Search

Regulated secretion of insulin from β-cells, glucagon from α-cells, and somatostatin from δ-cells is necessary for the maintenance of glucose homeostasis. The release of these hormones from pancreatic islet cells requires the assembly and disassembly of the SNARE protein complex to control vesicle fusion and exocytosis. Complexin 2 (Cplx 2) is a small soluble synaptic protein that participates in the priming and release steps of vesicle fusion. It plays a dual role as a molecular switch that first clamps and prevents fusion pore opening, and subsequently undergoes a conformational change upon Ca 2+ binding to synaptotagmin to facilitate exocytosis. Using a Cplx 2 knockout (KO) mouse model, we show a direct inhibitory role of Cplx 2 for glucagon and somatostatin secretion, along with an indirect role in the paracrine inhibition of insulin secretion by somatostatin. Deletion of Cplx 2 increases glucagon and somatostatin secretion from intact mouse islets, while there is no difference in insulin secretion between WT and Cplx 2 KO islets. The normal paracrine inhibition of insulin secretion by somatostatin is disrupted in Cplx 2 KO islets. On the contrary, deletion of Cplx 2 did not affect the known role of somatostatin in the paracrine inhibition of glucagon at elevated glucose levels, since the paracrine inhibition of glucagon secretion by somatostatin is similar for both WT and Cplx 2 KO islets. In both β- and α-cells, the secretion profiles are parallel to Ca 2+ activity changes following somatostatin treatment of WT and Cplx 2 KO islets. The loss of paracrine inhibition of insulin secretion is substantiated by direct measurements of insulin vesicle fusion events in Cplx 2 KO islets. Together, these data show a differential role for Cplx 2 in regulating hormone secretion from pancreatic islets.

Just under the plasma membrane of most animal cells lies a dense meshwork of actin filaments called the cortical cytoskeleton. In insulin-secreting pancreatic β cells, a long-standing model posits that the cortical actin layer primarily acts to restrict access of insulin granules to the plasma membrane. Here we test this model and find that stimulating β cells with pro-secretory stimuli (glucose and/or KCl) has little impact on the cortical actin layer. Chemical perturbations of actin polymerization, by either disrupting or enhancing filamentation, dramatically enhance glucose-stimulated insulin secretion. Using scanning electron microscopy, we directly visualize the cortical cytoskeleton, allowing us to validate the effect of these filament-disrupting chemicals. We find the state of the cortical actin layer does not correlate with levels of insulin secretion, suggesting filament disruptors act on insulin secretion independently of the cortical cytoskeleton., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

A design and fabrication technique for making high-precision and large-format multifaceted mapping mirrors is presented. The method is based on two-photon polymerization, which allows more flexibility in the mapping mirror design. The mirror fabricated in this paper consists of 36 2D tilted square pixels, instead of the continuous facet design used in diamond cutting. The paper presents a detailed discussion of the fabrication parameters and optimization process, with particular emphasis on the optimization of stitching defects by compensating for the overall tilt angle and reducing the printing field of view. The fabricated mirrors were coated with a thin layer of aluminum (93 nm) using sputter coating to enhance the reflection rate over the target wave range. The mapping mirror was characterized using a white light interferometer and a scanning electron microscope, which demonstrates its optical quality surface (with a surface roughness of 12 nm) and high-precision tilt angles (with an average of 2.03% deviation). Finally, the incorporation of one of the 3D printed mapping mirrors into an image mapping spectrometer prototype allowed for the acquisition of high-quality images of the USAF resolution target and bovine pulmonary artery endothelial cells stained with three fluorescent dyes, demonstrating the potential of this technology for practical applications.

Hyperinsulinemia often precedes type 2 diabetes. Palmitoylation, implicated in exocytosis, is reversed by acyl-protein thioesterase 1 (APT1). APT1 biology was altered in pancreatic islets from humans with type 2 diabetes, and APT1 knockdown in nondiabetic islets caused insulin hypersecretion. APT1 knockout mice had islet autonomous increased glucose-stimulated insulin secretion that was associated with prolonged insulin granule fusion. Using palmitoylation proteomics, we identified Scamp1 as an APT1 substrate that localized to insulin secretory granules. Scamp1 knockdown caused insulin hypersecretion. Expression of a mutated Scamp1 incapable of being palmitoylated in APT1-deficient cells rescued insulin hypersecretion and nutrient-induced apoptosis. High-fat-fed islet-specific APT1-knockout mice and global APT1-deficient db/db mice showed increased β cell failure. These findings suggest that APT1 is regulated in human islets and that APT1 deficiency causes insulin hypersecretion leading to β cell failure, modeling the evolution of some forms of human type 2 diabetes., Competing Interests: Declaration of interests C.F.S. is a member of the Cell Metabolism advisory board., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The K + channel selectivity filter (SF) is defined by TxGYG amino acid sequences that generate four identical K + binding sites (S1-S4). Only two sites (S3, S4) are present in the non-selective bacterial NaK channel, but a four-site K + -selective SF is obtained by mutating the wild-type TVGDGN SF sequence to a canonical K + channel TVGYGD sequence (NaK2K mutant). Using single molecule FRET (smFRET), we show that the SF of NaK2K, but not of non-selective NaK, is ion-dependent, with the constricted SF configuration stabilized in high K + conditions. Patch-clamp electrophysiology and non-canonical fluorescent amino acid incorporation show that NaK2K selectivity is reduced by crosslinking to limit SF conformational movement. Finally, the eukaryotic K + channel TREK2 SF exhibits essentially identical smFRET-reported ion-dependent conformations as in prokaryotic K + channels. Our results establish the generality of K + -induced SF conformational stability across the K + channel superfamily, and introduce an approach to study manipulation of channel selectivity., (© 2023. The Author(s).)

Improvements in reproductive techniques have resulted in the live birth rates from IVF procedures increasing from 5% to approximately 30% in recent decades but has plateaued since. Emerging preclinical and clinical data implicates endometrial receptivity deficiencies in patients with recurrent implantation failure (RIF) as the predominant factor hindering successful implantation. Mechanisms on how local endometrial injury (LEI) improves implantation rates in patients with RIF are currently unknown. We hypothesized that LEI may influence perivascular endometrial mesenchymal stem/progenitor cells (eMSCs) which are thought to regenerate the stromal vascular component of the functional layer every month. Here, we assessed the effect of LEI on the proportion and function of eMSCs present in consecutive LEI biopsies. Consecutive paired mid-luteal phase endometrial biopsies obtained from patients with RIF were digested to single cells and the proportion of SUSD2-expressing cells determined. Growth kinetics and decidualization were compared between the consecutive LEI samples. A mid-luteal LEI altered the decidualization capacity of SUSD2 + eMSCs in women with RIF, but not their proportion or clonogenicity. With the potential of LEI to improve IVF outcomes in women with RIF, additional investigations are needed to understand the impact of the altered decidualization response in eMSCs., (© 2023. The Author(s).)

Glucagon hypersecretion from pancreatic islet α-cells exacerbates hyperglycemia in type 1 diabetes (T1D) and type 2 diabetes. Still, the underlying mechanistic pathways that regulate glucagon secretion remain controversial. Among the three complementary main mechanisms (intrinsic, paracrine, and juxtacrine) proposed to regulate glucagon release from α-cells, juxtacrine interactions are the least studied. It is known that tonic stimulation of α-cell EphA receptors by ephrin-A ligands (EphA forward signaling) inhibits glucagon secretion in mouse and human islets and restores glucose inhibition of glucagon secretion in sorted mouse α-cells, and these effects correlate with increased F-actin density. Here, we elucidate the downstream target of EphA signaling in α-cells. We demonstrate that RhoA, a Rho family GTPase, plays a key role in this pathway. Pharmacological inhibition of RhoA disrupts glucose inhibition of glucagon secretion in islets and decreases cortical F-actin density in dispersed α-cells and α-cells in intact islets. Quantitative FRET biosensor imaging shows that increased RhoA activity follows directly from EphA stimulation. We show that in addition to modulating F-actin density, EphA forward signaling and RhoA activity affect α-cell Ca2+ activity in a novel mechanistic pathway. Finally, we show that stimulating EphA forward signaling restores glucose inhibition of glucagon secretion from human T1D donor islets., (© 2022 by the American Diabetes Association.)

The time period before, during and after pregnancy represents a unique opportunity for interventions to cultivate sustained healthy lifestyle behaviors to improve the metabolic health of mothers and their offspring. However, the success of a lifestyle intervention is dependent on uptake and continued compliance. To identify enablers and barriers towards engagement with a lifestyle intervention, thematic analysis of 15 in-depth interviews with overweight or obese women in the preconception, pregnancy or postpartum periods was undertaken, using the integrated-Promoting Action on Research Implementation in Health Services framework as a guide to systematically chart factors influencing adoption of a novel lifestyle intervention. Barrier factors include time constraints, poor baseline knowledge, family culture, food accessibility, and lack of relevant data sources. Enabling factors were motivation to be healthy for themselves and their offspring, family and social support, a holistic delivery platform providing desired information delivered at appropriate times, regular feedback, goal setting, and nudges. From the findings of this study, we propose components of an idealized lifestyle intervention including (i) taking a holistic life-course approach to education, (ii) using mobile health platforms to reduce barriers, provide personalized feedback and promote goal-setting, and (iii) health nudges to cultivate sustained lifestyle habits., (© 2022. The Author(s).)

There is a paucity of effective intervention tools for overweight/obese women to assess, guide and monitor their eating behavior. This study aimed to develop a lifestyle intervention tool, assess its acceptability and usefulness, and verify its construct validity in overweight/obese women. The 6P tool (Portion, Proportion, Pleasure, Phase, Physicality, Psychology) was developed and 15 women with a body mass index (BMI) ≥ 25 kg/m 2 were interviewed to assess its perceived acceptability and usefulness. Subsequently, the revised 6P tool was tested in 46 women with a BMI ≥ 25 kg/m 2 . The Three-Factor Eating Questionnaire (TFEQ), International Physical Activity Questionnaire-Short (IPAQ), and weight were measured at baseline and one-month. Most participants were satisfied with the presentation of the 6P tool (86.8%), and agreed it was useful in guiding healthy eating (81.6%) and raising awareness of eating behavior (97.4%). There were significant improvements in cognitive restraint ( p = 0.010) and disinhibition ( p = 0.030) (TFEQ), portion size (P1), pleasure behaviors (P3), and total composite 6P score ( p < 0.001). However, there was no significant reduction in weight or increase in physical activity. The 6P tool is acceptable and presents with good validity for assessing lifestyle behaviors.

Use of GnRH antagonists in IVF stimulation protocols shortens controlled ovarian hyperstimulation (COH) and reduces the risk of ovarian hyperstimulation syndrome (OHSS). However, profound reduction in LH levels has been associated with use of GnRH antagonists. This study aims to determine if LH suppression during GnRH antagonist cycles results in poorer IVF outcomes. This was a prospective pilot longitudinal study where serum LH levels were measured on day 2/3 of the menstrual cycle before COH, 1/2 days following institution of GnRH antagonist and at the day of ovulation trigger. A threshold of LH <0.5 IU/L was used to define profound LH suppression. Data on IVF outcomes was collected. Logistic regression analysis was used to investigate risk factors associated with LH suppression following GnRH antagonist IVF treatment. Ninety-one eligible women were recruited. Women underwent a standard antagonist cycle with Puregon 200u and Ganirelix. No participant had LH <0.5 IU/L prior to GnRH antagonist treatment, and 27 participants (29.7%) had significant LH suppression at either time point. Predictors of profound LH suppression following GnRH antagonist treatment identified (P < 0.20) were age (OR = 0.80, P = 0.013), no previous ovulation induction (OR = 0.26, P = 0.033) and previous GnRH antagonist IVF cycle (OR = 4.32, P = 0.125). Numbers of oocytes, embryos and ongoing pregnancy rates at 12 weeks gestation in patients with and without LH suppression did not differ significantly. We found associations between clinical characteristics and risk of profound LH suppression in women undergoing GnRH antagonist IVF cycles, but no significant differences in IVF and pregnancy outcomes between women with and without significant LH suppression., (© 2021. Society for Reproductive Investigation.)

Blood glucose homeostasis requires proper function of pancreatic islets, which secrete insulin, glucagon, and somatostatin from the β-, α-, and δ-cells, respectively. Each islet cell type is equipped with intrinsic mechanisms for glucose sensing and secretory actions, but these intrinsic mechanisms alone cannot explain the observed secretory profiles from intact islets. Regulation of secretion involves interconnected mechanisms among and between islet cell types. Islet cells lose their normal functional signatures and secretory behaviors upon dispersal as compared to intact islets and in vivo. In dispersed islet cells, the glucose response of insulin secretion is attenuated from that seen from whole islets, coordinated oscillations in membrane potential and intracellular Ca 2+ activity, as well as the two-phase insulin secretion profile, are missing, and glucagon secretion displays higher basal secretion profile and a reverse glucose-dependent response from that of intact islets. These observations highlight the critical roles of intercellular communication within the pancreatic islet, and how these communication pathways are crucial for proper hormonal and nonhormonal secretion and glucose homeostasis. Further, misregulated secretions of islet secretory products that arise from defective intercellular islet communication are implicated in diabetes. Intercellular communication within the islet environment comprises multiple mechanisms, including electrical synapses from gap junctional coupling, paracrine interactions among neighboring cells, and direct cell-to-cell contacts in the form of juxtacrine signaling. In this article, we describe the various mechanisms that contribute to proper islet function for each islet cell type and how intercellular islet communications are coordinated among the same and different islet cell types. © 2021 American Physiological Society. Compr Physiol 11:2191-2225, 2021., (Copyright © 2021 American Physiological Society. All rights reserved.)

An accurate depiction of the convalescent COVID-19 immunome will help delineate the immunological milieu crucial for disease resolution and protection. Using mass cytometry, we characterized the immune architecture in patients recovering from mild COVID-19. We identified a virus-specific immune rheostat composed of an effector T (T eff ) cell recall response that is balanced by the enrichment of a highly specialized regulatory T (T reg ) cell subset. Both components were reactive against a peptide pool covering the receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. We also observed expansion of IFNγ + memory CD4 + T cells and virus-specific follicular helper T (T FH ) cells. Overall, these findings pinpoint critical immune effector and regulatory mechanisms essential for a potent, yet harmless resolution of COVID-19 infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yeo, Leong, Tay, Nadua, Anderson, Lim, Ng, Poh, Guo, Yaung, Kumar, Wasser, Hazirah, Sutamam, Chua, Qui, Foo, Gamage, Yeo, Ramakrishna, Arkachaisri, Young, Lye, Wang, Chong, Tan, Li, Kam, Ginhoux, Thoon, Chan, Yung and Albani.)

Super-resolution microscopy and single molecule fluorescence spectroscopy require mutually exclusive experimental strategies optimizing either temporal or spatial resolution. To achieve both, we implement a GPU-supported, camera-based measurement strategy that highly resolves spatial structures (~100 nm), temporal dynamics (~2 ms), and molecular brightness from the exact same data set. Simultaneous super-resolution of spatial and temporal details leads to an improved precision in estimating the diffusion coefficient of the actin binding polypeptide Lifeact and corrects structural artefacts. Multi-parametric analysis of epidermal growth factor receptor (EGFR) and Lifeact suggests that the domain partitioning of EGFR is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-EGFR interactions but is largely independent of EGFR-actin interactions. These results demonstrate that pixel-wise cross-correlation of parameters obtained from different techniques on the same data set enables robust physicochemical parameter estimation and provides biological knowledge that cannot be obtained from sequential measurements.

Background: Effective management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires large-scale testing to identify and isolate infectious carriers. Self-administered buccal swab and saliva collection are convenient, painless, and safe alternatives to the current healthcare worker (HCW)-collected nasopharyngeal swab (NPS)., Methods: A cross-sectional single-centre study was conducted on 42 participants who had tested positive for SARS-CoV-2 via an NPS within the past 7 days. Real-time polymerase chain reaction (RT-PCR) was performed and cycle threshold (Ct) values were obtained for each test. The positive percent agreement (PPA), negative percent agreement (NPA), and overall agreement (OA) were calculated for the saliva samples and buccal swabs, and compared with NPS., Results: Among the 42 participants, 73.8% (31/42) tested positive by any one of the three tests. With reference to NPS, the saliva test had PPA 66.7%, NPA 91.7%, and OA 69.0%; the buccal swab had PPA 56.7%, NPA 100%, and OA 73.8%., Conclusion: Self-collected saliva tests and buccal swabs showed only moderate agreement with HCW-collected NPS. Primary screening for SARS-CoV-2 may be performed with a saliva test or buccal swab, with a negative test warranting a confirmatory NPS to avoid false-negatives, minimize discomfort, and reduce the risk of spread to the community and HCWs., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Prion diseases are a group of neurodegenerative disorders that infect animals and humans with proteinaceous particles called prions. Prions consist of scrapie prion protein (PrP Sc ), a misfolded version of the cellular prion protein (PrP C ). During disease progression, PrP Sc replicates by interacting with PrP C and inducing its conversion to PrP Sc . Attachment of PrP C to cellular membranes via a glycosylphosphatidylinositol (GPI) anchor is critical for the conversion of PrP C into PrP Sc . However, the mechanisms governing PrP C conversion and replication on the membrane remain largely unclear. Here, a site-selectively modified PrP variant equipped with a fluorescent GPI anchor mimic (PrP-GPI) was employed to directly observe PrP at the cellular membrane in neuronal SH-SY5Y cells. PrP-GPI exhibits a cholesterol-dependent membrane accumulation and a cytoskeleton-dependent mobility. More specifically, inhibition of actin polymerization reduced the diffusion of PrP-GPI indicating protein clustering, which resembles the initial step of PrP aggregation and conversion into its pathogenic isoform. An intact actin cytoskeleton might therefore prevent conversion of PrP C into PrP Sc and offer new therapeutic angles., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Pancreatic islets regulate glucose homeostasis through coordinated actions of hormone-secreting cells. What underlies the function of the islet as a unit is the close approximation and communication among heterogeneous cell populations, but the structural mediators of islet cellular cross talk remain incompletely characterized. We generated mice specifically lacking β-cell primary cilia, a cellular organelle that has been implicated in regulating insulin secretion, and found that the β-cell cilia are required for glucose sensing, calcium influx, insulin secretion, and cross regulation of α- and δ-cells. Protein expression profiling in islets confirms perturbation in these cellular processes and reveals additional targets of cilia-dependent signaling. At the organism level, the deletion of β-cell cilia disrupts circulating hormone levels, impairs glucose homeostasis and fuel usage, and leads to the development of diabetes. Together, these findings demonstrate that primary cilia not only orchestrate β-cell-intrinsic activity but also mediate cross talk both within the islet and from islets to other metabolic tissues, thus providing a unique role of cilia in nutrient metabolism and insight into the pathophysiology of diabetes., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Purpose: To investigate the drug release profiles of a tacrolimus-loaded poly(D,L-lactide-co-ε-caprolactone) (PLC) microfilm, and to evaluate its efficacy on the treatment of allergic conjunctivitis using a mouse model., Methods: The in vitro and in vivo drug release profiles were first characterized. Balb/c mice were immunized with short ragweed (SRW) injection followed by re-challenges with topical SRW solution. The mice were divided into six groups (n = 12 in each): negative control (NC); positive control (PC); tacrolimus eye drops (Te); subconjunctival tacrolimus microfilm (Tm); dexamethasone eye drops (De); and tacrolimus + dexamethasone eye drops (Te+De). The mice were evaluated for 28 days by a scoring system for allergic conjunctivitis. Histopathologic and immunohistochemical staining with CD11c, CD4, and IL-4 were performed., Results: The microfilms were biocompatible and delivered clinically sufficient dose in a sustained manner, with a steady rate of 0.212 to 0.243 μg/day in vivo. Compared to the PC groups, the Te, Tm, De, and Te+De groups significantly reduced the allergic clinical scores throughout the study period (all P < 0.01; 0.0 ± 0.0, 5.6 ± 0.9, 3.3 ± 0.9, 3.2 ± 0.9, 1.9 ± 0.4 and 1.7 ± 0.8 for the NC, PC, Tm, Te, De, and Te+De groups, respectively, at 4 weeks after treatment). The suppressed eosinophils, CD11c, CD4, and IL-4 expression were also observed in all treatment groups, with more reduction in the Te+De group., Conclusions: Tacrolimus-loaded microfilms display good biocompatibility and desirable sustained drug release. It was as effective as conventional tacrolimus eye drops on the treatment of allergic conjunctivitis, providing a promising clinically applicable alternative for controlling allergic disease activity, or other immune-mediated ocular diseases.

There has been increasing interest in biophysical studies on live organisms to gain better insights into physiologically relevant biological events at the molecular level. Zebrafish (Danio rerio) is a viable vertebrate model to study such events due to its genetic and evolutionary similarities to humans, amenability to less invasive fluorescence techniques owing to its transparency and well-characterized genetic manipulation techniques. Fluorescence techniques used to probe biomolecular dynamics and interactions of molecules in live zebrafish embryos are therefore highly sought-after to bridge molecular and developmental events. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are two robust techniques that provide molecular level information on dynamics and interactions respectively. Here, we detail the steps for applying confocal FCS and FCCS, in particular single-wavelength FCCS (SW-FCCS), in live zebrafish embryos, beginning with sample preparation, instrumentation, calibration, and measurements on the FCS/FCCS instrument and ending with data analysis.

Purpose: We evaluate the toxicity and plasma toxicokinetic (TK) profile of a biodegradable subconjunctival microrod for sustained prednisolone acetate (PA) release over 12 weeks in a non-human primate model., Methods: The biodegradable copolymer poly(l-lactide-co-ε-caprolactone) (PLC) and 40-wt% PA microrods were used and fashioned into 8 and 16 mm lengths. Twelve monkeys were divided into two treatment groups of PA-loaded and blank microrods, with six monkeys each receiving either 8- or 16-mm microrods subconjunctively implanted into both eyes. TK and hematology parameters were analyzed. Ophthalmic clinical evaluation, including slit-lamp and ophthalmoscopy examinations, was performed., Results: Over the study period of 12 weeks, the mean area under the plasma concentration-time curve was 45.7% higher, and the maximum plasma concentration was 17.2% lower for the animals treated with 40-wt% PA 16-mm microrods compared to 8-mm microrods (251.44 versus 172.54 hours × nanograms per milliliter and 8.53 versus 10.30 ng/mL, respectively). The PA release was significantly below the levels of assumed toxicity. There was no significant difference in the time to reach maximum concentration between the 8- and 16-mm microrod groups (7.33 and 8 hours; P = 0.421). Findings from clinical evaluation, hematology, and histopathology showed no ocular side effects and no significant adverse systemic effects., Conclusion: The PA biodegradable microrods demonstrated safe toxicokinetics even with the larger size implant containing a higher amount of drug. The PA implant may be considered as a safe alternative to the application of topical PA eyedrops., Translational Relevance: The results provide the evidence of the safety of implanting a steroid delivery system subconjunctively, offering an alternative to topical PA eyedrops.

Background: The soluble receptor for advanced glycation end products (sRAGE) has been shown to play an important role in diabetic complications. We conducted genome-wide association study (GWAS) of sRAGE in Asian type 2 diabetes mellitus (T2DM) patient and validated the association in an independent cohort of T2DM., Methods: GWAS for sRAGE was performed in 2058 T2DM patients. Associations between single-nucleotide polymorphisms (SNPs) and plasma sRAGE level were analyzed in an additive model using a linear mixed model. To validate the associations, we performed de novo genotyping in an independent cohort (n = 1984). We selected the top SNP for assessment with diabetic kidney disease (DKD)., Results: The strongest SNP, rs2070600C>T (P = 1.21 × 10-52), was a genotyped, missense SNP located on chromosome 6, corresponding to the RAGE (AGER) gene locus, the gene encoding RAGE. Conditioning analysis on rs2070600 revealed that rs2071288C>T was the top genotyped independent SNP (P = 8.36 × 10-10). Both SNPs were strongly and dose-dependently correlated with sRAGE level (TT = 399.6 pg/mL, CT = 737.0 pg/mL and CC = 967.0 pg/mL, P < 0.001 for rs2070600; TT = 687.9 pg/mL, CT = 737.6 pg/mL and CC = 904.7 pg/mL, P < 0.001 for rs2072188). Both SNPs were robustly replicated in the independent cohort, especially among Chinese patients (P = 9.02 × 10-72 for rs2070600; P = 1.13 × 10-9 for rs2071288). Log-transformed sRAGE was associated with DKD after adjustment for age, gender and ethnicity in pooled cohorts [odds ratio 2.536 (95% confidence interval 1.864-3.450), P < 0.001]. However, we did not observe any significant association between rs2070600 and DKD., Conclusions: Common variants in RAGE are strongly associated with plasma sRAGE level, which is associated with DKD. However, we did not find a causal link between sRAGE and renal function by Mendelian randomization., (© The Author 2016. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

While the cytosolic events of Wnt/β-catenin signaling (canonical Wnt signaling) pathway have been widely studied, only little is known about the molecular mechanisms involved in Wnt binding to its receptors at the plasma membrane. Here, we reveal the influence of the immediate plasma membrane environment on the canonical Wnt-receptor interaction. While the receptors are distributed both in ordered and disordered environments, Wnt binding to its receptors selectively occurs in more ordered membrane environments which appear to cointernalize with the Wnt-receptor complex. Moreover, Wnt/β-catenin signaling is significantly reduced when the membrane order is disturbed by specific inhibitors of certain lipids that prefer to localize at the ordered environments. Similarly, a reduction in Wnt signaling activity is observed in Niemann-Pick Type C disease cells where trafficking of ordered membrane lipid components to the plasma membrane is genetically impaired. We thus conclude that ordered plasma membrane environments are essential for binding of canonical Wnts to their receptor complexes and downstream signaling activity., (© 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Malaria remains a significant infectious disease with even artemisinin-based therapies now facing resistance in the field. Development of new therapies is urgently needed, either by finding new compounds with unique modes of action, or by reversing resistance towards known drugs with 'chemosensitizers' or 'chemoreversal' agents (CRA). Concerning the latter, we have focused on the resistance mechanisms developed against chloroquine (CQ). We have synthesized a series of compounds related to previously identified CRAs, and found promising novel compounds. These compounds show encouraging results in a coumarin labeled chloroquine uptake assay, exhibiting a dose response in resensitising parasites to the antimalarial effects of chloroquine. Selected compounds show consistent potency across a panel of chloroquine and artemisinin sensitive and resistant parasites, and a wide therapeutic window. This data supports further study of CRAs in the treatment of malaria and, ultimately, their use in chloroquine-based combination therapies., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Wnt3 is a morphogen that activates the Wnt signaling pathway and regulates a multitude of biological processes ranging from cell proliferation and cell fate specification to differentiation over embryonic induction to neural patterning. Recent studies have shown that the palmitoylation of Wnt3 by Porcupine, a membrane-bound O-acyltransferase, plays a significant role in the intracellular membrane trafficking of Wnt3 and subsequently, its secretion in live zebrafish embryos, where chemical inhibition of Porcupine reduced the membrane-bound and secreted fractions of Wnt3 and eventually led to defective brain development. However, the membrane distribution of Wnt3 in cells remains not fully understood. Here, we determine the membrane organization of functionally active Wnt3-EGFP in cerebellar cells of live transgenic zebrafish embryos and the role of palmitoylation in its organization using single plane illumination microscopy-fluorescence correlation spectroscopy (SPIM-FCS), a multiplexed modality of FCS, which generates maps of molecular dynamics, concentration, and interaction of biomolecules. The FCS diffusion law was applied to SPIM-FCS data to study the subresolution membrane organization of Wnt3. We find that at the plasma membrane in vivo, Wnt3 is associated with cholesterol-dependent domains. This association reduces with increasing concentrations of Porcupine inhibitor (C59), confirming the importance of palmitoylation of Wnt3 for its association with cholesterol-dependent domains. Reduction of membrane cholesterol also results in a decrease of Wnt3 association with cholesterol-dependent domains in live zebrafish. This demonstrates for the first time, to our knowledge, in live vertebrate embryos that Wnt3 is associated with cholesterol-dependent domains., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Imaging fluorescence correlation spectroscopy (FCS) and the related FCS diffusion law have been applied in recent years to investigate the diffusion modes of lipids and proteins in membranes. These efforts have provided new insights into the membrane structure below the optical diffraction limit, new information on the existence of lipid domains, and on the influence of the cytoskeleton on membrane dynamics. However, there has been no systematic study to evaluate how domain size, domain density, and the probe partition coefficient affect the resulting imaging FCS diffusion law parameters. Here, we characterize the effects of these factors on the FCS diffusion law through simulations and experiments on lipid bilayers and live cells. By segmenting images into smaller 7  ×  7 pixel areas, we can evaluate the FCS diffusion law on areas smaller than 2 µm and thus provide detailed maps of information on the membrane structure and heterogeneity at this length scale. We support and extend this analysis by deriving a mathematical expression to calculate the mean squared displacement (MSD ACF ) from the autocorrelation function of imaging FCS, and demonstrate that the MSD ACF plots depend on the existence of nanoscopic domains. Based on the results, we derive limits for the detection of domains depending on their size, density, and relative viscosity in comparison to the surroundings. Finally, we apply these measurements to bilayers and live cells using imaging total internal reflection FCS and single plane illumination microscopy FCS.

Objective: To examine the relationship between inflammation and central arterial stiffness in a type 2 diabetes Asian cohort., Method: Central arterial stiffness was estimated by carotid-femoral pulse wave velocity and augmentation index. Linear regression model was used to evaluate the association of high-sensitivity C-reactive protein and soluble receptor for advanced glycation end products with pulse wave velocity and augmentation index. High-sensitivity C-reactive protein was analysed as a continuous variable and categories (<1, 1-3, and >3 mg/L)., Results: There is no association between high-sensitivity C-reactive protein and pulse wave velocity. Augmentation index increased with high-sensitivity C-reactive protein as a continuous variable (β = 0.328, p = 0.049) and categories (β = 1.474, p = 0.008 for high-sensitivity C-reactive protein: 1-3 mg/L and β = 1.323, p = 0.019 for high-sensitivity C-reactive protein: >3 mg/L) after multivariable adjustment. No association was observed between augmentation index and soluble receptor for advanced glycation end products. Each unit increase in natural log-transformed soluble receptor for advanced glycation end products was associated with 0.328 m/s decrease in pulse wave velocity after multivariable adjustment (p = 0.007)., Conclusion: Elevated high-sensitivity C-reactive protein and decreased soluble receptor for advanced glycation end products are associated with augmentation index and pulse wave velocity, respectively, suggesting the potential role of systemic inflammation in the pathogenesis of central arterial stiffness in type 2 diabetes., (© The Author(s) 2016.)

Resistance to antimalarial therapies, including artemisinin, has emerged as a significant challenge. Reversal of acquired resistance can be achieved using agents that resensitize resistant parasites to a previously efficacious therapy. Building on our initial work describing novel chemoreversal agents (CRAs) that resensitize resistant parasites to chloroquine (CQ), we herein report new hybrid single agents as an innovative strategy in the battle against resistant malaria. Synthetically linking a CRA scaffold to chloroquine produces hybrid compounds with restored potency toward a range of resistant malaria parasites. A preferred compound, compound 35, showed broad activity and good potency against seven strains resistant to chloroquine and artemisinin. Assessment of aqueous solubility, membrane permeability, and in vitro toxicity in a hepatocyte line and a cardiomyocyte line indicates that compound 35 has a good therapeutic window and favorable drug-like properties. This study provides initial support for CQ-CRA hybrid compounds as a potential treatment for resistant malaria., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Newly synthesized proteins constitute an important subset of the proteome involved in every cellular process, yet existing chemical tools used to study them have major shortcomings. Herein we report a suite of cell-permeable puromycin analogues capable of being metabolically incorporated into newly synthesized proteins in different mammalian cells, including neuronal cells. Subsequent labeling with suitable bioorthogonal reporters, in both fixed and live cells, enabled direct imaging and enrichment of these proteins. By taking advantage of the mutually orthogonal reactivity of these analogues, we showed multiplexed labeling of different protein populations, as well as quantitative measurements of protein dynamics by fluorescence correlation spectroscopy, could be achieved in live-cell environments., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The use of drug-eluting coronary stents has led to significant reduction in in-stent restenosis (ISR), but led to delayed endothelialization, necessitating the prolonged use of expensive anti-thrombotic drugs with their side-effects. Cenderitide (CD-NP) is a novel anti-proliferative chimeric peptide of semi-endothelial origin. Our previous work in vitro has demonstrated; that the smooth muscle cells were inhibited significantly more than endothelial cells which is the desirable feature of an anti-restenosis drug. This work reports the effects of implantation of a centeritide-eluting stent (CES) on ISR and endothelialization in an in vivo model. CESs were produced by coating bare metallic stents with CD-NP entrapped in biodegradable poly(ε-caprolactone) using an ultrasonic spray coater. A total of 32 stents were successfully implanted into 16 pigs, and all animal survived for 28 days. The plasma levels of CD-NP were significantly higher in the CES group than in the control group (bare metal stents and polymer-coated stent) at post-stenting, indicating the successful release of CD-NP from the stent in vivo. Furthermore, SEM analysis results showed the greater endothelial coverage of the stent struts, as well as between the struts in CES group. Moreover, histological results showed mild inflammation, and low fibrin score at 28 days. However, plasma cGMP (second messenger, cyclic 3',5' guanosine monophosphate) does not show a significant difference, and the CES is also unable to show significant difference in terms on neointimal area and stenosis, in comparison to BMS at 28 days.

Timolol maleate (TM) has been used for many years for the reduction of intraocular pressure (IOP) in glaucoma patients. However, the topical mode of administration (eyedrops) is far from optimal because of the issues of low bioavailability, high drug wastage, and lack of patient compliance. Suboptimal control of the IOP leads to disease progression and eventually to blindness. Ideally, TM is delivered to the patient so that its action is both localized and sustained for 3 months or more. In this work, we developed a subconjunctival TM microfilm for sustained, long-term delivery of TM to the eyes, using the biodegradable elastomer poly(lactide-co-caprolactone) (PLC). The copolymer is biocompatible and has flexibility and mechanical characteristics suitable for a patient-acceptable implant. Controlling the release of TM for 3 months is challenging, and this work describes how, by using a combination of multilayering and blending with poly(ethylene glycol) (PEG) copolymers, we were able to develop a TM-incorporated biodegradable film that can deliver TM at a therapeutic dose for 90 days in vitro. The data was further confirmed in a diseased primate model, with sustained IOP-lowering effects for 5 months with a single implant, with acceptable biocompatibility and partial degradation.

Objective: We previously reported ethnic disparity in adverse outcomes among Asians with type 2 diabetes (T2DM) in Singapore. Central arterial stiffness can aggravate systemic vasculopathy by propagating elevated systolic and pulse pressures forward, thereby accentuating global vascular injury. We aim to study ethnic disparity in central arterial stiffness and its determinants in a multi-ethnic T2DM Asian cohort., Methods: Arterial stiffness was estimated by carotid-femoral pulse wave velocity (PWV) and augmentation index (AI) using applanation tonometry method in Chinese (N = 1045), Malays (N = 458) and Indians (N = 468). Linear regression model was used to evaluate predictors of PWV and AI., Results: PWV was higher in Malays (10.1 ± 3.0 m/s) than Chinese (9.7 ± 2.8 m/s) and Indians (9.6 ± 3.1 m/s) (P = 0.018). AI was higher in Indians (28.1 ± 10.8%) than Malays (25.9 ± 10.1%) and Chinese (26.1 ± 10.7%) (P < 0.001). Malays remain associated with higher PWV (β = 0.299, P = 0.048) post-adjustment for age, gender, duration of diabetes, hemoglobin A1c, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), soluble receptor for advanced glycation end-products, urinary albumin-to-creatinine ratio, and insulin usage, which were all independent predictors of PWV. Indians remain associated with higher AI (β = 2.776, P < 0.001) post-adjustment for age, gender, BMI, SBP, DBP, and height, which were independent predictors of AI. These variables explained 27.7% and 33.4% of the variance in PWV and AI respectively., Conclusions: Malays and Indians with T2DM have higher central arterial stiffness, which may explain their higher risk for adverse outcomes. Modifying traditional major vascular risk factors may partially alleviate their excess cardiovascular risk through modulating arterial stiffness., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background and Aims: The association of adhesion molecules ICAM-1 and VCAM-1 with cardiovascular diseases has been well-studied. However, their roles in diabetic kidney disease (DKD) are incompletely understood. We aim to study the association of plasma ICAM-1 and VCAM-1 with DKD in Asians with type 2 diabetes (T2DM)., Subjects and Methods: A total of 1950 Asians with T2DM were included in this cross-sectional study. Plasma ICAM-1 and VCAM-1 were measured by immunoassays., Results: Renal filtration function (eGFR) declined and urinary albumin-to-creatinine ratio (ACR) levels increased progressively with the increase in plasma VCAM-1 levels. In contrast, no significant changes in eGFR and ACR were observed in subjects across different plasma ICAM-1 levels. Both ICAM-1 and VCAM-1 were correlated with ACR (rho = 0.153, p < 0.001 for VCAM-1 and ACR; rho = 0.053, p = 0.020 for ICAM-1 and ACR) in bivariate correlation analysis. However, only VCAM-1 was correlated with eGFR (rho = -0.228, p < 0.001). Multivariable linear regression models revealed that VCAM-1, but not ICAM-1, was independently associated with eGFR and albuminuria. Backward linear regression suggested that plasma VCAM-1 variability was mainly determined by eGFR whereas plasma ICAM-1 level was mainly determined by C-reactive protein in patients with T2DM., Conclusions: Plasma VCAM-1 level, but not ICAM-1 level, was independently associated with prevalent DKD in Asians with T2DM. High level of ICAM-1 may be indicative of systemic inflammation and portends increase risk of incipient DKD., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: Diabetes mellitus (DM) is a major cause of chronic kidney disease (CKD). The epidemiology of CKD secondary to type 2 DM (T2DM) (i.e. diabetic nephropathy (DN)) has not been well studied in Singapore, a multi-ethnic Asian population. We aimed to determine the prevalence of CKD in adult patients with T2DM., Materials and Methods: We conducted a cross-sectional study on patients (n = 1861) aged 21 to 89 years with T2DM who had attended the DM centre of a single acute care public hospital or a primary care polyclinic between August 2011 and November 2013. Demographic and clinical data were obtained from patients using a standard questionnaire. Spot urine and fasting blood samples were sent to an accredited hospital laboratory for urinary albumin, serum creatinine, HbA1c and lipid measurement. CKD was defined and classified using the 2012 Kidney Disease: Improving Global Outcomes (KDIGO) guidelines and classification., Results: The distribution by risk of adverse CKD outcomes was: low risk, 47%; moderate risk, 27.2%; high risk, 12.8%; and very high risk, 13%. The prevalence of CKD in patients with T2DM was 53%. Variables significantly associated with CKD include neuropathy, blood pressure ≥140/80 mmHg, triglycerides ≥1.7 mmol, body mass index, duration of diabetes, HbA1c ≥8%, age, cardiovascular disease, and proliferative retinopathy., Conclusion: CKD was highly prevalent among patients with T2DM in Singapore. Several risk factors for CKD are well recognised and amenable to intervention. Routine rigorous screening for DN and enhanced programme for global risk factors reduction will be critical to stem the tide of DN.

Cardiovascular disease is the leading cause of morbidity and mortality in type 2 diabetes mellitus. We evaluated the predictive ability of the recently developed body adiposity index for aortic stiffness, an intermediate endpoint of cardiovascular disease, in a cross-sectional multi-ethnic Asian type 2 diabetes mellitus cohort (N = 1408). AS was estimated using carotid-femoral pulse wave velocity measured by applanation tonometry. Body adiposity index was computed as hip circumference/(height)(1.5) - 18. Compared to body mass index, waist circumference and visceral fat area, body adiposity index displayed the weakest association with pulse wave velocity (r = 0.077, 0.096, 0.134 and 0.058, respectively; all p < 0.05). Interestingly, the relationship between measurements of obesity and pulse wave velocity was ethnic dependent - body mass index, body adiposity index, waist circumference and visceral fat area consistently predicted pulse wave velocity only in Indians but not others. In multi-variable analysis, body mass index was a significant determinant of pulse wave velocity in all ethnicities. In conclusion, body adiposity index is a weak predictor of aortic stiffness (when compared with body mass index) in Asians with type 2 diabetes mellitus., (© The Author(s) 2014.)

The plasma membrane organization of live cells defines a plethora of cellular processes important for cell functionality. Many membrane structures that define this organization exist at a spatial resolution below the optical diffraction limit and are highly dynamic. Therefore, a method with millisecond time resolution and nanometer spatial resolution is required for the investigation of plasma membrane organization. However, spatial and temporal resolutions of the currently available biophysical techniques are often mutually exclusive. In a novel realization, Lenne and coworkers developed a spot-variation modality of fluorescence correlation spectroscopy (FCS), also known as FCS diffusion law, to harvest nanoscopic information from microscopic measurements. The FCS diffusion law, so far, has been instrumental to decode the physico-chemical origin of membrane organization and its relationship with biological processes. Overall, the structural information of plasma membrane obtained by FCS diffusion law provides a better understanding of its coupling to the underlying cellular processes.

Nanocarriers have been explored for delivering drugs and other bioactive molecules for well over 35years. Since the introduction of Doxil®, a nanoliposomal delivery system for the cancer drug doxorubicin, several products have been approved worldwide. The majority of these products focus on cancer chemotherapy, and utilize the size advantage of nanocarriers to obtain a favourable distribution of the drug carrier in the human body. In general, such carriers do not sustain drug release over more than a few days at best. In this review, we explore the reasons for this, and present an overview of successful research that is capable of generating sustained-release products in non-cancer applications. A variety of nanocarriers have been studied, and their advantages and shortcomings are highlighted in this review. The achievement of sustained release of bioactive molecules opens new doors in nanotherapeutics., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This paper reviews the latest research and development of drug-eluting stents. The emphasis is on coronary stenting, and both biostable and bioerodible stents are covered in this review. The advantages and shortcomings of the bioactive molecules used in these stents are analyzed, along with the rationale for using bioerodible coatings. The overall emphasis is on the performance of these stents in the clinic. Based on the evaluation of the different stent types, we conclude that fully-erodible stents with a coating of antiproliferative drug will slowly gain market share in the near future, and that the search for a more selective anti-proliferative compound will continue. Dual-drug eluting stents (DDESs) will have their market share but possibly a much smaller one than that for single-drug eluting stents due to the complexities and costs of DDES unless significantly superior performance is demonstrated in the clinic., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Despite the success that drug-eluting stents (DESs) have achieved for minimizing in-stent restenosis (ISR), the antirestenotic agents used in DES have been implicated in delayed endothelial healing and impairment of endothelial functions. Cenderitide (CD-NP) is a novel antiproliferation chimeric peptide of semiendothelial origin; thus, this paper aims to demonstrate the selectivity aspect of this new peptide via in vitro evaluation on key players in ISR-smooth muscle cells (SMCs) and endothelial cells. The microbicinchoninic acid protein assay was used to investigate the CD-NP release from films and stents. Cenderitide-containing films blended with poly(ethylene glycol) and its copolymer exhibited higher release kinetics compared with neat poly(ε-caprolactone) (PCL) formulation. Cenderitide-eluting stents (CES) was produced by coating bare metallic stents with CD-NP entrapped PCL using an ultrasonic spray coater. The investigation of CD-NP on in vitro cells revealed that CD-NP inhibits human coronary smooth muscle cells (HCaSMCs) proliferation but exhibits no effects on human umbilical vein endothelial cells (HUVECs) proliferation. Moreover, CD-NP released up to 7 days displayed inhibitory effects on SMCs proliferation. The CES produced in this work shows that the released CD-NP inhibits HCaSMCs proliferation but did not hamper HUVECs proliferation in vitro, suggesting that it has potential to reduce ISR without retarding the endothelialization healing in vivo., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Background: High normal albuminuria is associated with higher cardiovascular risk in patients with diabetes. Increased aortic stiffness is an established risk factor of vascular events. However, the relationship between albuminuria within the normal range (0-30 mg/g) and aortic stiffness in patients with Type 2 diabetes is unknown., Methods: A total of 614 normoalbuminuric subjects with Type 2 diabetes with spot urinary albumin:creatinine ratio ≤ 30 mg/g and estimated glomerular filtration rate ≥ 60 ml min⁻¹ 1.73 m⁻² were included in the study. Aortic stiffness was assessed by carotid-femoral pulse wave velocity., Results: Pulse wave velocity increased progressively with the increase of albumin:creatinine ratio within the normoalbuminuric range (0-30 mg/g). Only 2.6% of the subjects with an albumin:creatinine ratio in the lowest quartile (0.7-3.4 mg/g) were classified as having aortic stiffness (pulse wave velocity ≥12 m/s). In contrast, the proportion of subjects with aortic stiffness increased significantly with the increase of albumin:creatinine ratio level (11.0%, 10.4% and 13.6% in albumin:creatinine ratio quartiles 2, 3 and 4, respectively, P = 0.008). A logistic regression model revealed that the odds of having aortic stiffness were increased by 56% with a 1-SD increase of log albumin:creatinine ratio after adjustment for age, gender, duration of diabetes, HbA1c , blood pressure, HDL and LDL cholesterol, estimated glomerular filtration rate, BMI, usage of renin-angiotensin system antagonists, statins and insulin., Conclusions: High normal albuminuria is associated with aortic stiffness in patients with Type 2 diabetes, which may in part explain their increased cardiovascular risk., (© 2014 The Authors. Diabetic Medicine © 2014 Diabetes UK.)

Objective: Type 2 diabetes (T2DM) among the young population has become a serious concern globally, presumably due to the rising trend of obesity. Compared to other forms of diabetes, young-onset T2DM experiences more cardiovascular events and other vascular complications although the underlying mechanisms remain largely unknown. Increased arterial stiffness is a hallmark of vasculopathy. We aim to study the clinical and metabolic determinants of arterial stiffness in a cohort of multi-ethnic Asians with young-onset T2DM., Methods: 179 subjects with T2DM onset age below 30 years old were selected in this cross sectional study. Arterial stiffness was assessed by carotid-femoral pulse wave velocity (PWV)., Results: PWV was correlated with age, duration of diabetes, systolic blood pressure, alanine aminotransferase, urinary albumin-to-creatinine ratio (ACR) and eGFR in bivariate correlation analysis. However, PWV was only significantly correlated with body mass index (BMI), waist circumference, urinary ACR and eGFR after adjustment for age. Overweight individuals with young-onset T2DM had significantly higher PWV levels compared to their lean counterparts (7.3 ± 2.4 m/s vs 6.4 ± 2.3 m/s, p = 0.072 and p < 0.0001 without and with adjustment for age, respectively). Multivariable regression models revealed that age, BMI, eGFR and usage of insulin were independently associated with PWV. These 4 variables explained 35.5% variance in PWV levels., Conclusion: Age, BMI, renal function and insulin usage are the main determinants of PWV levels in Asians with young-onset T2DM. Notably, obesity is a modifiable determinant of arterial stiffness independent of high blood pressure, dyslipidemia and hyperglycemia in this population., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Aims: Osteoprotegerin (OPG) is a glycoprotein from tumour necrosis factor receptor superfamily, responsible for osteoclastogenesis inhibition and associated with arterial calcification and stiffness. We describe the association between metabolic syndrome (MS) and OPG in type 2 diabetes mellitus patients., Methodology: We consecutively enrolled 1220 patients from our institution's Diabetes Centre from August 2011. Anthropometric data such as fasting blood/urine were obtained, and OPG was measured by enzyme-linked immunosorbent assay (ELISA)., Results: Mean (standard deviation (SD)) of age and diabetes duration was 57.4 (10.9) years and 11.2 (8.9) years, respectively. Prevalence of MS was 64.3% (95% confidence interval (CI): 61.3%-67.2%) and associated with significantly higher OPG (5.44 vs 4.47 pmol/L) and microvascular complications. The presence of microvascular complications was associated with higher OPG: nephropathy (5.54 (2.20) vs 4.65 (1.70) pmol/L, p < 0.0001), neuropathy (6.33 (2.64) vs 5.06 (1.91) pmol/L, p < 0.0001) and retinopathy (6.08 (2.47) vs 5.00 (1.95) pmol/L, p < 0.0001). After adjusting for age, gender, ethnicity, glucose and microvascular complications, OPG remained an independent predictor of MS: (odds ratio (OR) = 1.102 (95% CI: 1.015-1.196), p = 0.021)., Conclusion: Higher OPG levels were associated with risk of MS and microvascular complications. Studies are needed to test whether OPG could be a useful biomarker identifying patients at risk of vascular complications and whether further exploration of this pathway may lead novel therapeutic options., (© The Author(s) 2014.)

In this work, we focused on the development and investigation of controlled release matrices for a novel cardiotherapeutic peptide, cenderitide (CD-NP) that has shown to be useful for control of ventricular remodeling. To circumvent the hydrophilicity disparity between CD-NP and hydrophobic polymer matrix, a cosolvent system (water/dichloromethane) was selected for investigation. The effect of emulsification conditions, addition of poly(ethylene glycol) (PEG) and its copolymer on the release mechanism and profile were investigated. To verify the retention of bioactivity of entrapped CD-NP in different formulations, the generation of 3',5' cyclic guanosine monophospate (cGMP) and the inhibition of human cardiac fibroblast (HCF) were evaluated. The results showed that neat poly(ε-caprolactone) matrices carried out via two distinct emulsification conditions had either an unacceptably high burst or incomplete release of CD-NP; and the addition of PEG and its copolymer obtained intermediate profiles. Our confocal laser scanning microscopy and surface morphological investigations showed that the copolymer excipient was superior in playing stabilizer role by colocalizing and redistributing peptide throughout the matrix, making the release less sensitive to emulsification conditions. Furthermore, the released CD-NP is able to generate the cGMP and inhibit the HCF proliferation. Our investigations showed that CD-NP-loaded platforms can be a feasible option to provide sustained antifibrotic moderation of fibrotic scar formation and be potentially used to alleviate the adverse effects of cardiac remodeling., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Cenderitide, also known as CD-NP, is a designer peptide developed by combining native mammalian c-type natriuretic peptide (CNP) and the C-terminus isolated from the dendroapis natriuretic peptide (DNP) of the venom from the green mamba. In early studies, intravenous and subcutaneous infusion of cenderitide was reported to reduce left ventricular (LV) mass and ameliorate cardiac remodelling. In this work, biodegradable polymeric films encapsulating CD-NP were developed and were investigated for their in vitro release and degradation characteristics. Subsequently, the bioactivity of released peptide and its effects on human cardiac fibroblast (HCF) were explored. We achieved sustained release from three films with low, intermediate and high release profiles for 30 days. Moreover, the bioactivity of released peptide was verified from the elevated production of cyclic guanosine monophospate (cGMP). The CD-NP released from films was able to inhibit the proliferation of hypertrophic HCF as well as suppress DNA synthesis in HCF. Furthermore, the sustained delivery from films showed comparable or superior suppressive actions on hypertrophic HCF compared to daily infusion of CD-NP. The results suggest that these films could be used to inhibit fibrosis and reduce cardiac remodelling via local delivery as cardiac patches.

Aims: Irisin is a novel myokine secreted in response to PPAR-γ co-activator-1α (PGC-1α) activation. Earlier studies suggested that PGC-1α expression and activity were lower in myocytes in type 2 diabetes mellitus (T2DM). Therefore, we hypothesize that circulating irisin levels are lower in T2DM patients., Methods: In this observational study, we recruited 96 T2DM subjects and 60 non-diabetic control subjects. Among T2DM subjects, 38% were on insulin treatment, 78% were taking statins and 72% were taking renin-angiotensin system antagonists. Circulating irisin was quantified by ELISA and its association with markers of metabolic phenotype was analyzed by Pearson bivariate correlation and multiple linear regression., Results: Circulating irisin was significantly lower in individuals with T2DM compared with non-diabetic controls (T2DM 204 ± 72 ng/ml vs. non-diabetic control 257 ± 24 ng/ml, p < 0.0001). In non-diabetic subjects, circulating irisin was correlated with age (r = 0.398, p < 0.01), BMI (r = 0.387, p < 0.01), total cholesterol (r = 0.341, p < 0.01), total triglycerides (r = 0.299, p < 0.05), fasting blood glucose (r = 0.430, p < 0.01) and diastolic blood pressure (r = 0.306, p < 0.05). Multiple linear regression model revealed that BMI (β = 0.407, p = 0.012) and FBG (β = 0.315, p = 0.034) were associated with irisin in non-diabetic subjects after adjusting for multiple co-variates. However, similar analysis in T2DM subjects didn't reveal significant association between circulating irisin and major markers of metabolic phenotype., Conclusions: Circulating irisin is lower in T2DM compared with non-diabetic controls. Plasma irisin levels appear to be associated with important metabolic factors in non-diabetic subjects but not in individuals with type 2 diabetes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Correction of perinatally lethal neurogenetic diseases requires efficient transduction of several cell types within the relatively inaccessible CNS. Intravenous AAV9 delivery in mouse has achieved development stage-specific transduction of neuronal cell types, with superior neuron-targeting efficiency demonstrated in prenatal compared with postnatal recipients. Because of the clinical relevance of the non-human primate (NHP) model, we investigated the ability of AAV9 to transduce the NHP CNS following intrauterine gene therapy (IUGT). We injected two macaque fetuses at 0.9 G with 1 × 10(13) vg scAAV9-CMV-eGFP through the intrahepatic continuation of the umbilical vein. Robust green fluorescent protein (GFP) expression was observed for up to 14 weeks in the majority of neurons (including nestin-positive cells), motor neurons and oligodendrocytes throughout the CNS, with a significantly lower rate of transduction in astrocytes. Photoreceptors and neuronal cell bodies in the plexiform and ganglionic retinal layers were also transduced. In the peripheral nervous system (PNS), widespread transduction of neurons was observed. Tissues harvested at 14 weeks showed substantially lower vector copy number and GFP levels, although the percentage of GFP-expressing cells remained stable. Thus, AAV9-IUGT in late gestation efficiently transduces both the CNS and PNS with neuronal predilection, of translational relevance to hereditary disorders characterized by perinatal onset of neuropathology.

Quantification of protein-polymer colocalization in a phase-separated polymer blend gives important insights into the protein release mechanism. Here, we report on the first visualization of protein-poly(ethylene glycol) (protein-PEG) colocalization in poly(ε-caprolactone)/poly(ethylene glycol) (PCL/PEG) blend films using a combined application of confocal Raman mapping and confocal laser scanning microscopy (CLSM) imaging. The degree of protein-PEG colocalization was further quantified via a novel image processing technique. This technique also allowed us to characterize the 3-D protein distribution within the films. Our results showed that the proteins were homogeneously distributed within the film matrix, independent of PEG content. However, the degree of protein-PEG colocalization was inversely proportional to PEG content, ranging from 65 to 94%. This quantitative data on protein-PEG colocalization was used along with in vitro PEG leaching profile to construct a predictive model for overall protein release. Our prediction matched well with the experimental protein release profile, which is characterized by an initial burst release and a subsequent slower diffusional release. More importantly, the success of this predictive model has highlighted the influence of protein-PEG colocalization on the protein release mechanism.