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When the genomic integrity of a cell is challenged, its fate is determined in part by signals conveyed by the p53 tumour suppressor protein. It was observed recently that such signals are not simple gradations of p53 concentration, but rather a counter-intuitive limit-cycle behaviour. Based on a careful mathematical interpretation of the experimental body of knowledge, we propose a model for the p53 signalling network and characterise the p53 stability and oscillatory dynamics. In our model, ATM, a protein that senses DNA damage, activates p53 by phosphorylation. In its active state, p53 has a decreased degradation rate and an enhanced transactivation of Mdm2, a gene whose protein product Mdm2 tags p53 for degradation. Thus the p53-Mdm2 system forms a negative feedback loop. However, the feedback in this loop is delayed, as the pool of Mdm2 molecules being induced by p53 at a given time will mark for degradation the pool of p53 molecules at some later time, after the Mdm2 molecules have been transcribed, exported out of the nucleus, translated and transported back into the nucleus. The analysis of our model demonstrates how this time lag combines with the ATM-controlled feedback strength and effective dampening of the negative feedback loop to produce limit-cycle oscillations. The picture that emerges is that ATM, once activated by DNA damage, makes the p53-Mdm2 oscillator undergo a supercritical Hopf bifurcation. This approach yields an improved understanding of the global dynamics and bifurcation structure of our time-delayed, negative feedback model and allows for predictions of the behaviour of the p53 system under different perturbations.

We developed a model of 545 components (nodes) and 1259 interactions representing signaling pathways and cellular machines in the hippocampal CA1 neuron. Using graph theory methods, we analyzed ligand-induced signal flow through the system. Specification of input and output nodes allowed us to identify functional modules. Networking resulted in the emergence of regulatory motifs, such as positive and negative feedback and feedforward loops, that process information. Key regulators of plasticity were highly connected nodes required for the formation of regulatory motifs, indicating the potential importance of such motifs in determining cellular choices between homeostasis and plasticity.

Introduction: MR imaging has emerged as an important modality in the non-invasive evaluation of osseous and soft-tissue structures in the post-traumatic knee. However, it is sometimes impossible to determine with confidence if a focus of high signal intensity in the meniscus is confined to the substance of the meniscus or if it extends to involve the joint surface. This is a critical differentiation because the latter represents meniscal tears that can be found and treated arthroscopically, whereas the former represents degeneration, intrasubstance tears or perhaps normal variants that are not amenable to arthroscopic intervention. The aim of this study was to investigate the occurrence of such borderline findings in relation to the posterior horn of the medial meniscus and to correlate the arthroscopic results., Materials and Methods: Sixty-four patients with suspected post-traumatic internal derangements of the knee who underwent MR imaging prior to arthroscopy were evaluated retrospectively. There were 48 men and 16 women. Their mean age was 28.2 years., Results: Tears of the posterior horn of the medial meniscus were diagnosed unequivocally (grade 3 signal) in 18 patients and equivocally (grade 2/3 signal) in 10 patients. Arthroscopic correlation revealed 16 tears (89%) in the unequivocal group and only 1 tear (10%) in the equivocal group., Conclusion: A meniscal tear is unlikely when MR shows a focus of high signal intensity in the posterior horn of the medial meniscus that does not unequivocally extend to involve the inferior or superior joint surface. An appropriate trial of conservative treatment is recommended in such questionable cases. MR is a useful diagnostic tool-however, it should be used selectively, and in conjunction with history and clinical examination in evaluating internal derangements of the knee.

Motivation: One of the present challenges in biological research is the organization of the data originating from high-throughput technologies. One way in which this information can be organized is in the form of networks of influences, physical or statistical, between cellular components. We propose an experimental method for probing biological networks, analyzing the resulting data and reconstructing the network architecture., Methods: We use networks of known topology consisting of nodes (genes), directed edges (gene-gene interactions) and a dynamics for the genes' mRNA concentrations in terms of the gene-gene interactions. We proposed a network reconstruction algorithm based on the conditional correlation of the mRNA equilibrium concentration between two genes given that one of them was knocked down. Using simulated gene expression data on networks of known connectivity, we investigated how the reconstruction error is affected by noise, network topology, size, sparseness and dynamic parameters., Results: Errors arise from correlation between nodes connected through intermediate nodes (false positives) and when the correlation between two directly connected nodes is obscured by noise, non-linearity or multiple inputs to the target node (false negatives). Two critical components of the method are as follows: (1) the choice of an optimal correlation threshold for predicting connections and (2) the reduction of errors arising from indirect connections (for which a novel algorithm is proposed). With these improvements, we can reconstruct networks with the topology of the transcriptional regulatory network in Escherichia coli with a reasonably low error rate.

A robust bacterial display methodology was developed that allows the rapid isolation of peptides that bind to arbitrarily selected targets with high affinity. To demonstrate the utility of this approach, a large library (5 x 10(10) clones) was constructed composed of random 15-mer peptide insertions constrained within a flexible, surface exposed loop of the Escherichia coli outer membrane protein A (OmpA). The library was screened for binding to five unrelated proteins, including targets previously used in phage display selections: human serum albumin, anti-T7 epitope mAb, human C-reactive protein, HIV-1 GP120 and streptavidin. Two to four rounds of enrichment (2-4 days) were sufficient to enrich peptide ligands having high affinity for each of the target proteins. Strong amino acid consensus sequences were apparent for each of the targets tested, with up to seven consensus residues. Isolated peptide ligands remained functional when expressed as insertional fusions within a monomeric fluorescent protein. This bacterial display methodology provides an efficient process for identifying peptide affinity reagents and should be useful in a variety of molecular recognition applications.

While the primary function of the heart is a pump, ironically, the development of myofilament models that predict developed force have generally lagged behind the modeling of the electrophysiological and Ca2+-handling aspects of heart cells. A major impediment is that the basic events in force generating actin-myosin interactions are still not well understood and quantified despite advanced techniques that can probe molecular levels events and identify numerous energetic states. As a result, the modeler must decide how to best abstract the many identified states into useful models with an essential tradeoff in the level of complexity. Namely, complex models map more directly to biophysical states but experimental data does not yet exist to well constrain the rate constants and parameters. In contrast, parameters can be better constrained in simpler, lumped models, but the simplicity may preclude versatility and extensibility to other applications. Other controversies exist as to why the activation of the actin-myosin is so steeply dependent on activator Ca2+. More specifically steady-state force-[Ca2+] (F-Ca) relationships are similar to Hill functions, presumably as the result of cooperative interactions between neighboring crossbridges and/or regulatory proteins. We postulate that mathematical models must contain explicit representation of nearest-neighbor cooperative interactions to reproduce F-Ca relationships similar to experimental measures, whereas spatially compressing, mean-field approximation used in most models cannot. Finally, a related controversy is why F-Ca relationships show increased Ca2+ sensitivity as sarcomere length (SL) increases. We propose a model that suggests that the length-dependent effects can result from an interaction of explicit nearest-neighbor cooperative mechanisms and the number of recruitable crossbridges as a function of SL., (Copyright 2004 Elsevier Ltd.)

Motivation: Despite the growing literature devoted to finding differentially expressed genes in assays probing different tissues types, little attention has been paid to the combinatorial nature of feature selection inherent to large, high-dimensional gene expression datasets. New flexible data analysis approaches capable of searching relevant subgroups of genes and experiments are needed to understand multivariate associations of gene expression patterns with observed phenotypes., Results: We present in detail a deterministic algorithm to discover patterns of multivariate gene associations in gene expression data. The patterns discovered are differential with respect to a control dataset. The algorithm is exhaustive and efficient, reporting all existent patterns that fit a given input parameter set while avoiding enumeration of the entire pattern space. The value of the pattern discovery approach is demonstrated by finding a set of genes that differentiate between two types of lymphoma. Moreover, these genes are found to behave consistently in an independent dataset produced in a different laboratory using different arrays, thus validating the genes selected using our algorithm. We show that the genes deemed significant in terms of their multivariate statistics will be missed using other methods., Availability: Our set of pattern discovery algorithms including a user interface is distributed as a package called Genes@Work. This package is freely available to non-commercial users and can be downloaded from our website (http://www.research.ibm.com/FunGen).

We have developed a model of cardiac thin filament activation using an Ising model approach from equilibrium statistical physics. This model explicitly represents nearest-neighbor interactions between 26 troponin/tropomyosin units along a one-dimensional array that represents the cardiac thin filament. With transition rates chosen to match experimental data, the results show that the resulting force-pCa (F-pCa) relations are similar to Hill functions with asymmetries, as seen in experimental data. Specifically, Hill plots showing (log(F/(1-F)) vs. log [Ca]) reveal a steeper slope below the half activation point (Ca(50)) compared with above. Parameter variation studies show interplay of parameters that affect the apparent cooperativity and asymmetry in the F-pCa relations. The model also predicts that Ca binding is uncooperative for low [Ca], becomes steeper near Ca(50), and becomes uncooperative again at higher [Ca]. The steepness near Ca(50) mirrors the steep F-pCa as a result of thermodynamic considerations. The model also predicts that the correlation between troponin/tropomyosin units along the one-dimensional array quickly decays at high and low [Ca], but near Ca(50), high correlation occurs across the whole array. This work provides a simple model that can account for the steepness and shape of F-pCa relations that other models fail to reproduce.

This study employs two modeling approaches to investigate short-term interval-force relations. The first approach is to develop a low-order, discrete-time model of excitation-contraction coupling to determine which parameter combinations produce the degree of postextrasystolic potentiation seen experimentally. Potentiation is found to increase 1) for low recirculation fraction, 2) for high releasable fraction, i.e., the maximum fraction of Ca(2+) released from the sarcoplasmic reticulum (SR) given full restitution, and 3) for strong negative feedback of the SR release on sarcolemmal Ca(2+) influx. The second modeling approach is to develop a more detailed single ventricular cell model that simulates action potentials, Ca(2+)-handling mechanisms, and isometric force generation by the myofilaments. A slow transition from the adapted state of the ryanodine receptor produces a gradual recovery of the SR release and restitution behavior. For potentiation, a small extrasystolic release leaves more Ca(2+) in the SR but also increases the SR loading by two mechanisms: 1) less Ca(2+)-induced inactivation of L-type channels and 2) reduction of action potential height by residual activation of the time-dependent delayed rectifier K(+) current, which increases Ca(2+) influx. The cooperativity of the myofilaments amplifies the relatively small changes in the Ca(2+) transient amplitude to produce larger changes in isometric force. These findings suggest that short-term interval-force relations result mainly from the interplay of the ryanodine receptor adaptation and the SR Ca(2+) loading, with additional contributions from membrane currents and myofilament activation.

A model of the functional release unit (FRU) in rat cardiac muscle consisting of one dihydropyridine receptor (DHPR) and eight ryanodine receptor (RyR) channels, and the volume surrounding them, is formulated. It is assumed that no spatial [Ca2+] gradients exist in this volume, and that each FRU acts independently. The model is amenable to systematic parameter studies in which FRU dynamics are simulated at the channel level using Monte Carlo methods with Ca2+ concentrations simulated by numerical integration of a coupled system of differential equations. Using stochastic methods, Ca(2+)-induced Ca2+ release (CICR) shows both high gain and graded Ca2+ release that is robust when parameters are varied. For a single DHPR opening, the resulting RyR Ca2+ release flux is insensitive to the DHPR open duration, and is determined principally by local sarcoplasmic reticulum (SR) Ca2+ load, consistent with experimental data on Ca2+ sparks. In addition, single RyR openings are effective in triggering Ca2+ release from adjacent RyRs only when open duration is long and SR Ca2+ load is high. This indicates relatively low coupling between RyRs, and suggests a mechanism that limits the regenerative spread of RyR openings. The results also suggest that adaptation plays an important modulatory role in shaping Ca2+ release duration and magnitude, but is not solely responsible for terminating Ca2+ release. Results obtained with the stochastic model suggest that high gain and gradedness can occur by the recruitment of independent FRUs without requiring spatial [Ca2+] gradients within a functional unit or cross-coupling between adjacent functional units.

Length-dependent steady-state and dynamic responses of five models of isometric force generation in cardiac myofilaments were compared with similar experimental data from the literature. The models were constructed by assuming different subsets of three putative cooperative mechanisms. Cooperative mechanism 1 holds that cross-bridge binding increases the affinity of troponin for Ca2+. In the models, cooperative mechanism 1 can produce steep force-Ca2+ (F-Ca) relations, but apparent cooperativity is highest at midlevel Ca2+ concentrations. During twitches, cooperative mechanism 1 has the effect of increasing latency to peak as the magnitude of force increases, an effect not seen experimentally. Cooperative mechanism 2 holds that the binding of a cross bridge increases the rate of formation of neighboring cross bridges and that multiple cross bridges can maintain activation of the thin filament in the absence of Ca2+. Only cooperative mechanism 2 can produce sarcomere length (SL)-dependent prolongation of twitches, but this mechanism has little effect on steady-state F-Ca relations. Cooperativity mechanism 3 is designed to simulate end-to-end interactions between adjacent troponin and tropomyosin. This mechanism can produce steep F-Ca relations with appropriate SL-dependent changes in Ca2+ sensitivity. With the assumption that tropomyosin shifting is faster than cross-bridge cycling, cooperative mechanism 3 produces twitches where latency to peak is independent of the magnitude of force, as seen experimentally.

Instrumented fusion of the collapsing spine has gained widespread acceptance for patients with Duchenne Muscular Dystrophy but controversy still exists on the issue of extending the surgical fusion to sacrum in these patients. This retrospective study reviews the long-term outcome of a group of patients with spinal deformity associated with Duchenne Muscular Dystrophy who were managed with long spinal fusion to L5 and ongoing wheelchair seating attention. The clinical notes and radiographs of 19 consecutive patients were reviewed. Fifteen patients attended for clinical and radiological assessment at a mean of 28 months post operatively. The surgery for these patients involved a mean anaesthetic time of 3.5 h and a mean transfusion requirement of 5 units of red cell concentrate. At long-term follow-up 15 patients continued to sit in a well-balanced position. Surgical fusion of the spine to L5 combined with ongoing attention to seating is associated with good long-term functional results in these patients.

We construct a detailed mathematical model for Ca2+ regulation in the ventricular myocyte that includes novel descriptions of subcellular mechanisms based on recent experimental findings: 1) the Keizer-Levine model for the ryanodine receptor (RyR), which displays adaptation at elevated Ca2+; 2) a model for the L-type Ca2+ channel that inactivates by mode switching; and 3) a restricted subspace into which the RyRs and L-type Ca2+ channels empty and interact via Ca2+. We add membrane currents from the Luo-Rudy Phase II ventricular cell model to our description of Ca2+ handling to formulate a new model for ventricular action potentials and Ca2+ regulation. The model can simulate Ca2+ transients during an action potential similar to those seen experimentally. The subspace [Ca2+] rises more rapidly and reaches a higher level (10-30 microM) than the bulk myoplasmic Ca2+ (peak [Ca2+]i approximately 1 microM). Termination of sarcoplasmic reticulum (SR) Ca2+ release is predominately due to emptying of the SR, but is influenced by RyR adaptation. Because force generation is roughly proportional to peak myoplasmic Ca2+, we use [Ca2+]i in the model to explore the effects of pacing rate on force generation. The model reproduces transitions seen in force generation due to changes in pacing that cannot be simulated by previous models. Simulation of such complex phenomena requires an interplay of both RyR adaptation and the degree of SR Ca2+ loading. This model, therefore, shows improved behavior over existing models that lack detailed descriptions of subcellular Ca2+ regulatory mechanisms.

Mechano-electrical feedback is studied by incorporating linear, instantaneously activating mechano-sensitive conductances into single cardiac cell models, as well as one- and two-dimensional cardiac network models. The models qualitatively reproduce effects of maintained mechanical stretch on experimentally measured action potential characteristics such as amplitude, maximum diastolic potential, peak upstroke velocity, and conduction velocity. Models are also used to simulate stretch-induced depolarizations, action potentials, and arrhythmias produced by pulsatile volume changes in left ventricle of dog. The mechano-sensitive conductance threshold for a stretch-induced action potential is closely related to the magnitude of the time-independent K+ current, IK1, which offsets inward mechano-sensitive current. Activation of mechano-sensitive conductances in small, spatially localized region of cells can evoke graded depolarizations, propagating ectopic beats, and if timed appropriately, spiral reentrant waves. Mechano-sensitive conductance changes required to evoke these responses are well within the physiologically plausible range. Results therefore indicate that many mechano-electrical feedback effects can be modeled using linear, instantaneously activating mechano-sensitive conductances. As an example of how stretch can occur in real human hearts, magnetic resonance images with saturation tagging are used to reconstruct the three-dimensional left ventricular wall motion. In patients with infarcts or recent ischemic events, "paradoxical deformation" is observed in that regions of myocardium are stretched rather than contracted during systole. In contrast, normal hearts contract uniformly with no stretch during systole. Paradoxical deformations in ischemic hearts may therefore present one possible substrate for the mechanically induced arrhythmias modeled above., (Copyright 1998 Academic Press Limited.)

To measure the effects of the pinna position on spectral sound localization cues, the head-related transfer function (HRTF) from the free-field to a point in the ear canal was measured for anesthetized cats with their pinnae in three positions: the relaxed, anesthetized position; pulled forward into an approximation of the "alert cat" posture; and pulled back. The general features of HRTFs are not changed by moving the pinna, although the mapping of particular HRTF features onto directions in space is changed. As an approximation, the pinna behaves like a fixed-shaped sound collector, so that HRTFs shift with the pinna when it moves; however, pinna movement changes some quantitative details of HRTFs beyond what is predicted by this approximation. When viewed as directional gain, pinna movements serve to optimize listening conditions. However, when considering sound localization, pinna movements lead to ambiguities regarding source location. If pinna position is not incorporated into the computation, and spectral cues alone are used for localization, the ambiguity is about 60 degrees in azimuth and 30 degrees in elevation. Pinna movements produce similar azimuthal ambiguity in interaural level differences. Interaural time difference cues could be used to reduce the ambiguity in azimuth, but a knowledge of pinna position seems to be necessary to resolve ambiguities in elevation.

The incidence of penetrating skin wounds and needle penetration of gloves during operation was studied in orthopaedic surgeons. Significant hand wounds were found in 11% of surgeons before operations. Glove penetration during closure of the deep tissues occurred in 16% of outer gloves and 6% of inner gloves when standard needle points were used. The surgeon sustained a needle-stick injury in 6% of this group. When a needle with a protective point was used, there were no glove perforations. This simple precaution reduces the risk of transmission of blood-borne disease during operation.

The elevation of sound sources and their front-back position is encoded in spectral cues produced by direction-dependent filtering in the pinna. Auditory-nerve (AN) fiber population recordings were used to analyze the neural representation of the acoustic features which carry this information. The most prominent pinna-produced spectral features occur at frequencies greater than 5 kHz, so this information must be encoded in AN discharge rates and not in measures of phase locking. However, profiles of discharge rate versus fiber best frequency (BF) reveal a poor representation of the spectra of the stimuli, primarily because of fiber-to-fiber variation in rate. The variation is not controlled by rate normalization, but a clear representation of the ratio of the magnitude spectra of two stimuli is seen when responses are plotted as the difference between the rates in response to the two stimuli. This results suggests that precise information about stimulus spectrum is present in discharge rate, which could be revealed in rate profiles constructed with suitable normalization. When binaural stimuli are presented, a weak inhibitory effect, due to the olivocochlear bundle or the middle ear muscle reflex, is observed. The rate changes are small and are not correlated with the spectrum of the contralateral ear stimulus.

The authors describe a variable compensation (VC) technique in which an x-ray equalizer and a computed radiography system are used. The VC technique allows retrospective alteration of equalized chest appearance with maintenance of improved signal-to-noise ratio in dense regions. Two imaging plates are used: one upstream of the patient to record the incident beam profile and one down-stream to record the equalized image. Subtraction of a weighted version of the upstream image from the down-stream image permits alteration of the appearance of the VC image, from the extremes of stimulated-unequalized to highly equalized. VC image appearance was optimized with a real-time workstation. The quality of VC images obtained in 33 patients was evaluated by three chest radiologists. Mediastinal appearance was better on VC equalized images than on conventional screen-film images. The stimulation of the appearance of a conventional radiograph with VC proved useful in interpretation of lung appearances on equalized radiographs.

The dorsal division of the cochlear nucleus (DCN) is the most complex of its subdivisions in terms of both anatomical organization and physiological response types. Hypotheses about the functional role of the DCN in hearing are as yet primitive, in part because the organizational complexity of the DCN has made development of a comprehensive and predictive model of its input-output processing difficult. The responses of DCN cells to complex stimuli, especially filtered noise, are interesting because they demonstrate properties that cannot be predicted, without further assumptions, from responses to narrow band stimuli, such as tones. In this paper, we discuss the functional organization of the DCN, i.e. the morphological organization of synaptic connections within the nucleus and the nature of synaptic interactions between its cells. We then discuss the responses of DCN principal cells to filtered noise stimuli that model the spectral sound localization cues produced by the pinna. These data imply that the DCN plays a role in interpreting sound localization cues; supporting evidence for such a role is discussed.

Images of a phantom obtained with computed radiography and standard screen-film imaging were compared to evaluate observer threshold perception performance with a modified contrast-detail technique. Optimum exposure necessary for performance with the imaging plate technique to match that with screen-film techniques was determined, as was comparative performance with variation in kilovoltages, plate type, spatial enhancement, and hard-copy interpolation method. It was found that computed radiography necessitates about 75%-100% more exposure than screen-film radiography to optimally match performance with Ortho-C film with Lanex regular or medium screens (Eastman Kodak, Rochester, NY) for detection of objects 0.05-2.0 cm in diameter. However, only minimal loss of detection performance (approximately 10% overall) was experienced if standard screen-film exposures were used with computed radiography. Little change in observer performance was found with variation in plate type, spatial enhancement, or method of hard-copy interpolation. However, perception performance with computed radiographic images was better at lower kilovoltages.

The directional dependence of the transfer function from free field plane waves to a point near the tympanic membrane (TM) was measured in anesthetized domestic cats. A probe tube microphone was placed approximately 3 mm from the TM from beneath the head in order to keep the pinna intact. Transfer functions were computed as the ratio of the spectrum of a click recorded near the TM to the spectrum of the click in freefield. We analyze the transfer functions in three frequency ranges: low frequencies (less than 5 kHz) where interaural level differences vary smoothly with azimuth; midfrequencies (5-18 kHz) where a prominent spectral notch is observed; and high frequencies (greater than 18 kHz) where the transfer functions vary greatly with source location. Because no two source directions produce the same transfer function, the spectrum of a broadband sound at the TM could serve as a sound localization cue for both elevation and azimuth. In particular, we show that source direction is uniquely determined, for source directions in front of the cat, from the frequencies of the midfrequency spectral notches in the two ears. The validity of the transfer functions as measures of the acoustic input to the auditory system is considered in terms of models of sound propagation in the ear canal.

A specific, stability-indicating, high-performance liquid chromatographic assay was developed for the determination of cephapirin, desacetyl cephapirin and cephapirin lactone in sodium cephapirin (cefadyl) bulk and injectables. The procedure uses a muBondapak C18 column and a mobile phase of dimethylformamide-acetic acid-potassium hydroxide in water. UV detection at 254 nm is used for quantitation with acetanilide used as the internal standard. The assay is precise, accurate and linear over the range of 100-300 micrograms/ml for cephapirin and over the range of 2-6 micrograms/ml for desacetyl cephapirin and cephapirin lactone. The assay is also stability-indicating for the described thermal, acid, base, aqueous and accelerated light degradations.