Search

Nodulin-26 intrinsic proteins (NIPs) are plant-specific multifunctional aquaporin-like channels that are phylogenetically and structurally segregated into three subfamilies: NIP I, II, and III. Each subfamily has a characteristic selectivity filter sequence (the “aromatic-arginine” region, or ar/R) that controls substrate transport specificity based on steric constraints, hydrophobicity, and the spatial orientation of hydrogen bonding moieties. All three NIP subfamilies transport metalloid hydroxides, both beneficial as well as toxic, but with different selectivities. Here we investigated the B, As, and water selectivity of representative Arabidopsis thaliana NIP I and II proteins as well as their ar/R mutants in transport assays as well as through B complementation analysis in the B sensitive nip5;1 mutant background. All NIP proteins, and their ar/R mutants, showed equal permeability to arsenite, but showed differences in boric acid and aquaporin activities that was linked to the amino acid at the helix 2 (H2) position of the ar/R filter (Ala for NIP II and Trp for NIP I). The presence of an alanine at this position in NIP II proteins enhances boric acid permeability and drastically reduces the aquaporin/water permeability of the channel. A NIP II structural model generated from the AlphaFold2 resource and evaluated by MD simulation shows that the alanine results in a wider ar/R pore that accommodates the trigonal boric acid molecule and may allow gating of the pore in a manner that affects water permeability. In contrast, NIP I proteins adopt a more classical aquaporin/glyceroporin arrangement in the ar/R that allows metalloid permeability, although with greater selectivity, as well as permeation by water.

Under anaerobic stress, Arabidopsis thaliana induces the expression of a collection of core hypoxia genes that encode proteins for an adaptive response. Among these genes is NIP2;1, which encodes a member of the “Nodulin 26-like Intrinsic Protein” (NIP) subgroup of the aquaporin superfamily of membrane channel proteins. NIP2;1 expression is limited to the “anoxia core” region of the root stele under normal growth conditions, but shows substantial induction (up to 1,000-fold by 2–4 h of hypoxia) by low oxygen stress, and accumulation in all root tissues. During hypoxia, NIP2;1-GFP accumulates predominantly on the plasma membrane by 2 h, is distributed between the plasma and internal membranes during sustained hypoxia, and remains elevated in root tissues through 4 h of reoxygenation recovery. In response to hypoxia challenge, T-DNA insertion mutant nip2;1 plants exhibit elevated lactic acid within root tissues, reduced efflux of lactic acid, and reduced acidification of the external medium compared to wild-type plants. Previous biochemical evidence demonstrates that NIP2;1 has lactic acid channel activity, and our work supports the hypothesis that NIP2;1 prevents lactic acid toxicity by facilitating release of cellular lactic acid from the cytosol to the apoplast, supporting eventual efflux to the rhizosphere. In evidence, nip2;1 plants demonstrate poorer survival during argon-induced hypoxia stress. Expressions of the ethanolic fermentation transcript Alcohol Dehydrogenase1 and the core hypoxia-induced transcript Alanine Aminotransferase1 are elevated in nip2;1, and expression of the Glycolate Oxidase3 transcript is reduced, suggesting NIP2;1 lactic acid efflux regulates other pyruvate and lactate metabolism pathways.

Margeaux M Schade,1,* Gina Marie Mathew,1,* Daniel M Roberts,2 Daniel Gartenberg,2 Orfeu M Buxton1 1Biobehavioral Health, Pennsylvania State University, University Park, PA, USA; 2Proactive Life, Inc., New York, NY, USA*These authors contributed equally to this workCorrespondence: Orfeu M BuxtonThe Pennsylvania State University, 221 Biobehavioral Health Building, University Park, PA 16802, USATel +1 814 867-5707Email orfeu@psu.eduPurpose: In non-rapid eye movement (NREM) stage 3 sleep (N3), phase-locked pink noise auditory stimulation can amplify slow oscillatory activity (0.5– 1 Hz). Open-loop pink noise auditory stimulation can amplify slow oscillatory and delta frequency activity (0.5– 4 Hz). We assessed the ability of pink noise and other sounds to elicit delta power, slow oscillatory power, and N3 sleep.Participants and Methods: Participants (n = 8) underwent four consecutive inpatient nights in a within-participants design, starting with a habituation night. A registered polysomnographic technologist live-scored sleep stage and administered stimuli on randomized counterbalanced Enhancing and Disruptive nights, with a preceding Habituation night (night 1) and an intervening Sham night (night 3). A variety of non-phase-locked pink noise stimuli were used on Enhancing night during NREM; on Disruptive night, environmental sounds were used throughout sleep to induce frequent auditory-evoked arousals.Results: Total sleep time did not differ between conditions. Percentage of N3 was higher in the Enhancing condition, and lower in the Disruptive condition, versus Sham. Standard 0.8 Hz pink noise elicited low-frequency power more effectively than other pink noise, but was not the most effective stimulus. Both pink noise on the “Enhancing” night and sounds intended to Disrupt sleep administered on the “Disruptive” night increased momentary delta and slow-wave activity (ie, during stimulation versus the immediate pre-stimulation period). Disruptive auditory stimulation degraded sleep with frequent arousals and increased next-day vigilance lapses versus Sham despite preserved sleep duration and momentary increases in delta and slow-wave activity.Conclusion: These findings emphasize sound features of interest in ecologically valid, translational auditory intervention to increase restorative sleep. Preserving sleep continuity should be a primary consideration if auditory stimulation is used to enhance slow-wave activity.Keywords: electroencephalographic spectral analysis, neurobehavioral performance, slow-wave sleep, sleep fragmentation, delta power, slow oscillation

Water and ligand binding play critical roles in the structure and function of proteins, yet their binding sites and significance are difficult to predict a priori. Multiple solvent crystal structures (MSCS) is a method where several X-ray crystal structures are solved, each in a unique solvent environment, with organic molecules that serve as probes of the protein surface for sites evolved to bind ligands, while the first hydration shell is essentially maintained. When superimposed, these structures contain a vast amount of information regarding hot spots of protein-protein or protein-ligand interactions, as well as conserved water-binding sites retained with the change in solvent properties. Optimized mining of this information requires reliable structural data and a consistent, objective analysis tool. Detection of related solvent positions (DRoP) was developed to automatically organize and rank the water or small organic molecule binding sites within a given set of structures. It is a flexible tool that can also be used in conserved water analysis given multiple structures of any protein independent of the MSCS method. The DRoP output is an HTML format list of the solvent sites ordered by conservation rank in its population within the set of structures, along with renumbered and recolored PDB files for visualization and facile analysis. Here, we present a previously unpublished set of MSCS structures of bovine pancreatic ribonuclease A (RNase A) and use it together with published structures to illustrate the capabilities of DRoP.

Relatively little is known about the relation between subthreshold error corrections and post-error behavioral compensations. The present study utilized lateralized beta power, which has been shown to index response preparation, to examine subthreshold error corrections in a task known to produce response conflict, the Simon task. We found that even when an overt correction is not made, greater activation of the corrective response, indexed by beta suppression ipsilateral to the initial responding hand, predicted post-error speeding, and enhanced post-error accuracy at the single-trial level. This provides support for the notion that response conflict associated with errors can be adaptive, and suggests that subthreshold corrections should be taken into account to fully understand error-monitoring processes. Furthermore, we expand on previous findings that demonstrate that post-error slowing and post-error accuracy can be dissociated, as well as findings that suggest that frontal midline theta oscillations and the error-related negativity (ERN) are dissociable neurocognitive processes.

In response to the energy crisis resulting from submergence stress and hypoxia, Arabidopsis limits non-essential mRNAs translation, and accumulate cytosolic stress granules (SG). SGs are phase-separated mRNA-protein particles that partition transcripts for various fates: storage, degradation, or return to translation after stress alleviation. Here, it is shown that RNA stress granules are dynamically regulated during hypoxia stress and aerobic recovery via two phases of autophagy that require the AAA+ ATPase CDC48 and the calcium sensor Calmodulin-like 38 (CML38). CML38 is a core hypoxia response-protein that associates with hypoxia-induced SGs. We show that CML38 is essential for SG autophagy during extended hypoxia. Further, cml38 mutants show disorganized SG morphology during extended hypoxia, suggesting a role in SG formation and maintenance. We also show that upon the return of aerobic conditions, intracellular calcium and CML38 are necessary for SG breakdown and turnover, and for upregulating autophagy. cml38 mutants not only lose these responses, but also have aberrant, sustained autophagosome accumulation during the reoxygenation recovery phase. The findings suggest that CDC48 RNA granule autophagy (“granulophagy”) is conserved in plants, and that the hypoxia-induced calcium sensor CML38 regulates SG autophagy during anaerobic stress as well as during the reprogramming phase associated with reoxygenation.

Under anaerobic stress Arabidopsis thaliana induces the expression of a collection of core hypoxia genes that encode proteins associated with an adaptive response. Included in these core hypoxia genes is NIP2;1, which encodes a member of the “Nodulin-like Intrinsic Protein” (NIP) subgroup of the aquaporin superfamily of membrane channel proteins. Under normal growth, NIP2;1 expression is limited to the “anoxia core” region of the root stele, but shows substantial induction in response to low oxygen stress (as high as 1000-fold by 2-4 hr of hypoxia challenge), and accumulates in all root tissues. During hypoxia, NIP2;1-GFP, accumulates on the cell surface by 2 hr and then is distributed between the cell surface and internal membranes during sustained hypoxia, and remains elevated in root tissues through 4 hrs of reoxygenation recovery. T-DNA insertional mutant nip2;1 plants show elevation of lactic acid within root tissues, and a reduced efflux of lactic acid and acidification of the external medium. Together with previous biochemical evidence demonstrating that NIP2;1 has lactic acid permease activity, the present work supports the hypothesis that the protein facilitates the release of cellular lactate to the rhizosphere to prevent lactic acid toxicity. In support of this, nip2;1 plants show poorer survival to argon-induced hypoxia stress. Nip2;1 mutant plants also show elevated expression of ethanolic fermentation transcripts, as well as reduced expression the lactate metabolic enzyme GOX3, suggesting that the altered efflux of lactate through NIP2;1 regulates other pyruvate and lactate metabolism pathways.One-sentence SummaryThe NIP2;1 lactic acid permease is necessary for an optimum response to low oxygen stress through the release of lactate from roots during hypoxia stress.

The present study investigated the neural dynamics of error processing in both the time and frequency domains, as well as associated behavioral phenomena, at the single-trial level. We used a technique that enabled us to separately investigate the evoked and induced aspects of the EEG signal (Cohen & Donner, 2013, Journal of Neurophysiology, 110[12], 2752-2763). We found that at the single-trial level, while the (evoked) error-related negativity (ERN) predicted only post-error slowing (PES)-and only when errors occurred on incongruent trials-induced frontal midline theta power served as a robust predictor of both PES and post-error accuracy (PEA) regardless of stimulus congruency. Mediation models of both electrophysiological indices demonstrated that although the relationship between theta and PEA was mediated by PES, there was not a relationship between the ERN and PEA. Our data suggest that although the ERN and frontal midline theta index functionally related underlying cognitive processes, they are not simply the same process manifested in different domains. In addition, our findings are consistent with the adaptive theory of post-error slowing, as PES was positively associated with post-error accuracy at the single-trial level. More generally, our study provides additional support for the inclusion of a time-frequency approach to better understand the role of medial frontal cortex in action monitoring.

Introduction Wrist-worn research-grade actigraphy devices are commonly used to identify sleep and wakefulness in freely-living people. However, common existing algorithms were developed primarily to classify sleep-wake within a defined in-bed period with PSG, and exhibit relatively high sensitivity (accuracy on sleep epochs) but relatively low specificity (accuracy on wake epochs). This classification imbalance results in the algorithms performing poorly when attempting to classify data that does not have a predefined sleep period, such as over a 24-hour interval. Here, we develop a 24-hour actigraphy classifier to overcome limitations in specificity (accuracy on wake epochs), which typically afflict in-bed focused algorithms. Methods Four datasets scored via either PSG or direct observation of daytime wakefulness were combined (n=52 participants of mean age 49.8yrs, age range 19 - 86; 52% male; 221 total days/nights). Actigraphy (counts) and PSG (RPSGT-staged epochs) were temporally aligned. A model was trained to transform a time-series actigraphy counts to a time series of sleep-wake classifications, using the TensorFlow library for Python. 5-fold cross-validation was used to train and evaluate the model. Classification performance was compared to the output of the Spectrum device (Philips-Respironics) using the Oakley algorithm with a wake threshold of ‘medium’. Results The developed classifier was compared to the Spectrum classifications across the 24-hour intervals. The developed classifier had higher accuracy (95.4% vs. 76.8%), higher specificity (95.9% vs. 68.9%) and higher balanced-accuracy (95.2% vs. 81.6%) relative to the Spectrum classifications, each assessed via paired-sample t-test. Sensitivity did not statistically differ (94.5% vs. 94.4%). Conclusion The model trained and evaluated on 24-hour data outperformed the existing algorithm output in terms of overall accuracy, specificity, and balanced accuracy, while sensitivity did not significantly differ. A model trained on 24-hour data may be more appropriate for analyses of freely living people, or older populations where napping is more common. Developing an accurate 24-hour sleep/wake classifier fosters new opportunities to evaluate sleep patterns in the absence of self-reports or assumptions about time in bed. Support (If Any) UL1TR002014, NSF#1622766, R43/44-AG056250

Boron is an essential plant micronutrient that plays a structural role in the rhamnogalacturonan II component of the pectic cell wall. To prevent boron deficiency under limiting conditions, its uptake, distribution, and homeostasis are mediated by boric acid transporters and channel proteins. Among the membrane channels that facilitate boric acid uptake are the type II nodulin intrinsic protein (NIP) subfamily of aquaporin-like proteins. Arabidopsis (Arabidopsis thaliana) possesses three NIP II genes (NIP5;1, NIP6;1, and NIP7;1) that show distinct tissue expression profiles (predominantly expressed in roots, stem nodes, and developing flowers, respectively). Orthologs of each are represented in all dicots. Here, we show that purified and reconstituted NIP7;1 is a boric acid facilitator. By using native promoter-reporter fusions, we show that NIP7;1 is expressed predominantly in anthers of young flowers in a narrow developmental window, floral stages 9 and 10, with protein accumulation solely within tapetum cells, where it is localized to the plasma membrane. Under limiting boric acid conditions, loss-of-function T-DNA mutants (nip7;1-1 and nip7;1-2) show reduced fertility, including shorter siliques and an increase in aborted seeds, compared with the wild type. Under these conditions, nip7;1 mutant pollen grains show morphological defects, increased aggregation, defective exine cell wall formation, reduced germination frequency, and decreased viability. During stages 9 and 10, the tapetum is essential for supplying materials to the pollen microspore cell wall. We propose that NIP7;1 serves as a gated boric acid channel in developing anthers that aids in the uptake of this critical micronutrient by tapetal cells.

Mind wandering is a poorly understood phenomenon that can undermine driving safety. Driving performance measures have been found to be associated with mind wandering (e.g., steering wheel movements, standard deviation of lateral position, and speed variation). However, no one measure can fully describe the driver behavior associated with mind wandering. Therefore, in this paper we explore the effect of mind wandering on nine steering measures with data collected from a study that included nine drivers over two sessions of driving over five days. Participants were periodically probed to report their attentional state–whether they were mind wandering or focusing on the task. We used two dimensionality-reduction techniques—Principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE)—to visualize the dimensions underlying the nine measures. Comparing PCA to t-SNE highlights the benefits of t-SNE in revealing the fine structure that differentiates driving behavior. These visualizations show that a) driver engagement increased during roadway curve segments, and b) mind wandering manifests itself through several types of steering behavior.

Iron (Fe) is an essential micronutrient for symbiotic nitrogen fixation in legume nodules, where it is required for the activity of bacterial nitrogenase, plant leghemoglobin, respiratory oxidases, and other Fe proteins in both organisms. Fe solubility and transport within and between plant tissues is facilitated by organic chelators, such as nicotianamine and citrate. We have characterized a nodule-specific citrate transporter of the multidrug and toxic compound extrusion family, MtMATE67 of Medicago truncatula The MtMATE67 gene was induced early during nodule development and expressed primarily in the invasion zone of mature nodules. The MtMATE67 protein was localized to the plasma membrane of nodule cells and also the symbiosome membrane surrounding bacteroids in infected cells. In oocytes, MtMATE67 transported citrate out of cells in an Fe-activated manner. Loss of MtMATE67 gene function resulted in accumulation of Fe in the apoplasm of nodule cells and a substantial decrease in symbiotic nitrogen fixation and plant growth. Taken together, the results point to a primary role of MtMATE67 in citrate efflux from nodule cells in response to an Fe signal. This efflux is necessary to ensure Fe(III) solubility and mobility in the apoplasm and uptake into nodule cells. Likewise, MtMATE67-mediated citrate transport into the symbiosome space would increase the solubility and availability of Fe(III) for rhizobial bacteroids.

There is extensive evidence that the contralateral delay activity (CDA), a scalp recorded event-related brain potential, provides a reliable index of the number of objects held in visual working memory. Here we present evidence that the CDA not only indexes visual object working memory, but also the number of locations held in spatial working memory. In addition, we demonstrate that the CDA can be predictably modulated by the type of encoding strategy employed. When individual locations were held in working memory, the pattern of CDA modulation mimicked previous findings for visual object working memory. Specifically, CDA amplitude increased monotonically until working memory capacity was reached. However, when participants were instructed to group individual locations to form a constellation, the CDA was prolonged and reached an asymptote at two locations. This result provides neural evidence for the formation of a unitary representation of multiple spatial locations.

As semiautonomous driving systems are becoming prevalent in late model vehicles, it is important to understand how such systems affect driver attention. This study investigated whether measures from low-cost devices monitoring peripheral physiological state were comparable to standard EEG in predicting lapses in attention to system failures. Twenty-five participants were equipped with a low-fidelity eye-tracker and heart rate monitor and with a high-fidelity NuAmps 32-channel quick-gel EEG system and asked to detect the presence of potential system failure while engaged in a fully autonomous lane changing driving task. To encourage participant attention to the road and to assess engagement in the lane changing task, participants were required to: (a) answer questions about that task; and (b) keep a running count of the type and number of billboards presented throughout the driving task. Linear mixed effects analyses were conducted to model the latency of responses reaction time (RT) to automation signals using the physiological metrics and time period. Alpha-band activity at the midline parietal region in conjunction with heart rate variability (HRV) was important in modeling RT over time. Results suggest that current low-fidelity technologies are not sensitive enough by themselves to reliably model RT to critical signals. However, that HRV interacted with EEG to significantly model RT points to the importance of further developing heart rate metrics for use in environments where it is not practical to use EEG.

Empirical evidence indicates that detecting one's own mistakes can serve as a signal to improve task performance. However, little work has focused on how task constraints, such as the response–stimulus interval (RSI), influence post-error adjustments. In the present study, event-related potential (ERP) and behavioral measures were used to investigate the time course of error-related processing while humans performed a difficult visual discrimination task. We found that error commission resulted in a marked reduction in both task performance and sensory processing on the following trial when RSIs were short, but that such impairments were not detectable at longer RSIs. Critically, diminished sensory processing at short RSIs, indexed by the stimulus-evoked P1 component, was predicted by an ERP measure of error processing, the Pe component. A control analysis ruled out a general lapse in attention or mind wandering as being predictive of subsequent reductions in sensory processing; instead, the data suggest that error detection causes an attentional bottleneck, which can diminish sensory processing on subsequent trials that occur in short succession. The findings demonstrate that the neural system dedicated to monitoring and improving behavior can, paradoxically, at times be the source of performance failures.SIGNIFICANCE STATEMENTThe performance-monitoring system is a network of brain regions dedicated to monitoring behavior to adjust task performance when necessary. Previous research has demonstrated that activation of the performance monitoring system following incorrect decisions serves to improve future task performance. However, the present study provides evidence that, when perceptual decisions must be made rapidly (within approximately half a second of each other), activation of the performance-monitoring system is predictive of impaired task-related attention on the subsequent trial. The data illustrate that the cognitive demands imposed by error processing can interfere with, rather than enhance, task-related attention when subsequent decisions need to be made quickly.

Objective: The goal for this study was to develop an English translation of the Attention-Related Driving Errors Scale (ARDES-US) and to determine its potential relationship with driver history and other demographic variables. Background: Individual differences in performance on vigilance and cognitive tasks are well documented, but less is known about susceptibility to attention-related errors while driving. The ARDES has been developed and administered in both Spanish and Chinese but to our knowledge has never been administered or examined in an English-speaking population. Method: Two hundred ninety-six English-speaking individuals completed a series of self-report measures, including the ARDES-US, Attention-Related Cognitive Errors Scale, Mindful Attention Awareness Scale, and Cognitive Failures Questionnaire. Results: A confirmatory factor analysis using maximum-likelihood estimates with robust standard errors revealed results largely consistent with previous versions of the ARDES, namely, the ARDES-Spain and ARDES-Argentina. Additionally, a number of new results emerged. Specifically, women, drivers who received traffic tickets within the previous 2 years, and those with a lower level of education all had a greater propensity toward self-reported driver inattention as measured by the ARDES-US. Further analyses revealed that these findings were independent of age, years of driving experience, and driving frequency. Conclusion: These results suggest that the ARDES-US is a valid and reliable measure of driver inattention with an English-speaking American sample. Application: Potential applications of the ARDES-US include identifying individuals who are at greater risk of attention-related errors while driving and suggesting individually tailored training and safety countermeasures.

Picloram is an auxinic herbicide that is widely used for controlling broad leaf weeds. However, its mechanism of transport into plants is poorly understood. In a genetic screen for picloram resistance, we identified three Arabidopsis mutant alleles of PIC30 (PICLORAM RESISTANT30) that are specifically resistant to picolinates, but not to other auxins. PIC30 is a previously uncharacterized gene that encodes a major facilitator superfamily (MFS) transporter. Similar to most members of MFS, PIC30 contains 12 putative transmembrane domains, and PIC30-GFP fusion protein selectively localizes to the plasma membrane. In planta transport assays demonstrate that PIC30 specifically transports picloram, but not indole-3-acetic acid (IAA). Functional analysis of Xenopus laevis oocytes injected with PIC30 cRNA demonstrated PIC30 mediated transport of picloram and several anions, including nitrate and chloride. Consistent with these roles of PIC30, three allelic pic30 mutants are selectively insensitive to picolinate herbicides, while pic30-3 is also defective in chlorate (analogue of nitrate) transport and also shows reduced uptake of 15NO3- . Overexpression of PIC30 fully complements both picloram and chlorate insensitive phenotypes of pic30-3. Despite the continued use of picloram as an herbicide, a transporter for picloram was not known until now. This work provides insight into the mechanisms of plant resistance to picolinate herbicides and also shed light on the possible endogenous function of PIC30 protein.

Empirical research demonstrates that when the time following error commission is constrained, subsequent sensory processing can be impaired (Buzzell et al., 2017). This reduction in sensory processing is presumably due to a bottleneck for cognitive resources produced by an overlap between error processing and subsequent stimulus processing. This finding suggests that the system dedicated to improving task performance can actually sometimes be the source of performance failures. Although this finding established that data-limited errors lead to a reduction in sensory processing at short response stimulus intervals (RSIs), it remains unclear if the relationship between error processing and subsequent sensory processing can be modulated by speeded-response errors. In the present study, event-related potentials and behavioral measures were recorded while participants performed a modified version of a Simon task, in which RSI duration was varied. We found that sensory processing, indexed by the P1 component, was reduced following errors at short (200–533 ms), but not long (866–1200 ms), RSIs. Moreover, the magnitude of error processing differentially influenced subsequent sensory processing as a function of RSI. However, whereas prior work demonstrated that the error positivity (Pe) modulated sensory processing on the subsequent trial, only the error-related negativity (ERN) did so within the Simon task. This suggests that although both data-limited errors and speeded-response errors can impact subsequent sensory processing, different stages of error processing appear to mediate this phenomenon.

During waterlogging and the associated oxygen deprivation stress, plants respond by the induction of adaptive programs, including the redirected expression of gene networks toward the synthesis of core hypoxia-response proteins. Among these core response proteins in Arabidopsis (Arabidopsis thaliana) is the calcium sensor CML38, a protein related to regulator of gene silencing calmodulin-like proteins (rgsCaMs). CML38 transcripts are up-regulated more than 300-fold in roots within 6 h of hypoxia treatment. Transfer DNA insertional mutants of CML38 show an enhanced sensitivity to hypoxia stress, with lowered survival and more severe inhibition of root and shoot growth. By using yellow fluorescent protein (YFP) translational fusions, CML38 protein was found to be localized to cytosolic granule structures similar in morphology to hypoxia-induced stress granules. Immunoprecipitation of CML38 from the roots of hypoxia-challenged transgenic plants harboring CML38pro::CML38:YFP followed by liquid chromatography-tandem mass spectrometry analysis revealed the presence of protein targets associated with messenger RNA ribonucleoprotein (mRNP) complexes including stress granules, which are known to accumulate as messenger RNA storage and triage centers during hypoxia. This finding is further supported by the colocalization of CML38 with the mRNP stress granule marker RNA Binding Protein 47 (RBP47) upon cotransfection of Nicotiana benthamiana leaves. Ruthenium Red treatment results in the loss of CML38 signal in cytosolic granules, suggesting that calcium is necessary for stress granule association. These results confirm that CML38 is a core hypoxia response calcium sensor protein and suggest that it serves as a potential calcium signaling target within stress granules and other mRNPs that accumulate during flooding stress responses.

Recent neuroimaging work has demonstrated that the ventral striatum (VS) encodes confidence in perceptual decisions. However, it remains unclear whether perceptual uncertainty can signal the need to adapt behavior (such as by responding more cautiously) and whether such behavioral changes are related to uncertainty-dependent activity within the VS. Changes in response strategy have previously been observed following errors and are associated with both medial frontal cortex (MFC) and VS, two components of the performance-monitoring network. If uncertainty can elicit changes in response strategy (slowing), then one might hypothesize that these changes rely on the performance-monitoring network. In the present study, we investigated the link between perceptual uncertainty and task-related behavioral adaptations (response slowing and accuracy increases), as well as how such behavioral changes relate to uncertainty-dependent activity within MFC and VS. Our participants performed a two-choice perceptual decision-making task in which perceptual uncertainty was reported on each trial while behavioral and event-related functional magnetic resonance imaging data were collected. Analysis of the behavioral data revealed that uncertain (but correct) responses led to slowing on subsequent trials, a phenomenon that was positively correlated with increased accuracy. Critically, post-uncertainty slowing was negatively correlated with the VS activity elicited by uncertain responses. In agreement with previous reports, increases in MFC activation were observed for uncertain responses, although MFC activity was not correlated with post-uncertainty slowing. These results suggest that perceptual uncertainty can serve as a signal to adapt one's response strategy and that such behavioral changes are closely tied to the VS, a key node in the performance-monitoring network.

National examinations are the principle method for tracking school quality and selecting students for successive educational levels in Tanzania. A qualitative approach is used to investigate the effects of high-stakes testing at two government primary schools with high passing rates in rural northern Tanzania. Extensive interviews and observations reveal that teachers’ interactions with students including their instruction and management strategies are compromised by national exam preparation. In follow-up interviews, the majority of participants desired changes in the content and structure of national exams and the teaching methods used to prepare students for exams.

Access to primary and secondary education in Tanzania has drastically expanded in the past two decades. In response to this success, its ministry is now targeting improvements in educational quality through additional reform. Yet teacher absenteeism, physical abuse, overcrowding, lecture-based pedagogy and a system of accountability based primarily on state and district examinations centered on rote memorization impose great obstacles to students being prepared for future employment and further education. This study draws insights into how traditional teaching and learning methods in one rural village in Tanzania can be changed through examining a collaboratively created intensive after-school program that focused on three content areas and used a participatory action research approach centered on cooperative inquiry. The key elements of the participatory action research approach drawn upon were using a participatory model to create a community of co-learners, designing the curriculum collectively, studen...

An experiment utilizing an auditory-spatial Stroop paradigm was created to assess whether participants are better able to attend to spatial or semantic information across near and far regions of space. Participants were instructed to attend to either the semantic information of a stimulus or identify the location of where the stimulus came from, depending on the condition. The sounds came from speakers that were physically located in either near space (peripersonal region of space) or far space (extrapersonal region of space) and the words were either “near” or “far.” Results indicate that participants in general were quicker at responding to the semantic condition than the location condition. Furthermore, consistent with findings of many other Stroop-like experiments, there was a significant difference between congruent and incongruent trials in both task conditions. The results of this investigation provide additional insight into how people process different types of information across near and far regions of space.

For the FAA’s Next Generation Air Transportation System (NextGen), the ability to reliably measure the effects of automation changes or new task demands on controller/pilot workload and performance is critical. Much of the EEG research has focused on identifying specific cognitive states associated with levels of workload during post processing. However, there are potential benefits for researchers to having EEG results as a continuous measure that can be observed during simulation experiments. This demonstration is a workload gauge that processes EEG data in real time as the Air Traffic Controller (ATC) participant is performing basic cognitive and operational ATC tasks. The visualizations demonstrated are also available during post processing to enable investigation of the continuous relationships between ATC situational variables, controller performance, and EEG.

Participants drove a simulated vehicle while completing an n-back task of high or low cognitive demand. Concurrently, they responded via brake press to signals presented in either a single modality (auditory, visual or tactile) or a bimodal combination. Participants were asked to indicate the perceived urgency of each signal by adjusting the force applied in their brake response. Signals were designed to be of “high” or “low” urgency based on previous research for both unimodal and bimodal combinations. Participants were capable of subjectively judging perceived urgency independent of response time and responded to bimodal (relative to unimodal) and high (relative to low) urgency stimuli with significantly greater brake force. However, both factors interacted with cognitive demand indicating that the magnitude of these differential responses was reduced under high demand. Implications for warning evaluation in an in-vehicle driving context are discussed.

Nodulin intrinsic proteins (NIPs) represent a land plant-specific subfamily of the major intrinsic protein/aquaporin superfamily. NIPs are named for the first member of the family discovered, soybean nodulin 26 of symbiotic nitrogen-fixing root nodules. Evolutionarily, NIPs appear in early nonvascular and vascular land plant lineages, with the family undergoing substantial diversification and sub-functionalization during subsequent evolution of seed plants. Structurally, most NIPs can be divided into three “pore” families based on the composition of amino acids comprising the predicted aromatic-arginine selectivity region of the channel pore. Functionally, two of these families (NIP II and NIP III) serve as channels for metalloid nutrients (boric acid and silicic acid respectively), while the biological role of NIP I channels remains more open. Biochemical functions for NIP proteins are diverse, with transport selectivities ranging from metalloid hydroxides to glycerol, lactic acid, urea, and hydrogen peroxide. Some NIPs retain their aquaporin function, while others have lost this signature activity of the aquaporin family. In the present chapter, the evolutionary origins, structural and functional properties, and potential biological functions, particularly beyond their roles as metalloid facilitators, are reviewed.

Complex in-vehicle technology and safety systems are finding their way into many cars on the road today. These systems require alerts and warnings that appropriately convey multiple levels of urgency, but if these are deemed excessively annoying, then their implementation may be of little consequence. In this study we used a well-documented psychophysical approach to identify the relationship between specific auditory parameters, perceived urgency and perceived annoyance. In agreement with existing literature, increases in all parameters led to increases in both urgency and annoyance - although differentially. Of the parameters investigated, only pulse rate exhibited a stronger psychophysical relationship with urgency than annoyance. The tradeoff between urgency and annoyance is of practical concern and results from this study provide a potential guideline to determine the viability of future in vehicle alerts based on this relationship.

Tactile vibrations are potentially useful in a variety of environments to communicate information to visually and auditorily overloaded people. However, since vibrotactile signals must come into physical contact with the skin, they may also be perceived as highly urgent and annoying. The current study examined whether scalable levels of perceived urgency could be obtained with tactile signals by measuring the relationship between changes in vibrotactile pulse rate and ratings of urgency and annoyance. In two separate experiments, changes in pulse rate resulted in changes in ratings of perceived urgency with faster pulse rates being perceived as more urgent. Importantly, in both studies pulse rate had a greater impact on perceived urgency than it did on annoyance suggesting that scalable levels of urgency can be achieved without similarly annoying operators. Results are discussed in terms of their implications for multimodal display design.

The occipital-temporal N1 component of the event-related potential (ERP) has previously been shown to index a stimulus discrimination process. However, the N1 has not consistently been shown to be sensitive to the difficulty of stimulus discrimination. Here, we manipulated the difficulty of stimulus discrimination by modulating the similarity between serially presented targets and nontargets. The same target stimulus was employed in both easy and difficult discrimination contexts, and these physically identical target stimuli elicited a larger N1 and smaller P3b in the difficult task context. Moreover, when targets were incorrectly categorized, N1 amplitude was diminished and a P3b was not elicited. These findings provide evidence that the N1 component reflects a sensory discrimination process that is modulated by executive control, and that this component can index discrimination errors when stimulus discrimination is difficult.

Age-related difficulties in speech processing remain a concern, especially as technology continues to depend heavily on successful speech comprehension on the part of users. Event-related potentials (ERPs) have frequently been used to assess age-related changes in the processing of language. Specifically, the amplitude of the ERP is often compared between conditions or groups of interest. In constructing ERPs, many neurophysiologic responses are averaged together to reduce the contribution of uncorrelated background activity in the electroencephalogram (EEG). However, if variability in the timing of each potential on a trial-by-trial basis (i.e., “latency jitter”) is confounded with a variable of interest, then the presence of amplitude differences observed in the average ERP might not be solely the result of genuine amplitude differences, but also timing variability. We examined the role latency jitter may play in the well-established observation of age-related changes in the processing of natural speech...

Plant nodulin-26 intrinsic proteins (NIPs) are members of the aquaporin superfamily that serve as multifunctional transporters of uncharged metabolites. In Arabidopsis thaliana, a specific NIP pore subclass, known as the NIP II proteins, is represented by AtNIP5;1 and AtNIP6;1, which encode channel proteins expressed in roots and leaf nodes, respectively, that participate in the transport of the critical cell wall nutrient boric acid. Modeling of the protein encoded by the AtNIP7;1 gene shows that it is a third member of the NIP II pore subclass in Arabidopsis. However, unlike AtNIP5;1 and AtNIP6;1 proteins, which form constitutive boric acid channels, AtNIP7;1 forms a channel with an extremely low intrinsic boric acid transport activity. Molecular modeling and molecular dynamics simulations of AtNIP7;1 suggest that a conserved tyrosine residue (Tyr81) located in transmembrane helix 2 adjacent to the aromatic arginine (ar/R) pore selectivity region stabilizes a closed pore conformation through interaction with the canonical Arg220 in ar/R region. Substitution of Tyr81 with a Cys residue, characteristic of established NIP boric acid channels, results in opening of the AtNIP7;1 pore that acquires a robust, transport activity for boric acid as well as other NIP II test solutes (glycerol and urea). Substitution of a Phe for Tyr81 also opens the channel, supporting the prediction from MD simulations that hydrogen bond interaction between the Tyr81 phenol group and the ar/R Arg may contribute to the stabilization of a closed pore state. Expression analyses show that AtNIP7;1 is selectively expressed in developing anther tissues of young floral buds of A. thaliana, principally in developing pollen grains of stage 9-11 anthers. Because boric acid is both an essential nutrient as well as a toxic compound at high concentrations, it is proposed that Tyr81 modulates transport and may provide an additional level of regulation for this transporter in male gametophyte development.

Electroencephalography (EEG) has the prospect of providing a means to gauge operator workload in a manner that does not intrude on the task being performed. Specifically, it has been proposed that the technique could be used as a method to speed the learning of a task, by adjusting the task to suit the state of the learner. The present study recorded EEG while participants performed a simulated Unmanned Aerial Vehicle (UAV) reconnaissance task. Analysis of power in three EEG frequency bands of interest found differences between the types of task being performed; however more complex analysis may be necessary to discern levels of difficulty within the task.

Nodulin 26 (nod26) is a major intrinsic protein that constitutes the major protein component on the symbiosome membrane (SM) of N(2)-fixing soybean nodules. Functionally, nod26 forms a low energy transport pathway for water, osmolytes, and NH(3) across the SM. Besides their transport functions, emerging evidence suggests that high concentrations of major intrinsic proteins on membranes provide interaction and docking targets for various cytosolic proteins. Here it is shown that the C-terminal domain peptide of nod26 interacts with a 40-kDa protein from soybean nodule extracts, which was identified as soybean cytosolic glutamine synthetase GS(1)beta1 by mass spectrometry. Fluorescence spectroscopy assays show that recombinant soybean GS(1)beta1 binds the nod26 C-terminal domain with a 1:1 stoichiometry (K(d) = 266 nm). GS(1)beta1 also binds to isolated SMs, and this binding can be blocked by preincubation with the C-terminal peptide of nod26. In vivo experiments using either a split ubiquitin yeast two-hybrid system or bimolecular fluorescence complementation show that the four cytosolic GS isoforms expressed in soybean nodules interact with full-length nod26. The binding of GS, the principal ammonia assimilatory enzyme, to the conserved C-terminal domain of nod26, a transporter of NH(3), is proposed to promote efficient assimilation of fixed nitrogen, as well as prevent potential ammonia toxicity, by localizing the enzyme to the cytosolic side of the symbiosome membrane.

Boron (B) in soil is taken up by roots through NIP5;1, a boric acid channel, and is loaded into the xylem by BOR1, a borate exporter. Here, the function of Arabidopsis thaliana NIP6;1, the most similar gene to NIP5;1, was studied. NIP6;1 facilitates the rapid permeation of boric acid across the membrane but is completely impermeable to water. NIP6;1 transcript accumulation is elevated in response to B deprivation in shoots but not in roots. NIP6;1 promoter–β-glucuronidase is predominantly expressed in nodal regions of shoots, especially the phloem region of vascular tissues. Three independently identified T-DNA insertion lines for the NIP6;1 gene exhibited reduced expansion of young rosette leaves only under low-B conditions. B concentrations are reduced in young rosette leaves but not in the old leaves of these mutants. Taken together, these data strongly suggest that NIP6;1 is a boric acid channel required for proper distribution of boric acid, particularly among young developing shoot tissues. We propose that NIP6;1 is involved in xylem–phloem transfer of boric acid at the nodal regions and that the water-tight property of NIP6;1 is important for this function. It is proposed that during evolution, NIP5;1 and NIP6;1 were diversified in terms of both the specificity of their expression in plant tissues and their water permeation properties, while maintaining their ability to be induced under low B and their boric acid transport activities.

There is increasing interest in real-time brain-computer interfaces (BCIs) for passive monitoring of human cognitive state, including cognitive workload. Too often, however, effective BCIs based on machine learning may function as “black boxes” that are difficult to analyze or interpret. In an effort toward more interpretable BCIs, we studied a family of N-back working memory tasks using a machine learning model, Gaussian Process Regression (GPR), that was both powerful and amenable to analysis. Participants performed the N-back task with three stimulus variants, auditory-verbal, visual-spatial, and visual-numeric, each at three working memory loads. GPR models were trained and tested on data from all three task variants combined, in an effort to identify a model that could be predictive of mental workload demand regardless of stimulus modality. To provide a comparison for GPR performance, a model was additionally trained using multiple linear regression (MLR).The model was effective when trained on individual human participant data, resulting in an average standardized mean squared error (SMSE) between true and predicted n-back levels of 0.44. In comparison, the MLR model using the same data resulted in an average SMSE of 0.55. We demonstrate how GPR can be used to identify which EEG features are highly influential in predictions of cognitive workload in an individual participant. A fraction of EEG features account for the predictive power, and only the top 25% of features were sufficient for near for maximum accuracy. Subsets of features identified by linear models (ANOVA) were not as efficient as subsets identified by GPR. Similarly, multiple linear regression was less effective than GPR for accurately predicting task load. This raises the possibility of real-time BCIs that are simpler (requiring fewer model features) but that still capture all the neurophysiological information needed to achieve high predictive accuracy. This study sought to establish effectiveness and interpretability for a rapidly trainable, real-time, passive BCI for cognitive monitoring in individuals, without regard for specific theories of neural function. Potential valuable applications beyond the scope of the present study include characterizing the neural basis of workloading or other cognitive states, in individual participants and across groups of human participants.

Objective: Most youth in Sub-Saharan Africa are not enrolled in school. Although more youth are enrolling inschool, they are increasingly less likely to complete their primary and secondary schooling. Similarly to many otherAfrican nations, Tanzania has an estimated out-of-school population of 49% of youth despite massivepost-millennium investments which have significantly raised enrollment rates. Due to a high unemployment rate,rural youth commonly opt to pursue employment in cities or carry out agricultural work at home. However, theirability to procure formal employment is hindered by competition due to a high youth population density. This studysought to understand how youth with different levels of formal educational attainment (no schooling, primaryschooling, secondary schooling) learn information in their community and formal schooling to improve theirlivelihoods and develop sustainable practices for generating income.Methods: Through semi-structured qualitative interviews and focus group discussions, the study examinesdifferences between the groups in what they seek to understand and the different pathways they use to learn.Results: Although there were positive effects of schooling among the participants in this study in terms of boostingtheir self-confidence and self-esteem, youth dropouts with schooling experience overwhelmingly reported that theydid not learn approaches and information in their schools that were beneficial for improving their livelihoods.Conclusions: Since the majority of Tanzanian students, and particularly those from rural areas, will be unable tostudy in higher education institutions, educational programs for youth in school and for school dropouts are moreeffective which center on methods for learning new information that are applicable to their intended occupationwithin their community and which also prepare them for participating in the workforce.

We are currently investigating factors that influence intracellular ice formation (IIF) in mouse oocytes and oocytes of the frog Xenopus. A major reason for choosing these two species is that while their eggs normally do not possess aquaporin channels in their plasma membranes, these channels can be made to express. We wish to see whether IIF is affected by the presence of these channels. The present Xenopus study deals with control eggs not expressing aquaporins. The main factor studied has been the effect of a cryoprotective agent [ethylene glycol (EG) or glycerol] and its concentration. The general procedure was to (a) cool the oocytes on a cryostage to slightly below the temperatures at which extracellular ice formation occurs, (b) warm them to just below the melting point, and (c) then re-cool them to -50 degrees C at 10 degrees C/min. In the majority of cases, IIF occurs well into step (c), but a sizeable minority undergo IIF in steps (a) or (b). The former group we refer to as low-temperature flashers; the latter as high-temperature flashers. IIF is manifested as abrupt blackening of the egg, which we refer to as "flashing." Observations on the Linkam cryostage are restricted to Stage I and II oocytes, which have diameters of 200 300 microm. In the absence of a cryoprotective agent, that is in frog Ringers, the mean flash temperature for the low-temperature freezers is -11.4 degrees C, although a sizeable percentage flash at temperatures much closer to that of the EIF (-3.9 degrees C). When EG is present, the flash temperature for the low-temperatures freezers drops significantly to approximately -20 degrees C for EG concentrations ranging from 0.5 to 1.5 M. The presence of 1.5 M glycerol also substantially reduces the IIF temperature of the low-temperature freezers; namely, to -29 degrees C, but 0.5 and 1 M glycerol exert little or no effect. The IIF temperatures observed using the Linkam cryostage agree well with those estimated by calorimetry [F.W. Kleinhans, J.F. Guenther, D.M. Roberts, P. Mazur, Analysis of intracellular ice nucleation in Xenopus oocytes by differential scanning calorimetry, Cryobiology 52 (2006) 128-138]. The IIF temperatures in Xenopus are substantially higher than those observed in mouse oocytes [P. Mazur, S. Seki, I.L. Pinn, F.W. Kleinhans, K. Edashige, Extra- and intracellular ice formation in mouse oocytes, Cryobiology 51 (2005) 29-53]. Perhaps that is a reflection of their much larger size.

Intracellular ice formation (IIF) plays a central role in cell damage during cryopreservation. We are investigating the factors which trigger IIF in Xenopus oocytes, with and without aquaporin water channels. Here, we report differential scanning calorimeter studies of Xenopus control oocytes which do not express aquaporins. Stage I to VI oocytes (which increase progressively in size) were investigated with emphasis on stage I and II because they are translucent and can also be studied under the cryomicroscope. Measurements were made in 1, 1.5, and 2M ethylene glycol (EG) in frog Ringers plus SnoMax. A multistep freezing protocol was used in which the samples were cooled until extracellular ice formation (EIF) occurred, partially remelted, slowly recooled through the EIF temperature, and then rapidly (10 degrees C/min) cooled. EIF in the 1, 1.5, and 2M EG occurred at -6.4, -7.8, and -8.9 degrees C, respectively. Freezing exotherms of individual stage I-VI oocytes were readily visible. A general trend was observed in which the IIF temperature of the early stage oocytes (I-III) was well below T(EIF) while the later stages (IV-VI) froze at temperatures much closer to T(EIF). Thus, in 1.5M EG, T(IIF) was -21.1, -25, and -26.6 degrees C in stages I-III, but was -17 and -8.5 degrees C for stage IV and V-VI. Concurrently, the percentage of oocytes in which IIF was observed fell dramatically from a high of 40 to 72% in early stages (I-III) to a low of only 7% in stage V-VI because, particularly in the later stages, IIF was hidden in the EIF exotherm. We conclude that early stage oocytes are a good model system in which to investigate modulators of IIF, but that late stage oocytes are damaged during EIF and infrequently supercool.

Major intrinsic proteins (MIPs) are a diverse class of integral membrane proteins that facilitate the transport of water and some small solutes across cellular membranes. X-ray structures of MIPs indicate that a tetrad of residues (the ar/R region) form a narrow pore constriction that constitutes the selectivity filter. In comparison with mammalian and microbial species, plants have a greater number and diversity of MIPs with greater than 30 genes encoding four phylogenetic subfamilies with eight different classes of ar/R sequences. The nodulin 26-like intrinsic protein (NIP) subfamily in Arabidopsis can be subdivided into two ar/R subgroups: the NIP subgroup I, which resembles the archetype of the family, soybean nodulin 26, and the NIP subgroup II, which is represented by the Arabidopsis protein AtNIP6;1. These two NIPs differ principally by the substitution of a conserved alanine (NIP subgroup II) for a conserved tryptophan (NIP subgroup I) in the helix 2 position (H2) of the ar/R filter. A comparison of the water and solute tranport properties of the two proteins was performed by expression in Xenopus laevis oocytes. Nodulin 26 is an aquaglyceroporin with a modest osmotic water permeability (P(f)) and the ability to transport uncharged solutes such as glycerol and formamide. In constrast, AtNIP6;1 showed no measurable water permeability but transported glycerol, formamide, as well as larger solutes that were impermeable to nodulin 26. By site-directed mutagenesis, we show that the H2 position is the crucial determinant that confers these transport behaviors. A comparison of the NIPs and tonoplast-intrinsic proteins (TIP) shows that the H2 residue can predict the transport profile for water and glycerol with histidine found in TIP-like aquaporins, tryptophan found in aquaglyceroporins (NIP I), and alanine found in water-impermeable glyceroporins (AtNIP6;1).

A cDNA was isolated from soybean (Glycine max) nodules that encodes a putative transporter (GmN70) of the major facilitator superfamily. GmN70 is expressed predominantly in mature nitrogen-fixing root nodules. By western-blot and immunocytochemical analyses, GmN70 was localized to the symbiosome membrane of infected root nodule cells, suggesting a transport role in symbiosis. To investigate its transport function, cRNA encoding GmN70 was expressed in Xenopus laevis oocytes, and two-electrode voltage clamp analysis was performed. Ooctyes expressing GmN70 showed outward currents that are carried by anions with a selectivity of nitrate > nitrite ≫ chloride. These currents showed little sensitivity to pH or the nature of the counter cation in the oocyte bath solution. One-half maximal currents were induced by nitrate concentrations between 1 to 3 mm. No apparent transport of organic anions was observed. Voltage clamp records of an ortholog of GmN70 from Lotus japonicus (LjN70; K. Szczyglowski, P. Kapranov, D. Hamburger, F.J. de Bruijn [1998] Plant Mol Biol 37: 651–661) also showed anion currents with a similar selectivity profile. Overall, these findings suggest that GmN70 and LjN70 are inorganic anion transporters of the symbiosome membrane with enhanced preference for nitrate. These transport activities may aid in regulation of ion and membrane potential homeostasis, possibly in response to external nitrate concentrations that are known to regulate the symbiosis.

Major intrinsic proteins (MIPs) are a family of membrane channels that facilitate the bidirectional transport of water and small uncharged solutes such as glycerol. The 35 full-length members of the MIP family in Arabidopsis are segregated into four structurally homologous subfamilies: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin 26-like intrinsic membrane proteins (NIPs), and small basic intrinsic proteins (SIPs). Computational methods were used to construct structural models of the putative pore regions of various plant MIPs based on homology modeling with the atomic resolution crystal structures of mammalian aquaporin 1 and the bacterial glycerol permease GlpF. Based on comparisons of the narrow selectivity filter regions (the aromatic/Arg [ar/R] filter), the members of the four phylogenetic subfamilies of Arabidopsis MIPs can be classified into eight groups. PIPs possess a uniform ar/R signature characteristic of high water transport aquaporins, whereas TIPs are highly diverse with three separate conserved ar/R regions. NIPs possess two separate conserved ar/R regions, one that is similar to the archetype, soybean (Glycine max) nodulin 26, and another that is characteristic of Arabidopsis NIP6;1. The SIP subfamily possesses two ar/R subgroups, characteristic of either SIP1 or SIP2. Both SIP ar/R residues are divergent from all other MIPs in plants and other kingdoms. Overall, these findings suggest that higher plant MIPs have a common fold but show distinct differences in proposed pore apertures, potential to form hydrogen bonds with transported molecules, and amphiphilicity that likely results in divergent transport selectivities.

Soybean nodulin 26 is expressed and targeted to the symbiosome membrane of nitrogen-fixing nodules, where it forms an aquaporin channel with a modest water transport rate. In this study, we show that the phosphorylation of nodulin 26 on Ser-262, which is catalyzed by a symbiosome membrane–associated calcium-dependent protein kinase, stimulates its intrinsic water transport rate. Furthermore, using a phosphospecific antibody, we have elucidated the developmental appearance and regulation of nodulin 26 phosphorylation in vivo. Although nodulin 26 protein is detected first in differentiating infected cells (16 days), phosphorylated nodulin 26 does not become pronounced until infected cell maturation (25 days). Phosphorylation is sustained at steady state levels until entry into senescence. Nodulin 26 phosphorylation is enhanced further by osmotic stresses (water deprivation and salinity). Thus, the phosphorylation of nodulin 26 coincides with the establishment of mature nitrogen-fixing symbiosomes, is regulated by osmotic stresses that induce calcium-signaling pathways, and appears to be part of the adaptive responses of infected cells to osmotic challenge.

Calmodulin is trimethylated at lysine 115 by a highly specific methyltransferase that utilizes S-adenosylmethionine as a co-substrate. Lysine 115 is found within a highly conserved six-amino acid loop (LGEKLT) that forms a 90 degrees turn between EF-hand III and EF-hand IV in the carboxyl-terminal lobe. In the present work a mutagenesis approach was used to investigate the structural features of the carboxyl-terminal lobe that lead to the specificity of calmodulin methylation. Three structural regions within the carboxyl-terminal lobe appear to be involved in methyltransferase recognition: the highly conserved six-amino acid loop-turn region that contains lysine 115 as well as the adjacent alpha-helices (helix 6 and helix 7) from EF-hands III and IV. Site-directed mutagenesis of residues in the loop show that three residues, glycine 113, glutamate 114, and leucine 116 are essential for methylation. In addition, subdomain (individual helix or Ca(2+) binding loop) exchange mutants show that the substitutions of either helix 6 (EF-hand III) with helix 2 (EF-hand I) or helix 7 (EF-hand IV) with helix 3 (EF-hand II) compromises methylation. Charge-to-alanine mutations in helix 7 show that substitution of conserved charged residues at positions 118, 120, 122, 126, and 127 reduced lysine 115 methylation rates, suggesting possible electrostatic interactions between this helix and the methyltransferase. Single substitutions in helix 6 did not affect calmodulin methylation, suggesting this region may play a more indirect role in stabilizing the conformation of the methyltransferase recognition sequence.

By using reverse transcriptase-polymerase chain reaction, two cDNAs were isolated that encode major intrinsic membrane proteins (MIPs) that are expressed in nitrogen-fixing root nodules of Lotus japonicus. Lotus intrinsic membrane protein 1 (LIMP 1) is expressed at high levels in both nodule and root tissues and shows highest sequence similarity to members of the tonoplast intrinsic protein (TIP) subfamily of plant MIPs. Functional analysis of LIMP 1 by expression in Xenopus laevis oocytes show that it is a water-specific aquaporin. In contrast, LIMP 2 shows the highest sequence similarity to soybean nodulin 26 (67.8% amino acid sequence identity). LIMP 2 is also a nodulin, showing expression only in mature nitrogen fixing nodules of L. japonicus. LIMP 2 is a multifunctional aquaglyceroporin, and displays the ability to flux both water as well as glycerol upon expression in Xenopus oocytes. Additionally, the carboxyl terminal region of LIMP 2 has a conserved phosphorylation motif that is phosphorylated by a calmodulin-like domain protein kinase. Overall, the data show that L. japonicus nodules contain two structurally and functionally distinct MIP proteins: one (LIMP 2) which appears to be the nodulin 26 ortholog of L. japonicus and another (LIMP 1) which appears to be a member of the TIP subfamily.

Sucrose synthase (SS) is a known phosphoserine-containing enzyme in legume root nodules and various other plant "sink" tissues. In order to begin to investigate the possible physiological significance of this posttranslational modification, we have cloned a full-length soybean nodule SS (nodulin-100) cDNA and overexpressed it in Escherichia coli. Authentic nodule SS and recombinant wild-type and mutant forms of the enzyme were purified and characterized. We document that a conserved serine near the N-terminus (Ser(11)) is the primary phosphorylation site for a nodule Ca(2+)-dependent protein kinase (CDPK) in vitro. Related tryptic digestion and mass spectral analyses indicated that this target residue was also phosphorylated in planta in authentic nodulin-100. In addition, a secondary phosphorylation site(s) in recombinant nodule SS was implicated given that all active mutant enzyme forms (S11A, S11D, S11C, and N-terminal truncation between Ala(2) and Arg(13)) were phosphorylated, albeit weakly, by the CDPK. This secondary site(s) likely resides between Glu(14) and Met(193) as evidenced by CNBr cleavage and phosphopeptide mapping. Phosphorylation of the recombinant and authentic nodule Ser(11) enzymes in vitro by the nodule CDPK had no major effect on the sucrose-cleavage activity and/or kinetic properties. However, phosphorylation decreased the apparent surface hydrophobicity of the recombinant wild-type enzyme, suggesting that this covalent modification could potentially play some role in the documented partitioning of nodulin-100 between the nodule symbiosome/plasma membranes and cytosol in planta.

Calmodulin is trimethylated by a specific methyltransferase on Lys115, a residue located in a six amino acid loop (LGEKLT) between EF hands III and IV. To investigate the structural requirements for methylation, domain exchange mutants as well as single point mutations of conserved methylation loop residues (E114A, Glu114 → Ala; L116T, Leu116 → Thr) were generated. E114A and L116T activated cyclic nucleotide phosphodiesterase (PDE) and NAD+ kinase (NADK) similar to wild-type calmodulin, but lost their ability to be methylated. Domain exchange mutants in which EF hand III or IV was replaced by EF hand I or II respectively (CaM1214 and CaM1232 respectively) showed a modest effect on PDE and NADK activation (50 to 100% of wild-type), but calmodulin methylation was abolished. A third domain exchange mutant, CaMEKL, has the methylation loop sequence placed at a symmetrical position between EF hands I and II in the N-terminal lobe [residues QNP(41-43) replaced by EKL]. CaMEKL activated PDE normally, but did not activate NADK. However, CaMEKL retained the ability to bind to NADK and inhibited activation by wild-type calmodulin. Site-directed mutagenesis of single residues showed that Gln41 and Pro43 substitutions had the strongest effect on NADK activation. Additionally, CaMEKL was not methylated, suggesting that the introduction of the methylation loop between EF hands I and II is not adequate for methyltransferase recognition. Overall the data indicate that residues in the methylation loop are essential but not sufficient for methyltransferase recognition, and that additional residues unique to EF hands III and IV are required. Secondly, the QNP sequence in the loop between EF hands I and II is necessary for NADK activation.

Transgenic tobacco (Nicotiana tabacum L. cv. Wisconsin 38) lines expressing a mutant calmodulin (VU-3) that hyperactivates NAD kinase exhibit an enhanced elicitor-stimulated oxidative-burst reaction (S.A. Harding et al., 1997, EMBO J. 16: 1137–1144). VU-3 transgenic tobacco was used in the present study to investigate the relationship between calmodulin signalling, the production of active oxygen species and cell death in response to infection with an incompatible pathogen. Following P. syringae pv. syringae 61 infection, suspension cells derived from VU-3 transgenic plants exhibited a stronger oxidative burst (3- to 4-fold higher primary and secondary burst reactions), greater media alkalinization (3-fold) and more rapid cell death (4-fold greater mortality at 20 h post infection) than did infected control tobacco cells. Infection of leaf tissues with P. syringae pv. syringae 61 also resulted in an enhanced cell death response compared to control tobacco tissues. This cell death response of VU-3 leaf tissues, but not control leaf tissues, was further enhanced by the presence of 50 μM salicylic acid, suggesting that this transgenic line is more sensitive to the effects of this agent. Overall, the data support the model that calmodulin signalling pathways are involved in the plant oxidative burst and contribute to the regulation of cell death in infected plant tissues undergoing the hypersensitive response.

Recent work has demonstrated that the occipital–temporal N1 component of the ERP is sensitive to the difficulty of visual discrimination, in a manner that cannot be explained by simple differences in low-level visual features, arousal, or time on task. These observations provide evidence that the occipital–temporal N1 component is modulated by the application of top–down control. However, the timing of this control process remains unclear. Previous work has demonstrated proactive, top–down modulation of cortical excitability for cued spatial attention or feature selection tasks. Here, the possibility that a similar top–down process facilitates performance of a difficult stimulus discrimination task is explored. Participants performed an oddball task at two levels of discrimination difficulty, with difficulty manipulated by modulating the similarity between target and nontarget stimuli. Discrimination processes and cortical excitability were assessed via the amplitude of the occipital–temporal N1 component and prestimulus alpha oscillation of the EEG, respectively. For correct discriminations, prestimulus alpha power was reduced, and the occipital–temporal N1 was enhanced in the hard relative to the easy condition. Furthermore, within the hard condition, prestimulus alpha power was reduced, and the occipital–temporal N1 was enhanced for correct relative to incorrect discriminations. The generation of ERPs contingent on relative prestimulus alpha power additionally suggests that diminished alpha power preceding stimulus onset is related to enhancement of the occipital–temporal N1. As in spatial attention, proactive control appears to enhance cortical excitability and facilitate discrimination performance in tasks requiring nonspatial, feature-based attention, even in the absence of competing stimulus features.