In Vitro Formation of β Cell Pseudoislets Using Islet-Derived Endothelial Cells

β cell pseudoislets (PIs) are used for the in vitro study of β-cells in a three-dimensional (3-D) configuration. Current methods of PI induction require unique culture conditions and extensive mechanical manipulations. Here we report a novel co-culture system consisting of high passage β-cells and islet-derived endothelial cells (iECs) that results in a rapid and spontaneous formation of free-floating PIs. PI structures were formed as early as 72 h following co-culture setup and were preserved for more than 14 d. These PIs, composed solely of β-cells, were similar in size to that of native islets and showed an increased percentage of proinsulin-positive cells, increased insulin gene expression in response to glucose stimulation, and restored glucose-stimulated insulin secretion when compared to β-cells cultured as monolayers. Key extracellular matrix proteins that were absent in β-cells cultured alone were deposited by iECs on PIs and were found in and around the PIs. iEC-induced PIs are a readily available tool for examining β cell function in a native 3-D configuration and can be used for examining β-cell/iEC interactions in vitro.     

Short and long-term safety of the 2009 AS03-adjuvanted pandemic vaccine

Background

This study assessed the short and the long term safety of the 2009 AS03 adjuvanted monovalent pandemic vaccine through an active web-based electronic surveillance. We compared its safety profile to that of the seasonal trivalent inactivated influenza vaccine (TIV) for 2010–2011.

Methodology/Principal Findings

Health care workers (HCW) vaccinated in 2009 with the pandemic vaccine (Arepanrix ® from GSK) or HCW vaccinated in 2010 with the 2010–2011 TIV were invited to participate in a web-based active surveillance of vaccine safety. They completed two surveys the day-8 survey covered the first 7 days post-vaccination and the day-29 survey covered events occurring 8 to 28 days after vaccination. Those who reported a problem were called by a nurse to obtain details. The main outcome was the occurrence of a new health problem or the worsening of an existing health condition that resulted in a medical consultation or work absenteeism. For the pandemic vaccine, a six-month follow-up for the occurrence of serious adverse events (SAE) was conducted. Among the 6242 HCW who received the pandemic vaccine, 440 (7%) reported 468 events compared to 328 of the 7645 HCW (4.3%) who reported 339 events after the seasonal vaccine. The 2009 pandemic vaccine was associated with significantly more local reactions than the 2010–2011 seasonal vaccine (1% vs. 0.03%, p<0.001). Paresthesia was reported by 7 HCW (0.1%) after the pandemic vaccine but by none after the seasonal vaccine. For the pandemic vaccine, no clustering of SAE was found in the 6 month follow-up.

Conclusion

The 2009 pandemic vaccine seems to have a good safety profile, similar to the 2010–2011 TIV, with the exception of local reactions. This surveillance was adequately powered to identify AE associated with an excess risk ≥1 per 1000 vaccinations but is insufficient to detect rare AE.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01289418","term_id":"NCT01289418"}}NCT01289418, {"type":"clinical-trial","attrs":{"text":"NCT01318876","term_id":"NCT01318876"}}NCT01318876     

Expression of voltage-gated K+ channels in insulin-producing cells. Analysis by polymerase chain reaction.

We have used the polymerase chain reaction (PCR) with primers against the S5 and S6 regions of voltage-gated K+ channels to identify 8 different specific amplification products using poly(A)+ RNA isolated from islets of Langerhans from obese hyperglycemic (ob/ob) mice and from the two insulin-producing cell lines HIT T15 and RINm5F. Sequence analysis suggests that they derive from mRNAs coding for a family of voltage-gated K+ channels; 5 of these have been recently identified in mammalian brain and 3 are novel. These hybridize in classes to different mRNAs which distribute differently to a number of tissues and cell lines including insulin-producing cells.     

Cutaneous blood supply of the penis

Twelve male cadaver specimens were injected with a latex solution to define the cutaneous blood supply of the penis. The cutaneous blood supply of the penile shaft is derived solely from a pair of axial arteries running in the dartos layer. Additional deep perforating arteries from the dorsal penile artery and corporal vessels supply the glans and subcoronal region. An understanding of this anatomy allows one to develop safely a variety of penile skin flaps for difficult reconstructive problems.     

Copper Modulation of Ion Channels of PrP[106–126] Mutant Prion Peptide Fragments

We have shown previously that the protease-resistant and neurotoxic prion peptide fragment PrP[106-126] of human PrP incorporates into lipid bilayer membranes to form heterogeneous ion channels, one of which is a Cu(2+)-sensitive fast cation channel. To investigate the role of PrP[106-126]'s hydrophobic core, AGAAAAGA, on its ability to form ion channels and their regulation with Cu(2+), we used the lipid-bilayer technique to examine membrane currents induced as a result of PrP[106-126] (AA/SS) and PrP[106-126] (VVAA/SSSS) interaction with lipid membranes and channel formation. Channel analysis of the mutant (VVAAA/SSS), which has a reduced hydrophobicity due to substitution of hydrophobic residues with the hydrophilic serine residue, showed a significant change in channel activity, which reflects a decrease in the beta-sheet structure, as shown by CD spectroscopy. One of the channels formed by the PrP[106-126] mutant has fast kinetics with three modes: burst, open and spike. The biophysical properties of this channel are similar to those of channels formed with other aggregation-prone amyloids, indicating their ability to form the common beta sheet-based channel structure. The current-voltage (I-V) relationship of the fast cation channel, which had a reversal potential, E(rev), between -40 and -10 mV, close to the equilibrium potential for K(+) ( E(K) = -35 mV), exhibited a sigmoidal shape. The value of the maximal slope conductance (g(max)) was 58 pS at positive potentials between 0 and 140 mV. Cu(2+) shifted the kinetics of the channel from being in the open and "burst" states to the spike mode. Cu(2+) reduced the probability of the channel being open (P(o)) and the mean open time (T(o)) and increased the channel's opening frequency (F(o)) and the mean closed time (T(c)) at a membrane potential ( V(m)) between +20 and + 140 mV. The fact that Cu(2+) induced changes in the kinetics of this channel with no changes in its conductance, indicates that Cu(2+) binds at the mouth of the channel via a fast channel block mechanism. The Cu(2+)-induced changes in the kinetic parameters of this channel suggest that the hydrophobic core is not a ligand Cu(2+) site, and they are in agreement with the suggestion that the Cu(2+)-binding site is located at M(109) and H(111) of this prion fragment. Although the data indicate that the hydrophobic core sequence plays a role in PrP[106-126] channel formation, it is not a binding site for Cu(2+). We suggest that the role of the hydrophobic region in modulating PrP toxicity is to influence PrP assembly into neurotoxic channel conformations. Such conformations may underlie toxicity observed in prion diseases. We further suggest that the conversions of the normal cellular isoform of prion protein (PrP(c)) to abnormal scrapie isoform (PrP(Sc)) and intermediates represent conversions to protease-resistant neurotoxic channel conformations.     

7-(4H-1,2,4-Triazol-3-yl)benzo[c][2,6]naphthyridines: a novel class of Pim kinase inhibitors with potent cell antiproliferative activity

A novel class of pan-Pim kinase inhibitors was designed by modifying the CK2 inhibitor CX-4945. Introduction of a triazole or secondary amide functionality on the C-7 position and 2'-halogenoanilines on C-5 resulted in potent inhibitors of the Pim-1 and Pim-2 isoforms, with many analogs active at single digit nanomolar concentrations. The molecules inhibited the phosphorylation at Serine 112 of the apoptosis effector BAD, and had potent antiproliferative effects on the AML cell line MV-4-11 (IC(50) <30 nM). This work delivers an excellent lead-optimization platform for Pim targeting anticancer therapies.     

Islet Endothelial Cells Induce Glycosylation and Increase Cell-surface Expression of Integrin β1 in β Cells

The co-culturing of insulinoma and islet-derived endothelial cell (iEC) lines results in the spontaneous formation of free-floating pseudoislets (PIs). We previously showed that iEC-induced PIs display improved insulin expression and secretion in response to glucose stimulation. This improvement was associated with a de novo deposition of extracellular matrix (ECM) proteins by iECs in and around the PIs. Here, iEC-induced PIs were used to study the expression and posttranslational modification of the ECM receptor integrin β1. A wide array of integrin β subunits was detected in βTC3 and NIT-1 insulinomas as well as in primary islets, with integrin β1 mRNA and protein detected in all three cell types. Interestingly, the formation of iEC-induced PIs altered the glycosylation patterns of integrin β1, resulting in a higher molecular weight form of the receptor. This form was found in native pancreas but was completely absent in monolayer β-cells. Fluorescence-activated cell sorting analysis of monolayers and PIs revealed a higher expression of integrin β1 in PIs. Antibody-mediated blocking of integrin β1 led to alterations in β-cell morphology, reduced insulin gene expression, and enhanced glucose secretion under baseline conditions. These results suggest that iEC-induced PI formation may alter integrin β1 expression and posttranslational modification by enhancing glycosylation, thereby providing a more physiological culture system for studying integrin-ECM interactions in β cells.     

Trophic ecology of parabiotic ants: Do the partners have similar food niches?

Organisms associated with another species may experience both costs and benefits from their partner. One of these costs is competition, which is the more likely if the two species are ecologically similar. Parabioses are associations between two ant species that share a nest and often attend the same food sources. Albeit parabioses are probably mutualistic, parabiotic partners may compete for food. We therefore investigated feeding niches and dietary overlap of two parabiotically associated ants in Borneo using cafeteria experiments and stable isotope analyses. The two species strongly differed in their food choices. While Crematogaster modiglianii mostly foraged at carbohydrate‐rich baits, Camponotus rufifemur preferred urea‐rich sources. Both species also consumed animal protein. The ¹⁵N concentration in Ca. rufifemur workers was consistently lower than in Cr. modiglianii. Camponotus rufifemur but not Cr. modiglianii possesses microbial endosymbionts, which can metabolize urea and synthesize essential amino acids. Its lower ¹⁵N signature may result from a relatively higher intake of plant‐based or otherwise ¹⁵N‐depleted nitrogen. Isotopic signatures of the two partners in the same parabiosis showed strongly parallel variation across nests. As we did not find evidence for spatial autocorrelation, this correlation suggests an overlap of food sources between the two ant species. Based on model simulations, we estimated a diet overlap of 22–66% for nitrogen sources and 45–74% for carbon sources. The overlap may arise from either joint exploitation of the same food sources or trophallactic exchange of food. This suggests an intense trophic interaction and potential for competition between the parabiotic partners.