Kinetics of Extracellular ATP from Goldfish Hepatocytes: A Lesson from Mathematical Modeling

In goldfish hepatocytes, hypotonic exposure leads to cell swelling, followed by a compensatory shrinkage termed RVD. It has been previously shown that ATP is accumulated in the extracellular medium of swollen cells in a non-linear fashion, and that extracellular ATP (ATPe) is an essential intermediate to trigger RVD. Thus, to understand how RVD proceeds in goldfish hepatocytes, we developed two mathematical models accounting for the experimental ATPe kinetics reported recently by Pafundo et al. in Am. J. Physiol. 294, R220–R233, 2008. Four different equations for ATPe fluxes were built to account for the release of ATP by lytic (J _L ) and nonlytic mechanisms (J _NL ), ATPe diffusion (J _D ), and ATPe consumption by ectonucleotidases (J _V ). Particular focus was given to J _NL , defined as the product of a time function (J _R ) and a positive feedback mechanism whereby ATPe amplifies J _NL . Several J _R functions (Constant, Step, Impulse, Gaussian, and Lognormal) were studied. Models were tested without (model 1) or with (model 2) diffusion of ATPe. Mathematical analysis allowed us to get a general expression for each of the models. Subsequently, by using model dependent fit (simulations) as well as model analysis at infinite time, we observed that:

Asymmetry in the Osmotic Response of a Rat Cortical Collecting Duct Cell Line: Role of Aquaporin-2

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride (Cl⁻) channel known to influence the function of other channels, including connexin channels. To further study potential functional interactions between CFTR and gap junction channels, we have co-expressed CFTR and connexin45 (Cx45) in Xenopus oocytes and monitored junctional conductance and voltage sensitivity by dual voltage clamp electrophysiology. In single oocytes expressing CFTR, an increase in cAMP caused by forskolin application induced a Cl⁻ current and increased membrane conductance; application of diphenylamine carboxylic acid (CFTR blocker) readily blocked the Cl⁻ current. With co-expression of CFTR and Cx45, application of forskolin to paired oocytes induced a typical outward current and increased junctional conductance (G_j). In addition, the presence of CFTR reduced the transjunctional voltage sensitivity of Cx45 channels without affecting the kinetics of junctional current inactivation. The drop in voltage sensitivity was further enhanced by forskolin application. The data indicate that CFTR influences cell-to-cell coupling mediated by Cx45 channels.     

Two nematodes, Dentinema trichomycteri n. g., n. sp. (Cosmocercidae) and Procamallanus chimusensis Freitas & Ibáñez, 1968 (Camallanidae), from catfishes Trichomycterus spp. (Pisces) in Colombia

Light and scanning electron microscopical examinations of nematode samples collected from the stomachs and intestines of catfishes Trichomycterus spp. (Trichomycteridae, Siluriformes) from three streams in Colombia revealed the presence of two species, Dentinema trichomycteri n. g., n. sp. (Cosmocercidae) (type-host Trichomycterus sp.) and Procamallanus (Spirocamallanus) chimusensis Freitas &; Ibáñez, 1968 (Camallanidae). The new, monotypic genus Dentinema is characterised mainly by the presence of a triangular mouth surrounded by three poorly developed lips, four submedian cephalic papillae, three conical teeth in the well-developed buccal cavity, an elongate oesophageal isthmus which is clearly separated from the corpus, precloacal oblique muscle bands, and by the absence of a ventral sucker. P. chimusensis, recorded from Colombia for the first time, is redescribed from specimens collected from Trichomycterus chapmani (Eigenmann) (a new host record) and Trichomycterus sp.; new observations show that this species belongs to a small group of Procamallanus spp. exhibiting a distinct sexual diversity in the structure of the buccal capsule. P. pexatus Pinto, Fábio, Noronha &; Rolas, 1976 is synonymised with P. chimusensis.     

BRAFV600E Mutation Analysis in Patients with Metastatic Colorectal Cancer (mCRC) in Daily Clinical Practice: Correlations with Clinical Characteristics,and Its Impact on Patients’ Outcome

Background

To prospectively evaluate the usefulness of the BRAFV600E mutation detection in daily clinical practice in patients with metastatic Colorectal Cancer (mCRC).

Patients and Methods

504 mCRC patients treated with systemic chemotherapy ± biologics were analyzed.

Results

A statistically significant higher incidence of the BRAF mutation was observed in patients with ECOG-PS 2 (p=0.001), multiple metastatic sites (p=0.002),> 65 years old (p=0.004), primary tumors located in the colon (p<0.001), high-grade tumors (p=0.001) and in those with mucinous features (p=0.037). Patients with BRAFV600E mutated tumors had a statistically significantly reduced progression-free survival (PFS) compared to wild-type (wt) ones (4.1 and 11.6 months, respectively; p<0.001) and overall survival (OS) (14.0 vs. 34.6 months, respectively; p<0.001). In the multivariate analysis the BRAFV600E mutation emerged as an independent factor associated with reduced PFS (HR: 4.1, 95% CI 2.7–6.2; p<0.001) and OS (HR: 5.9, 95% CI 3.7–9.5; p<0.001). Among the 273 patients treated with salvage cetuximab or panitumumab, the BRAFV600E mutation was correlated with reduced PFS (2.2 vs. 6.0 months; p<0.0001) and OS (4.3 vs. 17.4 months; p<0.0001).

Conclusions

The presence of BRAFV600E-mutation in mCRC characterizes a subgroup of patients with distinct biologic, clinical and pathological features and is associated with very poor patients’ prognosis.     

Microrna expression signatures predict patient progression and disease outcome in pediatric embryonal central nervous system neoplasms

Background

Although, substantial experimental evidence related to diagnosis and treatment of pediatric central nervous system (CNS) neoplasms have been demonstrated, the understanding of the etiology and pathogenesis of the disease remains scarce. Recent microRNA (miRNA)-based research reveals the involvement of miRNAs in various aspects of CNS development and proposes that they might compose key molecules underlying oncogenesis. The current study evaluated miRNA differential expression detected between pediatric embryonal brain tumors and normal controls to characterize candidate biomarkers related to diagnosis, prognosis and therapy.

Methods

Overall, 19 embryonal brain tumors; 15 Medulloblastomas (MBs) and 4 Atypical Teratoid/Rabdoid Tumors (AT/RTs) were studied. As controls, 13 samples were used; The First-Choice Human Brain Reference RNA and 12 samples from deceased children who underwent autopsy and were not present with any brain malignancy. RNA extraction was carried out using the Trizol method, whilst miRNA extraction was performed with the mirVANA miRNA isolation kit. The experimental approach included miRNA microarrays covering 1211 miRNAs. Quantitative Real-Time Polymerase Chain Reaction was performed to validate the expression profiles of miR-34a and miR-601 in all 32 samples initially screened with miRNA microarrays and in an additional independent cohort of 30 patients (21MBs and 9 AT/RTs). Moreover, meta-analyses was performed in total 27 embryonal tumor samples; 19 MBs, 8 ATRTs and 121 control samples. Twelve germinomas were also used as an independent validation cohort. All deregulated miRNAs were correlated to patients’ clinical characteristics and pathological measures.

Results

In several cases, there was a positive correlation between individual miRNA expression levels and laboratory or clinical characteristics. Based on that, miR-601 could serve as a putative tumor suppressor gene, whilst miR-34a as an oncogene. In general, miR-34a demonstrated oncogenic roles in all pediatric embryonal CNS neoplasms studied.

Conclusions

Deeper understanding of the aberrant miRNA expression in pediatric embryonal brain tumors might aid in the development of tumor-specific miRNA signatures, which could potentially afford promising biomarkers related to diagnosis, prognosis and patient targeted therapy.

     

A “Krokodil” emerges from the murky waters of addiction. Abuse trends of an old drug

“Krokodil” is the street name for the semi-synthetic opioid derivative desomorphine. Although an old drug, it re-staged on “drug arena” during the last decade causing detrimental effects to its users. Despite the fact that Russia and other former Soviet Republics were the initial plagued countries, “krokodil” arrived in Europe and United States lately, as a substitute of the relative expensive, and in many cases unavailable, heroin. It can be easily manufactured in home-environment from codeine and causes significant health problems, even deaths worldwide. The aim of this review is to summarize the current knowledge about this drug, concerning its chemistry, synthesis, pharmacology and toxicology. Published or reported “krokodil” related cases, fatalities or intoxications, as well as self reports from drug users are reviewed. The existing analytical methodologies for the determination of desomorphine in biological samples as well as its legal status are also presented.     

Negative feedback of extracellular ADP on ATP release in goldfish hepatocytes: A theoretical study

A mathematical model was built to account for the kinetic of extracellular ATP (ATPe) and extracellular ADP (ADPe) concentrations from goldfish hepatocytes exposed to hypotonicity. The model was based on previous experimental results on the time course of ATPe accumulation, ectoATPase activity, and cell viability [Pafundo et al., 2008].The kinetic of ATPe is controlled by a lytic ATP flux, a non-lytic ATP flux, and ecto-ATPase activity, whereas ADPe kinetic is governed by a lytic ADP flux and both ecto-ATPase and ecto-ADPase activities. Non-lytic ATPe efflux was included as a diffusion equation modulated by ATPe activation (positive feedback) and ADPe inhibition (negative feedback).The model yielded physically meaningful and stable steady-state solutions, was able to fit the experimental time evolution of ATPe and simulated the concomitant kinetic of ADPe. According to the model during the first minute of hypotonicity the concentration of ATPe is mainly governed by both lytic and non-lytic ATP efflux, with almost no contribution from ecto-ATPase activity. Later on, ecto-ATPase activity becomes important in defining the time dependent decay of ATPe levels. ADPe inhibition of the non-lytic ATP efflux was strong, whereas ATPe activation was minimal. Finally, the model was able to predict the consequences of partial inhibition of ecto-ATPase activity on the ATPe kinetic, thus emulating the exposure of goldfish cells to hypotonic medium in the presence of the ATP analog AMP-PCP. The model predicts this analog to both inhibit ectoATPase activity and increase non-lytic ATP release.