Topiramate is an insulin-sensitizing compound in vivo with direct effects on adipocytes in female ZDF rats

We have studied the in vivo and in vitro effects of Topiramate (TPM) in female Zucker diabetic fatty (ZDF) rats. After weight matching, drug treatment had a marked effect to lower fasting glucose levels of relatively normoglycemic animals as well as during an oral glucose tolerance test. The glucose clamp studies revealed a approximately 30% increased glucose disposal, increased hepatic glucose output (HGO) suppression from approximately 30 to 60%, and an increased free fatty acid suppression from 40 to 75%. Therefore, TPM treatment led to enhanced insulin sensitivity at the level of tissue glucose disposal (increased ISGDR), liver (increased inhibition of HGO), and adipose tissue (enhanced suppression of lipolysis). When soleus muscle strips of control or TPM-treated ZDF rats were studied ex vivo, insulin-stimulated glucose transport was not enhanced in the drug-treated animals. In contrast, when isolated adipocytes were studied ex vivo, a marked increase (+55%) in insulin-stimulated glucose transport was observed. In vitro treatment of muscle strips and rat adipocytes showed no effect on glucose transport in muscle with a 40% increase in insulin-stimulated adipocyte glucose transport. In conclusion, 1) TPM treatment leads to a decrease in plasma glucose and increased in vivo insulin sensitivity; 2) insulin sensitization was observed in adipocytes, but not muscle, when tissues were studied ex vivo or in vitro; and 3) TPM directly enhances insulin action in insulin-resistant adipose cells in vitro. Thus the in vivo effects of TPM treatment appear to be exerted through adipose tissue.     

Synthesis and evaluation of 3-anilino-quinoxalinones as glycogen phosphorylase inhibitors

A series of 3-anilino-quinoxalinones has been identified as a new class of glycogen phosphorylase inhibitors. The lead compound 1 was identified through high throughput screening as well as through pharmacophore-based electronic screening. Modifications were made to the scaffold of 1 to produce novel analogues, some of which are 25 times more potent than the lead compound.     

Induction of homozygous Spathiphyllum wallisii genotypes through gynogenesis

In Spathiphyllum wallisii the production of doubled haploids was attempted. Different combinations of growth regulators were tested, as well as different cultivars. The use of TDZ (0.25–1 M) in ovary cultures of Spathiphyllum was required. On the contrary, cytokinins were not crucial during ovule culture; in fact, the use of a too high TDZ concentration induced diploid parthenogenesis in ovules of `Alfa'. The use of the imidazole fungicides IMA, PRO or TRI during ovary culture was not critical, though they enhanced the swelling of ovules during ovary culture, making the isolation of the ovules easier. The ovule cultures yielded different plantlets. Flow cytometry and AFLP-patterns showed that two doubled haploid genotypes could be obtained from `Stefanie'. These plants showed a normal phenotype. We concluded that the induction of homozygous Spathiphyllum through gynogenesis is possible and is genotype dependent.     

Accuracy and Reliability of a Novel Method for Fusion of Digital Dental Casts and Cone Beam Computed Tomography Scans

Several methods have been proposed to integrate digital models into Cone Beam Computed Tomography scans. Since all these methods have some drawbacks such as radiation exposure, soft tissue deformation and time-consuming digital handling processes, we propose a new method to integrate digital dental casts into Cone Beam Computed Tomography scans. Plaster casts of 10 patients were randomly selected and 5 titanium markers were glued to the upper and lower plaster cast. The plaster models were scanned, impressions were taken from the plaster models and the impressions were also scanned. Linear measurements were performed on all three models, to assess accuracy and reproducibility. Besides that, matching of the scanned plaster models and scanned impressions was done, to assess the accuracy of the matching procedure. Results show that all measurement errors are smaller than 0.2 mm, and that 81% is smaller than 0.1 mm. Matching of the scanned plaster casts and scanned impressions show a mean error between the two surfaces of the upper arch of 0.14 mm and for the lower arch of 0.18 mm. The time needed for reconstructing the CBCT scans to a digital patient, where the impressions are integrated into the CBCT scan of the patient takes about 15 minutes, with little variance between patients. In conclusion, we can state that this new method is a reliable method to integrate digital dental casts into CBCT scans. As far as radiation exposure, soft tissue deformation and digital handling processes are concerned, it is a significant improvement compared to the previously published methods.     

Fab-based bispecific antibody formats with robust biophysical properties and biological activity

A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with C_H1/C_L domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.     

Trolox and Ascorbic Acid Reduce Direct and Indirect Oxidative Stress in the IPEC-J2 Cells,an In Vitro Model for the Porcine Gastrointestinal Tract

Oxidative stress in the small intestinal epithelium is a major cause of barrier malfunction and failure to regenerate. This study presents a functional in vitro model using the porcine small intestinal epithelial cell line IPEC-J2 to examine the effects of oxidative stress and to estimate the antioxidant and regenerative potential of Trolox, ascorbic acid and glutathione monoethyl ester. Hydrogen peroxide and diethyl maleate affected the tight junction (zona occludens-1) distribution, significantly increased intracellular oxidative stress (CM-H₂DCFDA) and decreased the monolayer integrity (transepithelial electrical resistance and FD-4 permeability), viability (neutral red) and wound healing capacity (scratch assay). Trolox (2 mM) and 1 mM ascorbic acid pre-treatment significantly reduced intracellular oxidative stress, increased wound healing capacity and reduced FD-4 permeability in oxidatively stressed IPEC-J2 cell monolayers. All antioxidant pre-treatments increased transepithelial electrical resistance and viability only in diethyl maleate-treated cells. Glutathione monoethyl ester (10 mM) pre-treatment significantly decreased intracellular oxidative stress and monolayer permeability only in diethyl maleate-treated cells. These data demonstrate that the IPEC-J2 oxidative stress model is a valuable tool to screen antioxidants before validation in piglets.     

NAD+ supplementation prevents STING‐induced senescence in ataxia telangiectasia by improving mitophagy

Senescence phenotypes and mitochondrial dysfunction are implicated in aging and in premature aging diseases, including ataxia telangiectasia (A‐T). Loss of mitochondrial function can drive age‐related decline in the brain, but little is known about whether improving mitochondrial homeostasis alleviates senescence phenotypes. We demonstrate here that mitochondrial dysfunction and cellular senescence with a senescence‐associated secretory phenotype (SASP) occur in A‐T patient fibroblasts, and in ATM‐deficient cells and mice. Senescence is mediated by stimulator of interferon genes (STING) and involves ectopic cytoplasmic DNA. We further show that boosting intracellular NAD⁺ levels with nicotinamide riboside (NR) prevents senescence and SASP by promoting mitophagy in a PINK1‐dependent manner. NR treatment also prevents neurodegeneration, suppresses senescence and neuroinflammation, and improves motor function in Atm^−/− mice. Our findings suggest a central role for mitochondrial dysfunction‐induced senescence in A‐T pathogenesis, and that enhancing mitophagy as a potential therapeutic intervention.     

Encapsulation performance of layer-by-layer microcapsules for proteins

This study reports on the encapsulation efficiency of proteins in dextran sulfate/poly-L-arginine-based microcapsules, fabricated via layer-by-layer assembly (LbL). For this purpose, radiolabeled proteins are entrapped in CaCO(3) microparticles, followed by LbL coating of the CaCO(3) cores and subsequent dissolving of the CaCO(3) using EDTA. To allow to improve protein encapsulation in LbL microcapsules, we studied all steps in the preparation of the microcapsules where loss of protein load might occur. The encapsulation efficiency of proteins in LbL microcapsules turns out to be strongly dependent on both the charge and molecular weight of the protein as well as on the number of polyelectrolyte bilayers the microcapsules consist of.