Reducing complexity and unidentifiability when modelling human atrial cells.

Mathematical models of a cellular action potential (AP) in cardiac modelling have become increasingly complex, particularly in gating kinetics, which control the opening and closing of individual ion channel currents. As cardiac models advance towards use in personalized medicine to inform clinical decision-making, it is critical to understand the uncertainty hidden in parameter estimates from their calibration to experimental data. This study applies approximate Bayesian computation to re-calibrate the gating kinetics of four ion channels in two existing human atrial cell models to their original datasets, providing a measure of uncertainty and indication of potential issues with selecting a single unique value given the available experimental data. Two approaches are investigated to reduce the uncertainty present: re-calibrating the models to a more complete dataset and using a less complex formulation with fewer parameters to constrain. The re-calibrated models are inserted back into the full cell model to study the overall effect on the AP. The use of more complete datasets does not eliminate uncertainty present in parameter estimates. The less complex model, particularly for the fast sodium current, gave a better fit to experimental data alongside lower parameter uncertainty and improved computational speed. This article is part of the theme issue 'Uncertainty quantification in cardiac and cardiovascular modelling and simulation'.

The novel CaMKII inhibitor GS-680 reduces diastolic SR Ca leak and prevents CaMKII-dependent pro-arrhythmic activity.

RATIONALE: Ca/calmodulin-dependent protein kinase II (CaMKII) was shown to increase diastolic sarcoplasmic reticulum (SR) Ca leak, which can result in delayed afterdepolarizations and triggered arrhythmias. Since increased CaMKII expression and activity has been mechanistically linked to arrhythmias in human heart failure (HF) and atrial fibrillation (AF), specific strategies aimed at CaMKII inhibition may have therapeutic potential. OBJECTIVE: We tested the antiarrhythmic and inotropic effects of a novel selective and ATP-competitive CaMKII inhibitor (GS-680). METHODS AND RESULTS: Trabeculae were isolated from right atrial appendage biopsies of patients undergoing cardiac surgery. Premature atrial contractions (PACs) were induced by stimulation with isoproterenol (ISO, 100 nM) at increased [Ca]o (3.5 mM). Interestingly, compared to vehicle, PACs were significantly inhibited by exposure to GS-680 (at 100 and 300 nM). GS-680 also significantly decreased early and delayed afterdepolarizations in isolated human atrial myocytes. Moreover, GS-680 (at 100 or 300 nM) significantly inhibited diastolic SR Ca leak, measured as frequency of spontaneous SR Ca release events (Ca sparks) in isolated human atrial myocytes (Fluo-4 loaded) similar to the well-established peptide CaMKII inhibitor AIP. In accordance, GS-680 significantly reduced CaMKII autophosphorylation (Western blot) but enhanced developed tension after 10 or 30 s pause of electrical stimulation (post-rest behavior). Surprisingly, we found a strong negative inotropic effect of GS-680 in atrial trabeculae at 1 Hz stimulation rate, which was not observed at 4 Hz and abolished by beta-adrenergic stimulation. In contrast, GS-680 did not impair systolic force of isolated ventricular trabeculae from explanted hearts of heart transplant recipients at 1 Hz, blunted the negative force-frequency relationship (1-3 Hz) and significantly increased the Ca transient amplitude. CONCLUSION: The novel ATP-competitive and selective CaMKII inhibitor GS-680 inhibits pro-arrhythmic activity in human atrium and improves contractility in failing human ventricle, which may have therapeutic implications.

Implementing a fracture liaison service open model of care utilizing a cloud-based tool.

PURPOSE: Although half of women and one-quarter of men aged 50 and older will sustain an acute low-trauma fracture, less than a quarter receive appropriate secondary fracture prevention. The goal of this quality improvement demonstration project was to implement a Fracture Liaison Service (FLS) focused on secondary prevention of an osteoporotic fracture in three open health care systems aided by a cloud-based tool. METHODS: The pre-post study design examined the proportion of men and women over age 50 who received appropriate assessment (bone mineral density, vitamin D levels) and treatment (calcium/vitamin D, pharmacologic therapy) in the six months following a recently diagnosed fracture. The pre-study (Pre FLS) included a retrospective chart review for baseline data (N = 344 patients) within each health care system. In the post-evaluation (Post FLS, N = 148 patients), the FLS coordinator from each health care system examined these parameters following enrollment and for 6 months following the recently diagnosed fracture. Data were managed in the cloud-based FLS application tool. RESULTS: Ninety-three participants completed the program. The FLS program increased the percentage of patients receiving bone mineral density testing from 21% at baseline to 93% (p < 0.001) Post FLS implementation. Assessments of vitamin D levels increased from 25 to 84% (p < 0.001). Patients prescribed calcium/vitamin D increased from 36% at baseline to 93% (p < 0.001) and those prescribed pharmacologic treatment for osteoporosis increased on average from 20 to 54% (p < 0.001) Post FLS. CONCLUSIONS: We conclude that the FLS model of care in an open health care system, assisted by a cloud-based tool, significantly improved assessment and/or treatment of patients with a recently diagnosed osteoporotic fracture. Future studies are necessary to determine if this model of care is scalable and if such programs result in prevention of fractures. Mini-Abstract: The goal was to implement a Fracture Liaison Service (FLS) focused on secondary prevention of an osteoporotic fracture in open health care systems aided by a cloud-based tool. This model significantly improved assessment and/or treatment of patients with a recently diagnosed fracture.

Comparison of otolith and scale readings for age and growth estimation of common dentex Dentex dentex.

Three methods of age estimation were compared for Dentex dentex. Based on sectioned otoliths, scales appeared to be relevant only up to 5 years and whole otoliths up to 12 years. The maximum estimated age was 36 years, which constitutes to date the oldest age reported.

Preparation of biologically active single-chain variable antibody fragments that target the HIV-1 gp120 V3 loop.

KD-247 is a humanized monoclonal antibody that targets the third hypervariable (V3) loop of gp120. It can efficiently neutralize a broad panel of clade B, but not non-clade B, HIV-1 isolates. To overcome this limitation, we are seeking to prepare genetically-engineered single-chain variable fragments (scFvs) of KD-247 that will have broader neutralizing activity against both clade B and non-clade B HIV-1 isolates. Initial attempts of optimizing the expression of KD-247 scFv have resulted in the formation of insoluble protein. Therefore, we have established purification protocols to recover, purify, and refold the KD-247 scFv from inclusion bodies. The protocol involved step-wise refolding of denatured scFv by dilution, dialysis, and on-column nickel-affinity purification. Monomeric scFv was further purified by size-exclusion chromatography. Using far UV circular dichroism (CD) spectroscopy we confirmed the expected beta-sheet profile of the refolded KD-247 scFv. Importantly, the refolded KD-247 scFv showed neutralizing activity against replication-competent HIV-1 BaL and JR-FL Env pseudotyped HIV-1, at potency comparable to that of the native full-size KD-247 antibody. Ongoing studies focus on the application of this system in generating KD-247 scFv variants with the ability to neutralize clade B and non-clade B HIV-1 isolates.

Effect of translocation defective reverse transcriptase inhibitors on the activity of N348I, a connection subdomain drug resistant HIV-1 reverse transcriptase mutant.

4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a highly potent inhibitor of HIV-1 reverse transcriptase (RT). We have previously shown that its exceptional antiviral activity stems from a unique mechanism of action that is based primarily on blocking translocation of RT; therefore we named EFdA a Translocation Defective RT Inhibitor (TDRTI). The N348I mutation at the connection subdomain (CS) of HIV-1 RT confers clinically significant resistance to both nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). In this study we tested EFdA-triphosphate (TP) together with a related compound, ENdA-TP (4'-ethynyl-2-amino-2'-deoxdyadenosine triphosphate) against HIV-1 RTs that carry clinically relevant drug resistance mutations: N348I, D67N/K70R/L210Q/T215F, D67N/K70R/L210Q/T215F/N348I, and A62V/V5I/F77L/F116Y/Q151M. We demonstrate that these enzymes remain susceptible to TDRTIs. Similar to WT RT, the N348I RT is inhibited by EFdA mainly at the point of incorporation through decreased translocation. In addition, the N348I substitution decreases the RNase H cleavage of DNA terminated with EFdA-MP (T/P(EFdA-MP)). Moreover, N348I RT unblocks EFdA-terminated primers with similar efficiency as the WT enzyme, and further enhances EFdA unblocking in the background of AZT-resistance mutations. This study provides biochemical insights into the mechanism of inhibition of N348I RT by TDRTIs and highlights the excellent efficacy of this class of inhibitors against WT and drug-resistant HIV-1 RTs.

Ultrastructural study of spermiogenesis and the spermatozoon of Acanthocephaloides incrassatus (Molin, 1858) (Acanthocephala, Paleacanthocephala, Arhythmacanthidae) from Anguilla anguilla (Pisces, Teleostei) in Urbino ponds (Corsica Island).

This study deals with first ultrastructure features of Acanthocephaloides incrassatus (Paleacanthocephala, Arhythmacanthidae), a parasite of the fish Anguilla anguilla, reported for the first time in a Mediterranean pond. The spermiogenesis of A. incrassatus shows original specificities which have never been pointed out to this day in ultrastructural studies on spermiogenesis: the centriolar derivative is divided into two parts of different densities: an electron-dense, and the other, electron-lucent; a ring form has been observed on each side of the axoneme; a centriole with one central element. After the elaboration of a flagellum of 9+2 pattern, the centriole migrates in a nuclear groove. Rapidly, the centriole disappears. Then, the flagellum migration occurs by a series of processes and gives rise to a spermatozoon. The spermatozoon of A. incrassatus presents its own specificities: it exhibits an evolution of the centriolar derivative characterized by only nine peripheral elements deprived of a central element in the anterior part, then nine peripheral and one central element, and finally, nine peripheral elements with two central elements. An assumption is emitted on a probable correspondence of the evolution of the derivative centriolar during the spermiogenesis and the evolution that occurs in the spermatozoon. Protein granules also show different sizes and forms, full or emptied of their contents compared with data on other Acanthocephala.

The sugar ring conformation of 4'-ethynyl-2-fluoro-2'-deoxyadenosine and its recognition by the polymerase active site of HIV reverse transcriptase.

4' Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is the most potent inhibitor of HIV reverse transcriptase (RT). We have recently named EFdA a Translocation Defective RT Inhibitor (TDRTI) because after its incorporation in the nucleic acid it blocks DNA polymerization, primarily by preventing translocation of RT on the template/primer that has EFdA at the 3'-primer end (T/PEFdA). The sugar ring conformation of EFdA may also influence RT inhibition by a) affecting the binding of EFdA triphosphate (EFdATP) at the RT active site and/or b) by preventing proper positioning of the 3'-OH of EFdA in T/PEFdA that is required for efficient DNA synthesis. Specifically, the North (C2'-exo/C3'-endo), but not the South (C2'-endo/C3'-exo) nucleotide sugar ring conformation is required for efficient binding at the primer-binding and polymerase active sites of RT. In this study we use nuclear magnetic resonance (NMR) spectroscopy experiments to determine the sugar ring conformation of EFdA. We find that unlike adenosine nucleosides unsubstituted at the 4'-position, the sugar ring of EFdA is primarily in the North conformation. This difference in sugar ring puckering likely contributes to the more efficient incorporation of EFdATP by RT than dATP. In addition, it suggests that the 3'-OH of EFdA in T/PEFdA is not likely to prevent incorporation of additional nucleotides and thus it does not contribute to the mechanism of RT inhibition. This study provides the first insights into how structural attributes of EFdA affect its antiviral potency through interactions with its RT target.

The influence of altitude, hydrographic network and season on brown trout parasites in Corsica using indicator species analysis.

The parasites of 484 brown trout, Salmo trutta, were studied between 2004 and 2007. An indicator value (IndVal) method was used for analysis, which combines measures of fidelity and specificity. Because of its resilience to detect changes in abundance, IndVal is an effective ecological bio-indicator. The IndVal method demonstrated that altitude, hydrographic network and season could influence the occurrence of parasite species in brown trout. A randomization test identified three parasite species as having a significant indicator value for altitude (the trematode Nicolla wisniewskii, and the nematodes Spinitectus gordoni and Rhabdochona gnedini); five parasite species for hydrographic network (the trematodes Nicolla testiobliquum, N. wisniewskii, Plagioporus stefanskii, and the nematodes S. gordoni and R. gnedini) and two parasite species for season (the nematodes S. gordoni, and R. gnedini). Data for species composition and infection levels should help to improve the monitoring and management of parasitism in salmonid populations.

[A new type of flagellar structure. Type 9+n]. / Un type nouveau de structure flagellaire. Type 9+n

The ultrastructural study of the Eoacanthocephala sperm cell shows a variation from 0 to 5 in the number of the axial fibers in the axoneme. All the species of the order Eoacanthocephala available to us show this variation; moreover, every individual possesses simultaneously several different structural types. So, we are dealing with a new flagellar organization: 9+n, with 0 less than or equal to n less than or equal to 5. In the Quadrigyridae and the Tenuisentidae families, n varies from 0 to 4, with a maximum of 2 for most individuals, exceptionally at 1 for some individuals. In the Neoechinorhynchidae family, n varies from 0 to 5 with a conspicuous prevalence of 3 (from 84 to 99%, according to the individual). These results prompted us to reexamine the two other orders of Acanthocephala in which the structural types 9+2 or 9+0 have been considered as fixed. Indeed, we have found a few flagella the structure of which is different from the prevalent one. It seems, therefore, that the number of the central fibers of the axoneme in the Acanthocephala sperm cell is never absolutely fixed.

Site influence in parasite distribution from fishes of the Bonifacio Strait Marine Reserve (Corsica Island, Mediterranean Sea).

The spatial distribution of parasite communities from teleost fishes (Diplodus vulgaris, Mullus surmuletus, Pagellus erythrinus, Phycis phycis, Scorpaena scrofa and Symphodus tinca) was studied in the Bonifacio Strait Marine Reserve (Corsica Island) with different protection levels. Canonical correspondence analysis was used to study spatial patterns and to quantify the importance of site influence on parasite communities. To supplement these results, we used the indicator species analysis (Indval) to identify parasite species as bioindicators of a given ecosystem. Moreover, fish spatial distribution in the marine reserve was evaluated in order to link parasite species occurrence and fish abundance. Several parasite species were associated with a specific site, and some of them were considered as site bioindicators, especially in the western part of the protected area (Bruzzi and Moines Islands). However, the relative abundance of fish followed a different spatial pattern. Stronger fish abundance was observed in the eastern part of the reserve (Lavezzi Islands). The parasite species were correlated to sites inside as well outside the reserve. The parasite distribution seemed not to be influenced by the protection status of the site. Furthermore, in our study, most of the parasite species were not linked to a site. The most important factor influencing their distribution was host specificity. This suggested that the parasite life traits history remained a fundamental determinant in the community composition.

Sperm ultrastructure of the digenean Siphoderina elongata (Platyhelminthes, Cryptogonimidae) intestinal parasite of Nemipterus furcosus (Pisces, Teleostei).

The ultrastructure of the spermatozoon of Siphoderina elongata was studied by transmission electron microscopy. A description and drawings of the mature spermatozoon are presented in this paper. Several ultrastructural elements of this male gamete have been observed: a nucleus, two mitochondria, two axonemes of 9 + "1" pattern, external ornamentation of the plasma membrane, spine-like bodies and cortical microtubules. The presence, the location or the number of these elements have been compared with other digenean spermatozoa. Moreover, a close attention was paid to the organization of the external ornamentation region. This zone presents a single row of cortical microtubules disposed in a semi-circle around a mitochondrion and associated with external ornamentation and spine-like bodies. The aim of this study is to highlight criteria which can be interesting in Platyhelminthes phylogeny.

Comparison of arsenic and antimony contents in tissues and organs of brown trout caught from the river Presa polluted by ancient mining practices and from the river Bravona in Corsica (France): a survey study.

The present study evaluates the concentrations of arsenic (As) and antimony (Sb) in the intestine, liver, muscle, gonads, gills, and kidney of Salmo trutta subsp. from the Presa River in Corsica (France; n = 10), which crosses an abandoned arsenic mine, and from the Bravona River (reference site; n = 10). Both metalloids were analyzed by means of ICP-MS. The relationships between fish size (length and weight) and metalloid concentrations in their tissues were investigated by linear regression analysis. In all fish samples concentrations of As and Sb (expressed as micrograms per gram fresh weight) were highest in the kidney. Lowest Sb concentrations were found in the muscle, whereas lowest As concentrations were found in the gonads of S. trutta. Two organotropisms were revealed: one for As-kidney (21.4656) > intestine (3.9535) > gills (3.0404) > liver (1.1743) > muscle (0.9976) > gonads (0.8081); and the other for Sb-kidney (0.70067) > gills (0.6181) > intestine (0.2576) > gonads (0.1673) > liver (0.9625) > muscle (0.0753). Results of linear regression analysis in most cases showed a significant negative correlation between metalloid concentration and fish size. Highly significant (p < 0.05) negative correlations were found between fish length and As concentration in the gonads, as well as between fish length and Sb concentrations in the gills. Arsenic concentrations in female fish were significantly higher than those in males in the kidney, gonads, gills, and liver. The same results were found for Sb, except in the liver, where the tendency was reversed.

Parasitofauna study of the brown trout, Salmo trutta (Pisces, Teleostei) from Corsica (Mediterranean island) rivers.

Corsica is a Mediterranean island characterised by a great number of rivers. Salmonides are the main fishes which populate these rivers. Very appreciated by fishermen, Salmonides are represented by three species in the insular hydrographical network, among which an autochthonous species, the brown trout (Salmo trutta). In the present work, we have analysed the parasitofauna of this species. According to our knowledge, this research has never been carried out in Corsica. In a first step, we drew up an inventory of the parasites found in this freshwater fish. In a second step, we studied the differences which appeared in the composition of parasite communities of this species.

Ultrastructure of the spermatozoon of Helicometroides atlanticus (Digenea, Monorchiidae), an intestinal parasite of Parapristipoma octolineatum (Pisces, Teleostei) in Senegal.

The mature Helicometroides atlanticus spermatozoon exhibits an anterior electron-dense material, two axonemes of the 9+"1" pattern, a nucleus, two mitochondria, extramembranous ornamentations located at the level of the first mitochondrion and cortical microtubules arranged into two fields: a ventral field corresponding to the mitochondrial side and a dorsal field corresponding to the nuclear side. It lacks of spine-like body and a cytoplasmic expansion as in Monorchis parvus the only species of Monorchioidea which spermatozoon has been described until now. Nevertheless, it is distinguished from M. parvus by the presence of an anterior electron-dense material and two axonemes which appear one after the other.

Double-head haptens. Synthesis of and experimentally induced contact sensitivity to substances containing two unrelated haptens, pyrocatechol and alpha-methylene-gamma-butyrolactone, in the same molecule.

A "double-head" hapten containing a pyrocatechol and an alpha-methylene-gamma-butyrolactone moiety (3) and "monofunctional haptens" with either one of these moieties (18 and 19) connected by a six-carbon chain have been synthesized, and their sensitizing capacity was tested on guinea pigs. In the "double-head" hapten, only the pyrocatechol end is "recognized". A possible interpretation of the biological results is offered.

Nonradioactive detection of retroviral-associated RNase H activity in a microplate-based, high-throughput format.

None of the available antiretroviral drugs that are currently used in the clinic to treat infection with HIV-1 is directed against the RNase H active site of the reverse transcriptase. Here we developed a nonradioactive, 96-well plate assay designed to be used for high-throughput screening of compounds capable of inhibiting the RNase H activity of HIV-1 reverse transcriptase. We employed a tRNA as substrate that was labeled with digoxygenin-modified reporter residues. The labeled tRNA was prehybridized with a DNA oligonucleotide that contained a single biotinylated residue at its 5'-terminus to ensure its attachment to streptavidin-coated microplates. The uncleaved, immobilized DNA/tRNA substrate was detected through the use of established ELISA protocols. Incubation with purified HIV-1 reverse transcriptase initiated RNase H degradation and caused a signal reduction to negligible background levels. In contrast, the signal intensity remained unaffected when using an RNase H deficient mutant enzyme. The assay was validated using the hydrazone derivative BBNH that was previously shown to inhibit RNase H degradation below concentrations of 10 microM.

Ultrastructure of spermiogenesis and the spermatozoon of Aporina delafondi (Cyclophyllidea, Anoplocephalidae), intestinal parasite of turtle doves in Senegal.

Spermiogenesis in Aporina delafondi begins with the formation of a differentiation zone bordered by cortical microtubules and containing from the beginning a portion of nucleus and two parallel centrioles. One of the centrioles aborts, the other gives rise to a flagellum. The cortical microtubules elongate and spiralize while the nucleus migrates along the axoneme and crest-like bodies form at the level of the differentiation zone. The old spermatid separates from the residual cytoplasm by strangulation of the ring of arched membranes. The mature spermatozoon lacks mitochondria, is filiform and tapered at both its extremities. Its anterior extremity is capped by an apical cone of electron-dense material and exhibits five crest-like bodies of unequal lengths on its periphery. Its cortical microtubules are regularly spiralized except at their posterior extremity where they become parallel to the spermatozoon axis. The cytoplasm is slightly dense in the anterior regions (I and II) and exhibits many protein granules and patches of electron-lucent material in the middle (III) and posterior zones (IV and V). The nucleus is an electron-dense cord coiled in a spiral around the middle region (III) of the axoneme. This is of the 9+ "1" pattern and ends before the posterior extremity of the gamete. Spermiogenesis in Aporina delafonidi differs from that of the other Cyclophyllidea by the very early movement of the nucleus into the differentiation zone, the formation of a ring of arched membranes in the distal part of the differentiation zone, the appearance of crest-like bodies during migration of the nucleus and the formation of a cytoplasmic bud which contains the abortive centriole and develops to temporarily form a large lateral extension. The mature spermatozoon differs from that of the other Cyclophyllidea in the presence of lucent patches in its cytoplasm and of five helicoidal crest-like bodies. The systematic position of the genus Aporina is also debated.

Ultrastructure of spermiogenesis and the spermatozoon of Raillietina (Raillietina) tunetensis (Cyclophyllidea, Davaineidae), intestinal parasite of turtle doves in Senegal.

Spermiogenesis in Raillietina (Raillietina) tunetensis begins with the formation of a differentiation zone equipped with cortical microtubules and containing two centrioles. One of the centrioles very rapidly gives rise to a flagellum which fuses with a median cytoplasmic extension, the cortical microtubules elongate and arched membranes appear. After the migration of the nucleus two crest-like bodies form and the old spermatid becomes detached from the residual cytoplasm. The mature spermatozoon of R. (R.) tunetensis exhibits an apical cone of electron-dense material and two helicoidal crest-like bodies 100 to 200 nm thick. The cortical microtubules are spiralized and make an angle of about 60 degrees to the spermatozoon axis. The axoneme is of the 9 + "1" pattern and does not reach the posterior extremity of the gamete. The nucleus is a fine, compact cord wound in a spiral which may make as much as two complete coils round the axoneme. The cytoplasm is electron-dense in region V of the spermatozoon. Over the rest of the gamete it is made up of lucent material divided into irregular compartments by electron-dense material. The latter consists of a fine, discontinuous peri-axonemal sheath, a fine granular sub-microtubular layer situated in regions I and II, and irregularly spaced partitions localized in regions III and IV. A nucleus with an annular cross section has never been described in a cestode spermatozoon; nor have two crest-like bodies of different length and thickness. In addition we report for the first time the existence of crest-like bodies in the Davaineidae.