Identification of secondary targets of N-containing bisphosphonates in mammalian cells via parallel competition analysis of the barcoded yeast deletion collection

Background

Nitrogen-containing bisphosphonates are the elected drugs for the treatment of diseases in which excessive bone resorption occurs, for example, osteoporosis and cancer-induced bone diseases. The only known target of nitrogen-containing bisphosphonates is farnesyl pyrophosphate synthase, which ensures prenylation of prosurvival proteins, such as Ras. However, it is likely that the action of nitrogen-containing bisphosphonates involves additional unknown mechanisms. To identify novel targets of nitrogen-containing bisphosphonates, we used a genome-wide high-throughput screening in which 5,936 Saccharomyces cerevisiae heterozygote barcoded mutants were grown competitively in the presence of sub-lethal doses of three nitrogen-containing bisphosphonates (risedronate, alendronate and ibandronate). Strains carrying deletions in genes encoding potential drug targets show a variation of the intensity of their corresponding barcodes on the hybridization array over the time.

Results

With this approach, we identified novel targets of nitrogen-containing bisphosphonates, such as tubulin cofactor B and ASK/DBF4 (Activator of S-phase kinase). The up-regulation of tubulin cofactor B may explain some previously unknown effects of nitrogen-containing bisphosphonates on microtubule dynamics and organization. As nitrogen-containing bisphosphonates induce extensive DNA damage, we also document the role of DBF4 as a key player in nitrogen-containing bisphosphonate-induced cytotoxicity, thus explaining the effects on the cell-cycle.

Conclusions

The dataset obtained from the yeast screen was validated in a mammalian system, allowing the discovery of new biological processes involved in the cellular response to nitrogen-containing bisphosphonates and opening up opportunities for development of new anticancer drugs.     

Rock weathering creates oases of life in a High Arctic desert

During primary colonization of rock substrates by plants, mineral weathering is strongly accelerated under plant roots, but little is known on how it affects soil ecosystem development before plant establishment. Here we show that rock mineral weathering mediated by chemolithoautotrophic bacteria is associated to plant community formation in sites recently released by permanent glacier ice cover in the Midtre Lovénbreen glacier moraine (78°53′N), Svalbard. Increased soil fertility fosters growth of prokaryotes and plants at the boundary between sites of intense bacterial mediated chemolithotrophic iron‐sulfur oxidation and pH decrease, and the common moraine substrate where carbon and nitrogen are fixed by cyanobacteria. Microbial iron oxidizing activity determines acidity and corresponding fertility gradients, where water retention, cation exchange capacity and nutrient availability are increased. This fertilization is enabled by abundant mineral nutrients and reduced forms of iron and sulfur in pyrite minerals within a conglomerate type of moraine rock. Such an interaction between microorganisms and moraine minerals determines a peculiar, not yet described model for soil genesis and plant ecosystem formation with potential past and present analogues in other harsh environments with similar geochemical settings.     

A new simple HPLC method for measuring mitotane and its two principal metabolites Tests in animals and mitotane-treated patients

A new C18 reversed-phase column and UV HPLC method for the detection of mitotane, its principal metabolites, dichlorodiphenylacetate and dichlrodiphenylethene, and its precursor DDT is described. In this article mitotane, dichlorodiphenylacetate, and dichlrodiphenylethene concentrations in organs of rats fed on a mitotane diet, and the effects of erythromycin and grapefruit juice as cytochrome P450 common inhibitors are presented. Tissue accumulation of mitotane and dichlrodiphenylethene, the acquired ability to eliminate dichlorodiphenylacetate, and inhibition of beta-hydroxylation by both inhibitors are illustrated here. Blood samples from mitotane-treated patients revealed two correlations: plasma mitotane/dichlrodiphenylethene and plasma mitotane/red cell mitotane.     

Cdc42 GTPase-activating proteins (GAPs) regulate generational inheritance of cell polarity and cell shape in fission yeast

The highly conserved small GTPase Cdc42 regulates polarized cell growth and morphogenesis from yeast to humans. We previously reported that Cdc42 activation exhibits oscillatory dynamics at cell tips of Schizosaccharomyces pombe cells. Mathematical modeling suggests that this dynamic behavior enables a variety of symmetric and asymmetric Cdc42 activation distributions to coexist in cell populations. For individual wild-type cells, however, Cdc42 distribution is initially asymmetrical and becomes more symmetrical as cell volume increases, enabling bipolar growth activation. To explore whether different patterns of Cdc42 activation are possible in vivo, we examined S. pombe rga4∆ mutant cells, lacking the Cdc42 GTPase-activating protein (GAP) Rga4. We found that monopolar rga4∆ mother cells divide asymmetrically leading to the emergence of both symmetric and asymmetric Cdc42 distributions in rga4∆ daughter cells. Motivated by different hypotheses that can mathematically reproduce the unequal fate of daughter cells, we used genetic screening to identify mutants that alter the rga4∆ phenotype. We found that the unequal distribution of active Cdc42 GTPase is consistent with an unequal inheritance of another Cdc42 GAP, Rga6, in the two daughter cells. Our findings highlight the crucial role of Cdc42 GAP localization in maintaining consistent Cdc42 activation and growth patterns across generations.     

Allosteric modulation by single enantiomers of a C3-chiral 1,4-benzodiazepine of the gamma aminobutyric acid type A receptor channel expressed in Xenopus oocytes

Xenopus laevis oocytes injected with Poly(A)⁺-RNA isolated from neuronal tissue express membrane proteins peculiar to the origin of mRNA. The translation of gamma aminobutyric acid type A (GABA_A) receptors has been shown by dose/ response behavior of GABA and the reversible blockade of the GABA-induced current by picrotoxin. This current was analyzed quantitatively under two electrode voltage-clamp conditions. This methodology has been applied for the first time to study the functional properties of the receptor as a function of the stereochemistry of the ligands. The (+)-S and (-)-R enantiomers of a water-soluble benzodiazepine derivative, 7-chloro-1,3-dihydro-3-hemisuccinyloxy-5-phenyl-1,4-benzodiazepin-2-one (OXHEM), obtained by preparative high performance liquid chromatographic (HPLC) resolution on chiral stationary phase, act as agonists in the in vitro modulation of the chloride channel. The (+)-S-OXHEM enantiomer was the more active. Chirality 9:286–290, 1997. © 1997 Wiley-Liss, Inc.     

Benzodipyrazoles: a new class of potent CDK2 inhibitors

The synthesis and the preliminary expansion of this new class of CDK2 inhibitors are presented. The synthesis was accomplished using a solution-phase protocol amenable to rapid parallel expansion and suitable to be scaled-up in view of possible lead development. Following a medicinal chemistry program aimed at improving cell permeability and selectivity, a series of compounds with nanomolar activity in the biochemical assay and able to efficiently inhibit tumor cell proliferation has been obtained.     

Acetylcholine and Dopamine Promote the Production of Platelet Activating Factor in Immature Cells of Chick Embryonic Retina

We have previously shown that platelet-activating factor (PAF), a naturally occurring lipid mediator of cell-to-cell communication, was produced by 3-day-old chick retina stimulated with acetylcholine (ACh) and dopamine (DA), but not with other neurotransmitters. ACh and DA stimulated PAF synthesis via a dithiothreitol (DTT)-insensitive cholinephosphotransferase, without affecting the acetyltransferase pathway, which was stimulated only by the calcium ionophore A23187. Therefore, we attempted to study the effects of neurotransmitters on PAF production and on the activities of the DTT-insensitive cholinephosphotransferase and acetyltransferase in the developing chick embryo retina up to hatching. Our results show that PAF was produced already at 8 days of development, when retinal cells are still rather immature and ganglion and Mueller cells are the only differentiated cells. The stimulation of PAF production occurred with ACh and not with other neurotransmitters. In older stages, DA also stimulated PAF production, as already described in the chick after hatching. DTT-insensitive cholinephosphotransferase and acetyltransferase activities were present in 8-day-old embryos, the earliest stage analyzed. Both enzymatic activities increased with age; DTT-insensitive cholinephosphotransferase increased rapidly from day 12 up to day 18, whereas acetyltransferase activity increased linearly up to the time of hatching. To promote PAF production, ACh and DA activate DTT-insensitive cholinephosphotransferase, but not acetyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)     

European Society of Biomechanics S.M. Perren Award 2014: Safety factor of the proximal femur during gait: A population-based finite element study

It has been suggested that the mechanical competence of the proximal femur is preserved with respect to physiological loading conditions rather than accidental overloading, but the consequences of this adaptation for fracture risk in the elderly remain unclear. The goal of the present study was to analyse the safety factor of the human femur in the two most frequent daily activities, level walking and stair climbing, and to understand the dependence, if any, of this safety factor on age, volumetric bone mineral density (vBMD), and gender.     

Sensitivity of a subject-specific musculoskeletal model to the uncertainties on the joint axes location

Subject-specific musculoskeletal models have become key tools in the clinical decision-making process. However, the sensitivity of the calculated solution to the unavoidable errors committed while deriving the model parameters from the available information is not fully understood. The aim of this study was to calculate the sensitivity of all the kinematics and kinetics variables to the inter-examiner uncertainty in the identification of the lower limb joint models. The study was based on the computer tomography of the entire lower-limb from a single donor and the motion capture from a body-matched volunteer. The hip, the knee and the ankle joint models were defined following the International Society of Biomechanics recommendations. Using a software interface, five expert anatomists identified on the donor's images the necessary bony locations five times with a three-day time interval. A detailed subject-specific musculoskeletal model was taken from an earlier study, and re-formulated to define the joint axes by inputting the necessary bony locations. Gait simulations were run using OpenSim within a Monte Carlo stochastic scheme, where the locations of the bony landmarks were varied randomly according to the estimated distributions. Trends for the joint angles, moments, and the muscle and joint forces did not substantially change after parameter perturbations. The highest variations were as follows: (a) 11° calculated for the hip rotation angle, (b) 1% BW × H calculated for the knee moment and (c) 0.33 BW calculated for the ankle plantarflexor muscles and the ankle joint forces. In conclusion, the identification of the joint axes from clinical images is a robust procedure for human movement modelling and simulation.