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The growing zone (GZ) of the unicellular coenocytic sporangiophore of Phycomyces blakesleeanus represents the site of stimulus reception (light, gravity, gas) and stimulus response, i.e., local modulations of the elongation growth, which may result, in dependence of the stimulus direction, in tropic bending. Until now, evidence for a possible participation of the columella in sensory reception is absent. We confirm with light microscopy earlier studies that show that the GZ and the columella are not separated by a membrane or cell wall, but rather form a spatial continuum that allows free exchange of cytoplasm and organelle transport. Evidence is presented that the columella is responsive to external stimuli. Columellae, from which spores and sporangial cell wall had been removed, respond to exogenous auxin with a local depolarization of the membrane potential and an increased growth rate of the GZ. In contrast, auxin applied to the GZ causes a decrease of the growth rate irrespective of the presence or absence of sporangia. The response pattern is specific and relevant for the sensory reception of Phycomyces, because the light-insensitive mutant C148carAmadC, which lacks the RAS-GAP protein MADC, displays abnormal IAA sensitivity and membrane depolarization. We argue that the traditional concept of the GZ as the only stimulus-sensitive zone should be abandoned in favor of a model in which GZ and columella operate as a single entity capable to orchestrate a multitude of stimulus inputs, including auxin, to modulate the membrane potential and elongation growth of the GZ.

The sustained increase in leaf photosynthesis may increase crop yield. Due to many limitations, plants use much less photosynthetic capacity than is theoretically possible. Plant nanobionics investigates nanoparticle application in living plants, which improves certain plant functions. We synthesized and tested nontoxic orange carbon dots (o-CDs) for the photosynthetic efficiency increase in maize (Zea mays L.). We applied o-CDs foliarly or by adding to the growth solution. The photosynthetic parameters and content of photosynthetic pigments were recorded. The total phenolic content (TPC) and total antioxidant activity (TAA) were measured to monitor the plant antioxidant response to o-CDs. The photosynthetic parameters' values were higher for foliar than for solution application. The 1 mg/L o-CDs applied foliarly and 5 mg/L in solution increased photosynthetic parameters in leaves. The o-CDs raised photosynthetic pigments. The TAA and TPC results indicate reduced antioxidant activity in the plant organs more exposed to o-CDs, depending on the way of application. ©

Fish erythrocytes released from erythropoietic sites are immature. During circulation, they increase their own surface, while the cell body and nuclei begin to become more elliptical and less spherical during maturation. The relative abundance of different developmental stages represents an erythron profile which could be a more sensitive indicator of contamination than classic hematological indices. In this study, we analyzed blood smears of the common nase (Chondrostoma nasus) and vimba bream (Vimba vimba) with the main goal to identify the determinants of the developmental stage of erythrocytes. Based on parameters developmental stages, erythrocytes are categorized into immature, intermediate or mature. In this investigation we have used four classification methods: the Two Step Cluster analysis, the K-Means Cluster analysis, and Neural Networks–Multilayer Perceptron and an Ordinal Regression model. Our findings clearly justify that Multilayers Perceptron and OLR models are appropriate to classify the developmental stage of fish erythrocytes in both species.

The main objective of this study was to evaluate the concentrations and seasonal variations of trace elements in surface sediments of three major rivers in Serbia—the Danube, the Zapadna Morava (ZM), and the Južna Morava (JM)—according to sediment quality guidelines. The ZM and the JM create the Velika Morava River, one of the most important tributaries of the Danube, which has been characterized as a source of heavy metal pollution. The total concentrations of 15 elements (Al, As, B, Ba, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Sr, and Zn) were determined in surface sediments (0–15 cm depth) collected during three seasons using inductively coupled plasma spectroscopy (ICP-OES). Principle component analysis (PCA) was used to identify the main variations in metal concentrations and grain size distribution. Scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM-EDS) was used for grain analysis. PCA and three-way MANOVA results showed significant differences in element concentrations and grain size distribution between the rivers, and significant seasonal differences for each river. The concentrations of Cu and Ni exceeded sediment quality guideline levels in the ZM and the Danube, respectively, while excess Hg was detected in all three rivers. Concentrations of Al, Ba, Cu, Fe, Sr, and Zn significantly varied between seasons in the Danube and the ZM, being the highest in the summer. In the JM, concentrations of Al, As, Fe, Mn, and Zn varied with season, with the lowest values in the summer. The ZM had the highest percentage of silt and clay, and SEM-EDS analysis of ZM sediments showed associations of Cu with carbonate hydroxides and/or iron oxides in particles

Metal content was evaluated in the sediment, macrophytes and fish in the Medjuvrsje reservoir (Western Serbia). Concentrations of 16 trace elements (Ag; Al; As; B; Ba; Cd; Co; Cr; Cu; Fe; Li; Mn; Ni; Pb; Sr; Zn) were analysed in the sediment, macrophytes and fish of an aquatic ecosystem. Five macrophyte species and three fish tissues (liver, muscle, gills) from five fish species (freshwater bream, common nase, Prussian carp, chub, wels catfish) were sampled and the metal content was analysed with ICP-OES. The sediment concentrations of Cu, Cd, and Zn exceeded the Canadian sediment quality guidelines while concentrations of Cr and Ni were above the Netherlands’ target values. Bioaccumulation factors (BSAF) were calculated for analysed macrophytes and fish tissue. The BSAF had higher values for macrophytes for all investigated elements except for Cu and Zn; Cu had a higher value in the liver of the freshwater bream (0.823) and Zn had a higher value in the liver of freshwater bream (0.914) and chub (0.834) as well as in gills of Prussian carp (2.58) and chub (1.26). Potamogeton pectinatus, Ceratophylum demersum and the root of Phragmites communis showed higher accumulation of elements than Trapa natans and Potamogeton fluitans and the body of P. communis. The highest BSAF values for Ba, Mn, Sr and Ni were recorded in the gills. Cd and Cu had the highest BSAF values in the liver. Results confirmed that particular macrophyte and fish species could be a good indicator of reservoir water and sediment pollution.

We propose a new numerical method for calculating 2D fractal dimension ( DF ) of a surface. This method represents a generalization of Higuchi’s method for calculating fractal dimension of a planar curve. Using a family of Weierstrass–Mandelbrot functions, we construct Weierstrass–Mandelbrot surfaces in order to test exactness of our new numerical method. The 2D fractal analysis method was applied to the set of histological images collected during direct shoot organogenesis from leaf explants. The efficiency of the proposed method in differentiating phases of organogenesis is proved.

The key method for therapies of various cancer types could be the molecular-targeted therapy, based on individual gene profile for each patient. One of the main procedures used for genetic testing is the real-time polymerase chain reaction (real-time PCR). Physical principle behind real-time PCR procedure is the fluorescence. Fluorescence labeled probes (primers) is attached to quenchers. Upon reaction of polymerization, quenchers are removed, and the fluorescence emission intensity increases in time. Emission spectra shape and its maximum position can differ if the fluorophore was present in different microenvironment. That property is widely exploited in fluorescence spectroscopy and chromatography. This paper, for the first time, describes utilization of full spectroscopic potential of multichannel excitation/emission filter sets in real-time PCR device. Instead of monitoring fluorescence intensity in time for a single fluorescence emission channel, the ratio values of three different kinetics curves were calculated and analyzed by applying k-means clustering and dendrogram analysis. Obtained results have shown that described analytical improvement provides identification of nine different groups of mutations if the commercial QIAGEN® EGFR PCR Kit was used. Method can be applied to any kit, capable to simultaneously detect several different mutations.

We applied a combination of fractal analysis and Independent Component Analysis (ICA) method to detect the sources of fractal complexity in snail Br neuron activity induced by static magnetic field of 2.7 mT. The fractal complexity of Br neuron activity was analyzed before (Control), during (MF), and after (AMF) exposure to the static magnetic field in six experimental animals. We estimated the fractal dimension (FD) of electrophysiological signals using Higuchi's algorithm, and empirical FD distributions. By using the Principal Component Analysis (PCA) and FastICA algorithm we determined the number of components, and defined the statistically independent components (ICs) in the fractal complexity of signal waveforms. We have isolated two independent components of the empirical FD distributions for each of three groups of data by using FastICA algorithm. ICs represent the sources of fractal waveforms complexity of Br neuron activity in particular experimental conditions. Our main results have shown that there could be two opposite intrinsic mechanisms in single snail Br neuron response to static magnetic field stimulation. We named identified ICs that correspond to those mechanisms - the component of plasticity and the component of elasticity. We have shown that combination of fractal analysis with ICA method could be very useful for the decomposition and identification of the sources of fractal complexity of bursting neuronal activity waveforms.

The complexity, entropy and other non-linear measures of the electroencephalogram (EEG), such as Higuchi fractal dimension (FD), have been recently proposed as the measures of anesthesia depth and sedation. We hypothesized that during unconciousness in rats induced by the general anesthetics with opposite mechanism of action, behaviorally and poligraphically controlled as appropriately achieved stable anesthesia, we can detect distinct inter-structure brain dynamic using mean FDs. We used the surrogate data test for nonlinearity in order to establish the existence of nonlinear dynamics, and to justify the use of FD as a nonlinear measure in the time series analysis. The surrogate data of predefined probability distribution and autocorrelation properties have been generated using the algorithm of statically transformed autoregressive process (STAP). FD then is applied to quantify EEG signal complexity at the cortical, hippocampal and pontine level during stable general anesthesia (ketamine/xylazine or nembutal anesthesia). Our study showed for the first time that global neuronal inhibition caused by different mechanisms of anesthetic action induced distinct brain inter-structure complexity gradient in Sprague Dawley rats. EEG signal complexities were higher at cortical and hippocampal level in ketamine/xylazine vs. nembutal anesthesia, with the dominance of hippocampal complexity. In nembutal anesthesia the complexity dominance moved to pontine level, and ponto-hippocampo-cortical decreasing complexity gradient was established. This study has proved the Higuchi fractal dimension as a valuable tool for measuring the anesthesia induced inter-structure EEG complexity.

The ILF (Image Landscapes’ Fractal Dimension) method and [Formula: see text] method obtained by a 2D generalization of Higuchi’s algorithm were applied to a set of 120 digital histological images of Anal Intraepithelial Neoplasia (AIN). The main goal of this research was to examine accuracy that means sensitivity and specificity of these methods and compare the applicability of both methods in the quantitative characterization and differentiation of clinical cases of AIN. Histological examination by an experienced pathologist revealed three grades of AIN tumors in the 120 histological slices: 36 of AIN1, 56 of AIN2 and 28 of AIN3. Statistical tests showed significant differences between calculated fractal dimension values in three datasets (AIN1, AIN2 and AIN3) using ILF and [Formula: see text] methods at the level of significance of 0.05. Application of the ILF and [Formula: see text] methods has an advantage when it comes to speed, accuracy, simplicity and time necessary for analysis. Both methods can be successfully applied for differentiation between AIN stages giving practically the same results. They can easily be adapted to other histological specimen.

The aim of our study was to investigate the effect of camphor essential oil on rat cerebral cortex activity by fractal analysis. Fractal dimension (FD) values of the parietal electrocortical activity were calculated before and after intra-peritoneal administration of camphor essential oil (450-675 ?l/kg) in anesthetized rats. Camphor oil induced seizure-like activity with single and multiple spiking of high amplitudes in the parietal electrocorticogram and occasional clonic limb convulsions. The FD values of cortical activity after camphor oil administration increased on the average. Only FD values of cortical ECoG sequences were lower than those before camphor oil administration.

The article reviews the applications of Higuchi's fractal dimension (HFD) in basic and clinical neurophysiology.This article summarizes and critically reviews a broad literature and major findings concerning the applications of HFD.Many studies on the presented topics demonstrated the advantage of HFD for measuring the complexity of neuronal activity. Background and objectiveFor more than 20 years, Higuchi's fractal dimension (HFD), as a nonlinear method, has occupied an important place in the analysis of biological signals. The use of HFD has evolved from EEG and single neuron activity analysis to the most recent application in automated assessments of different clinical conditions. Our objective is to provide an updated review of the HFD method applied in basic and clinical neurophysiological research. MethodsThis article summarizes and critically reviews a broad literature and major findings concerning the applications of HFD for measuring the complexity of neuronal activity during different neurophysiological conditions. The source of information used in this review comes from the PubMed, Scopus, Google Scholar and IEEE Xplore Digital Library databases. ResultsThe review process substantiated the significance, advantages and shortcomings of HFD application within all key areas of basic and clinical neurophysiology. Therefore, the paper discusses HFD application alone, combined with other linear or nonlinear measures, or as a part of automated methods for analyzing neurophysiological signals. ConclusionsThe speed, accuracy and cost of applying the HFD method for research and medical diagnosis make it stand out from the widely used linear methods. However, only a combination of HFD with other nonlinear methods ensures reliable and accurate analysis of a wide range of neurophysiological signals.

We propose a new method for calculating fractal dimension (DF) of a signal y(t), based on coefficients [Formula: see text], mean absolute values of its nth order derivatives (consecutive finite differences for sampled signals). We found that logarithms of [Formula: see text], n = 2,3,…,n max , exhibited linear dependence on n: [Formula: see text] with stable slopes and Y-intercepts proportional to signal DF values. Using a family of Weierstrass functions, we established a link between Y-intercepts and signal fractal dimension: [Formula: see text] and calculated parameters A(n max ) and B(n max ) for n max = 3,…,7. Compared to Higuchi's algorithm, advantages of this method include greater speed and eliminating the need to choose value for k max , since the smallest error was obtained with n max = 3.

The aim of this study was to reveal the nature and meaning of interspike background activity (RBA) recorded in Prukinje cell layer of rat cerebellum. We compared Fourier amtplitude spectra of recorded, extracted and averaged simple spike(s)—SS and complex spike(s)—CS with the mean amplitude spectrum of the remaining interspike RBA. A much greater similarity of spectral characteristics was noticed between SS and RBA, than between CS and RBA. Then, we simulated background activity (SBA), by superimposing averaged SS with randomized amplitudes and time delays. There was a significant correlation (p

River sediments are a major source of metal contamination in aquatic food webs. Due to the ability of metals to move up the food chain, fishes, occupying higher trophic levels, are considered to be good environmental indicators of metal pollution. The aim of this study was to analyze the metal content in tissues of the common barbel (Barbus barbus), a rheophilous cyprinid fish widely distributed in the Danube Basin, in order to find out if it can be used as a bioindicator of the metal content in the river sediment. We analyzed bioavailable concentrations of 15 elements (Al, As, B, Ba, Co, Cr, Cu, Hg, Mn, Mo, Ni, Pb, Se, Sr, and Zn) in sediments of the Danube (D), the Zapadna Morava (ZM), and the Južna Morava (JM) using the inductively coupled plasma spectroscopy (ICP-OES). The barbel specimens were collected in the proximity of sediment sampling sites for the analysis of metals in four tissues, gills, muscle, intestine, and liver. The sediment analysis indicated that the ZM is the most polluted with Cu, Ni, and Zn compared to other two rivers. The JM had the lowest concentrations of almost all observed elements, while the Danube sediments were mainly characterized by higher concentrations of Pb. The fish from the ZM had the highest concentration of Cu and Ni in the liver and intestine, and of Zn in the muscle tissue, which was in accordance with the concentrations of these metals in the sediment. Scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM-EDS) was used for further analyses of metal interactions with fish tissues. The results suggest that the barbel can potentially be used as a bioindicator of sediment quality with respect to metal contamination.

To study the influence of a static magnetic field (SMF, 2 mT) and extremely low frequency magnetic field (ELF MF, 50 Hz, 2 mT) on the neuronal population activity, the experiments were performed on adult longhorn beetles Morimus funereus (Coleoptera, Cerambycidae). Based on a wavelet analysis of the local field potentials (LFPs), our study showed for the first time that the effects of prolonged and repeated exposure to the ELF MF on the LFPs were irreversible within investigated time frame. The relative wavelet energy (RWE) of 4-8 Hz frequency band was significantly increased after sine ELF MF (SnMF)/square ELF MF (SqMF) in comparison to the control value. The RWE of slower oscillations (1-2 Hz) was significantly decreased after the repeated exposures to either SnMF or SqMF. The SqMF induced decreasing of the faster waves in the range of 64-128 Hz. However, we did not prove with presented methods that exposure to the SMF for 5 min produces any effects on the neuronal population activity. This study has proved the wavelet transform as a valuable tool for measuring the effects of SMF and ELF MF on the neuronal population activity in M. funereus antennal lobe. Ministry of Education, Science, and Technological Development of the Republic of Serbia {[}173027, 173045]

Aim of this study was to investigate the application of normalized mean of the empirical Higuchi fractal dimension (FD) distributions, as a new approach to analyze the spontaneous bioelectrical activity of garden snail (Helix pomatia) Br neuron. The effect of ouabain on modulation of Br neuron bursting activity before and after the exposure to 10 mT static magnetic field (SMF) was observed by analyzing the following parameters: action potential (AP), interspike interval (ISI) and interbursting interval (IBI) components. Normalized mean of the empirical FD distributions were formed for the following experimental conditions: Control 1, Ouabain 1, Control 2, SMF 2, ASMF 2, Control 3, SMF 3 and Ouabain ASMF 3. Our main results have shown that ouabain without SMF induced increase in participation of AP and a decrease in participation of IBI components compared to the first control condition. However, in the presence of 10 mT SMF, ouabain-induced changes of measured parameters of Br neuron activity were less pronounced compared to the third control condition. We have shown that normalized mean of the empirical FD distributions is a useful method for detecting the changes in AP, ISI, and IBI components of complex bursting activity in altered physiological states. The Serbian Ministry of Education, Science and Technological Development {[}173027, 173045]

University of Belgrade, Department of Neurobiology, Institute for Biological Research-Sinisa Stankovic, 11060, Belgrade, Serbia Abstract. Thisstudywasaimedtoexplorethesleep/wakestatesrelatedcortico-pontinethetacarrierfrequency phase shiftfollowingasystemicallyinducedchemicalaxotomyofthemonoaminergicafferentswithinabrainofthefreelymovingrats.Ourexperimentswereperformedin14adult,male Sprague Dawley rats, chronically implanted for sleep recording. We recorded sleep during baseline condition, following sham injection (saline i.p. 1 ml/kg), and every week for 5 weeks fol-lowing injection of the systemic neurotoxins (DSP-4 or PCA; 1 ml/kg, i.p.) for chemical axotomy of the locus coeruleus (LC) and dorsal raphe (DR) axon terminals. Aftersleep/wakestatesiden-tification, FFT analysis was performed on 5s epochs. Theta carrier frequency phase shift (∆Φ)was calculated for each epoch by averaging theta Fourier component phase shifts, and the ∆Φvalues were plotted for each rat in control condition and 28 days following the monoaminergic lesions, as a time for permanently established DR or LC chemical axotomy. Calculated group averages have shown that ∆Φ increased between pons and cortex significantlyinallsleep/wakestates (Wake, NREM and REM) following the monoaminergic lesions, with respect to controls. Monoaminergic lesions established the pontine leading role in the brain theta oscillations during all sleep/wake states.Key words: Monoaminergic lesion — Sleep/wake states — Theta carrier frequency phase shift— Locus coeruleus — Nucleus dorsalis raphe IntroductionNeuronal network oscillations in the brain functions, and neuronal synchrony within the same network or between interrelated networks, have been extensively investigated on different oscillation bands (Singer 1993). However, theexact mechanisms of brain oscillations and synchroniza-tion of neuronal activity within single or between differentbrain structures are not well understood (Penttonen and Buzsaki 2003). The hippocampal theta rhythm is the most studiedrhythm in rat. Theta oscillations occur across many brain

In the present work we report a new combination of fractal analysis and some advanced statistical methods and their application for the quantitative detection of the effects of a static magnetic field of 2.7 mT on fractal complexity changes of Br neuron activity in the subesophageal ganglia of the garden snail Helix pomatia. We used factor analysis (FA) in the analysis of the empirical distribution of fractal dimension (FD). FA showed that there are two factors in the empirical distribution of FD. Results indicated that the significant changes in the fractal complexity of Br neuron activity occurred during treatment with a magnetic field, were extended to the post exposure period.

The amplitude power spectra and fractal dimensions of surrogate data designed as coloured noise were compared with spectra and FDs of biosignals in order to improve the tools for brain activity analysis. It is interesting if biosignals yielded spectral indices and fractal dimension values different from almost all of those of surrogate data, could we have more confidence that original biosignals (brain activities) arised from a nonlinear process.

The cerebellum, even when not directly damaged, is potentially interesting for understanding the adaptive responses to brain injury. Cerebellar electrocortical activity (ECoG) in rats was studied using spectral and fractal analysis after single and repeated unilateral injury of the parietal cortex. Local field potentials of cerebellar paravermal cortex were recorded before brain injury, in the acute phase (up to 2.5 hours) after a first injury of anesthetized rats, and then before and after second, third, and, in some cases, fourth injury. Relative gamma power (32.1–128.0 Hz) and fractal dimension of ECoGs were temporarily increased after the first injury. However, there was a permanent mild increase in gamma activity and a mild increase in the fractal dimension of cerebellar activity as a chronic change after repeated remote brain injury. There was a negative linear correlation between the normalized difference in fractal dimensions and normalized difference in gamma powers of cerebellar activity only in the case of repeated brain injury. This is the first study showing that correlation between the parameters of spectral and fractal analyses of cerebellar activity can discriminate between single and repeated brain injuries, and is, therefore, a promising approach for identifying specific pathophysiological states.

We recorded electrocortical activity in anesthetized rats and constructed k(max) new self-similar time series, applying Higuchi's algorithm. The aim of this study was to estimate value of the parameter k(max) in order to obtain fractal dimension values as an optimum measure of biosignal change. After our analysis, electrocortical activity recordings resulted in a family of curves f(k(max)). Three regions could be distinguished 2or = k(max)8, with a U-shape; 8or = k(max)or = 30, with a steeper quasilinear increase; and k(max)or = 30, with a smaller slope quasilinear increase. We suggest the optimum region for k(max): 8k(max)18, specifically k(max) = 8.

Activity of neuronal populations is usually measured with multielectrode systems. In this paper a procedure is described for estimating population activity changes in rat cerebellar cortex, using one microelectrode. Signals consisted of simple, complex spikes and interspike recorded background activity (RBA). After their separation, simple spikes were averaged, forming a simple spike template (SST). The remaining RBA was simulated (SBA), by superimposing SST waveforms with random time delays and intensities. A series of SBA was formed, differing in the superposition frequency ( f sup ) of individual SST. Mean amplitude spectra ( Amp SBA ) were calculated and Amp SBA = f ( f sup ) treated as a calibration line for estimating activity level of the surrounding neuronal population. Since the uniform probability distribution of SST intensities proved inadequate, we derived a new one, based on the power function for spike intensity vs. electrode distance attenuation. A family of new lines emerged, depending on the model parameters. Since all were linear in the log–log plots, with slopes not varying significantly, we proposed a method for estimating population activity changes in different experimental conditions, using two measured values of Amp RBA . Relative nature of the results makes this method suitable for comparative studies.