Back to nature: herbal treatment, environmental enrichment, and social play can protect against unpredictable chronic stress in Long-Evans rats (Rattus norvegicus).

The importance of integrative biobehavioral responses to complex challenges cannot be overlooked. In this study, the synergetic effects of icariin (a flavonoid present in the plant Epimedium brevicornum), natural enrichment (NaEn), and play behavior were investigated. Rats (n = 60) were assigned to standard housing or NaEn; these two groups were subsequently divided into controls, rats receiving icariin treatments, and rats receiving icariin and allowed to play with an individual from another cage. All rats were exposed to unpredictable mild stressors for 4 weeks. At the end of the treatment, a Forced Swim Task (FST) was conducted to assess emotional regulation during an inescapable acute challenge. Biological samples were collected weekly and before and after the FST to monitor endocrine changes. Corticosterone (CORT), dehydroepiandrosterone (DHEA), and testosterone (T) were assayed. We found that icariin had a significant effect on DHEA/CORT ratios and T levels. NaEn also had a significant effect on both CORT and DHEA, but not on T levels. Play did not appear to be significantly related to the endocrine changes. The strongest positive effects on emotional resilience were observed in NaEn rats that also received icariin. Our results confirmed that using multiple channels to stimulate adaptive responses can be effective in increasing the ability of an organism to face uncertainty. Considering how quickly our life can change due to unpredictable events, our data is instrumental to a better comprehension of the many aspects of integrative biobehavioral responses.

Story hour at a family homeless center: contribution to graduate students' preparation and confidence in preliteracy skills.

PURPOSE: This study evaluated the potential benefit to graduate students' of participating in a service-learning program conducting a storybook reading program for children in a family homeless shelter. METHOD: Ten graduate students in the second year of a two-year master's degree program in communication science and disorders participated in the storybook reading program. The graduate students engaged in reflective writing about their experiences and completed self-ratings of confidence in preliteracy skills before and after program participation. Twenty graduate students in two comparison groups (10 students in a pre-program comparison group, and 10 in a post-program comparison group) also completed questionnaires. The mixed-methods study used quantitative analyses to analyze questionnaire ratings and qualitative methods to analyze reflective writings. RESULTS: Together, the quantitative and qualitative results indicated positive outcomes from the service-learning experience with regard to graduate students' perceived confidence in preliteracy skills and preparation for careers as speech-language pathologists. The results provide empirical data showing that service-learning experiences with at-risk populations can contribute to graduate students' clinical education and preparation as speech-language pathologists. CONCLUSION: The results support the value of service-learning experiences in communication sciences and disorders. Clinical preparation in preliteracy development also supports the American Speech-Language-Hearing Association statement on the roles and responsibilities of speech-language pathologists in relation to reading and writing in children.

Equipping Learners to Evaluate Online Health Care Resources: Longitudinal Study of Learning Design Strategies in a Health Care Massive Open Online Course.

BACKGROUND: The digital revolution has led to a boom in the number of available online health care resources. To navigate these resources successfully, digital literacy education is required. Learners who can evaluate the reliability and validity of online health care information are likely to be more effective at avoiding potentially dangerous misinformation. In addition to providing health care education, massive open online courses (MOOCs) are well positioned to play a role in providing digital literacy education in this context. OBJECTIVE: This study focused on learners enrolled in a MOOC on cancer genomics. The aim of this study was to evaluate the efficacy of a series of digital literacy-related activities within this course. This was an iterative study, with changes made to digital literacy-related activities in 4 of the 8 runs of the course. METHODS: This mixed methods study focused on learner engagement with the digital literacy-related activities, including the final course written assignment. Quantitative data including the number of references listed in each written assignment were compared between successive runs. Qualitative data in the form of learner comments on discussion forums for digital literacy-related tasks were evaluated to determine the impact of these educational activities. RESULTS: Using the number of references included for each final course assignment as an indicator of digital literacy skills, the digital literacy-related activities in the final 2 runs were judged to be the most successful. We found a statistically significant increase in the number of references cited by learners in their final written assignments. The average number of references cited in Run 8 was significantly higher (3.5) than in Run 1 (1.8) of the MOOC (P=.001). Learner comments in Runs 7 and 8 showed that a poll in which learners were asked to select which of 4 online resources was reliable was effective in stimulating learner discussion about how to evaluate resource reliability. CONCLUSIONS: Similar to many health care MOOCs, the course studied here had a heterogeneous group of learners, including patients (and their families), the public, health care students, and practitioners. Carefully designing a range of digital literacy-related activities that would be beneficial to this heterogenous group of learners enabled learners to become more effective at evaluating and citing appropriate online resources within their written assignments.

Student-Created Online Teaching Resources for Students.

Students have long been creating their own visualisations of concepts taught through presentations, posters, figures in essays etc. However, with the introduction of technology, the content created by students is now easily shared with future cohorts of students. This chapter explores the history and advantages of student-created resources for students, then describes the examples of e-tutorials and MOOCs in more detail. Finally, future challenges are identified and discussed.

Phosphorylation of the Par-1 polarity kinase by protein kinase D regulates 14-3-3 binding and membrane association.

The Par-1 protein kinases are conserved from yeast to humans, where they function as key polarity determinants. The mammalian Par-1 family is comprised of 4 members (Par-1a, -b, -c, and -d). Previously, we demonstrated that atypical protein kinase C (aPKC) phosphorylates the Par-1 kinases on a conserved threonine residue (T595) to regulate localization and kinase activity. Here, we demonstrate that Par-1b is also regulated by another arm of the PKC pathway, one that involves novel PKCs (nPKC) and protein kinase D. Treatment of cells with the PKC activator phorbol-12-myristate-13-acetate (PMA) potently stimulated phosphorylation of Par-1b on serine 400 (S400), a residue that is conserved in all 4 mammalian Par-1 kinases as well as the fly ortholog. We demonstrate that PMA stimulates nPKC to activate PKD, which in turn directly phosphorylates Par-1b on S400 to positively regulate 14-3-3 binding and to negatively regulate membrane association. Thus, 2 arms of the PKC pathway regulate interactions between Par-1b and 14-3-3 proteins: one involving aPKC and the other nPKC/PKD.

Phosphorylation and 14-3-3 binding of Arabidopsis trehalose-phosphate synthase 5 in response to 2-deoxyglucose.

Trehalose-6-phosphate is a 'sugar signal' that regulates plant metabolism and development. The Arabidopsis genome encodes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphatase (TPP) enzymes. It also encodes class II proteins (TPS isoforms 5-11) that contain both TPS-like and TPP-like domains, although whether these have enzymatic activity is unknown. In this paper, we show that TPS5, 6 and 7 are phosphoproteins that bind to 14-3-3 proteins, by using 14-3-3 affinity chromatography, 14-3-3 overlay assays, and by co-immunoprecipitating TPS5 and 14-3-3 isoforms from cell extracts. GST-TPS5 bound to 14-3-3s after in vitro phosphorylation at Ser22 and Thr49 by either mammalian AMP-activated protein kinase (AMPK) or partially purified plant Snf1-related protein kinase 1 (SnRK1s). Dephosphorylation of TPS5, or mutation of either Ser22 or Thr49, abolished binding to 14-3-3s. Ser22 and Thr49 are both conserved in TPS5, 7, 9 and 10. When GST-TPS5 was expressed in human HEK293 cells, Thr49 was phosphorylated in response to 2-deoxyglucose or phenformin, stimuli that activate the AMPK via the upstream kinase LKB1. 2-deoxyglucose stimulated Thr49 phosphorylation of endogenous TPS5 in Arabidopsis cells, whereas phenformin did not. Moreover, extractable SnRK1 activity was increased in Arabidopsis cells in response to 2-deoxyglucose. The plant kinase was inactivated by dephosphorylation and reactivated by phosphorylation with human LKB1, indicating that elements of the SnRK1/AMPK pathway are conserved in Arabidopsis and human cells. We hypothesize that coordinated phosphorylation and 14-3-3 binding of nitrate reductase (NR), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (F2KP) and class II TPS isoforms mediate responses to signals that activate SnRK1.

A novel repressor domain is required for maximal growth inhibition by the IRF-1 tumor suppressor.

Interferon regulatory factor-1 (IRF-1) is a transcription factor and tumor suppressor that can regulate gene expression in a manner requiring either its sequence specific DNA binding activity or its ability to bind the p300 coactivator. We show that IRF-1-mediated growth inhibition is dependent on the integrity of a C-terminal transcriptional enhancer domain. An enhancer subdomain (amino acids 301-325) that differentially regulates IRF-1 activity has been identified and this region mediates the repression of Cdk2. The repressor domain encompasses an LXXLL coregulator signature motif and mutations or deletions within this region completely uncouple transcriptional activation from repression. The loss of growth suppressor activity when the Cdk2-repressor domain of IRF-1 is mutated implicates repression as a determinant of its maximal growth inhibitory potential. The data link IRF-1 regulatory domains to its growth inhibitory activity and provide information about how differential gene regulation may contribute to IRF-1 tumor suppressor activity.

Lack of PTEN sequesters CHK1 and initiates genetic instability.

Pten-/- cells display a partially defective checkpoint in response to ionizing radiation (IR). The checkpoint defect was traced to the ability of AKT to phosphorylate CHK1 at serine 280, since a nonphosphorylated mutant of CHK1 (S280A) complemented the checkpoint defect and restored CDC25A degradation. CHK1 phosphorylation at serine 280 led to covalent binding of 1 to 2 molecules of ubiquitin and cytoplasmic CHK1 localization. Primary breast carcinomas lacking PTEN expression and having elevated AKT phosphorylation had increased cytoplasmic CHK1 and displayed aneuploidy (p <0.005). We conclude that loss of PTEN and subsequent activation of AKT impair CHK1 through phosphorylation, ubiquitination, and reduced nuclear localization to promote genomic instability in tumor cells.

Comprehensive proteomic analysis of interphase and mitotic 14-3-3-binding proteins.

14-3-3 proteins regulate the cell division cycle and play a pivotal role in blocking cell cycle advancement after activation of the DNA replication and DNA damage checkpoints. Here we describe a global proteomics analysis to identify proteins that bind to 14-3-3s during interphase and mitosis. 14-3-3-binding proteins were purified from extracts of interphase and mitotic HeLa cells using specific peptide elution from 14-3-3 zeta affinity columns. Proteins that specifically bound and eluted from the affinity columns were identified by microcapillary high pressure liquid chromatography tandem mass spectrometry analysis. Several known and novel 14-3-3-interacting proteins were identified in this screen. Identified proteins are involved in cell cycle regulation, signaling, metabolism, protein synthesis, nucleic acid binding, chromatin structure, protein folding, proteolysis, nucleolar function, and nuclear transport as well as several other cellular processes. In some cases 14-3-3 binding was cell cycle-dependent, whereas in other cases the binding was shown to be cell cycle-independent. This study adds to the growing list of human 14-3-3-binding proteins and implicates a role for 14-3-3 proteins in a plethora of essential biological processes.

Phosphorylation and 14-3-3 binding of Arabidopsis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Fructose 2,6-bisphosphate (fru-2,6-P2) is a signalling metabolite that regulates photosynthetic carbon partitioning in plants. The content of fru-2,6-P2 in Arabidopsis leaves varied in response to photosynthetic activity with an abrupt decrease at the start of the photoperiod, gradual increase through the day, and modest decrease at the start of the dark period. In Arabidopsis suspension cells, fru-2,6-P2 content increased in response to an unknown signal upon transfer to fresh culture medium. This increase was blocked by either 2-deoxyglucose or the protein phosphatase inhibitor, calyculin A, and the effects of calyculin A were counteracted by the general protein kinase inhibitor K252a. The changes in fru-2,6-P2 at the start of dark period in leaves and in the cell experiments generally paralleled changes in nitrate reductase (NR) activity. NR is inhibited by protein phosphorylation and binding to 14-3-3 proteins, raising the question of whether fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase protein from Arabidopsis thaliana (AtF2KP), which both generates and hydrolyses fru-2,6-P2, is also regulated by phosphorylation and 14-3-3s. Consistent with this hypothesis, AtF2KP and NR from Arabidopsis cell extracts bound to a 14-3-3 column, and were eluted specifically by a synthetic 14-3-3-binding phosphopeptide (ARAApSAPA). 14-3-3s co-precipitated with recombinant glutathione S-transferase (GST)-AtF2KP that had been incubated with Arabidopsis cell extracts in the presence of Mg-ATP. 14-3-3s bound directly to GST-AtF2KP that had been phosphorylated on Ser220 (SLSASGpSFR) and Ser303 (RLVKSLpSASSF) by recombinant Arabidopsis calcium-dependent protein kinase isoform 3 (CPK3), or on Ser303 by rat liver mammalian AMP-activated protein kinase (AMPK; homologue of plant SNF-1 related protein kinases (SnRKs)) or an Arabidopsis cell extract. We have failed to find any direct effect of 14-3-3s on the F2KP activity in vitro to date. Nevertheless, our findings indicate the possibility that 14-3-3 binding to SnRK1-phosphorylated sites on NR and F2KP may regulate both nitrate assimilation and sucrose/starch partitioning in leaves.

Dual phosphorylation controls Cdc25 phosphatases and mitotic entry.

Negative regulation of the Cdc25C protein phosphatase by phosphorylation on Ser 216, the 14-3-3-binding site, is an important regulatory mechanism used by cells to block mitotic entry under normal conditions and after DNA damage. During mitosis, Cdc25C is not phosphorylated on Ser 216 and ionizing radiation (IR) does not induce either phosphorylation of Ser 216, or binding to 14-3-3. Here, we show that Cdc25C is phosphorylated on Ser 214 during mitosis, which in turn prevents phosphorylation of Ser 216. Mutation of Ser 214 to Ala reconstitutes Ser 216 phosphorylation and 14-3-3 binding during mitosis. Introduction of exogenous Cdc25C(S214A) into HeLa cells depleted of endogenous Cdc25C results in a substantial delay to mitotic entry. This effect was fully reversed in a S214A/S216A double-mutant, implying that the inhibitory effect of S214A mutant was entirely dependent on Ser 216 phosphorylation. A similar regulatory mechanism may also apply to another mitotic phosphatase, Cdc25B, as well as mitotic phosphatases of other species, including Xenopus laevis. We propose that this pathway ensures that Cdc2 remains active once mitosis is initiated and is a key control mechanism for maintaining the proper order of cell-cycle transitions.

Purification of a plant nucleotide pyrophosphatase as a protein that interferes with nitrate reductase and glutamine synthetase assays.

An activity that inhibited both glutamine synthetase (GS) and nitrate reductase (NR) was highly purified from cauliflower (Brassica oleracea var. botrytis) extracts. The final preparation contained an acyl-CoA oxidase and a second protein of the plant nucleotide pyrophosphatase family. This preparation hydrolysed NADH, ATP and FAD to generate AMP and was inhibited by fluoride, Cu2+, Zn2+ and Ni2+. The purified fraction had no effect on the activity of NR when reduced methylviologen was used as electron donor instead of NADH; and inhibited the oxidation of NADH by both spinach NR and an Escherichia coli extract in a time-dependent manner. The apparent inhibition of GS and NR and the ability of ATP and AMP to relieve the inhibition of NR can therefore be explained by hydrolysis of nucleotide substrates by the nucleotide pyrophosphatase. We have no evidence that the nucleotide pyrophosphatase is a specific physiological regulator of NR and GS, but suggest that nucleotide pyrophosphatase activity may underlie some confusion in the literature about the effects of nucleotides and protein factors on NR and GS in vitro.