Phylogeography and connectivity of molluscan parasites: Perkinsus spp. in Panama and beyond

Panama is a major hub for commercial shipping between two oceans, making it an ideal location to examine parasite biogeography, potential invasions, and the spread of infectious agents. Our goals were to (i) characterise the diversity and genetic connectivity of Perkinsus spp. haplotypes across the Panamanian Isthmus and (ii) combine these data with sequences from around the world to evaluate the current phylogeography and genetic connectivity of these widespread molluscan parasites. We collected 752 bivalves from 12 locations along the coast of Panama including locations around the Bocas del Toro archipelago and the Caribbean and Pacific entrances to the Panama Canal, from December 2012 to February 2013. We used molecular genetic methods to screen for Perkinsus spp. and obtained internal transcribed spacer region (ITS) ribosomal DNA (rDNA) sequences for all positive samples. Our sequence data were used to evaluate regional haplotype diversity and distribution across both coasts of Panama, and were then combined with publicly available sequences to create global haplotype networks. We found 26 ITS haplotypes from four Perkinsus spp. (1–12 haplotypes per species) in Panama. Perkinsus beihaiensis haplotypes had the highest genetic diversity, were the most regionally widespread, and were associated with the greatest number of hosts. On a global scale, network analyses demonstrated that some haplotypes found in Panama were cosmopolitan (Perkinsus chesapeaki, Perkinsus marinus), while others were more geographically restricted (Perkinsus olseni, P. beihaiensis), indicating different levels of genetic connectivity and dispersal. We found some Perkinsus haplotypes were shared across the Isthmus of Panama and several regions around the world, including across ocean basins. We also found that haplotype diversity is currently underestimated and directly related to the number of sequences. Nevertheless, our results demonstrate long-range dispersal and global connectivity for many haplotypes, suggesting that dispersal through shipping probably contributes to these biogeographical patterns.     

The major metabolite of doxorubicin is a potent inhibitor of membrane-associated ion pumps. A correlative study of cardiac muscle with isolated membrane fractions

Doxorubicin (adriamycin) is a highly effective cancer chemotherapeutic drug but its clinical utility is limited by its cardiotoxicity. Doxorubicinol, the major metabolite of doxorubicin, is up to 10 times more potent than doxorubicin at inhibiting isometric contraction of the papillary muscle isolated from the right ventricle of rabbit heart. Doxorubicinol also increases resting tension of isolated cardiac muscle indicative of incomplete relaxation between contractions, a characteristic of doxorubicinol but not of doxorubicin. This study assesses the effect(s) of doxorubicinol on a variety of ion pumps which may explain, in part, the action of the metabolite in the intact muscle. We find the doxorubicinol is a potent inhibitor (IC50 less than 5 micrograms/ml) of calcium-stimulated ATPase activity of sarcoplasmic reticulum from canine heart and rabbit skeletal muscle. At comparable levels, doxorubicinol is also a potent inhibitor of (Na + K)-ATPase of cardiac sarcolemma and the Mg-dependent ATPase activity referable to the F0F1 proton pump of mitochondria. For each of these ion pumps, doxorubicinol is at least 80 times more potent an inhibitor than doxorubicin. Doxorubicinol, between 10 and 50 micrograms/ml, increases resting tension up to 4-fold in isolated papillary muscles cyclically contracting at 30 times/min. Resting stress is relatively insensitive to doxorubicin. Thus, doxorubicinol is a potent inhibitor of several key cationic pumps that directly or indirectly regulate cell calcium and inhibits relaxation in the isolated fiber preparation. These observations add a new dimension to understanding the cardiotoxicity of doxorubicin.     

Isolation of the ryanodine receptor from cardiac sarcoplasmic reticulum and identity with the feet structures

Ryanodine, a highly toxic alkaloid, reacts specifically with the Ca2+ release channels which are localized in the terminal cisternae of sarcoplasmic reticulum (SR). In this study, the ryanodine receptor from cardiac SR has been purified, characterized, and compared with that of skeletal muscle SR. The ryanodine receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of phospholipids. Purification was performed by sequential affinity chromatography followed by gel permeation chromatography in the presence of CHAPS and phospholipids. The enrichment of the receptor from cardiac microsomes was about 110-fold. The purified receptor contained a major polypeptide band of Mr 340,000 with a minor band of Mr 300,000 (absorbance ratio 100/8) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electron microscopy of the purified receptor from heart showed square structures of 222 +/- 21 A/side, which is the unique characteristic of feet structures of junctional face membrane of terminal cisternae of SR. Recently, we isolated the ryanodine receptor from skeletal muscle (Inui, M., Saito, A., and Fleischer, S. (1987) J. Biol. Chem. 262, 1740-1747). The ryanodine receptors from heart and skeletal muscle have similar characteristics in terms of protein composition, morphology, chromatographic behavior, and Ca2+, salt, and phospholipid dependence of ryanodine binding. However, there are distinct differences: 1) the Mr of the receptor is slightly larger for skeletal muscle (Mr approximately 360,000); 2) the purified receptor from heart contains two different affinities for ryanodine binding with Kd values in the nanomolar and micromolar ranges, contrasting with that of skeletal muscle SR which shows only the high affinity binding; 3) the affinity of the purified cardiac receptor for ryanodine was 4-5-fold higher than that of skeletal muscle, measured under identical conditions. The greater sensitivity in ryanodine in intact heart can be directly explained by the tighter binding of the ryanodine receptor from heart. The present study suggests that basically similar machinery (the ryanodine receptor and foot structure) is involved in triggering Ca2+ release from cardiac and skeletal muscle SR, albeit there are distinct differences in the sensitivity to ryanodine and other ligands in heart versus skeletal muscle.     

Smooth muscle and brain inositol 1,4,5-trisphosphate receptors are structurally and functionally similar

Inositol 1,4,5-trisphosphate (InsP3) mediates smooth muscle contraction by mobilizing intracellular calcium release. In this study we provide a direct comparison of the smooth muscle and brain InsP3 receptors in terms of InsP3 binding and primary structure. The KD for InsP3 binding for both receptors was found to be essentially the same. Sequences from 11 bovine smooth muscle receptor tryptic peptides (120 amino acids) were identified in the mouse brain receptor with two substitutions attributable to species differences. A cDNA (approximately 1-kilobase) encoding a portion of the mouse smooth muscle InsP3 receptor was cloned and found to be identical to that reported for the brain receptor. This cDNA was used as a probe to demonstrate that the approximately 10-kilobase InsP3 receptor mRNA is detected in brain, smooth muscle, heart, liver, and kidney but was not detected in skeletal muscle or skin.     

A Novel 3D Label-Free Monitoring System of hES-Derived Cardiomyocyte Clusters: A Step Forward to In Vitro Cardiotoxicity Testing

Unexpected adverse effects on the cardiovascular system remain a major challenge in the development of novel active pharmaceutical ingredients (API). To overcome the current limitations of animal-based in vitro and in vivo test systems, stem cell derived human cardiomyocyte clusters (hCMC) offer the opportunity for highly predictable pre-clinical testing. The three-dimensional structure of hCMC appears more representative of tissue milieu than traditional monolayer cell culture. However, there is a lack of long-term, real time monitoring systems for tissue-like cardiac material. To address this issue, we have developed a microcavity array (MCA)-based label-free monitoring system that eliminates the need for critical hCMC adhesion and outgrowth steps. In contrast, feasible field potential derived action potential recording is possible immediately after positioning within the microcavity. Moreover, this approach allows extended observation of adverse effects on hCMC. For the first time, we describe herein the monitoring of hCMC over 35 days while preserving the hCMC structure and electrophysiological characteristics. Furthermore, we demonstrated the sensitive detection and quantification of adverse API effects using E4031, doxorubicin, and noradrenaline directly on unaltered 3D cultures. The MCA system provides multi-parameter analysis capabilities incorporating field potential recording, impedance spectroscopy, and optical read-outs on individual clusters giving a comprehensive insight into induced cellular alterations within a complex cardiac culture over days or even weeks.     

Contacting the brain – aspects of a technology assessment of neural implants

The public interest in neural implants has grown considerably in recent years. Progress within related research areas in combination with increasing – albeit overly optimistic and indiscriminate – mass media coverage have led to the impression that the possibilities of neural prosthetics have grown enormously. But a closer look reveals that the reasons for the intensified interest are varied and cannot be attributed to technical progress alone. Some neural prostheses that have been under development for many years have not left the clinical development phase despite intensive research activities. Other implants, like cardiac pacemakers and cochlea implants, are mature products that have already been implanted in a large number of patients. From the public perspective and in media reports, progress in the development of neural implants is associated with new achievements in other fields of neuroscience. Communications on new applications of functional magnetic resonance imaging (fMRI) may suggest that a number of cognitive functions are now easily accessible with technological means. The fact that the interpretation of the results of fMRI studies depends on many conditions and is partly disputed also within the scientific community has been discussed in many publications but only very limited, in the general media. Besides this, research results and implementations in the area of electroencephalography and magnetoencephalography have sparked further debate on the question of free will, on determinism and indeterminism, and have attracted a large media response. The purpose of this paper is to discuss some societal and ethical aspects of neural implants from a technology assessment perspective. Technology assessment (TA) aims at providing knowledge about impacts and consequences of (new) technologies as well as about political and societal ways of dealing with them. It reflects about implementation conditions of technology and potential technology conflicts. Over the last years, neural implants became a subject for TA since they have gained a higher attention in both the political arena and the general public. Especially the ethical and social implications of technologies that electrically stimulate the brain and the possibilities of changing personality traits, changing moods, and perhaps enhancing human cognitive capabilities are central issues in related discussions. In this paper, we want to briefly summarize some of the key arguments as well as topics for future discussion and research.     

Mitochondrial apoptosis induced by BH3‐only molecules in the exclusive presence of endoplasmic reticular Bak

Bak and Bax are critical apoptotic mediators that naturally localize to both mitochondria and the endoplasmic reticulum (ER). Although it is generally accepted that mitochondrial expression of Bak or Bax suffices for apoptosis initiated by BH3‐only homologues, it is currently unclear whether their reticular counterparts may have a similar potential. In this study, we show that cells exclusively expressing Bak in endoplasmic membranes undergo cytochrome c mobilization and mitochondrial apoptosis in response to BimEL and Puma, even when these BH3‐only molecules are also targeted to the ER. Surprisingly, calcium was necessary but not sufficient to drive the pathway, despite normal ER calcium levels. We provide evidence that calcium functions coordinately with the ER‐stress surveillance machinery IRE1α/TRAF2 to transmit apoptotic signals from the reticulum to mitochondria. These results indicate that BH3‐only mediators can rely on reticular Bak to activate an ER‐to‐mitochondria signalling route able to induce cytochrome c release and apoptosis independently of the canonical Bak,Bax‐dependent mitochondrial gateway, thus revealing a new layer of complexity in apoptotic regulation.