosscha, Paul M. Werker, Carolina P. Schröder, Marlous Arjaans. Adipose tissue supernatant of overweight ER positive breast cancer patients promotes migration of MCF-7 breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4002.

Body weight-dependent and independent improvement in lipid metabolism after Roux-en-Y gastric bypass in ApoE*3Leiden.CETP mice.

The incidence of obesity and metabolic syndrome (MetS) has rapidly increased worldwide. Roux-en-Y gastric bypass (RYGB) achieves long-term weight loss and improves MetS-associated comorbidities. Using a mouse model with a humanized lipoprotein metabolism, we elucidated whether improvements in lipid and glucose metabolism after RYGB surgery are body weight loss-dependent or not. Male ApoE*3Leiden.CETP (ApoE3L.CETP) mice fed Western type diet for 6 weeks underwent RYGB or Sham surgery. Sham groups were either fed ad libitum or were body weight-matched (BWm) to the RYGB mice to discriminate surgical effects from body weight loss-associated effects. Before and after surgery, plasma was collected to assess the metabolic profile, and glucose tolerance and insulin sensitivity were tested. Twenty days after surgery, mice were sacrificed, and liver was collected to assess metabolic, histological and global gene expression changes after surgery. RYGB induced a marked reduction in body weight, which was also achieved by severe food restriction in BWm mice, and total fat mass compared to Sham ad libitum mice (Sham AL). Total cholesterol, non-high-density lipoprotein cholesterol (non-HDL-C) and ceramide were strongly reduced 20 days after surgery in RYGB compared to BWm mice. Glucose tolerance and insulin sensitivity improved 13 days after surgery similarly in RYGB and BWm mice. Liver histology confirmed lipid reduction in RYGB and BWm mice while the transcriptomics data indicated altered genes expression in lipid metabolism. RYGB surgery improves glucose metabolism and greatly ameliorates lipid metabolism in part in a body weight-dependent manner. Given that ApoE3L.CETP mice were extensively studied to describe the MetS, and given that RYGB improved ceramide after surgery, our data confirmed the usefulness of ApoE3L.CETP mice after RYGB in deciphering the metabolic improvements to treat the MetS.

Effects of Chitosan Oligosaccharides on Human Blood Components.

Chitosan oligosaccharide (COS) is known for its unique biological activities such as anti-tumor, anti-inflammatory, anti-oxidant, anti-bacterial activity, biological recognition, and immune enhancing effects, and thus continuous attracting many research interests in drug, food, cosmetics, biomaterials and tissue engineering fields. In comparison to its corresponding polymer, COS has much higher absorption profiles at the intestinal level, which results in permitting its quick access to the blood flow and potential contacting with blood components. However, the effects of COS on blood components remain unclear to date. Herein, two COS with different molecular weight (MW) were characterized by FTIR and 1H NMR, and then their effects on human blood components, including red blood cells (RBCs) (hemolysis, deformability, and aggregation), coagulation system [activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and the concentration of fibrinogen (Fib)], complement (C3a and C5a activation), and platelet (activation and aggregation), were comprehensively studied. In the case of RBCs, COS exhibited a low risk of hemolysis in a dose and molecular weight dependent manner and the irreversible aggregation was observed in their high concentration. For coagulation system, COS has a mild anticoagulation activity through blocking the intrinsic coagulation pathway. In addition, COS showed no effect on complement activation in C3a and C5a and on platelet activation while inhibition of platelet aggregation was evident. Finally, the mechanism that effects of COS on blood components was discussed and proposed.

Sulfated polysaccharide from sea cucumber modulates the gut microbiota and its metabolites in normal mice

Sulfated polysaccharide from sea cucumber (SCSP) has been demonstrated with various health effects, the mechanism of which, however, remains poorly understood. This study aimed to investigate the possible mechanism exhibited by gut microbiota in response to SCSP. BALB/c mice were fed diets supplemented with SCSP and depolymerized SCSP (d-SCSP) for 42 days. The microbiota composition, short chain fatty acids (SCFAs), lipopolysaccharide-binding protein (LBP), body weight and gut tissue index were analyzed. Results revealed that both SCSP and d-SCSP positively regulated the gut microbiota as indicated by the enriched microbiota diversity, SCFA-producing bacteria and sulfide-degrading bacteria, and decreased harmful bacteria. Moreover, SCSP and d-SCSP not only significantly improved the levels of microbial metabolites including SCFAs and LBP, but also effectively adjusted body weight and gut tissue index. The microbial metabolites were identified to strongly correlate with the growth performance using Pearson's correlation coefficient. We further showed that the modulating effect of SCSP on the gut microbiota was altered by free-radical depolymerization, while the microbial metabolites and related growth performance were not. These findings suggest that SCSP can be used as a gut microbiota manipulator for health promotion and alter the gut microbiota in a molecular weight (Mw) dependent manner.

Effect of Molecular Architecture on Cell Interactions and Stealth Properties of PEG.

PEGylation, covalent attachment of PEG to therapeutic biomolecules, in which suboptimal pharmacokinetic profiles limiting their therapeutic utility are of concern, is a widely applied technology. However, this technology has been challenged by reduced bioactivity of biomolecules upon PEGylation and immunogenicity of PEG triggering immune response and abrogating clinical efficacy, which collectively necessitate development of stealth polymer alternatives. Here we demonstrate that comb-shape poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA), a stealth polymer alternative, has a more compact structure than PEG and self-organize into nanoparticles in a molecular weight dependent manner. Most notably, we show that comb-shape POEGMA promotes significantly higher cellular uptake and exhibits less steric hindrance imposed on the conjugated biomolecule than PEG. Collectively, comb-shape POEGMA offers a versatile alternative to PEG for stealth polymer-biomolecule conjugation applications.

36\xa0h fasting of young men influences adipose tissue DNA methylation of LEP and ADIPOQ in a birth weight-dependent manner

BackgroundSubjects born with low birth weight (LBW) display a more energy-conserving response to fasting compared with normal birth weight (NBW) subjects. However, the molecular mechanisms explaining these metabolic differences remain unknown. Environmental influences may dynamically affect epigenetic marks, also in postnatal life. Here, we aimed to study the effects of short-term fasting on leptin (LEP) and adiponectin (ADIPOQ) DNA methylation and gene expression in subcutaneous adipose tissue (SAT) from subjects with LBW and NBW.MethodsTwenty-one young LBW men and 18 matched NBW controls were studied during 36 h fasting. Eight subjects from each group completed a control study (overnight fast). We analyzed SAT LEP and ADIPOQ methylation (Epityper MassARRAY), gene expression (q-PCR), and adipokine plasma levels.ResultsAfter overnight fast (control study), LEP and ADIPOQ DNA methylation levels were higher in LBW compared to those in NBW subjects (p ≤ 0.03) and increased with 36 h fasting in NBW subjects only (p ≤ 0.06). Both LEP and ADIPOQ methylation levels were positively associated with total body fat percentage (p ≤ 0.05). Plasma leptin levels were higher in LBW versus NBW subjects after overnight fasting (p = 0.04) and decreased more than threefold in both groups after 36 h fasting (p ≤ 0.0001).ConclusionsThis is the first study to demonstrate that fasting induces changes in DNA methylation. This was shown in LEP and ADIPOQ promoters in SAT among NBW but not LBW subjects. The altered epigenetic flexibility in LBW subjects might contribute to their differential response to fasting, adipokine levels, and increased risk of metabolic disease.

Molecular Weight-Dependent Immunostimulative Activity of Low Molecular Weight Chitosan via Regulating NF-κB and AP-1 Signaling Pathways in RAW264.7 Macrophages

Chitosan and its derivatives such as low molecular weight chitosans (LMWCs) have been found to possess many important biological properties, such as antioxidant and antitumor effects. In our previous study, LMWCs were found to elicit a strong immunomodulatory response in macrophages dependent on molecular weight. Herein we further investigated the molecular weight-dependent immunostimulative activity of LMWCs and elucidated its mechanism of action on RAW264.7 macrophages. LMWCs (3 kDa and 50 kDa of molecular weight) could significantly enhance the mRNA expression levels of COX-2, IL-10 and MCP-1 in a molecular weight and concentration-dependent manner. The results suggested that LMWCs elicited a significant immunomodulatory response, which was dependent on the dose and the molecular weight. Regarding the possible molecular mechanism of action, LMWCs promoted the expression of the genes of key molecules in NF-κB and AP-1 pathways, including IKKβ, TRAF6 and JNK1, and induced the phosphorylation of protein IKBα in RAW264.7 macrophage. Moreover, LMWCs increased nuclear translocation of p65 and activation of activator protein-1 (AP-1, C-Jun and C-Fos) in a molecular weight-dependent manner. Taken together, our findings suggested that LMWCs exert immunostimulative activity via activation of NF-κB and AP-1 pathways in RAW264.7 macrophages in a molecular weight-dependent manner and that 3 kDa LMWC shows great potential as a novel agent for the treatment of immune suppression diseases and in future vaccines.

The Effects of Super-Flux (High Performance) Dialyzer on Plasma Glycosylated Pro-B-Type Natriuretic Peptide (proBNP) and Glycosylated N-Terminal proBNP in End-Stage Renal Disease Patients on Dialysis

BackgroundPlasma BNP levels are predictive of prognosis in hemodialysis patients. However, recent studies showed that the current BNP immunoassay cross-reacts with glycosylated proBNP, and the NT-proBNP assay underestimates glycosylated NT-proBNP. In addition, the recently developed high performance dialyzer removes medium-sized molecular solutes such as β2-microgloburin. We therefore investigated the effects of high performance dialysis on measured levels of glycosylated proBNP, glycosylated NT-proBNP and other BNP-related peptides in end-stage renal disease (ESRD) patients on hemodialysis.MethodThe relationships between clinical parameters and BNP-related molecule were also investigated. We used our newly developed immunoassay to measure plasma total BNP and proBNP in 105 normal subjects and 36 ESRD patients before and after hemodialysis. Plasma NT-proBNP was measured using Elecsys II after treatment with or without deglycosylating enzymes. We also measured plasma ANP and cGMP using radioimmunoassays.ResultsAll the measured BNP-related peptides were significantly higher in ESRD patients than healthy subjects. Total BNP (−38.9%), proBNP (−29.7%), glycoNT-proBNP (−45.5%), nonglycoNT-proBNP (−53.4%), ANP (−50.4%) and cGMP (−72.1%) were all significantly reduced after hemodialysis, and the magnitude of the reduction appeared molecular weight- dependent. Both the proBNP/total BNP and glycoNT-proBNP/nonglycoNT-proBNP ratios were increased after hemodialysis. The former correlated positively with hemodialysis vintage and negatively with systolic blood pressure, while the latter correlated positively with parathyroid hormone levels.ConclusionThese results suggest that hemodialysis using super-flux dialyzer removes BNP-related peptides in a nearly molecular weight-dependent manner. The ProBNP/total BNP and glycoNT-proBNP/nonglycoNT-proBNP ratios appear to be influenced by hemodialysis-related parameters in ESRD patients on hemodialysis.

PPARGC1A DNA methylation in subcutaneous adipose tissue in low birth weight subjects — impact of 5 days of high-fat overfeeding

ObjectiveIncreased DNA methylation of the metabolic regulator peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) has been reported in skeletal muscle from type 2 diabetes (T2D) subjects and from low birth weight (LBW) subjects with an increased risk of T2D. High-fat overfeeding increases PPARGC1A DNA methylation in muscle in a birth weight dependent manner. However, PPARGC1A DNA methylation in subcutaneous adipose tissue (SAT) in LBW subjects has not previously been investigated. Our objective was to determine PPARGC1A DNA methylation and mRNA expression in basal and insulin-stimulated SAT from LBW and matched normal birth weight (NBW) subjects during control and high-fat overfeeding. Materials/MethodsNineteen young healthy men with LBW and 26 NBW controls were studied after both a 5-day high-fat overfeeding and a control diet in a randomized crossover setting. DNA methylation was assessed with bisulfite sequencing and mRNA expression with quantitative real-time PCR. ResultsFollowing high-fat overfeeding, increased SAT PPARGC1A DNA methylation was observed in LBW subjects but not in NBW controls. Basal SAT PPARGC1A mRNA expression was unaffected by diet and similar in the two groups. However, LBW subjects showed an increased SAT PPARGC1A mRNA expression during insulin-stimulation. SAT PPARGC1A methylation correlated inversely with mRNA expression during insulin-stimulation. ConclusionsThe study adds to the increasing awareness of PPARGC1A DNA methylation being flexible and influenced by high-fat overfeeding in a birth weight dependent manner with muscle and fat responding differently. Further data are needed to understand the role of PPARGC1A DNA methylation in insulin resistance and developmental programming of T2D.

Physical inactivity affects skeletal muscle insulin signaling in a birth weight-dependent manner

AimsWe investigated whether physical inactivity could unmask defects in insulin and AMPK signaling in low birth weight (LBW) subjects. MethodsTwenty LBW and 20 normal birth weight (NBW) subjects were investigated using the euglycemic–hyperinsulinemic clamp with excision of skeletal muscle biopsies pre and post 9days of bed rest. Employing Western blotting, we investigated skeletal muscle Akt, AS160, GLUT4, and AMPK signaling. ResultsPeripheral insulin action was similar in the two groups and was decreased to the same extent post bed rest. Insulin and AMPK signaling was unaffected by bed rest in NBW individuals. LBW subjects showed decreased insulin-stimulated Akt phosphorylation and increased AMPK α1 and γ3 protein expression post bed rest. Insulin response of AS160 phosphorylation was lower in LBW subjects both pre and post bed rest. ConclusionsBed rest-induced insulin resistance is not explained by impaired muscle insulin or AMPK signaling in subjects with or without LBW. Lower muscle insulin signaling in LBW subjects post bed rest despite similar degree of insulin resistance as seen in controls may to some extent support the idea that LBW subjects are at higher risk of developing type 2 diabetes when being physically inactive.

Effect of hemodyalysis on precursor proBNP and glycosylated NT-proBNP in end-stage renal disease patients

Background: Brain natriuretic peptide (BNP) is established biomarker for cardiovascular diseases. Recent studies have shown that unprocessed precursor proBNP circulates in the healthy individuals, and its levels in plasma are increased in severe heart failure patients. However, since current BNP immunoassay crossreacts proBNP, it is important to consider the significance of proBNP on clinical situation of BNP elevation. In addition, recent study has shown that most proBNP is glycosylated, and that NT-proBNP is also glycosylated in human plasma, and current NT-proBNP assay underestimates the glycosylated NT-proBNP. Recent developed super-flux dialyzer can remove middle-sized molecular solutes such as β2-microgloburin. In this study, we investigated the effects of hemodialysis using super-flux dialyzer on the glycosylated proBNP, glycosylated NT-proBNP, and BNP-related other peptide in patients with end-stage renal disease (ESRD). Method: Thirty-six ESRD patients with hemodialysis were studied. We measured plasma BNP (which was defined as BNP messured with current BNP assay system), proBNP, and mature BNP (which was defined as total BNP- proBNP), by our newly developed immunoassay before and after hemodialysis. Plasma glycosylated NT-proBNP and nonglycosylated NT-proBNP was measured by Elecsys II after deglycosylating enzyme treatment such as neuramoinidase and O-glycosydase. We also measured plasma ANP and cGMP by radioimmunoassay. Results: After hemodialysis, BNP (-28%), proBNP (-20%), mature BNP (-47%), glycosylated NT-proBNP (-33%), nonglycosylated NT-proBNP (-45%), ANP (-52%), and cGMP (-63%) significantly decreased. The decreasing ratio did not correlated with any indices of reduction of plasma volume; however, these peptides decreased after hemodialysis in a molecular weight dependent manner. ProBNP/total BNP ratio and glycosylated NT-proBNP/nonglycosylated NT-proBNP ratio increased after hemodialysis. ProBNP/total BNP ratio positively correlated with hemodialysis vintage and negatively correlated with left atrial dimater and systolic blood pressure, whereas glycosylated NT-proBNP/nonglycosylated positively correlated with parathyroid hormone levels. Conclusion: These results suggest that hemodialysis modulates profile of BNP and its related peptide, and it may be important to consider the effect of this modulation on the pathological conditions in ESRD patients in super-flux dialyzer era.

The effects of super-flux (high performance) dialyzer on the plasma glycosylated pro-B-type natriuretic peptide (proBNP) and glycosylated N-terminal proBNP in end-stage renal disease patients on dialysis

Background: Plasma BNP levels are predictive of prognosis in hemodialysis patients. However, recent studies showed that the current BNP immunoassay cross-reacts with glycosylated proBNP, and the NT-proBNP assay underestimates glycosylated NT-proBNP. In addition, the recently developed high performance dialyzer removes medium-sized molecular solutes such as b2-microgloburin. We therefore investigated the effects of high performance dialysis on measured levels of glycosylated proBNP, glycosylated NT-proBNP and other BNP-related peptides in end-stage renal disease (ESRD) patients on hemodialysis. Method: The relationships between clinical parameters and BNP-related molecule were also investigated. We used our newly developed immunoassay to measure plasma total BNP and proBNP in 105 normal subjects and 36 ESRD patients before and after hemodialysis. Plasma NT-proBNP was measured using Elecsys II after treatment with or without deglycosylating enzymes. We also measured plasma ANP and cGMP using radioimmunoassays. Results: All the measured BNP-related peptides were significantly higher in ESRD patients than healthy subjects. Total BNP (238.9%), proBNP (229.7%), glycoNT-proBNP (245.5%), nonglycoNT-proBNP (253.4%), ANP (250.4%) and cGMP (272.1%) were all significantly reduced after hemodialysis, and the magnitude of the reduction appeared molecular weightdependent. Both the proBNP/total BNP and glycoNT-proBNP/nonglycoNT-proBNP ratios were increased after hemodialysis. The former correlated positively with hemodialysis vintage and negatively with systolic blood pressure, while the latter correlated positively with parathyroid hormone levels. Conclusion: These results suggest that hemodialysis using super-flux dialyzer removes BNP-related peptides in a nearly molecular weight-dependent manner. The ProBNP/total BNP and glycoNT-proBNP/nonglycoNT-proBNP ratios appear to be influenced by hemodialysis-related parameters in ESRD patients on hemodialysis.

ECM modulated early kidney development in embryonic organ culture

The use of exogenous signals is gaining importance in renal regenerative therapies. We wanted to explore the role of extracellular matrix (ECM) constituents on renal structure formation during renal organogenesis. We used a recently established organ culture setup to expose embryonic kidney rudiments directly to a large set of surface-immobilized or dissolved ECM molecules and growth factors. Organ culture was also performed on immobilized adult kidney ECM extracts and on reactive polymer films without any biomolecular components. The applied conditions resulted in distinct differences of organ phenotypes, underlining the multifaceted role of exogenous signals during kidney development. Specific ECM components, including collagen I and laminin, supported nephronal and tubular structure formation of the developing organ. ECM biopolymers, e.g. hyaluronic acid, were found to determine the fate of developing explants in a concentration- and molecular weight-dependent manner. The organ culture system used was an effective and robust means to identify exogenous signals that direct kidney development. This system can provide valuable insight for future regenerative therapies of kidney diseases.

Iontophoresis-Mediated Transdermal Permeation of Peptide Dendrimers across Human Epidermis

Purpose of the Study: The overall aim of the present work was to elucidate the effects of iontophoresis on assisting permeation/deposition of peptide dendrimers across/within human skin. Procedures: A series of peptide dendrimers containing arginine and histidine as terminal acids were synthesized and characterized. These dendrimers were subjected to passive and iontophoretic permeation studies across human epidermis. Results: The synthesized peptide dendrimers were found to be stable in epidermal, dermal and skin extracts up to 6 h. Passive diffusion studies revealed that none of the synthesized peptide dendrimers permeated human epidermis up to 6 h, although minute concentrations of low molecular weight dendrimers were detected in receptor medium at the end of 24 h. Application of iontophoresis significantly increased the permeation of all the tested peptide dendrimers across human skin in a molecular weight-dependent manner compared to simple passive diffusion. Electromigration was found to be the dominant mechanism behind the iontophoretic permeation of peptide dendrimers across human skin. Conclusions: The present study demonstrates that iontophoresis is an effective technique in enhancing the transdermal permeation of peptide dendrimers. Message of the Paper: This study foresees the possibility of applying peptide dendrimers in iontophoretic delivery of drugs and macromolecules across/within the skin.

Serum under-carboxylated osteocalcin levels in women with polycystic ovary syndrome: weight-dependent relationships with endocrine and metabolic traits

BackgroundUnder-carboxylated osteocalcin (ucOC), the precursor substrate of bone biomarker OC is a potent regulator of energy metabolism by promoting insulin production and adiponectin synthesis and decreasing fat stores. The aim of the present study was to point out the potential role of ucOC in the physiopathology of polycystic ovary syndrome (PCOS), a common disorder defined by the constellation of anovulation, insulinresistance, hyperinsulinemia, obesity and androgen excess.MethodsIn this prospective case–control investigation, 78 young premenopausal women, i.e. 52 PCOS patients and 26 age- and body mass index (BMI)-matched healthy controls, were successively enrolled. Recruitment of PCOS patients was performed according to Androgen Excess-Polycystic Ovary Syndrome (AE-PCOS) Society 2006 criteria. All study participants were subjected to clinical examination, whole-body composition assessment and measurements of serum ucOC, OC (1-49), glucose and lipids, insulin, total testosterone (TT), estradiol, sex-hormone binding globulin (SHBG), high-sensitivity C-reactive protein (Hs-CRP) and β-CrossLaps.ResultsBMI-stratified multivariate analysis revealed significantly higher ucOC levels in PCOS vs. controls in lean (p = 0.001) but not overweight and obese study participants (p = 0.456). Notably, a positive correlation between ucOC and TT (p = 0.018), calculated free testosterone (cFT, p = 0.028) and serum insulin (p = 0.036), respectively, was found to be confined to the lean analysis subgroup. Furthermore, in stepwise multiple regression models, β-CrossLaps and cFT were able to predict 46.71% of serum ucOC variability. (1-43/49)OC failed to be significantly associated to any PCOS trait.ConclusionsCirculating ucOC concentration is related to key endocrine PCOS characteristics in a weight-dependent manner. Within the bone-pancreas loop, high ucOC may favor insulin release in lean hyperandrogenic women to compensate for impaired insulin sensitivity.

Noninvasive Detection of Passively Targeted Poly(ethylene glycol) Nanocarriers in Tumors

The present studies noninvasively investigate the passive tumor distribution potential of a series of poly(ethylene glycol) (PEG) nanocarriers using a SkinSkan spectrofluorometer and an In Vivo Imaging System (IVIS) 100. Fluorescein conjugated PEG nanocarriers of varying molecular weights (10, 20, 30, 40, and 60 kDa) were prepared and characterized. The nanocarriers were administered intravenously to female balb/c mice bearing subcutaneous 4T1 tumors. Passive distribution was measured in vivo (λ(exc), 480 nm; λ(em), 515-520 nm) from the tumor and a contralateral skin site (i.e., control site). The signal intensity from the tumor was always significantly higher than that from the contralateral site. Trends in results between the two methods were consistent with tumor distribution increasing in a molecular weight-dependent manner (10 < 20 < 30 ≪ 40 ≪ 60 kDa). The 10 kDa nanocarrier was not detected in tumors at 24 h, whereas 40-60 kDa nanocarriers were detected in tumors for up to 96 h. The 30, 40, and 60 kDa nanocarriers showed 2.1, 5.3, and 4.1 times higher passive distribution in tumors at 24 h, respectively, as compared to the 20 kDa nanocarrier. The 60 kDa nanocarrier exhibited 1.5 times higher tumor distribution than 40 kDa nanocarrier at 96 h. Thus, PEG nanocarriers (40 and 60 kDa) with molecular weights close to or above the renal exclusion limit, which for globular proteins is ≥45 kDa, showed significantly higher tumor distribution than those below it. The hydrodynamic radii of PEG polymers, measured using dynamic light scattering (DLS), showed that nanocarriers obtained from polymers with hydrodynamic radii ≥8 nm exhibited higher tumor distribution. Ex vivo mass balance studies revealed that nanocarrier tissue distribution followed the rank order tumor > lung > spleen > liver > kidney > muscle > heart, thus validating the in vivo studies. The results of the current studies suggest that noninvasive dermal imaging of tumors provides a reliable and rapid method for the initial screening of nanocarrier tumor distribution pharmacokinetics.