Characterization of Stimulus-Secretion Coupling in the Human Pancreatic EndoC-βH1 Beta Cell Line

Aims/Hypothesis

Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.

Methods

Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay), gene expression (Gene Chip array), metabolite levels (GC/MS), respiration (Seahorse XF24 Extracellular Flux Analyzer), glucose utilization (radiometric), lactate release (enzymatic colorimetric), ATP levels (enzymatic bioluminescence) and plasma membrane potential and cytoplasmic Ca²⁺ responses (microfluorometry) were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.

Results

Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.

Conclusions/Interpretation

Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca²⁺ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the advantage of carrying the human genome, allowing studies of human genetic variants, epigenetics and regulatory RNA molecules.     

Novel potent macrocyclic inhibitors of the hepatitis C virus NS3 protease: use of cyclopentane and cyclopentene P2-motifs

Several highly potent novel HCV NS3 protease inhibitors have been developed from two inhibitor series containing either a P2 trisubstituted macrocyclic cyclopentane- or a P2 cyclopentene dicarboxylic acid moiety as surrogates for the widely used N-acyl-(4R)-hydroxyproline in the P2 position. These inhibitors were optimized for anti HCV activities through examination of different ring sizes in the macrocyclic systems and further by exploring the effect of P4 substituent removal on potency. The target molecules were synthesized from readily available starting materials, furnishing the inhibitor compounds in good overall yields. It was found that the 14-membered ring system was the most potent in these two series and that the corresponding 13-, 15-, and 16-membered macrocyclic rings delivered less potent inhibitors. Moreover, the corresponding P1 acylsulfonamides had superior potencies over the corresponding P1 carboxylic acids. It is noteworthy that it has been possible to develop highly potent HCV protease inhibitors that altogether lack the P4 substituent. Thus the most potent inhibitor described in this work, inhibitor 20, displays a K(i) value of 0.41 nM and an EC(50) value of 9 nM in the subgenomic HCV replicon cell model on genotype 1b. To the best of our knowledge this is the first example described in the literature of a HCV protease inhibitor displaying high potency in the replicon assay and lacking the P4 substituent, a finding which should facilitate the development of orally active small molecule inhibitors against the HCV protease.     

Droplet digital PCR as a tool for investigating dynamics of cryptic symbionts

Interactions among symbiotic organisms and their hosts are major drivers of ecological and evolutionary processes. Monitoring the infection patterns among natural populations and identifying factors affecting these interactions are critical for understanding symbiont–host relationships. However, many of these interactions remain understudied since the knowledge about the symbiont species is lacking, which hinders the development of appropriate tools. In this study, we developed a digital droplet PCR (ddPCR) assay based on apicomplexan COX1 gene to detect an undescribed agamococcidian symbiont. We show that the method gives precise and reproducible results and enables detecting cryptic symbionts in low target concentration. We further exemplify the assay''s use to survey seasonally sampled natural host (Pygospio elegans) populations for symbiont infection dynamics. We found that symbiont prevalence differs spatially but does not show seasonal changes. Infection load differed between populations and was low in spring and significantly increased towards fall in all populations. We also found that the symbiont prevalence is affected by host length and population density. Larger hosts were more likely to be infected, and high host densities were found to have a lower probability of infection. The observed variations could be due to characteristics of both symbiont and host biology, especially the seasonal variation in encounter rates. Our findings show that the developed ddPCR assay is a robust tool for detecting undescribed symbionts that are otherwise difficult to quantify, enabling further insight into the impact cryptic symbionts have on their hosts.     

Individual-based model highlights the importance of trade-offs for virus-host population dynamics and long-term co-existence

Viruses play diverse and important roles in ecosystems. In recent years, trade-offs between host and virus traits have gained increasing attention in viral ecology and evolution. However, microbial organism traits, and viral population parameters in particular, are challenging to monitor. Mathematical and individual-based models are useful tools for predicting virus-host dynamics. We have developed an individual-based evolutionary model to study ecological interactions and evolution between bacteria and viruses, with emphasis on the impacts of trade-offs between competitive and defensive host traits on bacteria-phage population dynamics and trait diversification. Host dynamics are validated with lab results for different initial virus to host ratios (VHR). We show that trade-off based, as opposed to random bacteria-virus interactions, result in biologically plausible evolutionary outcomes, thus highlighting the importance of trade-offs in shaping biodiversity. The effects of nutrient concentration and other environmental and organismal parameters on the virus-host dynamics are also investigated. Despite its simplicity, our model serves as a powerful tool to study bacteria-phage interactions and mechanisms for evolutionary diversification under various environmental conditions.     

Analysis of protein expression in pure cell nuclei populations isolated from human breast cancer tissue by DNA flow cytometric sorting

In this study, cell nuclei from aneuploid breast cancer samples were sorted with respect to DNA content into pure diploid and aneuploid fractions using flow cytometry. The nuclear proteins were then separated by one-dimensional gel electrophoresis (1D-PAGE) and differences in protein expression patterns, between diploid and aneuploid nuclei from the same tumours, were compared. Using a combination of peptide finger printing and peptide identification by MALDI-TOF mass spectrometry, we identified proteins and confirmed that the proteins were of nuclear origins. The results in this study add further information to the knowledge about the breast cancer disease complexity and heterogeneity at molecular level. For some of the tumours studied different nuclei protein patterns were obtained, in the diploid respective aneuploid nuclei populations, whilst other tumours did not show these differences.     

Probing spinal circuits controlling walking in mammals

Locomotion in mammals is a complex motor act that involves the activation of a large number of muscles in a well-coordinated pattern. Understanding the network organization of the intrinsic spinal networks that control the locomotion, the central pattern generators, has been a challenge to neuroscientists. However, experiments using the isolated rodent spinal cord and combining electrophysiology and molecular genetics to dissect the locomotor network have started to shed new light on the network structure. In the present review, we will discuss findings that have revealed the role of designated populations of neurons for the key network functions including coordinating muscle activity and generating rhythmic activity. These findings are summarized in proposed organizational principles for the mammalian segmental CPG.     

Peptide binding and NMR analysis of the interaction between SAP97 PDZ2 and GluR-A: potential involvement of a disulfide bond

Synaptic delivery of GluR-A (GluR1) subunit-containing glutamate receptors depends on a C-terminal type I PDZ binding motif in GluR-A. Synapse-associated protein 97 (SAP97) is the only PDZ domain protein known to associate with GluR-A. We have used NMR spectroscopy and a biotinylated peptide binding assay to characterize the interaction between synthetic GluR-A C-terminal peptides and the PDZ2 domain of SAP97 (SAP97(PDZ2)), previously determined to be the dominant factor responsible for the interaction. The binding mode appeared to be strongly influenced by redox conditions. Chemical shift changes observed in NMR spectra indicate that under reducing conditions, the last four residues of GluR-A peptides bind to PDZ2 in a fashion typical of class I PDZ interactions. The binding is weak and relatively nonselective as it occurs similarly with a PDZ2 domain derived from PSD-95, a related protein not believed to directly interact with GluR-A. In the absence of reducing agents, conserved cysteine residues in SAP97(PDZ2) and the GluR-A C-terminus gave rise to an anomalous behavior in a microplate assay with a biotinylated GluR-A 18-mer peptide. A covalent disulfide-linked complex between SAP97(PDZ2) and the GluR-A peptide was seen in the binding assay and in the NMR experiments performed under oxidizing conditions. The results are consistent with a two-step binding mechanism consisting of an initial PDZ interaction followed by stabilization of the complex by a disulfide bond. The possible physiological relevance of redox regulation of SAP97-GluR-A interaction remains to be established.     

Predictors of infusion reactions during infliximab treatment in patients with arthritis

In the present study we evaluated the impact of baseline antinuclear antibody (ANA) status and use of methotrexate on development of infliximab-related infusion reactions in patients with rheumatoid arthritis (RA) or spondylarthropathies (SpAs), including psoriatic arthritis. All patients with RA (n = 213) or SpA (n = 76) treated with infliximab during the period 1999–2005 at the Department of Rheumatology in Lund, Sweden were included. ANAs were present in 28% and 25% of RA and SpA patients, respectively. Because of differences in baseline characteristics, we used a binary logistic regression model to calculate odds ratios (ORs), adjusting for age, sex and prednisolone dosage. Altogether 21% of patients with RA and 13% of patients with SpA developed infusion reactions (P = 0.126). The OR for development of infusion reactions in RA patients with baseline ANA positivity alone was 2.1. Infliximab without methotrexate and infliximab as monotherapy were associated with ORs of 3.1 and 3.6, respectively. Combining infliximab without methotrexate and ANA positivity yielded an OR for infusion reaction of 4.6. Lower age at disease onset and longer disease duration were associated with infusion reactions (P = 0.012 and P = 0.036, respectively), but age, sex, C-reactive protein, erythrocyte sedimentation rate, Health Assessment Questionnaire and Disease Activity Score-28 at baseline were not. No predictors of infusions reactions were identified in SpA patients. RA patients treated with infliximab without methotrexate, and who are positive at baseline for ANAs are at increased risk for developing infliximab-related infusion reactions.