The structural components of the Azotobacter vinelandii iron-only nitrogenase, AnfDKG, form a protein complex within the plant mitochondrial matrix.

A long-held goal of synthetic biology has been the transfer of a bacterial nitrogen-fixation pathway into plants to reduce the use of chemical fertiliser on crops such as rice, wheat and maize. There are three classes of bacterial nitrogenase, named after their metal requirements, containing either a MoFe-, VFe- or FeFe-cofactor, that converts N2 gas to ammonia. Relative to the Mo-nitrogenase the Fe-nitrogenase is not as efficient for catalysis but has less complex genetic and metallocluster requirements, features that may be preferable for engineering into crops. Here we report the successful targeting of bacterial Fe-nitrogenase proteins, AnfD, AnfK, AnfG and AnfH, to plant mitochondria. When expressed as a single protein AnfD was mostly insoluble in plant mitochondria, but coexpression of AnfD with AnfK improved its solubility. Using affinity-based purification of mitochondrially expressed AnfK or AnfG we were able to demonstrate a strong interaction of AnfD with AnfK and a weaker interaction of AnfG with AnfDK. This work establishes that the structural components of the Fe-nitrogenase can be engineered into plant mitochondria and form a complex, which will be a requirement for function. This report outlines the first use of Fe-nitrogenase proteins within a plant as a preliminary step towards engineering an alternative nitrogenase into crops.

Controlling the action of chlorine radical: from lab to environment.

The strength of Bz-Cl˙ complexation has been explored using density functional theory (DFT) calculations, including dispersion-corrected (DFT-D) calculations. Of the methods tested, the ωB97X-D method seems the best performing, along with the previously tested MPW1K method. The effect of substituent (X = NO(2), F, Cl, Br, H, CH(3), OCH(3), OH, NH(2) and N(CH(3))(2)) on the stabilities of the Ar-Cl˙π-like intermediates show a good correlation with the linear free energy relationships used experimentally, but this is not the case for Ar-Cl˙σ-complexes, suggesting the transition state of abstraction as being π-like in nature. The role of PAH and lignin derivatives in mediating chlorination reactions in nature is explored. Stable π-complexes were identified for lignin derivatives, indicating humic substances may mediate chlorine atom reactivity at the marine boundary layer, in addition to forming chlorolignins.

Auditory evoked potentials during speech perception.

Neural responses evoked by the same binaural speech signal were recorded from ten right-handed subjects during two auditory identification tasks. One task required analysis of acoustic parameters important for making a linguistic distinction, while the other task required analysis of an acoustic parameter which provides no linguistic information at the phoneme level. In the time interval between stimulus onset and the subjects' identification responses, evoked potentials from the two tasks were significantly different over the left hemisphere but identical over the right hemisphere. These results indicate that different neural events occur in the left hemisphere during analysis of linguistic versus nonlinguistic parameters of the same acoustic signal.

Electrical sources in human somatosensory cortex: identification by combined magnetic and potential recordings.

Magnetic fields and electrical potentials produced by neuronal activity have different properties that can be used for the identification of electrical sources in the human brain. Fields and potentials occurring 20 to 30 milliseconds after median nerve stimulation in human subjects were compared in order to investigate the sources of evoked potential components that have been attributed by different investigators to the thalamus or thalamocortical afferents, to separate radial sources in somatosensory cortex and motor cortex, or to a tangential source in somatosensory cortex. The magnetic and potential wave forms were highly similar in morphology, and their spatial distributions were centered over sensorimotor cortex, were dipolar in shape, and differed in orientation by approximately 90 degrees; distances between the minimum and maximum of the magnetic distributions were about 60 percent of those of the potential distributions. These results cannot be accounted for by thalamic sources or radial cortical sources alone, but are consistent with a tangential source in somatosensory cortex, with an additional smaller contribution from radial sources.

The computational stance in biology.

The goal of this article is to call attention to, and to express caution about, the extensive use of computation as an explanatory concept in contemporary biology. Inspired by Dennett's 'intentional stance' in the philosophy of mind, I suggest that a 'computational stance' can be a productive approach to evaluating the value of computational concepts in biology. Such an approach allows the value of computational ideas to be assessed without being diverted by arguments about whether a particular biological system is 'actually computing' or not. Because there is sufficient difference of agreement among computer scientists about the essential elements that constitute computation, any doctrinaire position about the application of computational ideas seems misguided. Closely related to the concept of computation is the concept of information processing. Indeed, some influential computer scientists contend that there is no fundamental difference between the two concepts. I will argue that despite the lack of widely accepted, general definitions of information processing and computation: (1) information processing and computation are not fully equivalent and there is value in maintaining a distinction between them and (2) that such value is particularly evident in applications of information processing and computation to biology. This article is part of the theme issue 'Liquid brains, solid brains: How distributed cognitive architectures process information'.

Modeling spatiotemporal covariance for magnetoencephalography or electroencephalography source analysis.

We propose a new model to approximate spatiotemporal noise covariance for use in neural electromagnetic source analysis, which better captures temporal variability in background activity. As with other existing formalisms, our model employs a Kronecker product of matrices representing temporal and spatial covariance. In our model, spatial components are allowed to have differing temporal covariances. Variability is represented as a series of Kronecker products of spatial component covariances and corresponding temporal covariances. Unlike previous attempts to model covariance through a sum of Kronecker products, our model is designed to have a computationally manageable inverse. Despite increased descriptive power, inversion of the model is fast, making it useful in source analysis. We have explored two versions of the model. One is estimated based on the assumption that spatial components of background noise have uncorrelated time courses. Another version, which gives closer approximation, is based on the assumption that time courses are statistically independent. The accuracy of the structural approximation is compared to an existing model, based on a single Kronecker product, using both Frobenius norm of the difference between spatiotemporal sample covariance and a model, and scatter plots. Performance of ours and previous models is compared in source analysis of a large number of single dipole problems with simulated time courses and with background from authentic magnetoencephalography data.

Extravasation reactions associated with the administration of pamidronate: 11 cases (2008-2013).

Pamidronate is a bisphosphonate drug widely utilized in veterinary oncologic practice for the palliation of malignant osteolysis. Pamidronate has not been previously reported to cause tissue injury upon extravasation in dogs. The medical records of 11 client-owned dogs undergoing palliative treatment for primary bone tumors with known or suspected pamidronate extravasation reactions were reviewed. The majority of adverse events were low grade in nature, however in some cases, the reactions were severe and led to euthanasia in one instance. Time to complete resolution of lesions ranged from within several days to greater than one and a half months. Aside from the dog that was euthanized, no long-term sequelae of extravasation were identified. Treatments employed to address the reactions varied widely. Pamidronate extravasation reaction appears to be an uncommon, but potentially serious complication of intravenous administration.

Improving source detection and separation in a spatiotemporal Bayesian inference dipole analysis.

Most existing spatiotemporal multi-dipole approaches for MEG/EEG source localization assume that the dipoles are active for the full time range being analysed. If the actual time range of activity of sources is significantly shorter than the time range being analysed, the detectability, localization and time-course determination of such sources may be adversely affected, especially for weak sources. In order to improve detectability and reconstruction of such sources, it is natural to add active time range information (starting time point and ending time point of source activation) for each candidate source as unknown parameters in the analysis. However, this adds additional nonlinear free parameters that could burden the analysis and could be unfeasible for some methods. Recently, we described a spatiotemporal Bayesian inference multi-dipole analysis for the MEG/EEG inverse problem. This approach treated the number of dipoles as a free parameter, produced realistic uncertainty estimates using a Markov chain Monte Carlo numerical sampling of the posterior distribution and included a method to reduce the unwanted effects of local minima. In this paper, our spatiotemporal Bayesian inference multi-dipole analysis is extended to incorporate active time range parameters of starting and stopping time points. The properties of this analysis in comparison to the previous one without active time range parameters are demonstrated through extensive studies using both simulated and empirical MEG data.

Efficiency of Ipratropium Bromide and Albuterol Deposition in the Lung Delivered via a Soft Mist Inhaler or Chlorofluorocarbon Metered-Dose Inhaler.

The propellant-free Combivent Respimat Soft Mist Inhaler (CVT-R) was developed to replace the chlorofluorocarbon-propelled Combivent metered-dose inhaler (CVT-MDI). This steady-state pharmacokinetic (PK) substudy evaluated drug lung-delivery efficiency, using data from two phase III safety and efficacy trials. PK parameters were obtained from well-controlled population PK analyses. Area under the plasma concentration-time curve (AUC), maximum observed plasma concentration (C(max)), and minimum observed plasma concentration (C(min)) showed systemic exposure to ipratropium bromide and albuterol delivered via the CVT-R was proportional to ex-mouthpiece delivered dose. Although the labeled dose of ipratropium bromide in the CVT-R was half that in the CVT-MDI, the systemic exposure was comparable. No PK interaction for the ipratropium bromide and albuterol Respimat drug components was demonstrated. Ipratropium bromide alone resulted in similar exposure to the combination of ipratropium bromide and albuterol. These results show that CVT-R delivers drug more efficiently to the lung than CVT-MDI.

Transplantation of thymic tissue into patients with AIDS. An attempt to reconstitute the immune system.

Thymic epithelial fragments were transplanted into 15 patients in an advanced stage of the acquired immunodeficiency syndrome (AIDS). One patient was given interleukin 2 in addition to thymic tissue. We demonstrated the following: Thymic epithelial fragments cultured before transplantation to remove T cells survived for months after transplantation in eight of 15 patients and seemed to be responsible for a partial, selective, but transient repopulation of the circulating T-cell pool. The absolute number of T8 cells, but not T4 cells, increased three to four weeks after the procedure in eight of the 15 subjects. This increase in T8 cells was associated with clinical improvement in some cases and increased T-cell responsiveness in vitro. Thymic tissue transplantation as a single therapeutic maneuver is unlikely to reconstitute the immune system of patients with AIDS, but the potential of the approach, used in combination with agents that block replication of human T-cell lymphotropic virus type III, deserves further study.

Single vs. paired visual stimulation: superposition of early neuromagnetic responses and retinotopy in extrastriate cortex in humans.

Neuromagnetic techniques were used in conjunction with magnetic resonance imaging (MRI) techniques to: (1) localize and characterize cortical sources evoked by visual stimuli presented at different locations in the lower right visual field; (2) examine the superposition of cortical responses by comparing the summation of responses to the presentation of single stimuli with responses to paired stimuli; and (3) examine the spatial resolution of magnetoencephalographic (MEG) techniques by comparing the identified source locations evoked by the presentation of single vs. paired stimuli. Using multi-dipole, non-linear minimization analyses, three sources were localized for each stimulus condition during the initial 80-170 ms poststimulus interval for all subjects. In addition to an occipital source, two extrastriate sources were identified: occipital-parietal and occipital-temporal. Each source evidenced a systematic shift in location associated with changes in stimulus placement parallel to the vertical meridian. To our knowledge, this is the first demonstration of retinotopic organization of extrastriate areas, using non-invasive neuromagnetic techniques. The paired presentation of stimuli reflected superposition of the responses evoked by single stimuli but only for early activity up to 150 ms poststimulus. Undersummation was evident after 150 ms. All sources identified for single stimuli were also identified in the paired-stimulus responses; but at the expense of larger errors for some of the estimated parameters.

Microcystic adnexal carcinoma. Immunohistologic observations suggesting dual (pilar and eccrine) differentiation.

Microcystic adnexal carcinoma (MAC) is a locally aggressive neoplasm that has recently been recognized as a clinicopathologic entity. Its histologic appearance includes both pilar and eccrine differentiation. We initially treated two patients with MAC of the cheek and of the nasolabial fold, respectively; by microscopically controlled excision because of the contiguous growth of the tumors. Despite the benign histologic appearance, there was deep and extensive infiltration of the subcutaneous tissue. Both patients responded favorably to initial treatment with microscopically controlled excision. In addition, immunoperoxidase staining for carcinoembryonic antigen supported the dual differentiation of this unusual neoplasm. We speculate that previous radiotherapy may be an important predisposing factor in the pathogenesis of MAC.

Noninvasive preoperative cortical localization by magnetic source imaging.

The authors successfully used magnetoencephalography and MR data to localize the sensorimotor cortex in two patients prior to neurosurgery; preoperative localization influenced surgical management.

Auditory and phonetic levels of processing in speech perception: neurophysiological and information-processing analyses.

Two new experimental operations were used to distinguish between auditory and phonetic levels of processing in speech perception: the first based on reaction time data in speeded classification tasks with synthetic speech stimuli, and the second based on average evoked potentials recorded concurrently in the same tasks. Each of four experiments compared the processing of two different dimensions of the same synthetic consonant-vowel syllables. When a phonetic dimensions was compared to an auditory dimension, different patterns of results were obtained in both the reaction time and evoked potential data. No such differences were obtained for isolated acoustic components of the phonetic dimension or for two purely auditory dimensions. Together with other recent evidence, the present results constitute additional converging operations on the distinction between auditory and phonetic processes in speech perception and on the idea that phonetic processing involves mechanisms that are lateralized in one cerebral hemisphere.

Mapping function in the human brain with magnetoencephalography, anatomical magnetic resonance imaging, and functional magnetic resonance imaging.

Integrated analyses of human anatomical and functional measurements offer a powerful paradigm for human brain mapping. Magnetoencephalography (MEG) and EEG provide excellent temporal resolution of neural population dynamics as well as capabilities for source localization. Anatomical magnetic resonance imaging (MRI) provides excellent spatial resolution of head and brain anatomy, whereas functional MRI (fMRI) techniques provide an alternative measure of neural activation based on associated hemodynamic changes. These methodologies constrain and complement each other and can thereby improve our interpretation of functional neural organization. We have developed a number of computational tools and techniques for the visualization, comparison, and integrated analysis of multiple neuroimaging techniques. Construction of geometric anatomical models from volumetric MRI data allows improved models of the head volume conductor and can provide powerful constraints for neural electromagnetic source modeling. These approaches, coupled to enhanced algorithmic strategies for the inverse problem, can significantly enhance the accuracy of source-localization procedures. We have begun to apply these techniques for studies of the functional organization of the human visual system. Such studies have demonstrated multiple, functionally distinct visual areas that can be resolved on the basis of their locations, temporal dynamics, and differential sensitivity to stimulus parameters. Our studies have also produced evidence of internal retinotopic organization in both striate and extrastriate visual areas but have disclosed organizational departures from classical models. Comparative studies of MEG and fMRI suggest a reasonable but imperfect correlation between electrophysiological and hemodynamic responses. We have demonstrated a method for the integrated analysis of fMRI and MEG, and we outline strategies for improvement of these methods. By combining multiple measurement techniques, we can exploit the complementary strengths and transcend the limitations of the individual neuro-imaging methods.

Localization of human sensorimotor cortex during surgery by cortical surface recording of somatosensory evoked potentials.

The traditional means of localizing sensorimotor cortex during surgery is Penfield's procedure of mapping sensory and motor responses elicited by electrical stimulation of the cortical surface. This procedure can accurately localize sensorimotor cortex but is time-consuming and best carried out in awake, cooperative patients. An alternative localization procedure is presented that involves cortical surface recordings of somatosensory evoked potentials (SEP's), providing accurate and rapid localization in patients under either local or general anesthesia. The morphology and amplitude of median nerve SEP's recorded from the cortical surface varied systematically as a function of spatial location relative to the sensorimotor hand representation area. These results were validated in 18 patients operated on under local anesthesia in whom the sensorimotor cortex was independently localized by electrical stimulation mapping; the two procedures were in agreement in all cases. Similar SEP results were demonstrated in an additional 27 patients operated on under general anesthesia without electrical stimulation mapping. The following three spatial relationships between SEP's and the anatomy of the sensorimotor cortex permit rapid and accurate localization of the sensorimotor hand area: 1) SEP's with approximately mirror-image waveforms are recorded at electrode sites in the hand area on opposite sides of the central sulcus (P20-N30 precentrally and N20-P30 postcentrally); 2) the P25-N35 is recorded from the postcentral gyrus as well as a small region of the precentral gyrus in the immediate vicinity of the central sulcus: this waveform is largest on the postcentral gyrus about 1 cm medial to the focus of the 20- and 30-msec potentials; and 3) regardless of component identification, maximum SEP amplitudes are recorded from the hand representation area on the precentral and postcentral gyri.

ERPs predictive of subsequent recall and recognition performance.

By exploiting measures of information processing complementary to those obtained from behavioral studies, electrophysiological studies of human memory may provide insights into the cognitive processes associated with encoding. In the present experiment, subjects viewed words under incidental learning conditions in which each word required a two-choice decision based on semantic criteria (interesting/uninteresting or edible/inedible). Memory for those words was subsequently assessed by a free recall test and then a recognition test. Event-related brain potentials elicited in response to the original presentation of each word were found to differ as a function of later memory performance. Over the 400-800 ms latency range, responses to remembered words were positive relative to responses to forgotten words, especially for recall. These electrophysiological differences are interpreted as reflections of processes that correlated with encoding.

Efficacy and safety of ipratropium bromide/albuterol delivered via Respimat inhaler versus MDI.

We compared the efficacy and safety of ipratropium bromide/albuterol delivered via Respimat inhaler, a novel propellant-free inhaler, versus chlorofluorocarbon (CFC)-metered dose inhaler (MDI) and ipratropium Respimat inhaler in patients with COPD. This was a multinational, randomized, double-blind, double-dummy, 12-week, parallel-group, active-controlled study. Patients with moderate to severe COPD were randomized to ipratropium bromide/albuterol (20/100mcg) Respimat inhaler, ipratropium bromide/albuterol MDI [36mcg/206mcg (Combivent Inhalation Aerosol MDI)], or ipratropium bromide (20mcg) Respimat inhaler. Each medication was administered four times daily. Serial spirometry was performed over 6h (0.15min, then hourly) on 4 test days. The primary efficacy variable was forced expiratory volume in 1s (FEV(1)) change from test day baseline at 12 weeks. A total of 1209 of 1480 randomized, treated patients completed the study; the majority were male (65%) with a mean age of 64 yrs and a mean screening pre-bronchodilator FEV(1) (percent predicted) of 41%. Ipratropium bromide/albuterol Respimat inhaler had comparable efficacy to ipratropium bromide/albuterol MDI for FEV(1) area under the curve at 0-6h (AUC(0-6)), superior efficacy to ipratropium Respimat inhaler for FEV(1) AUC(0-4) and comparable efficacy to ipratropium Respimat inhaler for FEV(1) AUC(4-6). All active treatments were well tolerated. This study demonstrates that ipratropium bromide/albuterol 20/100mcg inhaler administered four times daily for 12 weeks had equivalent bronchodilator efficacy and comparable safety to ipratropium bromide/albuterol 36mcg/206mcg MDI, and significantly improved lung function compared with the mono-component ipratropium bromide 20 mcg Respimat inhaler. [Clinical Trial Identifier Number: NCT00400153].