Drug2ways: Reasoning over causal paths in biological networks for drug discovery

Elucidating the causal mechanisms responsible for disease can reveal potential therapeutic targets for pharmacological intervention and, accordingly, guide drug repositioning and discovery. In essence, the topology of a network can reveal the impact a drug candidate may have on a given biological state, leading the way for enhanced disease characterization and the design of advanced therapies. Network-based approaches, in particular, are highly suited for these purposes as they hold the capacity to identify the molecular mechanisms underlying disease. Here, we present drug2ways, a novel methodology that leverages multimodal causal networks for predicting drug candidates. Drug2ways implements an efficient algorithm which reasons over causal paths in large-scale biological networks to propose drug candidates for a given disease. We validate our approach using clinical trial information and demonstrate how drug2ways can be used for multiple applications to identify: i) single-target drug candidates, ii) candidates with polypharmacological properties that can optimize multiple targets, and iii) candidates for combination therapy. Finally, we make drug2ways available to the scientific community as a Python package that enables conducting these applications on multiple standard network formats.     

Disruption of CENP antigen function perturbs dynein anchoring to the mitotic kinetochore

Injection of purified autoantibodies against human centromeric proteins into HeLa cells during interphase disrupts the organization of the kinetochore and interferes with chromosomal movements during the subsequent mitosis even though the chromosomes retain the ability to bind microtubules. We have investigated the hypothesis that this phenotype arises from effects on cytoplasmic dynein, the microtubule motor protein. In previous experiments we found that introduction of anticentromere antibodies into cell nuclei during the G₁- or S-phases causes a prometaphase-like arrest, while injections during G₂-phase cause a metaphase arrest. We show here that, in both cases, the level of detectable cytoplasmic dynein at kinetochores is significantly decreased. In contrast, when injected cells were permitted to enter mitosis in the absence of microtubules (conditions where trilaminar kinetochores could be detected by electron microscopy), the intensity of dynein labeling on the kinetochores was identical to that seen in uninjected control cells exposed to colcemid. Therefore, the loss of dynein label on mitotic kinetochores was correlated both with the injection of anticentromere antibodies and with the presence of intact spindle microtubules. We suggest that the injection of anticentromere antibodies somehow weakens the association of dynein with the kinetochore, so that when microtubules are present, these motor molecules are pulled away from the kinetochores as they generate force. This model offers an explanation for the failure of chromosomes of injected cells to move normally in mitosis even though they have attached microtubules.     

Ammonification rates and 210Pb in sediments from a lagoon under a wet tropical climate: Marica,Rio de Janeiro state,Brazil

The ammonification rates in surficial sediments of the Marica lagoon (Rio de Janeiro, Brazil) were estimated using two methods:

Effects of eel (Anguilla anguilla L.) removals from selected sites of a stream on its subsequent densities

We assessed the effect of eel (Anguilla anguilla) removal from three sites of a Cantabrian stream upon its subsequent densities. In the first sample (Sept. 1986) numbers and densities were estimated as 43, 45 and 84 ind and 3490, 3030 and 3750 ind ha ⁻¹. Removal of these eels reduced the subsequent numbers and densities which, except on two occasions, were never reached again during the two years (eleven estimates) of study. Highest densities were recorded in the uppermost site in May and July, 1987, coincident with a strong drought and the lowest densities occurred in 1988 during a normal wet year. We hypothesize first that, because of a selective underground homing behaviour of eels, electro-fishing is inefficient and results in underestimates of the population. Second, seasonal variations of water discharge and droughts may not influence the homing behaviour of'eels until a threshold of dryness is reached. If this occurs, eels abandon their refuges and move towards the stream bottom. It seems that in Arroyo Chabatchos this threshold was exceeded in the summer of 1987 when the highest densities were estimated. The re-colonization of these sites experimentally depleted of eels, is a slow procces that lasts for, at least, two years.     

Up-regulation of Na-coupled glucose transporter SGLT1 by caveolin-1

The Na⁺-coupled glucose transporter SGLT1 (SLC5A1) accomplishes concentrative cellular glucose uptake even at low extracellular glucose concentrations. The carrier is expressed in renal proximal tubules, small intestine and a variety of nonpolarized cells including several tumor cells. The present study explored whether SGLT1 activity is regulated by caveolin-1, which is known to regulate the insertion of several ion channels and carriers in the cell membrane. To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of caveolin-1 and electrogenic glucose transport determined by dual electrode voltage clamp experiments. In SGLT1-expressing oocytes, but not in oocytes injected with water or caveolin-1 alone, the addition of glucose to the extracellular bath generated an inward current (I_g), which was increased following coexpression of caveolin-1. Kinetic analysis revealed that caveolin-1 increased maximal I_g without significantly modifying the glucose concentration required to trigger half maximal I_g (K_M). According to chemiluminescence and confocal microscopy, caveolin-1 increased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5 μM) resulted in a decline of I_g, which was similar in the absence and presence of caveolin-1. In conclusion, caveolin-1 up-regulates SGLT1 activity by increasing carrier protein abundance in the cell membrane, an effect presumably due to stimulation of carrier protein insertion into the cell membrane.     

Tumor ablation with irreversible electroporation

We report the first successful use of irreversible electroporation for the minimally invasive treatment of aggressive cutaneous tumors implanted in mice. Irreversible electroporation is a newly developed non-thermal tissue ablation technique in which certain short duration electrical fields are used to permanently permeabilize the cell membrane, presumably through the formation of nanoscale defects in the cell membrane. Mathematical models of the electrical and thermal fields that develop during the application of the pulses were used to design an efficient treatment protocol with minimal heating of the tissue. Tumor regression was confirmed by histological studies which also revealed that it occurred as a direct result of irreversible cell membrane permeabilization. Parametric studies show that the successful outcome of the procedure is related to the applied electric field strength, the total pulse duration as well as the temporal mode of delivery of the pulses. Our best results were obtained using plate electrodes to deliver across the tumor 80 pulses of 100 micros at 0.3 Hz with an electrical field magnitude of 2500 V/cm. These conditions induced complete regression in 12 out of 13 treated tumors, (92%), in the absence of tissue heating. Irreversible electroporation is thus a new effective modality for non-thermal tumor ablation.     

Kinetic analysis of the slow ionization of glutathione by microsomal glutathione transferase MGST1

An important aspect of the catalytic mechanism of microsomal glutathione transferase (MGST1) is the activation of the thiol of bound glutathione (GSH). GSH binding to MGST1 as measured by thiolate anion formation, proton release, and Meisenheimer complex formation is a slow process that can be described by a rapid binding step (K(GSH)d = 47 +/- 7 mM) of the peptide followed by slow deprotonation (k2 = 0.42 +/- 0.03 s(-1). Release of the GSH thiolate anion is very slow (apparent first-order rate k(-2) = 0.0006 +/- 0.00002 s(-)(1)) and thus explains the overall tight binding of GSH. It has been known for some time that the turnover (kcat) of MGST1 does not correlate well with the chemical reactivity of the electrophilic substrate. The steady-state kinetic parameters determined for GSH and 1-chloro-2,4-dinitrobenzene (CDNB) are consistent with thiolate anion formation (k2) being largely rate-determining in enzyme turnover (kcat = 0.26 +/- 0.07 s(-1). Thus, the chemical step of thiolate addition is not rate-limiting and can be studied as a burst of product formation on reaction of halo-nitroarene electrophiles with the E.GS- complex. The saturation behavior of the concentration dependence of the product burst with CDNB indicates that the reaction occurs in a two-step process that is characterized by rapid equilibrium binding ( = 0.53 +/- 0.08 mM) to the E.GS- complex and a relatively fast chemical reaction with the thiolate (k3 = 500 +/- 40 s(-1). In a series of substrate analogues, it is observed that log k3 is linearly related (rho value 3.5 +/- 0.3) to second substrate reactivity as described by Hammett sigma- values demonstrating a strong dependence on chemical reactivity that is similar to the nonenzymatic reaction (rho = 3.4). Microsomal glutathione transferase 1 displays the unusual property of being activated by sulfhydryl reagents. When the enzyme is activated by N-ethylmaleimide, the rate of thiolate anion formation is greatly enhanced, demonstrating for the first time the specific step that is activated. This result explains earlier observations that the enzyme is activated only with more reactive substrates. Taken together, the observations show that the kinetic mechanism of MGST1 can be described by slow GSH binding/thiolate formation followed by a chemical step that depends on the reactivity of the electrophilic substrate. As the chemical reactivity of the electrophile becomes lower the rate-determining step shifts from thiolate formation to the chemical reaction.     

Long-term results and learning curve for radio frequency ablation of accessory pathways

Radio frequency (RF) catheter ablation of accessory pathways represents an interventional method in modern cardiology that has become the first-line treatment for patients with symptomatic WPW-syndrome. The aim of this study was to analyze: (1) the learning curve for the ablation procedure; (2) procedural parameters and success; and (3) personal assessment of the treatment by the patients. Learning curve analysis included 195 consecutive patients, who underwent ablation between 1991 and 1996. The follow-up survey included 65 consecutive patients. The analysis of the procedural parameters showed significant improvement after 100 cases, implying a completion of the learning curve at this point. Long-term follow-up showed a high success rate for all pathways (95.4%). All procedure parameters indicated significantly higher degree of difficulty for right free-wall and septal pathways, with lowest long-term success rate for right-sided pathways (78.6%). Personal assessment survey showed high acceptance of the treatment; the procedure was described as a significant improvement of overall quality-of-life by 92.3% of patients. The results of this study confirm the catheter ablation of accessory pathways--in particular after completion of the learning curve--as a low-risk and highly efficient treatment for symptomatic WPW-syndrome, with a high degree of patient-related acceptance.     

Degradation of tributyltin by the filamentous fungus Cunninghamella elegans, with involvement of cytochrome P-450

Cunninghamella elegans degraded tributyltin (TBT) at 20 mg l^–1 when grown in Sabouraud medium. Above this concentration, growth was inhibited. After 7 d 70% TBT (added at 10 mg l^–1) was converted to less toxic derivatives: dibutyltin and monobutyltin. TBT metabolism was totally blocked by cytochrome P-450 inhibitors, metyrapone and proadifen. Only in medium with 1-aminobenzotriazole, was dibutyltin (0.42 mg l^–1) found after 7 d of culturing. It is postulated that the significant resistance of C. elegans to TBT is associated with the capacity of the fungus to metabolise TBT.