In this paper, we present an automatic method for estimating the trajectories of Escherichia coli bacteria from in vivo phase-contrast microscopy. To address the low-contrast boundaries in cellular images, an adaptive kernel-based technique is applied to detect cells in each frame. In addition to intensity features, region homogeneity measure and class uncertainty are also applied in this detection technique. To track cells with complex motion, a novel matching gain measure is introduced to cope with the challenges, particularly the presence of low-contrast boundary, the variations of appearance, and the frequent overlapping and occlusion. For multicell tracking over time, an optimal matching strategy is introduced to improve the handling of cell collision and broken trajectories. The results of successful tracking of Escherichia coli from various phase-contrast sequences are reported and compared with manually determined trajectories, as well as those obtained from existing tracking schemes. The stability of the algorithm with different parameter values is also analyzed and discussed.

We describe an approach for automation of the process of reconstruction of neural tissue from serial section transmission electron micrographs. Such reconstructions require 3D segmentation of individual neuronal processes (axons and dendrites) performed in densely packed neuropil. We first detect neuronal cell profiles in each image in a stack of serial micrographs with multi-scale ridge detector. Short breaks in detected boundaries are interpolated using anisotropic contour completion formulated in fuzzy-logic framework. Detected profiles from adjacent sections are linked together based on cues such as shape similarity and image texture. Thus obtained 3D segmentation is validated by human operators in computer-guided proofreading process. Our approach makes possible reconstructions of neural tissue at final rate of about 5 microm3/manh, as determined primarily by the speed of proofreading. To date we have applied this approach to reconstruct few blocks of neural tissue from different regions of rat brain totaling over 1000microm3, and used these to evaluate reconstruction speed, quality, error rates, and presence of ambiguous locations in neuropil ssTEM imaging data.

The R‐specific alcohol dehydrogenase from Lactobacillus brevis (Lb‐ADH) catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. It is stable and has broad substrate specificity. These features make this enzyme an attractive candidate for biotechnological applications. A drawback is its preference for NADP(H) as a cofactor, which is more expensive and labile than NAD(H). Structure‐based computational protein engineering was used to predict mutations to alter the cofactor specificity of Lb‐ADH. Mutations were introduced into Lb‐ADH and tested against the substrate acetophenone, with either NAD(H) or NADP(H) as cofactor. The mutant Arg38Pro showed fourfold increased activity with acetophenone and NAD(H) relative to the wild type. Both Arg38Pro and wild type exhibit a pH optimum of 5.5 with NAD(H) as cofactor, significantly more acidic than with NADP(H). These and related Lb‐ADH mutants may prove useful for the green synthesis of pharmaceutical precursors.

Hormones coordinate developmental, physiological, and behavioral processes within and between all living organisms. They orchestrate and shape organogenesis from early in development, regulate the acquisition, assimilation, and utilization of nutrients to support growth and metabolism, control gamete production and sexual behavior, mediate organismal responses to environmental change, and allow for communication of information between organisms. Genes that code for hormones; the enzymes that synthesize, metabolize, and transport hormones; and hormone receptors are important targets for natural selection, and variation in their expression and function is a major driving force for the evolution of morphology and life history. Hormones coordinate physiology and behavior of populations of organisms, and thus play key roles in determining the structure of populations, communities, and ecosystems. The field of endocrinology is concerned with the study of hormones and their actions. This field is rooted in the comparative study of hormones in diverse species, which has provided the foundation for the modern fields of evolutionary, environmental, and biomedical endocrinology. Comparative endocrinologists work at the cutting edge of the life sciences. They identify new hormones, hormone receptors and mechanisms of hormone action applicable to diverse species, including humans; study the impact of habitat destruction, pollution, and climatic change on populations of organisms; establish novel model systems for studying hormones and their functions; and develop new genetic strains and husbandry practices for efficient production of animal protein. While the model system approach has dominated biomedical research in recent years, and has provided extraordinary insight into many basic cellular and molecular processes, this approach is limited to investigating a small minority of organisms. Animals exhibit tremendous diversity in form and function, life-history strategies, and responses to the environment. A major challenge for life scientists in the 21st century is to understand how a changing environment impacts all life on earth. A full understanding of the capabilities of organisms to respond to environmental variation, and the resilience of organisms challenged by environmental changes and extremes, is necessary for understanding the impact of pollution and climatic change on the viability of populations. Comparative endocrinologists have a key role to play in these efforts.

The genetically encoded calcium indicator GCaMP2 shows promise for neural network activity imaging, but is currently limited by low signal-to-noise ratio. We describe x-ray crystal structures as well as solution biophysical and spectroscopic characterization of GCaMP2 in the calcium-free dark state, and in two calcium-bound bright states: a monomeric form that dominates at intracellular concentrations observed during imaging experiments and an unexpected domain-swapped dimer with decreased fluorescence. This series of structures provides insight into the mechanism of Ca2+-induced fluorescence change. Upon calcium binding, the calmodulin (CaM) domain wraps around the M13 peptide, creating a new domain interface between CaM and the circularly permuted enhanced green fluorescent protein domain. Residues from CaM alter the chemical environment of the circularly permuted enhanced green fluorescent protein chromophore and, together with flexible inter-domain linkers, block solvent access to the chromophore. Guided by the crystal structures, we engineered a series of GCaMP2 point mutants to probe the mechanism of GCaMP2 function and characterized one mutant with significantly improved signal-to-noise. The mutation is located at a domain interface and its effect on sensor function could not have been predicted in the absence of structural data.

Ambulation after spinal cord injury is possible with the aid of neuroprosthesis employing functional electrical stimulation (FES). Individuals with incomplete spinal cord injury (iSCI) retain partial volitional control of muscles below the level of injury, necessitating careful integration of FES with intact voluntary motor function for efficient walking. In this study, the intramuscular electromyogram (iEMG) was used to detect the intent to step and trigger FES-assisted walking in a volunteer with iSCI via an implanted neuroprosthesis consisting of two channels of bipolar iEMG signal acquisition and 12 independent channels of stimulation. The detection was performed with two types of classifiers- a threshold-based classifier that compared the running mean of the iEMG with a discrimination threshold to generate the trigger and a pattern recognition classifier that compared the time-history of the iEMG with a specified template of activity to generate the trigger whenever the cross-correlation coefficient exceeded a discrimination threshold. The pattern recognition classifier generally outperformed the threshold-based classifier, particularly with respect to minimizing False Positive triggers. The overall True Positive rates for the threshold-based classifier were 61.6% and 87.2% for the right and left steps with overall False Positive rates of 38.4% and 33.3%. The overall True Positive rates for the left and right step with the pattern recognition classifier were 57.2% and 93.3% and the overall False Positive rates were 11.9% and 24.4%. The subject showed no preference for either the threshold or pattern recognition-based classifier as determined by the Usability Rating Scale (URS) score collected after each trial and both the classifiers were perceived as moderately easy to use.

The microtubule cytoskeleton has proven to be an effective target for cancer therapeutics. One class of drugs, known as microtubule stabilizing agents (MSAs), binds to microtubule polymers and stabilizes them against depolymerization. The prototype of this group of drugs, Taxol, is an effective chemotherapeutic agent used extensively in the treatment of human ovarian, breast, and lung carcinomas. Although electron crystallography and photoaffinity labeling experiments determined that the binding site for Taxol is in a hydrophobic pocket in beta-tubulin, little was known about the effects of this drug on the conformation of the entire microtubule. A recent study from our laboratory utilizing hydrogen-deuterium exchange (HDX) in concert with various mass spectrometry (MS) techniques has provided new information on the structure of microtubules upon Taxol binding. In the current study we apply this technique to determine the binding mode and the conformational effects on chicken erythrocyte tubulin (CET) of another MSA, discodermolide, whose synthetic analogues may have potential use in the clinic. We confirmed that, like Taxol, discodermolide binds to the taxane binding pocket in beta-tubulin. However, as opposed to Taxol, which has major interactions with the M-loop, discodermolide orients itself away from this loop and toward the N-terminal H1-S2 loop. Additionally, discodermolide stabilizes microtubules mainly via its effects on interdimer contacts, specifically on the alpha-tubulin side, and to a lesser extent on interprotofilament contacts between adjacent beta-tubulin subunits. Also, our results indicate complementary stabilizing effects of Taxol and discodermolide on the microtubules, which may explain the synergy observed between the two drugs in vivo.

Insufficient kinetic stability of exoinulinase (EI) restricts its application in many areas including enzymatic transformation of inulin for production of ultra-high fructose syrup and oligofructan, as well as fermentation of inulin into bioethanol. The conventional method for enzyme stabilization involves mutagenesis and therefore risks alteration of an enzyme’s desired properties, such as activity. Here, we report a novel method for stabilization of EI without any modification of its primary sequence. Our method employs domain insertion of an entire EI domain into a thermophilic scaffold protein. Insertion of EI into a loop of a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) resulted in improvement of kinetic stability (the duration over which an enzyme remains active) at 37 degrees C without any compromise in EI activity. Our analysis suggests that the improved kinetic stability at 37 degrees C might originate from a raised kinetic barrier for irreversible conversion of unfolded intermediates to completely inactivated species, rather than an increased energy difference between the folded and unfolded forms.

The excitability of individual dendritic branches is a plastic property of neurons. We found that experience in an enriched environment increased propagation of dendritic Na(+) spikes in a subset of individual dendritic branches in rat hippocampal CA1 pyramidal neurons and that this effect was mainly mediated by localized downregulation of A-type K(+) channel function. Thus, dendritic plasticity might be used to store recent experience in individual branches of the dendritic arbor.

Synaptic plasticity in adult neural circuits may involve the strengthening or weakening of existing synapses as well as structural plasticity, including synapse formation and elimination. Indeed, long-term in vivo imaging studies are beginning to reveal the structural dynamics of neocortical neurons in the normal and injured adult brain. Although the overall cell-specific morphology of axons and dendrites, as well as of a subpopulation of small synaptic structures, are remarkably stable, there is increasing evidence that experience-dependent plasticity of specific circuits in the somatosensory and visual cortex involves cell type-specific structural plasticity: some boutons and dendritic spines appear and disappear, accompanied by synapse formation and elimination, respectively. This Review focuses on recent evidence for such structural forms of synaptic plasticity in the mammalian cortex and outlines open questions.