An in vitro model of tumor heterogeneity resolves genetic, epigenetic, and stochastic sources of cell state variability.

Tumor heterogeneity is a primary cause of treatment failure and acquired resistance in cancer patients. Even in cancers driven by a single mutated oncogene, variability in response to targeted therapies is well known. The existence of additional genomic alterations among tumor cells can only partially explain this variability. As such, nongenetic factors are increasingly seen as critical contributors to tumor relapse and acquired resistance in cancer. Here, we show that both genetic and nongenetic factors contribute to targeted drug response variability in an experimental model of tumor heterogeneity. We observe significant variability to epidermal growth factor receptor (EGFR) inhibition among and within multiple versions and clonal sublines of PC9, a commonly used EGFR mutant nonsmall cell lung cancer (NSCLC) cell line. We resolve genetic, epigenetic, and stochastic components of this variability using a theoretical framework in which distinct genetic states give rise to multiple epigenetic "basins of attraction," across which cells can transition driven by stochastic noise. Using mutational impact analysis, single-cell differential gene expression, and correlations among Gene Ontology (GO) terms to connect genomics to transcriptomics, we establish a baseline for genetic differences driving drug response variability among PC9 cell line versions. Applying the same approach to clonal sublines, we conclude that drug response variability in all but one of the sublines is due to epigenetic differences; in the other, it is due to genetic alterations. Finally, using a clonal drug response assay together with stochastic simulations, we attribute subclonal drug response variability within sublines to stochastic cell fate decisions and confirm that one subline likely contains genetic resistance mutations that emerged in the absence of drug treatment.

Experimental Validation of an Inductive System for Magnesium Level Detection in a Titanium Reduction Reactor.

In order to precisely determine the magnesium level in a titanium reduction retort by inductive methods, many interfering influences have to be considered. By using a look-up-table method, the magnesium level can be reliably identified by taking into account the interfering effects of the titanium sponge rings forming at the walls with their unknown geometrical and electrical parameters. This new method uses a combination of numerical simulations and measurements, whereby the simulation model is calibrated so that it represents the experimental setup as closely as possible. Previously, purely theoretical studies on this method were presented. Here, the practical feasibility of that method is demonstrated by performing measurements on a model experiment. The method is not limited to the production of titanium but can also be applied to other applications in metal production and processing.

Local Heating Test for Detection of Microcirculation Abnormalities in Patients with Diabetes-Related Foot Complications.

OBJECTIVE: In this study, authors used a wavelet analysis of skin temperature (WAST) to assess the mechanisms of microvascular tone regulation during the local heating test in patients with diabetic foot syndrome (DFS). PARTICIPANTS: The participants included control subjects and 36 hospitalized patients with DFS between 52 and 79 years old (68 ± 8 years old). They were distributed among 5 groups: 15 control subjects, 8 patients with DFS who did not develop ulcerative or necrotic disorders, 10 patients who developed the neuroischemic form of DFS complicated by foot ulceration, 12 patients with DFS complicated by toe necrosis, and 6 patients with DFS and foot gangrene. OUTCOME MEASURES: A comparison among the groups was made using a nonparametric Mann-Whitney U test. Spearman correlation coefficients were used to assess the relationship between WAST results, aortoarteriography, and ultrasonic dopplerography data. MAIN RESULTS: In control subjects, a local increase in temperature (up to 42° C) causes a more than 3-fold increase in the amplitude of foot skin temperature oscillations. In patients with DFS, the response to the local heating test was much weaker. High correlations of WAST results to arterial patency of the lower extremities and the state of the vascular walls were established. CONCLUSIONS: The WAST technique may have considerable value in evaluating the progression of DFS and the effectiveness of therapeutic interventions. The low cost of an individual test makes the WAST technique suitable for routine use in most healthcare facilities.

Early differential diagnosis of the severity of acute pancreatitis.

There is a great need for early verification of the severity of acute pancreatitis (AP). The early stage of pathogenesis of AP is characterized by endothelial dysfunction which could be determined by wavelet analysis of skin temperature (WAST) technique. The aim is to investigate whether the dysregulation of microvascular tone caused by endothelial dysfunction and detected by WAST can be a significant indicator in early differential diagnosis of AP severity. The WAST performed in the frequency range of 0.0095-0.02 Hz during the contralateral cold test. Forty nine patients with AP aged 19-65 participated in this study. The control group included 12 healthy volunteers aged 20-65. Dysregulation of vascular tone during the contralateral cold test was observed in all patients with AP. The basal amplitudes of skin temperature oscillations in patients with AP were much lower than in healthy volunteers and progressively decreased as the disease severity increased. In patients with mild and moderate AP only the vasodilator component is destroyed, but vasoconstriction still operates. In patients with severe AP both mechanisms of endothelial vascular tone regulation are destroyed. Patients with AP have abnormal microvascular reactions related to the endothelial mechanism of vascular tone regulation. Based on the initial values of amplitudes and the indices of vasoconstriction and postcold vasodilatation, the WAST method makes it possible to evaluate two related but different characteristics of the endothelial dysfunction in patients with AP on admission which can be a significant indicator in early differential diagnosis of AP severity.

Relationship of oscillating and average components of laser Doppler flowmetry signal.

Signals from laser Doppler flowmeters widely used in intravital studies of skin blood flow include, along with a slowly varying average component, an oscillating part. However, in most clinical studies, pulsations are usually smoothed by data preprocessing and only the mean blood flow is analyzed. To reveal the relationship between average and oscillating perfusion components measured by a laser Doppler flowmeter, we examined the microvascular response to the contralateral cold pressor test recorded at two different sites of the hand: dorsal part of the arm and finger pad. Such a protocol makes it possible to provide a wide range of perfusion. The average perfusion always decreases during cooling, while the oscillating component demonstrates a differently directed response. The wavelet analysis of laser Doppler flowmetry (LDF) signals shows that the pulsatile component is nonlinearly related to the average perfusion. Under low perfusion, the amplitude of pulsations is proportional to its mean value, but, as perfusion increases, the amplitude of pulsations becomes lower. The type of response is defined by the basal perfusion and the degree of vasoconstriction caused by cooling. Interpretation of the results is complicated by the nonlinear transfer function of the LDF device, the contribution of which is studied using artificial examples.

An unbiased metric of antiproliferative drug effect in vitro.

In vitro cell proliferation assays are widely used in pharmacology, molecular biology, and drug discovery. Using theoretical modeling and experimentation, we show that current metrics of antiproliferative small molecule effect suffer from time-dependent bias, leading to inaccurate assessments of parameters such as drug potency and efficacy. We propose the drug-induced proliferation (DIP) rate, the slope of the line on a plot of cell population doublings versus time, as an alternative, time-independent metric.

Detection of Endothelial Dysfunction Using Skin Temperature Oscillations Analysis During Local Heating in Patients With Peripheral Arterial Disease.

OBJECTIVE: The purpose of this study was to examine correlations between laboratory markers of ED and the degree of endothelium-dependent vasodilation using WAST during a local heating test in patients with PAD. MATERIALS AND METHODS: The study population consisted of 17 healthy subjects and 38 patients with PAD. The ST on the plantar surface of the first toe was measured during the test, and the inverse wavelet transform was applied to reconstruct the ST signals in three frequency bands corresponding to myogenic, neurogenic, and endothelial mechanisms of vascular tone regulation. RESULTS: In healthy subjects, a local increase in temperature of up to 42°C caused a greater than threefold increase in the amplitudes of foot ST oscillations. Among patients with PAD, the response to the test was much weaker in all frequency ranges. The level of vasodilation dysfunction correlated with the level of artery stenosis in the lower extremities and with laboratory markers of ED (endothelin, homocysteine, and von Willebrand factor). CONCLUSION: WAST can be considered as a low cost, portable, and easy to use technique for the noninvasive assessment of ED.

Hindered Energy Cascade in Highly Helical Isotropic Turbulence.

The conventional approach to the turbulent energy cascade, based on Richardson-Kolmogorov phenomenology, ignores the topology of emerging vortices, which is related to the helicity of the turbulent flow. It is generally believed that helicity can play a significant role in turbulent systems, e.g., supporting the generation of large-scale magnetic fields, but its impact on the energy cascade to small scales has never been observed. We suggest, for the first time, a generalized phenomenology for isotropic turbulence with an arbitrary spectral distribution of the helicity. We discuss various scenarios of direct turbulent cascades with new helicity effect, which can be interpreted as a hindering of the spectral energy transfer. Therefore, the energy is accumulated and redistributed so that the efficiency of nonlinear interactions will be sufficient to provide a constant energy flux. We confirm our phenomenology by high Reynolds number numerical simulations based on a shell model of helical turbulence. The energy in our model is injected at a certain large scale only, whereas the source of helicity is distributed over all scales. In particular, we found that the helical bottleneck effect can appear in the inertial interval of the energy spectrum.

Quantifying the correlation between photoplethysmography and laser Doppler flowmetry microvascular low-frequency oscillations.

Photoplethysmography (PPG) and laser Doppler flowmetry (LDF) are two recognized optical techniques that can track low-frequency perfusion changes in microcirculation. The aim of this study was to determine, in healthy subjects, the correlation between the techniques for specific low-frequency bands previously defined for microcirculation. Twelve healthy male subjects (age range 18 to 50 years) were studied, with PPG and LDF signals recorded for 20 min from their right and left index (PPG) and middle (LDF) fingers. Wavelet analysis comprised dividing the low-frequency integral wavelet spectrum (IWS) into five established physiological bands relating to cardiac, respiratory, myogenic, neurogenic, and endothelial activities. The correlation between PPG and LDF was quantified using wavelet correlation analysis and Spearman correlation analysis of the median IWS amplitude. The median wavelet correlation between signals (right-left side average) was 0.45 (cardiac), 0.49 (respiratory), 0.86 (myogenic), 0.91 (neurogenic), and 0.91 (endothelial). The correlation of IWS amplitude values (right-left side average) was statistically significant for the cardiac (ρ = 0.64, p < 0.05) and endothelial (ρ = 0.62, p < 0.05) bands. This pilot study has shown good correlation between PPG and LDF for specific physiological frequency bands. In particular, the results suggest that PPG has the potential to be a low-cost replacement for LDF for endothelial activity assessments.

Quantifying heterogeneity and dynamics of clonal fitness in response to perturbation.

The dynamics of heterogeneous clonal lineages within a cell population, in aggregate, shape both normal and pathological biological processes. Studies of clonality typically relate the fitness of clones to their relative abundance, thus requiring long-term experiments and limiting conclusions about the heterogeneity of clonal fitness in response to perturbation. We present, for the first time, a method that enables a dynamic, global picture of clonal fitness within a mammalian cell population. This novel assay allows facile comparison of the structure of clonal fitness in a cell population across many perturbations. By utilizing high-throughput imaging, our methodology provides ample statistical power to define clonal fitness dynamically and to visualize the structure of perturbation-induced clonal fitness within a cell population. We envision that this technique will be a powerful tool to investigate heterogeneity in biological processes involving cell proliferation, including development and drug response.

Skin blood flow and temperature oscillations during cold pressor test.

We study the relationship between the blood flow and skin temperature variations under a cold pressor test (CPT). The simultaneous laser Doppler flowmetry (LDF) and skin temperature (ST) measurements were carried out for 8 healthy subjects on the skin surface of the distal phalanx of the second (LDF) and third (ST) fingers. The skin blood perfusion decreases stepwise about twice during contralateral CPT for all 8 subjects. The temperature of the finger pad decays monotonically during the test and dropped about 1°C in mean. The power spectral densities of LDF flow and ST variations are also affected by the CPT, but subjects under study demonstrate two different types of reaction. LDF pulsations at the frequency about 0.1 Hz, which corresponds to the myogenic mechanism of vascular tone regulation, decreases in 5 subjects and increases in other 3 subjects. However in all subjects the ST pulsations behave contradictory, namely, the changes in amplitude of blood perfusion and ST pulsations due to cold pressor test are strongly anticorrelated. We discuss possible mechanisms of vascular reaction that can cause the behavior observed.

Fractional proliferation: a method to deconvolve cell population dynamics from single-cell data.

We present an integrated method that uses extended time-lapse automated imaging to quantify the dynamics of cell proliferation. Cell counts are fit with a quiescence-growth model that estimates rates of cell division, entry into quiescence and death. The model is constrained with rates extracted experimentally from the behavior of tracked single cells over time. We visualize the output of the analysis in fractional proliferation graphs, which deconvolve dynamic proliferative responses to perturbations into the relative contributions of dividing, quiescent (nondividing) and dead cells. The method reveals that the response of 'oncogene-addicted' human cancer cells to tyrosine kinase inhibitors is a composite of altered rates of division, death and entry into quiescence, a finding that challenges the notion that such cells simply die in response to oncogene-targeted therapy.

Turbulent viscosity and turbulent magnetic diffusivity in a decaying spin-down flow of liquid sodium.

The free decay of a strong flow of liquid sodium (at Reynolds number defined via the maximal mean velocity and the radius of the channel cross section up to Re≈3×10(60) and the corresponding magnetic Reynolds number up to Rm≈30) generated by the sudden stop of a rapidly rotating toroidal channel is studied experimentally. The toroidal and poloidal components of velocity are measured using a potential probe. We describe the onset of motion, the evolution of strongly anisotropic fluctuations, and the homogenization and decay of turbulence in the final period. We analyze the statistical characteristics of velocity fields in relation to the behavior of effective magnetic diffusivity estimated from measurements of the phase shift between the induced and applied magnetic fields. For the late (self-similar) decay of turbulent flow, turbulent viscosity is shown to be dependent on the root-mean-square velocity pulsations and can be expressed as νt∼νRe1.3. The behavior of turbulent magnetic diffusivity depends on the magnetic Reynolds number defined in terms of the root-mean-square velocity pulsations. At low magnetic Reynolds numbers (Rmrms<1), turbulent magnetic diffusivity grows rapidly with increasing velocity pulsations (ηt∼ηRmrms2). If the magnetic Reynolds number exceeds unity, the behavior of turbulent magnetic diffusivity becomes similar to the behavior of turbulent viscosity. The highest values of turbulent magnetic diffusivity are achieved at the end of braking, which corresponds to the transient stage of a strongly anisotropic turbulent flow in which the poloidal velocity oscillations prevail.

Direct measurement of effective magnetic diffusivity in turbulent flow of liquid sodium.

The first direct measurements of effective magnetic diffusivity in turbulent flow of electroconductive fluids (the so-called ß effect) under the magnetic Reynolds number Rm≫1 are reported. The measurements are performed in a nonstationary turbulent flow of liquid sodium, generated in a closed toroidal channel. The peak level of the Reynolds number reached Re≈3×10(6), which corresponds to the magnetic Reynolds number Rm≈30. The magnetic diffusivity of the liquid metal was determined by measuring the phase shift between the induced and the applied magnetic fields. The maximal deviation of magnetic diffusivity from its laminar value reaches about 50%.

Wavelet-based correlations of skin temperature and blood flow oscillations.

The wavelet transform-based correlation analysis has been used to study skin temperature fluctuations caused by periodic changes in blood flow resulting from oscillations in vasomotor smooth muscle tone. We considered two cases, one in which temperature measurements and blood flow recordings by laser Doppler flowmetry are made simultaneously and another in which two temperature signals are measured concurrently. Twelve healthy subjects participated in our study. The gapped wavelet technique was used to suppress artifacts caused by boundary effects. Simultaneous recordings of skin temperature fluctuations and the signal of the laser Doppler flowmeter provided correlation coefficients essentially exceeding the values obtained for noise signals within three spectral ranges corresponding to myogenic (0.05-0.14 Hz), neurogenic (0.02-0.05 Hz), and endothelial (0.0095-0.02 Hz) regulation mechanisms. Within the frequency range from 0.14 to 2 Hz the values of the correlation function are compatible with the values of noise correlations. The same results were obtained for two concurrently measured temperature signals. Reduction in the amplitude of temperature fluctuations and in the level of correlations with the frequency arises because the skin has the properties of a low-frequency filter. As temperature fluctuations propagate their amplitude decays as an exponential function of frequency. Hence small oscillations in the spectral range reflecting the influence of heartbeat and respiration cannot be distinguished from external thermal noise.

Newly synthesized tetraoxa-diaza crown ether derivatives versus commercialized crown ethers in the separation of positional isomers with capillary electrophoresis.

Three new tetraoxa-diaza derivatives of 1,4,10,13-tetraoxa-7,16-diazacyclo-octadecane (R-1, R-2 and R-3) and three commercially available crown ethers, 18-crown-6 (18C6), (+)-18-crown-6-tetracarboxilic acid (18C6H4) and 1,4,10,13-tetraoxa-7,16-diazacyclo-octadecane, were investigated to separate the positional isomers of aminophenol, aminobenzoic acid and aminocresol. The running electrolyte, in which the crown ethers were dissolved, was a 50 mM Tris solution adjusted to pH 2.0 with hydrochloric acid. Using 50 mM H3PO4 buffer, whose pH was adjusted to 2.0 with Tris, or only hydrochloric acid solution with the same pH, did not allow good separations for the tested components. The effect of the crown ether concentration on the separation of the 11 positional isomers was studied in the concentration range of 10-50 mM. The best separations were achieved using the 18C6 and the 18C6H4 crown ethers: 9 isomers out of 11 could be separated within one run. The m- and p-aminophenol isomers could not be separated under the investigated experimental conditions. The newly synthesized tetraoxa-diaza crown ether derivatives were only found suitable for the separation of aminobenzoic acid positional isomers. The macrocyclic ring of the tetraoxa-diaza crown ethers was not able to form a stable inclusion complex with the tested positional isomers. Consequently, the aminophenol and aminocresol isomers were not separated, the isomers migrated with the same or very similar velocities.

Large- and small-scale interactions and quenching in an alpha2-dynamo.

The evolution of the large-scale magnetic field in a turbulent flow of conducting fluid is considered in the framework of a multiscale alpha2-dynamo model, which includes the poloidal and the toroidal components for the large-scale magnetic field and a shell model for the small-scale magnetohydrodynamical turbulence. The conjugation of the mean-field description for the large-scale field and the shell formalism for the small-scale turbulence is based on strict conformity to the conservation laws. The model displays a substantial magnetic contribution to the alpha effect. It was shown that a large-scale magnetic field can be generated by current helicity even solely. The alpha quenching and the role of the magnetic Prandtl number (Pm) are studied. We have determined the dynamic nature of the saturation mechanism of dynamo action. Any simultaneous cross correlation of alpha and large-scale magnetic field energy EB is negligible, whereas coupling between alpha and EB becomes substantial for moderate time lags. An unexpected result is the behavior of the large-scale magnetic energy with variation of the magnetic Prandtl number. Diminishing of Pm does not have an inevitable ill effect on the magnetic field generation. The most efficient large-scale dynamo operates under relatively low Prandtl numbers--then the small-scale dynamo is suppressed and the decrease of Pm can lead even to superequipartition of the large-scale magnetic field (i.e., EB>Eu). In contrast, the growth of Pm does not promote the large-scale magnetic field generation. A growing counteraction of the magnetic alpha effect reduces the level of mean large-scale magnetic energy at the saturated state.

Screw dynamo in a time-dependent pipe flow.

The kinematic dynamo problem is investigated for the flow of a conducting fluid in a cylindrical, periodic tube with conducting walls. The methods used are an eigenvalue analysis of the steady regime, and the three-dimensional solution of the time-dependent induction equation. The configuration and parameters considered here are close to those of a dynamo experiment planned in Perm, which will use a torus-shaped channel. We find growth of an initial magnetic field by more than three orders of magnitude. A marked field growth can be obtained if the braking time is less than 0.2 s and only one diverter is used in the channel. The structure of the seed field has a strong impact on the field amplification factor. Generation properties can be improved by adding ferromagnetic particles to the fluid in order to increase its relative permeability, but this will not be necessary for the success of the dynamo experiment. For higher magnetic Reynolds numbers, the nontrivial evolution of different magnetic modes limits the value of simple "optimistic" and "pessimistic" estimates.