Laser bio-stimulation is a well-established procedure in Medical Acupuncture. Nevertheless there is still a confusion as to whether it works or the effect is just placebo. Although a plethora of scientific papers published, showing positive clinical results, there is still a lack of objective scientific proofs about the bio-stimulation effect of lasers used in Acupuncture. The objective of this work was to design and build a body surface electrode and an amplifier for biopotential recording from acupuncture points, considered here as small localized bio-sources (SLB). The design is aimed for studying SLB potentials provoked by laser stimulus, in search for objective proofs of the bio-stimulation effect of lasers used in Medical Acupuncture.The active electrode presented features a new adjustable anchoring system and fractionation of the biopotential amplifier between the electrode and the cabinet's location. The new adjustable electrode anchoring system is designed to reduce the electrode-skin contact impedance, its variation and motion artifacts. That is achieved by increasing the electrode-skin tension and decreasing its relative movement. Additionally the sensing element provides local constant skin stretching thus eliminating the contribution of the skin potential artifact. The electrode is attached to the skin by a double-sided adhesive pad, where the sensing element is a stainless steel, 4 mm in diameter. The fractionation of the biopotential amplifier is done by incorporating the amplifier's front-end op-amps at the electrodes, thus avoiding the use of extra buffers. The biopotential amplifier features two selectable modes of operation: semi-AC-mode with a -3 dB bandwidth of 0.32–1000 Hz and AC-mode with a bandwidth of 0.16–1000 Hz.2 on an unprepared skin of the inner forearm. The peak-to-peak noise voltage measured at the amplifier output, with input terminals connected to common, was 10 mVp-p, or 2 μVp-p referred to the input. The common-mode rejection ratio of the amplifier was 96 dB at 50 Hz, measured with imbalanced electrodes' impedances. The prototype was also tested practically and sample records were obtained after a low intensity SLB laser stimulation. All measurements showed almost a complete absence of 50 Hz interference, although no electrolyte gel or skin preparation was applied.The average measured DC electrode-skin contact impedance of the proposed electrode was 450 kΩ, with electrode tension of 0.3 kg/cmThe results showed that the new active electrode presented significantly reduced the electrode-skin impedance, its variation and motion artifact influences. This allowed SLB signals with relatively high quality to be recorded without skin preparation. The design offers low noise and major reduction in parts, size and power consumption. The active electrode specifications were found to be better or at least comparable to those of other existing designs. The non-invasive nature of laser bio-stimulation have made lasers an attractive alternative in Medical Acupuncture at the last 25 years. Unfortunately there is still a confusion as to whether they work or their effect is just placebo. Although a plethora of scientific papers published, showing positive clinical results, there is still a lack of objective scientific proofs about the bio-stimulation effect of lasers used in Acupuncture. The objective of this work was to design and built a body surface electrode and an amplifier for biopotential recording from acupuncture points. The latter are considered here as small localized bio-sources (SLB). As discussed by other authors, SLB are small area body regions with specific electrical, physiological and anatomical properties (e.g. high density of gap junctions, relatively low impedance etc.) -4. They The active electrode is aimed for studying SLB potentials provoked by laser stimulus, in search for objective proofs of the bio-stimulation effect of lasers used in Medical Acupuncture.The attempt to define the optimal parameters of the active electrode was based on a set of preliminary measurements performed in our laboratory. Anatomical, physiological and electrical characteristics of the signal source were considered. The SLB AC signal level, after stimulation, varied from subject to subject, but did not exceed 1 mV peak-to-peak (p-p). Additionally SLB occasionally manifested a high DC potential up to 200 mV, implying the use of differential amplifier with optional DC coupling and wide DC input range. The frequency band of the signal of interest was found to be in the range 0–200 Hz. The preliminary experiments showed that SLB potentials were best recorded with small pasteless electrodes although their contact impedance depends strongly on sweat gland secretion. The application of electrolytic gel resulted in significant reduction of the SLB signal amplitude, probably due to smoothing of the potential caused by saturation of the epidermis with electrolyte. Moreover, potentials between closely spaced SLB might be shortened by the application of excessive gel or large surface electrodes. An additional difficulty is that the SLB are often situated at convex or concave body surface areas where large flat electrodes could not be easily affixed. Skin abrasion with sandpaper is also not recommended since it can cause skin irritation and SLB potential changes. However, the use of small passive dry electrodes on an unprepared skin results in high electrode-skin impedance, motion artifacts, high power-line cable interference and noise. When the electrodes are DC coupled to the amplifier, a motion induced interfering signal appears at the amplifier input, mainly due to:• Electrokinetic effect – the disturbance of the double layer of charge at the electrode-skin interface causes variations of the DC polarization potential .• Skin potential or skin stretch artifact – stretching of the skin causes a change of the potential of the barrier layer between the epidermis and the dermis .• Variation in the electrode-skin contact resistance – caused by the amplifier input bias current and the current flowing due to the polarization potential.d//Rd is the coupling impedance of the double layer at the electrode-skin interface, Ci//Ri is the amplifier input impedance, Rs is the minimum series contact resistance and Vpol is the DC polarization potential. Then the motion artifact signal at the amplifier input can be expressed asThe complex electrochemical interactions that take place at the electrode-skin interface have been subject to much study in order an equivalent electrical model to be developed -12. The Vmot = ΔVpol + ΔVskin + (ΔRd + ΔRs) (VPOL / Ri + ib)     (1)skin is the skin stretch artifact and ib is the amplifier input bias current. It was deduced that in order to keep the resistive interfering component less than 10 μV when DC coupling is employed and with both currents contributing equally to it, then ib<50 pA and Ri>1 GΩ [where ΔV Ri>1 GΩ . If an AFor dry electrodes the motion artifacts are mainly caused by changes of the polarization potential and the contact impedance due to the poor electrolyte layer at the electrode-skin interface. Therefore a firm electrode-skin contact is of primary importance. Thus a new adjustable electrode anchoring system was designed for the purpose, as shown in Fig. 0 and A1 were integrated at the electrodes  is the differential-mode (DM) gain and Ac1(s) is the common-mode (CM) gain of op-amp A1. If we take the usual definitions for the DM input signal, Vd=(E1-E0), and for the CM input signal, Vc=(E1 + E0)/2, then the output voltage can be also written aswhere AU1(s) = Ad(s)Vd + Ac(s)Vc     (3)d(s), and the CM gain Ac(s), are given byIt can be shown that the respective expressions for the DM gain A, τ0 and τ1 are the respective DM open loop gains, and the time constants of the first poles of op-amps A0 and A1 , and Ac1(s) is the CM gain of op-amp A1. It should be noted that the CM gain of op-amp A0 is omitted from (4) and (5), since its influence on both gains is insignificant.where Considering (4) and (5), then the common-mode rejection ratio CMRR(s) is0 and A1 are ideal then the only factor contributing to the CMRR is the mismatching of the resistors. Thus we can define a common-mode rejection ratio for the resistors, CMRRR. By taking 1/Ad0(s) = Ac(s) = 0 in (6) we obtainAssuming op-amps AR(s) approaches infinity if the relevant impedances are chosen according toTherefore CMRR0 and A1 are ideal, then (4) simplifies toIf the condition in (8) is fulfilled and op-amps AConsidering (7), equation (6) can be written asA1(s) is the CMRR of op-amp A1. Further if we assume that Z1, R2, R3 and ZE have tolerance t, then from (7) and (8) we can deduce that the worst case condition will be when Z1 = Z10(1-t), R2 = R20(1+t), R3 = R30(1-t) and ZE = ZE0(1+t) where Z10, R20, R30, and ZE0, are the respective nominal values. Equation (7) then can be written aswhere CMRR. This means that large differential gain is desirable since very small tolerance components are expensive. Therefore, considering (11), equation (10) can be written aswhere A1 has the formThe CMRRr is the frequency where CMRRA1 has decreased by 3 dB and is usually between 100 Hz and 1 kHz. The open loop gain Ad0(s), also decreases at higher frequencies with a corner frequency ω0 = 1/τ0 which is usually lower than ωr, if A0 and A1 are of the same type. Therefore the CMRR is mainly determent at low frequency by the matching of the resistors and the DM gain, and at high frequencies by the open loop response of op-amp A0, rather than its CMRR. If we take the advantage of the fact implicit in (10), and achievewhere ω1  will have the formthen theoretically the CMRR becomes infinite. In part this can be achieved by the use of a capacitor and resistor in parallel for the impedance Z1 Fig. , and the1 isIt has been shown  that a gA0 is the gain bandwidth product of op-am A0. Trimming R2 is a good solution for achieving an ultra high CMRR for demanding application, however it is not practical since the trimmer has to be incorporated in the electrode. Alternatively, ZE or R3 can be trimmed, which however will alter also the amplifier DM gain. Considering equation (12) it can be shown that for application with relatively high DM gain and proper op-amp selection, both trimming and compensation (C1) can be omitted, without significantly degrading the CMRR. For example, if the usual 1% tolerance resistors are used and op-amps with CMRR of 100 dB and DM open loop gain of 120 dB at 50 Hz, then for an amplifier with DM gain of 5000, a CMRR of 96 dB can be achieved without trimming.where GBPE is replaced with an active DC rejection/suppression circuit [2, Ri, Ci) and two potential dividers (R6, R5 and R4, R3). The amplifier can operate in AC-mode or in semi-AC-mode. The two modes are selectable by the switch S1: AC-mode with S1 open and semi-AC-mode with S1 closed. In AC-mode the DC signals are rejected, where in semi-AC-mode they are suppressed. If R6 = R6 *, R5 = R5 * and Ri = Ri *, then the respective expressions for the equivalent impedance ZE(s) for the two modes are given byIn the amplifier circuit shown in Fig.  circuit . It incli = RiCi is the time constant of the integrator,  and τ2 are the respectively the DM open loop gain and the time constant of the first pole of op-amp A2. Whenever Ri>>R5 then k ≈ (R6/R5+1), which is true with the time constants and voltage gains, typical in biopotential recordings. For signals bellow the amplifier high-pass cut-off frequency, ZE(s) decreases due to the active DC rejection/suppression circuit. For DC signals equation (8) is maximally imbalanced and thus CMRRR(0) ≈ Ad(0). Since for biopotential amplifiers Ad(0) is much lower than CMRRA1(0) and Ad0(0), therefore CMRR(0) ≈ Ad(0), which represents the worst case.where τ2 is ideal, then (17) and (18) simplify toIf we consider only the -3 dB bandwidth and assume that op-amp ATherefore, in this case (9) can be written aswhich represents the mid band DM gain for both modes. After substituting (17) and (18) in (9), it can be shown that the respective DC differential gains for the two modes are given byk is approximately the DC gain of the stopped integrator (A2, Ri, Ci, Ri *, R5 *, R6 *) in semi-AC-mode. Thus DC signals meet lower gain, in order to prevent saturation from large electrode offsets or other high DC potentials.where 2iE0 and RiE1, are not equal due to the different closed loop gains of op-amps A0 and A1, and can be expressed asThe active electrodes' input resistances RiA0 and RiA1 are the input resistances of op-amps A0 and A1. However, at higher frequencies, the electrodes' input impedances are much lower and about the same (assumed that A0 and A1 are of the same type), due to the op-amps' input and additional stray capacitance, being in parallel to the high op-amps' input resistance.where RThe output noise spectral density for the -3 dB bandwidth is approximately the same for both modes and can be written asn0, en1 and en2 are the respective voltage noises of op-amps A0, A1 and A2.where e0 is connected to common  and Ad1(s) in (24), it can be shown that H(s) has three poles and two zeros for both modes. However, with the time constants and voltage gain used in the current application, one pole almost coincides with one zero. Therefore, H(s) can be approximated very well by a transfer function with two poles and one zero. The respective approximations for AC-mode and semi-AC-mode are given byAfter substituting ZAC(s) and HsAC(s) are stable because all the poles are situated in the left half of the complex s-plane and there are no resonance effects as the poles are on the real s-axis.The circuits described by the transfer functions H1...EN) and the reference input E0 (monopolar configuration). The output voltage of op-amp A2 is equal to the DC input voltage, multiplied by the ratio (R3 + R4)/R3 for both modes. The choice of the resistor ratio (R3 + R4)/R3 is a trade-off between DC input range and noise, since a low ratio enhances the noise contribution of op-amp A2. The ratio R4/R3 was chosen so that to allow a DC input voltage range of ± 370 mV, without saturating op-amp A2. The offset voltage at the amplifier output (U1) is the input offset voltage of op-amp A2, times the resistor ratio (R5 + R6)/R5. In case of high DM gain, the output offset voltage would become unacceptably high. Thus, op-amp A2 was selected for its ultra-low offset voltage of 1 μV, low noise and high CM input range to prevent latch-up. Moreover A2 is a low input bias current type, which allows the use of high value input resistances without producing large offset voltages between its inputs. Op-amp A0, was selected with a relatively high GBP, in order to confine its influence on the CMRR at higher frequencies. Combining high DM gain, with high GBP op-amps, allowed Z1 to be implemented only with its active part R1, as thus the CMRR at 50 Hz was not significantly degraded. As shown in (23), the equivalent input noise is mainly determent by the noise of op-amps A0 and A1. They were implemented with the low-noise CMOS op-amp LMV751 in a SOT23-5 package.The schematic of a multichannel amplifier with active electrodes, built according to the design discussed is shown in Fig. i), caused by large signal disturbances. Thus a deblocking circuit was added at the cabinet's location, for temporary reduction of the time constant during overload [13, C2). The filter output controls two threshold triggers (A3, A4), which through D1,D2 control the MOS transistor T1, acting like a switch. When the output signal reaches its range limits (defined by R14, R15, R16), T1 opens and the new reduced time constant τi* = (R7//R11)C2, pulls the output signal to the zero level. This state is maintained for additional hundred milliseconds (R13C2) and then is switched back to its original value.Because of the large integrator's time constant, the amplifier has a very slow response after overloads , which theoretically should give a 50 dB extra CMRR at 50 Hz. In addition, in case of a faulty op-amp, the DRL circuit will limit the maximum patient current to a safe level of 50 μA.The connection between the amplifier common and the signal source is implemented by a driven right-leg (DRL) circuit. The CM voltage at the output of As = 300 Ω, Rd = 450 kΩ and Cd = 3 nF. The measuring technique is described by Bergey et al. [2 on an unprepared skin of the inner forearm. The impedance was lower when higher tension was applied or when sweat was present on the skin. The applied current density was 0.01 mA/cm2 and no current density impedance dependence was observed [The contact impedance of the proposed electrode, measured and averaged over five subjects, and its calculated model impedance are shown in Fig. y et al.  and was y et al. . The eleobserved . The eleobserved .i = RiCi = 1, R4/R3 = 6 and R6/R5 = 700. The amplifier frequency response plots, for both operating modes, are shown in Fig. st order pole at 0.32 Hz and zero at 0.2 mHz. In AC-mode the high-pass response is with 1st order pole at 0.16 Hz and zero at 0.16 μHz. The mid band DM gain is the same for both modes. Simulations were also carried out for the estimation of the power line interference due to induced displacement currents into the electrode leads. The increased stray capacitance between the power line and the amplifier, caused by the increased number of electrode leads, was taken into consideration in the simulation model. The results showed that the interference caused by the active electrode unshielded leads was insignificant.Simulations of the amplifier circuit were carried out using PSPICE. The op-amps used in the model were with gain-bandwidth product (GBP) of 5 MHz, DM open loop gain of 120 dB and CMRR of 100 dB. The integrator time constant and the resistor ratios of the feedback loop were: τp-p, or 2 μVp-p when referred to the input. The CMRR of the amplifier was 96 dB at 50 Hz, measured with imbalanced electrode impedances (ΔZe = 47 kΩ). The maximum measured CMRR with DRL and a CM input signal of 4 Vp-p, was 126 dB at 50 Hz, where the output signal level was approximately equal to the amplifier output voltage noise.Table th order low pass Bessel filter, and the signal was sampled with 1 kHz. The electrodes were connected with a high-density unshielded ribbon cable. One electrode (E1) was placed on SLB TH-23, near the eyebrow, where the reference (E0) was placed on the ear lobe. No electrolyte gel or skin preparation was applied. The measurements were performed in a typical laboratory room. All measurements showed almost complete absence of 50 Hz interference. The noise present in the signal is mainly compounded of artifacts from eye movements, electromyographic signals, and noise from the electrode-skin interface.A sample record from the practical application of the active electrode, obtained after a low intensity SLB laser stimulation, is shown in Fig. The best solution for an active electrode would be to perform the entire analog signal processing at the electrode site. This could be achieved with a custom made integrated circuit, but the cost would be much higher. We found a good alternative in using SMD technology and integrating only the front-end of the amplifier into the electrode.The ultra high input resistance of the electrode is degraded at higher frequencies by the op-amp's input capacitance in parallel with the stray capacitance due to the electrode Printed Circuit Board (PCB). Nevertheless, combining an op-amp with low input capacitance and a proper PCB design, allowed a relatively high input impedance to be achieved at 50 Hz. That decreased the amplifier sensitivity to high electrode-skin impedance imbalances, by reducing the transformation of the CM interference signal into unwanted DM signal. Unfortunately, most data sheets do not properly specify op-amp's input capacitance, neither DM nor CM.0 and A1. Because of the limited electrode space, it is preferable that the front-end op-amps feature internal electrostatic discharge protection circuitry, rather than building an external one.The active electrode presented is not suitable for applications requiring a low differential gain and large signal bandwidth due to the decreasing CMRR at higher frequencies, if not properly compensated. On the other hand, below the high-pass cut-off frequency, the CMRR is degraded by the active feedback circuit, and reaches its minimum value for DC signals, equal to the DM gain. The circuit can accept high value input filter resistances, which will also limit the patient auxiliary current in case of fault condition of op-amps AThe new electrode anchoring system significantly reduced the electrode-skin impedance, its variation and motion artifact influences. The proposed amplifier fractionation resulted in lower noise and less parts. Moreover splitting the amplifier between the electrodes and the cabinet's location allowed the use of an automatic DC deblocking system and mode switching. The prototype tests showed that with the active electrode presented, SLB signals with relatively high quality could be recorded without skin preparation. The 50 Hz interference pickup by the electrode leads was practically eliminated. Because high electrode-skin impedances are tolerated, no electrolytic gel is needed. This allows fast application of the electrodes, minimizes patient discomfort and eliminates the risk of infection.With proper op-amps selection, the active electrode specifications were found to be better or at least comparable to those of other existing designs. The design offers low noise and major reduction in parts, size and power consumption. It is currently used in studying laser provoked SLB potentials and their propagation, aiming to gain a better insight into the bio-stimulation effect of lasers in Medical Acupuncture.The authors contributed equally to this work


Ventricular tachycardia (VT) and ventricular fibrillation (VF) are ventricular cardiac arrhythmia that could be catastrophic and life threatening. Correct and timely detection of VT or VF can save lives.In this paper, a multiscale-based non-linear descriptor, the Hurst index, is proposed to characterize the ECG episode, so that VT and VF can be recognized as different from normal sinus rhythm (NSR) in the descriptor domain.This newly proposed technique was tested using MIT-BIH malignant ventricular arrhythmia database. The relationship between the ECG episode length and the corresponding recognition performance was studied. The experiments demonstrated good performance of the proposed descriptor. An accuracy rate as high as 100% was obtained for VT/VF to be recognized from NSR; for VT and VF to be recognized from each other, the recognition accuracy varies from 84.24% to 100%. In addition, the results were compared favorably against those obtained using Complexity measure.There is strong potential for using the Hurst index for malignant ventricular arrhythmia recognition in clinical applications. If a life-threatening ventricular tachycardia (VT) or ventricular fibrillation (VF) is detected promptly, a high energy electrical shock can be delivered to the heart, in an attempt to return the heart to a normal sinus rhythm (NSR). If a normal sinus rhythm is misinterpreted as VT or VF, leading to delivering of an unnecessary shock, it can damage the heart, causing fatal consequences to the patient. Therefore, correct and prompt detection of VT or VF is of great importance. However, the detection of these life-threatening cardiac arrhythmia is difficult because the waveform and frequency distribution of these life-threatening arrhythmia changes with the prolonged duration . FurtherTill now, many linear techniques for VT/VF detection have been developed, such as the probability density function method , rate anRecent studies ,13 have Some non-linear techniques -20 have ECG episodes with VT and VF from MIT-BIH malignant arrhythmia database  are testThe present paper is organized as follows. Mathematical background on the proposed non-linear descriptor is given in Section. Methodology for the recognition of ventricular arrhythmia is described in Section. Section covers the experimental results and discussions. Lastly, a conclusion of the proposed study is given in Section.Multiscale analysis is a useful framework for many signal processing tasks. Wavelet transform is a good tool for multiscale analysis, which allows the expansion of a signal from the time domain into the time-frequency domain. In this paper, the Hurst index, defined in multiscale space, is proposed for the characterization of ECG episodes.H, is a single scalar parameter describing the fractal Brownian motion (fBm) model, which is a useful model for nonstationary stochastic self-similar processes with long term dependencies over wide ranges of frequencies [BH(t), t ∈ ℝ, 0 <H < 1, [D is a commonly used parameter for measuring self-similarity. The relationship between the fractal dimension, D, and the Hurst index H is: D = S - H, where S is the topology dimension. For a one-dimensional signal, S = 2; for a two-dimensional image, S = 3 [The Hurst index, quencies . fBm is  <H < 1, . Self-sie, S = 3 . The fBm• It is non-stationary, which necessitates some time-dependent analysis.E(BH(t)BH(s)) = σ2/2(|t|2H + |s|2H - |t - s|2H)     (1)E(·) represents the expectation operator, σ is the standard deviation, t is a time variable, s is a time lag variable. Based on Equation (1), the variance of fBm, is computed as var(BH(t)) = σ2|t|H2.where • It is self-similar, which necessitates some scale-dependent analysis.BH(at)} ≜ aH BH(t), a ∈ ℝ+     (2){+ is the set of positive real numbers. ≜ means equality in distribution, which means that the fBm has stationary increments, and the probability properties of the process BH(t + s) - BH(t) only depend on the lag variable s. The scalar index H of fBm is related to the complexity and roughness of fBm samples.where ℝBH(t).Consider a discrete orthogonal wavelet decomposition of a given fBm, J, the wavelet mean-square representation of fBm is:For any given resolution 2aj[n] and detail coefficients dj[n]:Computing the corresponding wavelet coefficients amounts to evaluating the following approximate coefficients φ(t) is the corresponding smooth function of wavelet ψ(t).where Flandrin et al. in  have dedWavelet coefficients of fBm give rise to:Vψ(H) is constant, which depends on both the chosen wavelet and the fBm index H. It follows the power-law behavior of the wavelet coefficients' variance:where 2(var(dj[n])) = (2H + 1)j + constant     (9)logH  can be easily obtained from the slope of this variance plotted as a function of scale in a log-log plot.Therefore, the fBm index For each testing ECG episode, the following steps are performed:• Perform wavelet decomposition and computation of its detail coefficients at different scales.H according to Equation (9).• Compute the Hurst index H.• Detect the life-threatening ventricular arrhythmia in the feature space of In this study, the wavelet used is a quadratic spline wavelet with compact support and one vanishing moment. It is a first derivative of a smooth function , whose dL(ω) and G(ω) are respectively:The low-pass and high-pass filters f(n) can be calculated with Mallat's algorithm [The dyadic wavelet transform (WT) of a digital signal lgorithm  as follo is a smoothing operator.  is the wavelet transform of digital signal f(n). lk|k ∈ Z and gk|k ∈ Z are coefficients of a low-pass filter L(ω) and a high-pass filter G(ω), respectively, and, L(ω) = Σk∈Zlkeikω-, G(ω) = Σk∈Zgkeikω-. Based on the frequency analysis of the ECG characteristic waves [j (j = 1 to 4) are selected. For each experimental episode, its wavelet transform coefficient sets d1, d2, d3 and d4 corresponding to different scales 21, 22, 23, 24 are computed. The Hurst index H is then computed according to Equation (9). Smaller Hurst index corresponds to larger fractal dimension and more irregular signal.where ic waves , scale 2Hz. Typical waveforms of VT and VF as well as NSR are shown in Figure H, computed by Equation (9). The statistical distribution of the Hurst indexes for characterizing different types of episodes is studied so that VT and VF can be recognized in the feature domain of the Hurst index. Recognition performance is measured by Sensitivity (SE), Specificity (SP) and Accuracy (ACR). They are defined as: Sensitivity = ; Specificity = ; Accuracy = . Where TP is true positive, the abnormal case being correctly recognized as abnormal one; FN is false negative, the abnormal case being wrongly recognized as normal one; TN is true negative, the normal case being correctly recognized as normal one; and FP is false positive, the normal case being wrongly recognized as abnormal one. Lastly, results are compared with that of Complexity measure technique.The database used in this study is the MIT-BIH malignant ventricular arrhythmia database  with a ssec to 5.5 sec with a difference of 0.5 sec. For each length, the whole dataset was randomly divided into two equal parts for training and testing, respectively. From a clinical point of view, it is essential to recognize and diagnose malignant ventricular arrhythmia as soon as possible. This calls for detection with as short a length of the time series as possible.In this study, about 5076 ECG episodes are tested for performance evaluation of life-threatening ventricular arrhythmia recognition using the proposed Hurst index. Among them, 2588 cases are NSR episodes, 1390 cases are VT episodes, and 1098 are VF episodes. In order to explore the effect of the time series lengths on the recognition performance using the proposed Hurst index, analyzing was conducted using different lengths of ECG episodes from 1 The statistical results, viz, the means and standard deviations for characterizing NSR, VT and VF episodes using the Hurst index are given in Table From the results shown in Figure • As the episode length increases, the mean of Hurst index for every type of rhythm basically increases and tends to approach a relatively stable value, while the standard deviation decreases gradually.• For a particular episode length, from NSR to VT then to VF, the corresponding Hurst index increases gradually. The increase from NSR to VT is more than the increase from VT to VF.• As the episode length increases, the mean of Complexity measure for every type of rhythm basically decreases and tends to approach a relatively stable value, while the standard deviation decreases gradually.• For a particular episode length, from NSR to VT then to VF, both the Hurst index and the Complexity measure increase gradually, in which, the increase from NSR to VF is far more than the increase from VT to VF.sec to 5.5 sec. It is concluded that the Hurst index is more stable than the Complexity measure with respect to episode lengths.• The mean values of Hurst index vary slower than those of Complexity measure as the episode length increases from 1 sec, it has as good performance as the Hurst index; when the length of the ECG episode is 1 sec, there is 6 false negatives and 27 false positives; when the length of the ECG episode is 1.5 sec, there is 1 false negatives and 5 false positives. The statistical values of SE, SP and ACR for VT/VF recognition from NSR using the Hurst index are all 100%. Hence, the Hurst index can be used to detect VT and VT earlier.Using the Hurst index for VT or VF recognition from NSR with different episode lengths, there is no false detection, meaning that the VT/VF can be totally correctly recognized from NSR without exception. For the Complexity measure, when the length of ECG episode is longer than 1 SE, SP and ACR for different episode lengths using the Hurst index and the Complexity measure, are shown in Table As for VF differentiation from VT, the statistical values of • The performance on differentiating VT and VF is worse than the performance of VT/VF recognition from NSR, for both the Hurst index and the Complexity measure.• The recognition performance by either descriptors improves as the length of ECG episode increases.sec, the recognition performance for the Hurst index is better. When the length of ECG episode is longer than 2 sec and less than 5 sec, the recognition performance for the Complexity measure is better. When the length of ECG episode is longer than 5 sec, VT and VF can be 100% differentiated with either descriptor, the recognition performance for both descriptors are same.• When the length of ECG episode is less than or equal to 2 MHz workstation. The computational burden for the Hurst index is O(N log2 N), while the computational burden for the complexity is O(N2), where N is the length of ECG episode. It is noted that with more powerful computer programming in C, the computational speed will be further improved.According to Table Time is an important factor for saving lives in clinical situations, therefore, algorithm with less computational burden is obviously preferred. In addition, using short ECG episode length is preferred for earlier detection of arrhythmia (such as VT/VF). Based on the experimental results, it is observed that the Hurst index has a better potential for clinical adaptation than the Complexity measure.In this paper, a new technique based on multiscale analysis and non-linear dynamics was presented for VT and VF recognition. Hurst index defined across multiscale was proposed for characterizing ECG episode so that life-threatening arrhythmia can be recognized. Furthermore, upon applying to the MIT-BIH malignant ventricular arrhythmia database, the performance for malignant arrhythmia recognition using Hurst index was compared with that using Zheng's complexity measure. The Hurst index requires less computation and is more reliable in detecting VT and VF with short ECG episode. There is strong potential for using the Hurst index for malignant ventricular arrhythmia recognition in clinical applications.SY conceived the study, performed data analysis and drafted the manuscript. CKL and KSM guided the study, helped the analysis and interpretation of the results, and critically reviewed the manuscript. All authors read and approved the final script.


Cholesterol has a bad rap for its association with human heart disease. But actually cholesterol and other sterols are essential for a wide variety of organisms. For most eukaryotes—organisms whose cells have nuclei—sterols reside in the cell membrane and play major structural roles. Sterols keep cell membranes flexible, for example. These chemicals also hinder leakage of ions across the membrane, which is crucial in order for muscles to contract and nerves to conduct signals.Caenorhabditis elegans, sterols are a dietary staple. Worms can't make these chemicals from scratch, just as humans can't make vitamin C or the essential amino acids, so they have to harvest these chemicals from their surroundings. If nematodes hit hard times—they can't find enough sterols, say, or are starved or overcrowded—they can delay developing into adults. Instead, they enter a stage called a dauer in which they don't eat and hardly move a muscle. In this state, they can persist several months—many times their normal lifespan—and then revive when conditions improve.For the tiny (eukaryote) nematode worm C. elegans is extensively studied, there's still controversy over the role of cholesterol in this organism. To develop into adults, the nematodes need only small amounts of cholesterol in their diet, suggesting cholesterol does not play a major role in their membranes. Instead, nematodes—like many other eukaryotes—might use cholesterol to make hormones, which are typically active at very low concentrations. Such hormones could play a key role in the worms' development into either adults or dormant dauers. But no one had found any nematode hormones derived from cholesterol—until now.Though PLoS Biology, Teymuras Kurzchalia and colleagues show definitively that cholesterol does not play an essential structural role in C. elegans. Rather, cholesterol is the precursor for a hormone—or set of hormones—key in the worms' development into adulthood and thus key for reproduction. The researchers have partially purified this cholesterol derivative and named it gamravali, from the Georgian word for reproduction, “gamravleba.”In this issue of When on sterol-free diets, all larvae showed arrested development, becoming dormant dauers. But, surprisingly, the concentration of cholesterol they needed to develop into adults was miniscule, around 20 nanomoles. When given scant amounts of cholesterol, the worms converted some of it to a sterol called lophenol. The researchers found, however, that supplementing a sterol-free diet with lophenol was not enough to sustain development into adulthood. Apparently the worms need cholesterol, which is fed into two distinct pathways: one makes lophenol and another makes the hormone gamravali.C. elegans line, for instance, was unfazed by the cholesterol-free diet. These mutants were missing the daf-12 gene, one of a set of genes crucial in nematode development and aging. On the cholesterol-free, lophenol-supplemented diet, these mutants developed into normal adults. Other mutant lines that each lacked one of several other daf genes, however, developed into dauers when deprived of cholesterol. In this way the researchers found where gamravali acts in the worms' developmental pathway: the hormone gamravali likely comes into play before daf-12, but after the other daf genes. Kurzchalia and colleagues are currently working to further purify gamravali and identify exactly how it gives cholesterol such a crucial role in the worms' lifecycle.The researchers have only partially purified gamravali, so they don't yet know its molecular weight or composition or even whether it is a single molecule. But by working with mutant worms, they have begun to pin down where gamravali acts in the worms' developmental pathway. One mutant


The resolution of radiation hybrid (RH) maps is intermediate between that of the genetic and BAC  contig maps. Moreover, once framework RH maps of a genome have been constructed, a quick location of markers by simple PCR on the RH panel is possible. The chicken ChickRH6 panel recently produced was used here to construct a high resolution RH map of chicken GGA5. To confirm the validity of the map and to provide valuable comparative mapping information, both markers from the genetic map and a high number of ESTs (Expressed Sequence Tags) were used. Finally, this RH map was used for testing the accuracy of the chicken genome assembly for chromosome 5.A total of 169 markers (21 microsatellites and 148 ESTs) were typed on the ChickRH6 RH panel, of which 134 were assigned to GGA5. The final map is composed of 73 framework markers extending over a 1315.6 cR distance. The remaining 61 markers were placed alongside the framework markers within confidence intervals.The high resolution framework map obtained in this study has markers covering the entire chicken chromosome 5 and reveals the existence of a high number of rearrangements when compared to the human genome. Only two discrepancies were observed in relation to the sequence assembly recently reported for this chromosome. Chicken is the first major agricultural species for which the complete genome sequencing was undertaken. This is partly due to its position as a model species in various fields of biology including embryo development, oncology, immunology and evolution . MoreoveRadiation hybrid maps have a resolution power intermediate to that of the genetic and BAC contig maps and are also a powerful tool for the mapping of ESTs and genes by simple PCR. They are thus useful at two levels: first, they can be used constructively as scaffolds for a correct genome assembly or for detecting and correcting misassembled portions of the genome; second, before obtaining whole annotated genome sequences, they are very efficient tools for inter-species comparative genome analyses through the easy mapping of genes and ESTs -7.The successful production of a RH panel in chicken is quite recent , and theThe available human/chicken comparative mapping data indicated conservation of synteny between GGA5 and portions of HSA11, HSA14 and HSA15. In addition, two genes from HSA1 had also been shown to be located on GGA5 . This inst, 2004). The quality of both the GGA5 RH map and of the sequence assembly was therefore checked by alignment of all the markers by BLAST searches.While in the process of finishing our map, the first draft sequence assembly of the chicken genome was released  enabled a successful amplification and the subsequent mapping of the corresponding genes, confirming the high success rate obtained when using the ICCARE software for designing chicken PCR primers based on EST data [In addition to the 21 microsatellite markers from the genetic map, and 9 primer pairs chosen either from available primer data in the literature or designed using the gene sequence deposited in Genbank/EMBL, 156 primer pairs were chosen from chicken EST markers selected on the basis of the known conservations of synteny between human and chicken using the ICCARE (Interactive Comparative Clustering and Annotation foR Est) software EST data .6000 long, comprising a total of 73 markers including 12 microsatellites and 61 ESTs was obtained. The remaining 61 markers are located relative to the framework map within confidence intervals, to build a comprehensive map  and 21 microsatellites from the GGA5 genetic map. Two-point analysis using a LOD threshold of 6 enabled to constitute a group of 134 markers, including all the microsatellite markers from the genetic map. The remaining 35 markers correspond to the external boundaries of the regions of conserved synteny with human, from which ESTs were chosen for marker development and map either to other chromosomes for which RH maps were developed (GGA1, 10, 18 or 24) or to unknown regions (data not shown). After multipoint analysis, a 1000:1 framework map 1315.6 cRBRF1 . We compared our data with the GGA5 sequence, by using BLASTN searches and sequence alignments. The agreement between the RH framework map and the sequence orders is almost perfect (figure A preliminary data set based on the first draft chicken genome assembly has been deposited into public databases by a team led by R. Wilson and W. Warren, from the Washington University School of Medicine in St. Louis (1st March, 2004, GPR48, PAX6, SPON1 and CSTF3), that we developed on the basis of the conservation of synteny between GGA5 and HSA11, is assigned to GGA3 in the sequence assembly. Three of these markers are on the framework map and for all four, the RH genotypings obtained are very similar to those obtained with the flanking RH framework markers SLC17A6 and ARNTL (two-point LOD scores ranging from 7.1 to 15.7), both located on the GGA5 sequence assembly. Furthermore, when two-point analysis of the four markers was computed with the flanking markers LOC134957 (1.2 Mb to GPR48) and SLC22A3 (0.6 Mb to CSTF3) suggested in the GGA3 sequence assembly, LOD scores were equal to zero. This part of the genome assigned to a wrong chromosome on the sequence assembly covers a region at least 50 cR long, corresponding to a distance of 2 to 3 Mb, as estimated from the cR to Mb ratio. Indeed, the length of the sequence between the two extreme markers CSTF3 and GPR48 on the GGA3 assembly is 2.703 Mb. The retention frequency of these four markers is amongst the highest of all, suggesting that their location is close to the centromere and that the possible sequence assembly problems are related to this proximity, perhaps due to repetitive sequences.First, a group of markers  also appeared to belong to a region with no sequence available (no blast hit). This gene had previously been located on the cytogenetic map to the short arm of GGA5 [Third, several markers absent in the sequence assembly could be localised on the RH map figure . Most of of GGA5 , so we cGTF2A1. However, the difference of likelihood between our framework map and the same map with an inversion of these two markers is only 103.7. It is therefore difficult to conclude as to which between the sequence and the RH map presents the correct order.Fourth, we observed a discrepancy in the local order of the two framework map markers MCW238 and From these data we conclude that radiation hybrid maps can be useful to help detect errors in the draft sequence assembly and for mapping genes either absent or of unknown location in the assembly.The average cR/cM ratio is 6.5 when calculated over the whole map length. This relatively high value, as compared to the 4 cR/cM obtained for GGA7 , must be, the ratio is thus now closer to the value we obtain here for GGA5.The agreement between the gene order found on RH map and the sequence assembly is very high. Considering only the q arm of the chromosome, the cR/Mb ratio is 22.9, or 43.7 kb per cR. This ratio, similar to that obtained for GGA2 , is quite lower than the 63 kb/cR and 61 kb/cR values obtained for GGA15  respecti. The results indicate a high number of chromosomal rearrangements in the chicken and human lineages in the region corresponding to GGA5. The results presented in figures Figure We have built a high resolution RH map of chicken chromosome 5 using the ChickRH6 panel. In doing this, we fulfilled our objective of obtaining a detailed comparative map of GGA5, providing jointly a source of potential polymorphic markers and of candidate genes for QTL mapping on this chromosome.At the end of our work, the first draft chicken genome assembly was released and we aligned it to our GGA5 RH map. Although we detected a few errors to correct, this allowed us to demonstrate the high quality of the sequence assembly, which may have benefited from a low frequency of repeated elements.In the near future, the ChickRH6 panel will be used to assist in improving the chicken genome assembly. This is clearly needed in the regions for which the genetic map is still not complete, such as some microchromosomes, but also for parts of macrochromosomes, as shown in this study..Twenty one microsatellite markers distributed along GGA5 were chosen from the genetic map. Their primer sequences are available at CKB, IGF2 and RYR3 for which primers were chosen from the literature, and 6 other genes for which primers were designed from sequences deposited in Genbank/EMBL, primers pairs were designed from the available chicken EST sequence of orthologs defined using the ICCARE (Interspecific Comparative Clustering and Annotation foR ESTs) software (T. Faraut, ). The exonic structure of the genes was taken into account by extrapolating the information available from an alignment to the human genomic sequence. A link with the Primer3 software  allowed us to design the primers. Primer data for markers amplifying successfully and accession numbers of the sequences used as a basis for primer design, are indicated in Table 1 , 20 mM Tris-HCl pH 8.4, 50 mM KCl, 0.05% W-1 detergent, 2 mM MgCl2, 0.2 mM dNTP.Amplifications were carried out on a GeneAmp PCR System 9700 thermocycler (Applied Biosystem). The first 5 min denaturation was followed by 30 cycles, each of denaturation at 94°C for 30 s, annealing at Tm for 30 s and elongation at 72°C for 30 s. PCR products were analyzed on 2% agarose gels, electrophoresed in 1 X TBE buffer, and visualized by ethidium bromide staining.Each marker was genotyped twice and a third genotyping was performed in cases of discrepancies between the first two experiments.The genotyping data obtained was analyzed with the Carthagene software ,32. A grth January 2004 – ). RH maps were drawn with MapChart 2.0 [The human and mouse reference maps were built from data available through EnsMart v19.1  is shown on the left. Conserved blocks are indicated by coloured plain boxes. Empty boxes show HSA regions for which the chicken homologous part of the genome is not positioned on GGA5.Click here for filePrimer pairs for the studied gene fragments Accession numbers for the chicken EST sequences from which the primers were chosen are given in this Excel fileClick here for file


Uracil DNA glycosylase (UDG) plays a major role in repair of uracil formed due to deamination of cytosine. UDG in human cells is present in both the nucleus and mitochondrial compartments. Although, UDG's role in the nucleus is well established its role in mitochondria is less clear.In order to identify UDG's role in the mitochondria we expressed UGI (uracil glycosylase inhibitor) a natural inhibitor of UDG in the mitochondria. Our studies suggest that inhibition of UDG by UGI in the mitochondria does not lead to either spontaneous or induced mutations in mtDNA. Our studies also suggest that UGI expression has no affect on cellular growth or cytochrome c-oxidase activity.These results suggest that human cell mitochondria contain alternatives glycosylase (s) that may function as back up DNA repair protein (s) that repair uracil in the mitochondria. Mitochondrion plays an important role in various cellular functions ranging from synthesis of lipids to maintenance of ion homeostasis ,2. Howevin vivo mtDNA repair we targeted UGI to the mitochondria to inhibit UDG activity. Our studies suggest that mitochondrial inhibition of UDG is not mutagenic. This study indicates that alternative DNA glycosylase(s) may be operative in the mitochondria that might repair uracil in the mitochondrial genome.The uracil DNA glycosylase (UDG) removes misincorporated uracil or deaminated cytosine from DNA. Human UDG gene encodes two alternative spliced isoforms, UNG1 and UNG2 -13. Of tThe reading frame of uracil DNA glycosylase (UDG) that codes for functional UDG was amplified by PCR using forward primers (5'CCAGTGCCGCGCGCCAAGATCCATTCGTTGTTTGGAGAGAGCTGGAAGAAG) specific to human uracil DNA glycosylase that had a BssH II site at the 5' end and the reverse primers 5'TTGA TCTCGAGTCACAGCTCCTTCCAGTCAATGGG that had the Xho I site engineered at the 5' end. The template used for the amplification was pTUNGΔ84 . The PCRThe complete reading frame of uracil DNA glycosylase inhibitor gene was amplified using pTZUgi (a gift from Dr. Umesh Varshney) as a template with forward primers (5'CCAGTGCCGCGCGCCAAGATCC ATTCGTTGATGACAAA TTTATCTG ACATC) specific to uracil DNA glycosylase inhibitor from phage PBS2 that had a BssH II site at the 5' end and the reverse primer(5'CGCCCGTTTGATCTCGAGTTATAAC ATTTTAATCCATTAC) which had the Xho I site engineered at the 5' end. The PCR fragment was cloned into pCMV/myc/mito (Invitrogen). The construct was named as pCMV UGI.Stable transfectants of the above constructs were made in immortalized normal breast epithelial MCF 12A cells using lipofectin as a transfecting agent. Briefly, MCF12A cells were plated to 70 % confluency in a 35 mm dish and transfected with 2 ug of pCMV UNG and pCMV Ugi. The cells were selected using G418 as a selection medium. The clones were selected after plating the cells in a 96 well plate to single cell dilution and the clones were screened for integration using PCR. A pCMV/myc/mito/GFP that has a GFP protein fused to the mitochondrial signal was used as a control to assay the efficiency of transfection and the expression of the protein using the vector. An empty vector was stably transfected and used as a control in all the experiments.Each construct was assayed for stable integration after transfection using PCR. The primers were the same that were used for amplifying the gene for cloning namely UDG specific primers, forward primer: 5'CCAGTGC CGCGCGCCAAGATCCATTC GTTGTTTGGAGAGAGCTGGAAGAAG reverse primer 5'TTGATCTCGAGTCAC AGCTCCTTCCAGTCAATGGG, for screening UDG stable integrants and UGI specific primers, forward primer 5'CCAGTGCCGCGCGCCAAGATCCATTCGTTGATGACA AATTTATCTGACATC and reverse primer 5'CGCCCGTTTGATCTCGAGTTATAAC ATTTTAATCCATTAC for screening Ugi stable integrants. Briefly, the each clone was transferred from the 96 well plate to a 24 well plate and DNA was extracted when the wells were confluent using standard methods. 100 ng of the DNA was used to PCR amplify the DNA that was transfected. Clones that showed an intact gene were selected for further analysis.Stable clones and parental MCF12A cells were grown in T75 flask to seventy percent confluency. The cells were washed with 1X PBS and treated with 1.5 ml of 0.04% Digitonin solution (0.4 mg Digitonin /ml,2.5 mM EDTA,250 mM mannitol, 17 mM MOPS., pH 7.4). The cells were thoroughly resuspended and homogenized using ten strokes of a dounce homogenizer on ice. One ml of 2.5 X sucrose mannitol buffer (525 mM Mannitol, 175 mM Sucrose, 12.5 mM tris-HCl., pH 7.49) was added and homogenized further using 20 strokes of the homogenizer. Ten micro liter of the homogenate was visualized under the microscope to assess complete breakdown of the cells. The mitochondria were isolated by differential centrifugation . The homStable transfectants were assayed for production of the UDG protein in the mitochondria by western blotting. Twenty micrograms of the mitochondrial protein was electrophoresd on a 12% SDS polyacrylamide gel and transferred on a nitrocellulose membrane. The membrane was blocked overnight in a blocking solution containing 5% non-fat milk and probed with the primary antibody (1:1000 dilution) against UDG (a gift from Dr. Hans Krokan, Norway). The membrane was washed twice with TBST and probed with a secondary antibody linked to horseradish peroxidase. The bands were visualized using ECL (Amersham Pharmacia) kit. The membrane was then probed for the house keeper protein beta actin to assess for equal loading.-reverse transcriptase (Invitrogen). Two microlitres of the reverse transcribed products was used in the subsequent PCR reactions. Twenty-five microlitres of the PCR reactions contained 20 mM Tris-HCL, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTP and 10 picomoles of each primer (forward primer: 5'CCAGTGCCGCGCGCCAAGATCCATTCGTTGATGACAAATTTATCTGACATC and reverse primer 5'CGCCCG TTTGATCTCGAGT TATAACATTTTAATCCATTAC and one unit of Taq DNA polymerase (Invitrogen). The PCR profile consisted of an initial denaturation at 94°C for 5 minutes and 32 cycles of denaturation at 94°C for 45 sec, annealing at 58°C for 1 min and extension for 2 min at 72°C with a final extension at 72°C for 10 min. The PCR products were electrophoresed on a 1% agarose gel stained with ethidium bromide (0.5 μg/ml) and visualized under UV.RNA from Ugi stably transfected MCF 12A cells was extracted using TRIZOL reagent following the manufacturers instruction. One and a half micrograms of total RNA was used for reverse transcription using Superscript II Rnase H5 in a 60 mm dish and stained for 15 min using the fluorescent dye CFSE (Molecular Probes). Cells were fixed in 70% alcohol just after staining to have a 0 hour time point and after a period of 72 hours. Proliferation was then estimated using flow cytometry using a FACSvantage™, Becton Dickinson [Proliferation assay was done using a flourescent lipophilic molecule, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) that gets incorporated into live cells and gets diluted into daughter cells with every cell division. The dilution in the intensity of the dye as estimated by flow cytometry with respect to a "0" hour time point gives an indication of the proliferation of the cells. Cells were plated at a density of 1 × 10ickinson ,21.S-nitroso-N-acetylpenicillamine (SNAP), NO donors for a period of 1 hour after which the medium was changed and cells were harvested after 0, 2, 4, 6 hour period intervals. DNA was extracted from these cell lines and Cox I was PCR amplified and sequenced using an automated sequencer (ABI PRISM) for mutation analysis.MCF12A parental cells were used for dose optimization of the SIN1 and SNAP. An optimal dose was used for further experiments. The parental and the transfected cells were plated on a 60 mm dish to 70% confluency. Each of the cell lines were treated with 4 mM 3-morpholinosydnonimine (SIN-1) and 2 mM 32P using T4 polynucleotide kinase. The labeled products were precipitated and then resuspended in a lower volume of distilled water. These were directly used as single stranded substrates in the enzyme assay. To prepare double stranded substrates twenty picomoles of the labeled products were annealed to 10 pmoles of the unlabelled complementary or mismatch oligos by heating at 70°C and slowly cooling it down to room temperature for an hour. UDG assay was performed using 50 μg of mitochondrial extract in 1X UNG buffer (20 mM Tris-HCl pH8.0,1 mM EDTA,1 mM DTT) and 4 pmoles of labeled oligos. The reaction was carried out at 30°C for 45 min. The assay using commercially available Ugi (NEB) was performed using similar conditions. Ten units of Ugi per reaction was used. Apyrimidinic sites (AP-sites) generated by uracil removal from DNA substrates were hydrolyzed by the addition 0.1 N NaOH and incubating for 10 min at room temperature and terminated using a formamide buffer (80% formamide in 1XTBE) to generate single stranded products. Half of the reaction was electrophoresed using a 15% acrylamide gel containing 8.3 M urea and 1X TBE buffer. The gel was autoradiographed after electrophoresis to visualize the bands.Uracil DNA repair assay was conducted as described by Radany et al., . OligonuPrevious studies have shown that uracil DNA glycolyase can be inhibited by PBS2 phage protein UGI in a stoichiometric fashion ,18. ThisIt is possible that inhibition of UDG in the mitochondria may affect cell growth. To determine if UGI expression in the MCF12A cells resulted in a difference in cellular growth, cell cycle analysis was conducted using flow cytometry. The cell cycle distribution of parental MCF12A cells, wild type UNG and UGI transfected cell line and the cell line containing the control vector is shown in figure Our previous studies suggest that inactivation of UDG in yeast Saccharomyces cerevisae leads to mutations in mtDNA . We therIt has been previously reported that the UGI protein when targeted to the nucleus lowers the activity of the nuclear UDG enzyme . To analCells are exposed to DNA damaging agents generated both as a process of normal physiology as well through extrinsic mutagens. Cells repair damage done to the DNA by a variety of repair mechanisms each specific for the type of DNA damage . Many prUDG's role in the nucleus is well established . It is aOur results of UGI expression in the mitochondria are different when compared with UGI expression in the nucleus. A previous study has shown that expression of UGI results in inhibition of uracil DNA repair in the nucleus and subsequently mutation in the nuclear DNA . Our resin vivo and in the extract from untargeted mitochondria to carry out the uracil repair activity in vitro. However, these possibilities are ruled out because UGI expression did not result in lower level cytochrome C oxidase activity (data not shown).It is also possible that an extremely low level of mutant mtDNA may be present in the cells expressing UGI in the mitochondria and PCR technique used to identify mutant copies among a heterogeneous population of mtDNA was unable to detect mutant mtDNA molecules. It is also conceivable that targeted UGI is present in a subset of mitochondria and at any given time there is always enough active UDG


It acts through the phosphorylation of downstream effectors that are recruited to it by the binding partner Raptor. In Arabidopsis, Raptor activity is essential for postembryonic growth. Though comparative studies suggest potential downstream effectors, no Raptor binding partners have been described in plants.TOR, the Arabidopsis Mei2-like 1) protein in a yeast two-hybrid assay. This interaction is mediated by the N-terminal 219 residues of AML1, and marks AML1 as a candidate AtTOR kinase substrate in plants. The AML1 N-terminus additionally carries transcriptional activation domain activity. Plants homozygous for insertion alleles at the AML1 locus, as well as plants homozygous for insertion alleles at all five loci in the AML gene family, bolt earlier than wild-type plants.AtRaptor1B, a plant Raptor homologue, binds the AML1 (AML1 interacts with AtRaptor1B, homologue of a protein that recruits substrates for phosphorylation by the major cell-growth regulator TOR. Identification of AML1 as a putative downstream effector of TOR gives valuable insights into the plant-specific mode of action of this critical growth regulator. TOR is nslation  present l growth -7 – thoul growth . Raptor,l growth ,7. TOR al growth ,8,10.Disruption of the Arabidopsis TOR homologue AtTOR is lethal early in plant embryonic development . DisruptSchizosaccharomyces pombe [Mei2 is a putative TOR substrate and potent meiosis-signaling molecule identified in the fission yeast es pombe ,14. Mei2es pombe , and is es pombe ,17 – cones pombe ; TOR is es pombe ,18. The es pombe . AccumulMei2-like proteins are predicted in a wide range of organisms ,21 incluArabidopsis Mei2-like 1 (AML1), the first member of this family to be described, was isolated in a screen for plant cDNAs whose expression could complement defects in the fission yeast meiosis signaling pathway [mei2 locus itself. Terminal Ear 1 (te1), a more divergent member of the mei2-like family of genes, regulates leaf initiation in maize; the tassel of mutant plants is encased a whorl of leaves superficially resembling a maize ear [ pathway . Like Meaize ear .Given the potential role of downstream AtTOR effectors in post-embryonic growth, the intriguing signaling activity of Mei2 in fission yeast, and the known interaction between Mei2 and the fission yeast Raptor protein, we asked if the Mei2-Raptor interaction was conserved between its Arabidopsis orthologues AML1 and AtRaptor1B.ADE2 and HIS3. pGADT7 and pGBKT7 additionally carry the genes LEU2 and TRP1. When transformed into the yeast line AH109, which is leu2-trp1-ade2-his3-, co-transformed cells will grow on yeast medium lacking leucine and tryptophan. Cells co-transformed with two-hybrid constructs encoding proteins that interact will grow on media additionally lacking histidine and adenine (selective media).To test for an interaction between the Arabidopsis proteins AML1 and AtRaptor1B, the open reading frames (ORFs) of each of the transcripts predicted to encode these proteins were amplified via PCR with primers carrying suitable restriction sites for cloning into the yeast two hybrid vectors pGADT7 and pGBKT7. pGADT7 encodes a transcriptional activation domain (AD) which can recruit the yeast transcription machinery. pGBKT7 encodes a DNA binding-domain (BD) which binds to the promoters of AH109 yeast cells co-transformed with clones encoding AML1 and AtRaptor1B in complementary two-hybrid vectors grew on selective media. Co-transformations of control pGBKT7 and pGADT7 empty vectors, or either control vector co-transformed with its complement harboring the AML1 or AtRaptor1B ORF, yielded cells able to grow on media lacking leucine and tryptophan but not on selective media.mei2 locus [Evidence from other systems indicates that Raptor protein fragments lose the ability to bind substrates . Therefoi2 locus . Additioi2 locus . TherefoAML1 fragments N412 and N219, comprised of the first 412 or the first 219 residues of the 915 residue AML1 protein, restored growth on media lacking leucine, tryptophan, histidine and adenine when cloned into pGADT7 and co-transformed with pGBK-Raptor  and media additionally lacking adenine. The DNA binding-domain of the pGBKT7 tethers any C-terminally fused fragments to the AML1 N412 and AML1 N163, but not full length AML1, AML1 N219 or any C-terminal AML1 fragments, were able to restore growth on media lacking tryptophan and adenine .Additionally, AML insertion homozygote seedlings were assayed for a differential response to a range of signaling molecules. Seedlings were germinated on culture medium supplemented with the gibberellic acid GAmei2 locus [AML4 are disrupted at or near the 5' ends of their predicted coding regions. We therefore designed primers that anneal to the region downstream of the insertion site in each allele and performed polymerase chain reactions to assay for accumulation of fragments capable of encoding the C-terminal half of any of the AML proteins .The oral dip . TransfoAtRaptor loci show normal embryonic development but are unable to maintain shoot meristem activity [Raptor proteins in yeast and mammals function by recruiting substrates for TOR, a central regulator of cell growth in response to nutrients ,7,28. Anactivity . TOR subMei2 interacting protein 1) was the first Raptor homologue characterized in any eukaryote [An interaction between Mei2 and the fission yeast Raptor homologue Mip1 has been reported previously; indeed, Mip1  remains and may be sufficient to cause transcription of AML coding region DNA downstream of the insertion site.Transcripts originating downstream of the insertion sites but still capable of encoding the C-terminal half of the wild-type protein accumulate from all loci but Viewed in this light, the early flowering phenotype of AML insertion homozygotes may arise not from the total disruption of AML activity but from the accumulation of AML proteins which, due to the truncations in their N-termini caused by the insertions, are no longer bound by AtRaptor1B, no longer phosphorylated by AtTOR, or no longer able to activate transcription of floral repressors.Finally, these results provide a cautionary tale. RT-PCR performed using primers which span an insertion site may not be sufficient to conclude that all activity of a protein of interest is abolished.TOR is a major regulator of cell growth in eukaryotes, but little is known about its downstream effectors in plants. This work shows that AML1 binds AtRaptor1B, and suggests that the AML protein family may be phosphorylated by AtTOR in an AtRaptor1B-mediated interaction. The interaction with AtRaptor1B implicates AML1 as a downstream effector of AtTOR kinase signaling, and provides insight into the mode of action of this critical growth regulator.AML1 was cloned via RT-PCR. The cDNA template was reverse-transcribed using Omniscript (Qiagen) from RNA extracted from bulk shoot tissue using Trizol Reagent (Invitrogen). Restriction sites NcoI and XmaI/SmaI were added to the 5' and 3' ends of the ORF and of all smaller AML1 fragments via PCR using ExTaq high-fidelity polymerase (Takara). An EST clone (RZL03b06) tagging AtRaptor1B was obtained from Kazusa DNA institute and sequenced. Restriction sites NcoI and EcoRI were added at the 5' and 3' ends of the ORF via PCR. pGBKT7 and pGADT7 are distributed by BDBiosciences.leu2-trp1-ade2-his3-) were grown in YEPDA liquid plates or on YEPDA plates with 17 g/L Agar-Y (Bio101 Systems). Cells were transformed using the Yeastmaker Yeast Transformation System2 (BD Biosciences) and plated on medium lacking the appropriate macronutrients (Bio101 Systems). Colonies were observed 3–7 days after transformation.AH109 cells  ecotype background. The AML1 insertion was found in pool CSJ8-46-H35. The AML5 insertion was found in pool CSJ1091-H45. Insertions in the AML3 and AML4 loci were obtained from the University of Wisconsin Basta collection and are in the Ws background. The AML3 insertion was found in pool 67-6-F. The AML4 insertion was found in pool 18-2-H. The insertion in AML2 was obtained from the line 029713 from the Salk collection and is in the Columbia (Col-0) ecotype background. Aside from regions genetically linked to any of the insertion loci, the AML quintuple insertion allele homozygote lines are in the genomic background of a Col-0 × WS F3 individual once backcrossed to the WS background.Insertions in the free™ DNase (Ambion) and reverse transcribed using Omniscript reverse transcriptase (Qiagen) with an oligo-dT primer. Primer pairs spanning the insertion site for RT-PCR on all five lines are as follows: AML1 117–138 5'gtgatggatgcgattggataga3', AML1 556-534rc 5'attgtggcttcagctggtaactt3'; AML2 86–109 5'tttgcttctccgattctcttcctt3', AML2 456-435rc 5'agcatcgggttcaacatcttcc3'; AML3 548–568 5'gtagcggaggaggtcttgaat3', AML3 1060-1039rc 5'tctccttgatctcgccataaac3'; AML4 1932–1955 5'aagcggtagtccatataaatcctc3', AML4 2944-2927rc 5'tcccctgaatccgaccat3'; AML5 104–124 5'cgtgatcatcgtcggtgttgg3', AML5 1047-1024rc 5'ctcgacgaatttgtgatgcctctta3'. Reactions were performed using Takara ExTaq and 35 cycles of 94°C for 30 sec, 58°C annealing (AML1,2,4) or 60°C annealing (AML3,5) for 30 sec, and 72°C for one minute.RNA extraction was performed using TRIzol™ Reagent (Invitrogen) essentially according to manufacturer's instructions. Total RNA was treated with DNA-AML1, AML2, AML3 and AML5 insertion homozygotes are as follows: AML1 +1635 5'aggctctcgccgccctatta3', AML1 -2466 5'cgttgccaccttctcgctatt3'; AML2 +1779 5'accggggaacagtagtgaac3', AML2 -2107 5'ctgtcggcaagcatagaaag3'; AML3 +1756 5'tctggcctgctgctacaatgg3', AML3 -2326 5'cgccgacaagaagatgagaaaac3'; AML5 +1268 5'gcaacggcttccaacagtca3', AML5 -1869 5'acgaggcctaccattttcatacaa3'. All reactions were performed with a 59°C annealing temperature.Primer pairs amplifying a region 3' of the insertion site for RT-PCR on GHA conceived of the study and performed all experimental manipulations. MRH provided guidance and arranged funding for the project. GHA and MRH drafted the manuscript, and both authors approve the final manuscript.


A leading cause of death and disability, diabetes affects some 16 million Americans and up to 135 million people worldwide. While environmental factors such as diet and lifestyle are known to influence an individual's risk of getting adult-onset, or Type 2, diabetes, there is also a substantial inherited component, though many of the genetic pathways involved remain unidentified.The challenge of defining these genetic pathways lies in the fact that diabetes is what is known as a “complex trait”: not only is it likely that variations in many different genes or some combination of genes contribute to an increased risk, but there are probably different genes associated with diabetes in different populations. Tackling the monumental task of deciphering this genetic puzzle in the largest known study of its kind, Inês Barroso and colleagues confirm the genetic complexity of the disease and clearly demonstrate that untangling the genetics will require even larger studies.In diabetes, defects in both the secretion and function of insulin—which is produced by the pancreas—impair the body's ability to metabolize glucose. Based on what is known about the biology of pancreatic function and diabetes, the researchers chose 71 potential suspect genes that could reasonably be expected to contribute to the disease if they malfunctioned. Some of these genes are involved in the function of pancreatic beta-cells, which secrete insulin; a second group influences the function of insulin and glucose metabolism; and a third plays a broader role in energy metabolism. The results show that none of the genes on their own had a large effect, though a number of gene variations increased risk slightly. They also suggest the existence of variations in several genes that influence the risk of Type 2 diabetes.The dataset will be valuable for future studies of diabetes and supports the view that variation in genes affecting insulin production as well as insulin action can influence the risk of Type 2 diabetes. However, the genetic complexity of the disease—with a number of genes conveying a slightly increased risk— demands additional studies of larger populations to reliably identify the genes involved and the genetic variations that, alone or in combination, increase or lower an individual's risk of developing the disease.


What makes us human? From a philosophical perspective, the answer may lie in part in our apparently unique need—and self-awareness—to ask the question in the first place. From a biological perspective, the answer lies in part in the sequence of our DNA. While fossil evidence has provided a rough draft of the story of human evolution, much more remains to be learned about the path our genes followed, a path that diverged millions of years ago from our closest living hominid relatives, the chimp and bonobo. Charting differences between human genomes and those of our evolutionary relatives—both near and distant—has become a powerful tool for filling in the gaps in the human fossil record.Comparing the human genome to the genomes of other great apes can provide a window into the molecular changes that may ultimately spell the difference between human and nonhuman primates. That task was recently aided by the release of the draft sequence of the chimpanzee genome. Comparing the protein-coding sequences of human and chimp has identified molecular dissimilarities between us, which is to be expected. Though many differences between species can be explained at the molecular level by differences in protein structure, where and when a given protein is produced can be just as or even more important. Differences in protein expression arise from sequences in non-coding DNA that influence the timing and regulation of protein production and action.In a new study, Peter Keightley and colleagues conduct parallel comparative genomics studies—comparing regulatory regions in the chimp and human genome with those of mouse and rat—and make a startling discovery. The hominid lineages show a surprising lack of selective constraint—deleterious mutations have apparently accumulated—compared to the rodents, racking up an estimated additional 140,000 harmful mutations fixed, or retained, in the human and chimp lineages since they diverged. Such mutations have been selectively eliminated in mouse and rat.The authors focused on DNA sequences making up the bulk of gene-regulating elements—regions immediately preceding or following protein-coding sequences, as well as the first intron of each gene (an intron is a non-coding DNA sequence squeezed between two adjacent coding fragments). The degree of conservation in these areas was weighed against the conservation in other nearby non-coding sequences, which were assumed to be free of selective constraints. Keightley and colleagues found marked conservation in the regulatory regions between mice and rats, but nearly none between humans and chimps. This result suggests that the gene-regulating elements of hominids are subject to nearly unfettered mutation accumulation, likely due to an absence of natural selection forces strong enough to stabilize the ancestral sequences common to both human and chimpanzee.How can one explain these puzzling results? Keightley and colleagues propose that selection is ineffective against mildly unfavorable mutations in the gene-regulating regions because of the small effective population size in the evolutionary history of hominids.What do these results suggest for the future of human evolution? It's unlikely that the regulatory gatekeepers of our genome will allow mutations to spin out of control. Even if the number of unwanted mutations were to increase, stronger natural selection against them is likely to develop in parallel, Keightley and colleagues explain, protecting our fitness from a downward spiral. The authors' results support the notion that population size exerts a powerful influence on evolutionary changes at the molecular level and that many changes in gene control regions are under weak selection. With each new sequenced genome added to the comparative genomics lexicon, scientists are becoming increasingly conversant in the grammar and syntax of gene sequences—and filling in more and more gaps in the human story, letter by letter.


The telomeric protein TRF2 is required to prevent mammalian telomeres from activating DNA damage checkpoints. Here we show that overexpression of TRF2 affects the response of the ATM kinase to DNA damage. Overexpression of TRF2 abrogated the cell cycle arrest after ionizing radiation and diminished several other readouts of the DNA damage response, including phosphorylation of Nbs1, induction of p53, and upregulation of p53 targets. TRF2 inhibited autophosphorylation of ATM on S1981, an early step in the activation of this kinase. A region of ATM containing S1981 was found to directly interact with TRF2 in vitro, and ATM immunoprecipitates contained TRF2. We propose that TRF2 has the ability to inhibit ATM activation at telomeres. Because TRF2 is abundant at chromosome ends but not elsewhere in the nucleus, this mechanism of checkpoint control could specifically block a DNA damage response at telomeres without affecting the surveillance of chromosome internal damage. The human telomere-associated protein TRF2 inhibits ATM kinase, suggesting a mechanism by which the telomeric protein complex prevents the activation of this DNA damage response transducer Telomeres prevent the recognition of natural chromosome ends as double-stranded breaks (DSBs). When telomeres become dysfunctional due to shortening or loss of protective factors, chromosome ends activate a DNA damage response mediated (in part) by the ATM kinase . A majorΔBΔM, which removes the endogenous TRF2 complex from telomeres  repeats and recruits hRap1, ERCC1/XPF, WRN, and the Mre11/Rad50/Nbs1 complex to chromosome ends . TRF2 caelomeres . Upon exigase IV . The DNAigase IV . Primaryigase IV .An important transducer of the DNA damage signal is the ATM kinase , TRF2 overexpression partially abrogated this checkpoint response, increasing the percentage of cells entering mitosis from 0.3% to 8% . The inaWe then asked whether TRF2 overexpression inhibited other ATM-dependent readouts of the DNA damage response. ATM phosphorylates and stabilizes p53 in response to DNA damage  on the ATM kinase, we determined TRF2's effect on the activation of ATM itself. Phosphorylation of ATM on S1981, an early and essential step in the activation of this kinase, can be detected rapidly after IR, even when a low level of DNA damage is induced . To testIn order to understand the mechanism by which TRF2 inhibited ATM, we determined whether they interacted in vivo. IPs of the ATM kinase from primary human IMR90 fibroblasts resulted in recovery of a small fraction (approximately 1%) of endogenous TRF2 A. This aThe nature of the association of TRF2 with ATM was explored further using in vitro pulldown experiments. GST-tagged fragments of ATM were tested for their ability to bind purified TRF2 protein expressed in a baculovirus system. In parallel, we used baculovirus-derived TRF1, which was previously shown to interact with ATM by co-IP . TRF2 boBlunting of the DNA damage response was observed when TRF2 was overexpressed throughout the nucleus. Because TRF2 is chiefly present at telomeres, the simplest interpretation is that the observed activity reflected a telomeric function. However, we also considered the possibility that TRF2 may have a heretofore clandestine role in the general DNA damage response. If this were true, TRF2 might be expected to localize to IR-induced foci (IRIF), where it would be in a position to modulate ATM. Previous data had shown that the endogenous TRF2 does not relocate from telomeres to IRIF . SimilarNatural chromosome ends require mechanisms to prevent the activation of the DNA damage response. Inhibition of the ATM kinase at human telomeres is particularly important since the telomeric complex contains the Mre11 complex, one of the DNA damage sensors of the ATM pathway . The telWe feel that the interaction of TRF2 with ATM is likely to be relevant to the mechanism by which TRF2 blocks ATM signaling. TRF2 binds ATM in a region surrounding S1981, which is functionally linked to the oligomerization state of ATM. Phosphorylation on S1981 occurs concomitant with the dissociation of ATM dimers (or oligomers), forming the monomeric, active form of the kinase . TRF2 isIn this study we have expressed TRF2 at high levels throughout the nucleus, whereas endogenous TRF2 is localized primarily to telomeres . Our estPrevious studies have shown that overexpression of TRF2 can protect critically short telomeres generated by replicative aging . TRF2 reAs TRF2 can bind ATM, it has the inherent ability to recruit this protein to telomeres. ATM has not been observed at undamaged telomeres, but its abundance may be too low for detection. The idea that ATM could be recruited to telomeres by TRF2 is interesting considering that ATM-like kinases are necessary for telomere maintenance in Saccharomyces cerevisiae  and Schi5 cells were seeded in 5-cm culture dishes and exposed to a Ce137 source. Where indicated, the medium was replaced with medium containing 10 mM caffeine (Sigma, St. Louis, Missouri, United States) and 1 μg/ml colcemide (Sigma). Indirect immunofluorescence was performed as described (IMR90 primary lung fibroblasts (ATCC, Manassas, Virginia, United States) and AG02496 and AG04405 primary A-T fibroblasts  were grown and infected with retroviruses as described elsewhere . For γ-iescribed .6 293T cells were plated in 10-cm dishes. Cells were transfected with 1 μg of FLAG–ATM DNA . The NaCl concentration was raised to 400 mM, and the lysate was incubated on ice for 5 min. The NaCl concentration was reduced to 200 mM, cell debris was removed by centrifugation, and an equal volume of Laemmli buffer was added to the lysate.One day prior to transfection, approximately 5 10–ATM DNA  and 9 μg–ATM DNA  or TRF1 6 IMR90 fibroblasts were run on 7.5% precast polyacrylamide Bio-Rad  Ready Gels. PVDF ImmobilonTM Transfer Membrane (Millipore, Billerica, Massachusetts, United States) was prepared for protein transfer according to the manufacturer's instructions and the gel was transferred for 2 h at 90 V. Membranes were preincubated in 10% milk, 0.1% Tween-20 in PBS for 30 min at room temperature and subsequently incubated with primary antibodies: polyclonal rabbit ATM S1981–P (4 cells/μl. Lysates (10 μl) were separated on SDS-polyacrylamide gels (29:1 acrylamide: bisacrylamide, 8% for p53, TRF2, and γ-tubulin, 6% for pRB and Hdm2, 12% for Bax, p21, and p16) and transferred onto nitrocellulose membranes (Schleicher and Schuell) for 60 min at 90 V (Bio-Rad Mini-Protean II Cell). Membranes were preincubated in 10% nonfat dry milk, 0.1% Tween-20 in PBS for 30 min and subsequently incubated with primary antibodies: p53 D01 ; TRF2 serum 647 (TM 2200 using the SpotDenso function of AlphaEaseFCTM Software Version 3.1.2 (Witec, Littau, Switzerland).For ATM immunoblots in the ATM S1981–P suppression assays: 40 μl of 293T cell lysate or ATM immunoprecipitated from approximately 5 10 S1981–P  and mouserum 647 ; pRB #55erum 647  in 5% dr7 cells/0.1 ml buffer) in 20 mM HEPES (pH 7.9), 0.42 M KCl, 25% glycerol, 0.1 mM EDTA, 5 mM MgCl2, 0.2% NP40, 1 mM DTT, 0.5 mM PMSF, 1 μg/ml leupeptine, 1 μg/ml aprotinin, 10 μg/ml pepstatin on ice for 30 min. Debris was removed by centrifugation (14,000 rpm, 4 °C, 10 min). Protein concentration in the supernatant was determined using the Bradford assay and 400 μg of protein was diluted to 150 mM KCl and incubated for 20 min with 100 μl of protein G–Sepharose beads (Amersham), blocked with 1% fetal bovine serum in PBS. The beads were collected at 14,000 rpm for 1 min and the supernatant was incubated with 5 μg of anti-ATM antibody (AB3, Oncogene Research) or anti-Cyclin D1 antibody (#sc-6281, Santa Cruz Biotechnology) for 1 h at 4 °C on a nutator. Protein G–Sepharose beads (30 μl) were added, and the mixture was incubated 1 h at 4 °C on a nutator. The beads were collected at 4,000 rpm at 4 °C, washed three times with wash buffer (150 mM NaCl, 1% NP40, 50 mM Tris [pH 8.0] with protease inhibitors as described above) by vortexing the suspension for 10 s. The beads were resuspended in Laemmli buffer and boiled 5 min, and proteins were separated by polyacrylamide gel electrophoresis. For IP of endogenous ATM from IMR90 fibroblasts for the phosphorylation assay, cells were resuspended in lysis buffer (50 mM Hepes [pH 7.5], 150 mM NaCl, 50 mM NaF, 1% Tween-20, 0.2% NP40, 1mM PMSF, with 1 complete mini-protease inhibitor tablet [Roche, Basel, Switzerland] ) and centrifuged at 13,000 rpm for 10 min. 400 μl of lysate was incubated with 30 μl of blocked protein G beads and 100 μl of D16.11 monoclonal supernatant  was added. After 3 h at 30 °C, cells were harvested, resuspended in 8 ml of lysis buffer (50 mM Tris [pH 7.9], 100 mM KCl, 1% Triton X-100, 2 mM DTT, 0.1 mM PMSF, 1 complete protease inhibitor tablet [Roche]) and sonicated three times for 30 s on ice. The lysate was cleared by centrifugation at 50,000 g at 4 °C and incubated with 600 μl of equilibrated glutathione beads for 2 h at 4 °C. The beads were washed three times for 10 min each  and a fourth time in wash 4 (50 mM Tris [pH 7.9], 100 mM KCl, 10% glycerol, 2 mM DTT, 0.1 mM PMSF). Fusion proteins were eluted in 500 μl of wash 4 containing 15 mM glutathione (reduced form). Two subsequent elutions were collected. Five micrograms of GST fusion proteins or GST alone were incubated with 2 μg of baculoviral TRF1 or TRF2 in binding buffer (150 mM NaCl, 100 mM KCl, 50 mM Tris [pH 8.0], 1% NP40, 0.1% SDS, 100 g/ml BSA) at 4 °C for 1 h. Glutathione beads (20 μl) were added and incubated for 1 h at 4 °C. Beads were collected by centrifugation at 5,000 rpm at 4 °C and washed three times for 10 min each with binding buffer, and bound protein was eluted by boiling the samples in Laemmli buffer. GST fusion proteins, TRF1, and TRF2 were detected by immunoblotting.GST–ATM fusion plasmids  were traCells were seeded on microscope coverslips and irradiated with 4 Gy of γ-irradiation as described above. Cells were incubated in growth medium containing 1 μg/ml colcemid for 16 h. Cells were fixed, and phosphorylated histone H3 was detected by indirect immunofluorescence using a phosphospecific antibody . Cells in mitosis were counted and expressed as a percentage of total cell number.


Bioinformatics, Biocomputing and Perl" [omething in a Nutshell".The book "nd Perl"  attemptsThe book aims at both biology- and computation-oriented audiences and is designed as a number of 'crash-courses' quickly updating the reader on the basics of bioinformatics. It starts with a preface outlining main biological and technological concepts of the modern computational biology. The rest is organized into four sections consisting of 18 chapters elaborating on essential bioinformatics tools and skills.etc. By working through the section, the reader acquires basic skills for mySQL database use and DBI Perl programming.The section 'Working with Perl' presents an extended tutorial with practical tips and useful references for Perl beginners. Following this is 'Working with Data', which familiarizes the reader with some public genomic and proteomic databases and discusses important subjects of database formats, non-redundancy, cross-referencing and programmable access, Next, the authors offer Perl-based solutions for remote database access and for creation of WWW-based bioinformatics services using Perl functionalities in 'Working with the Web'.BioPerl applications.The final topic of the book, 'Working with Applications', features basic tools for sequence alignment, protein homology modeling and data visualization, all commonly used in bioinformatics practice. The section also offers recent and relevant examples of BioPerl and web-automation, are excellent. The presented material is rather comprehensive and yet easy to read – the authors spent appreciative efforts to make the book interesting and enjoyable. The authors also acknowledge the open-source nature of Perl and the bioinformatics community and offer on-line support and direct feedback to the readers.In general, the book reflects the state of bioinformatics field with its strengths and weaknesses. Many Perl chapters, such as Perl regular expressions, modular organization, DBI-programming, AcePerl [There are also certain aspects, in which the book could be further improved. Several sections may be too advanced for the beginner level (such as Perl basics and database downloading), while others may contain too excessive details (the Protein Databank section). In addition, it may be of advantage to mention AcePerl , Perl-prAcePerl  and distA very useful feature of the book is the use of maxims that highlight key points throughout the text. The authors also provide helpful technical comments where necessary and offer practical exercises at the end of each chapter. The book is concluded with six appendices covering the Linux basics, Perl installation, operators, on-line support and suggested reading materials which, in my mind, benefit the book tremendously.Bioinformatics, Biocomputing and Perl", serves well its purpose as an introductory textbook and a resource of reference materials for bioinformaticians.Thus, the overall product, the "AcePerl – is a Perl interface for the AceDB – a popular object-oriented bioinformatics database.DBI Perl – the primary interface for database programming by Perl.BioPerl – a collection of Perl modules specifically designed for several most common bioinformatics tasks.XML – Extensible Markup Language – a popular standard for documents containing structured information.SRS – the Sequence Retrieval System – a popular relational database for bioinformatics.


Rostatus quo of traditional publishing paradigms [Biomedical Digital Libraries.Biomedical scientific communities increasingly articulate concern about the unsustainable aradigms , feedingaradigms . SeizingWhile traditional biomedical library concern revolves around the role of collection stewardship and delivering published knowledge to students, scholars, researchers, and clinicians, information professionals in library or information center settings are also concerned with the cultivation of their own knowledge domains and evidence-based library practice ,5. PerhaOpen access scientific literature arrived in several pre-print guises over the previous two decades and in a recent contemporary commercial model with BioMed Central  (BMC). SBMC now has 425 institutional members in 40 countries . ResearcBiomedical Digital Libraries is the conviction that open access will push biomedical librarianship forward in new and improved ways. Biomedical Digital Libraries provides a legitimate alternative to traditional specialty journals in the field, which have subscription fees and assumption of copyright by the publisher. This journal will stress peer-reviewed open access to research and practice in digital collection and services settings and will permit rapid and unimpeded dissemination of knowledge, only weeks after manuscript submission. Over 30 information professionals and biomedical scholars will be engaged as editorial staff to conduct blinded peer review reports for original research, as well as integrate commentaries, resource reviews, and debates into a forum for the discussion of unique considerations of biomedical information needs. These include both opportunities and constraints presented by health care settings, including collaborative initiatives with information technology and informatics partners.The decision to found a BMC journal for the world of organized biomedical information was both spontaneous and practical. At the core of the founding of Biomedical Digital Libraries. Beyond our immediate narrow spheres of digital library practice and service, the community of open knowledge has the immediate and timely potential to inspire, inform, and create value on a global scale through permanent, uninhibited access.We hope the advocates, philosophers, caretakers, and architects of biomedical library digital content take immediate advantage of rapid peer-review and publication, extensive BMC content promotion, permanent URL, redundant public archiving, and retention of copyright when they submit to Some seek good in authority, others in scientific research, others in pleasure. Others, who are in fact nearer the truth, have considered it necessary that the universal good, which all men desire, should not consist in any of the particular things which can only be possessed by one man, and which, when shared, afflict their possessor more by the want of the part he has not, than they please him by the possession of what he has. They have learned that the true good should be such as all can possess at once, without diminution and without envy, and which no one can lose against his will .– Blaise Pascal(1623–1662)