Purinergic Signalling Mediates Aberrant Excitability of Developing Neuronal Circuits in the Fmr1 Knockout Mouse Model.

Neuronal hyperexcitability within developing cortical circuits is a common characteristic of several heritable neurodevelopmental disorders, including Fragile X Syndrome (FXS), intellectual disability and autism spectrum disorders (ASD). While this aberrant circuitry is typically studied from a neuron-centric perspective, glial cells secrete soluble factors that regulate both neurite extension and synaptogenesis during development. The nucleotide-mediated purinergic signalling system is particularly instrumental in facilitating these effects. We recently reported that within a FXS animal model, the Fmr1 KO mouse, the purinergic signalling system is upregulated in cortical astrocytes leading to altered secretion of synaptogenic and plasticity-related proteins. In this study, we examined whether elevated astrocyte purinergic signalling also impacts neuronal morphology and connectivity of Fmr1 KO cortical neurons. Here, we found that conditioned media from primary Fmr1 KO astrocytes was sufficient to enhance neurite extension and complexity of both wildtype and Fmr1 KO neurons to a similar degree as UTP-mediated outgrowth. Significantly enhanced firing was also observed in Fmr1 KO neuron-astrocyte co-cultures grown on microelectrode arrays but was associated with large deficits in firing synchrony. The selective P2Y2 purinergic receptor antagonist AR-C 118925XX effectively normalized much of the aberrant Fmr1 KO activity, designating P2Y2 as a potential therapeutic target in FXS. These results not only demonstrate the importance of astrocyte soluble factors in the development of neural circuitry, but also show that P2Y purinergic receptors play a distinct role in pathological FXS neuronal activity.

Fontan Fenestration Reduction with an Occlutech Atrial Flow Regulator: Unique Views from Three-Dimensional Intracardiac Echocardiography.

Three-dimensional intracardiac echocardiography (3D ICE) has gained popularity in interventional cardiology given its improved spatial and temporal imaging in assessing intracardiac anatomy pre- and post-intervention. We describe the use of 3D ICE in the reduction of a Fontan fenestration with an Occlutech atrial flow regulator (AFR) device.

Effect of Multiple-Patient Simulation on Baccalaureate Nursing Students' Anxiety and Self-confidence: A Pilot Study.

BACKGROUND: Multiple-patient simulation (MPS) allows nursing students to develop leadership skills. Limited research examining student outcomes following MPS exists. PURPOSE: This pilot study investigated the impact of MPS on (1) anxiety with transition to practice, (2) anxiety with clinical decision-making, (3) self-confidence with clinical decision-making, and (4) perceptions about MPS as a learning strategy. METHODS: Twenty-two senior baccalaureate nursing students participated in this 2-group mixed-methods study. Data were collected before and after a leadership course using the State-Trait Anxiety Inventory, Nursing Anxiety and Self-Confidence with Clinical Decision-Making Scale, and a researcher-developed perceptions survey. RESULTS: Self-confidence with clinical decision-making significantly increased for all participants regardless of group assignment. Anxiety and anxiety with clinical decision-making decreased without significant changes. No significant differences were found between groups. Qualitative findings yielded 3 themes: preparation for clinical practice, overcoming anxiety, and confidence. CONCLUSION: Research investigating additional student outcomes after MPS with larger, more diverse samples is needed.

HIV stigma beliefs and unprotected sex among teenagers and young adults in sub-Saharan Africa: The moderating role of mass media exposure.

Despite significant advances in the fight against HIV in sub-Saharan Africa, health experts remain concerned about new infections and risky sexual behavior among teenagers & young adults (T&YAs). These concerns have spurred efforts to buttress a voluminous literature on the social determinants of risky sexual behavior in Africa. Absent from this flurry of new scholarship is a consistent focus on associations between HIV stigma beliefs and risky sexual behavior, especially among T&YAs. Alongside health professionals' concerns about sexual behaviors is growing alarm about a dramatic expansion of sexual content in African mass media markets, which experts suspect may lead to T&YA risky sexual behavior. Yet, little work using multi-country data has confirmed whether mass media exposure increases the likelihood of risky sexual behavior. We fill these two gaps in the literature using a Demographic and Health Survey sample of unpartnered sub-Saharan African people, ages 15-24, in 30 countries. With this sample, we examine the direct relationships between HIV stigma beliefs, mass media exposure, and unprotected sex. We also explore whether the effect of stigma beliefs on unprotected sex is moderated by individual and regional-level exposure to mass media content. We first find that the effect of HIV stigma beliefs is harmful or associated with increases in the probability of unprotected sex. Second, contrary to past findings, individual-level mass media exposure is protective, or associated with declines in the likelihood of unprotected sex. Third, the harmful effect of stigma attitudes is weakest when individual and regional-level mass media exposure are low, but strongest when individual and regional-level mass media exposure are high. These findings suggest that stigma beliefs can shape the sexual behaviors of African T&YAs in counterintuitive ways. They also show that mass media exposure can be simultaneously protective and harmful for this population.

Functionally Clustered mRNAs Are Distinctly Enriched at Cortical Astroglial Processes and Are Preferentially Affected by FMRP Deficiency.

Mature protoplasmic astroglia in the mammalian CNS uniquely possess a large number of fine processes that have been considered primary sites to mediate astroglia to neuron synaptic signaling. However, localized mechanisms for regulating interactions between astroglial processes and synapses, especially for regulating the expression of functional surface proteins at these fine processes, are largely unknown. Previously, we showed that the loss of the RNA binding protein FMRP in astroglia disrupts astroglial mGluR5 signaling and reduces expression of the major astroglial glutamate transporter GLT1 and glutamate uptake in the cortex of Fmr1 conditional deletion mice. In the current study, by examining ribosome localization using electron microscopy and identifying mRNAs enriched at cortical astroglial processes using synaptoneurosome/translating ribosome affinity purification and RNA-Seq in WT and FMRP-deficient male mice, our results reveal interesting localization-dependent functional clusters of mRNAs at astroglial processes. We further showed that the lack of FMRP preferentially alters the subcellular localization and expression of process-localized mRNAs. Together, we defined the role of FMRP in altering mRNA localization and expression at astroglial processes at the postnatal development (P30-P40) and provided new candidate mRNAs that are potentially regulated by FMRP in cortical astroglia.SIGNIFICANCE STATEMENT Localized mechanisms for regulating interactions between astroglial processes and synapses, especially for regulating the expression of functional surface proteins at these fine processes, are largely unknown. Previously, we showed that the loss of the RNA binding protein FMRP in astroglia disrupts expression of several astroglial surface proteins, such as mGluR5 and major astroglial glutamate transporter GLT1 in the cortex of FMRP-deficient mice. Our current study examined ribosome localization using electron microscopy and identified mRNAs enriched at cortical astroglial processes in WT and FMRP-deficient mice. These results reveal interesting localization-dependent functional clusters of mRNAs at astroglial processes and demonstrate that the lack of FMRP preferentially alters the subcellular localization and expression of process-localized mRNAs.

Converging purinergic and immune signaling pathways drive IL-6 secretion by Fragile X cortical astrocytes via STAT3.

The symptoms of Fragile X syndrome (FXS) are driven in part by abnormal glial-mediated function. FXS astrocytes release elevated levels of immune-related factors interleukin-6 (IL-6) and tenascin C (TNC), and also demonstrate increased purinergic signaling, a pathway linked to signaling factor release. Here, in cortical astrocytes from the Fmr1 knockout (KO) FXS mouse model, purinergic agonism enhanced TNC secretion and STAT3 phosphorylation, two processes linked to elevated IL-6 secretion in FXS, while STAT3 knockdown and TLR4 antagonism normalized Fmr1 KO IL-6 release. We therefore suggest that purinergic signaling and immune regulatory pathways converge to drive FXS cortical pro-inflammatory responses.

An analysis of Medtronic MiniMed 670G insulin pump use in clinical practice and the impact on glycemic control, quality of life, and compliance.

AIMS: This study evaluated the use of the Medtronic MiniMed 670G system in adults with type 1 diabetes mellitus from a large endocrinology practice and its impact on glycemic control, quality of life (QoL), compliance and safety. METHODS: 84 participants completed one site visit for data collection. Percentage of time in range (TIR: 70-180 mg/dL), hyperglycemia or time above range (TAB) (>180 mg/dL), hypoglycemia or time below range (TBR) (<70 mg/dL), HbA1c, average blood glucose (ABG), and other metrics were evaluated at the last visit using the system (LVMM) and compared between the last visit on previous insulin therapy (LVPT). RESULTS: The mean percentage of TIR at the LVMM was 74.0 ± 12.1%, with an increase of 27.1% (p < 0.001) in TIR from the LVPT. The mean percentage of TAR was 22.9 ± 11.8% and the mean percentage of TBR was 3.2 ± 5.1%. CONCLUSIONS: The use of the Medtronic MiniMed 670G system in our practice resulted in a TIR above the recommended target with a high degree of treatment satisfaction and compliance in adults with type 1 diabetes. Furthermore, the system may be a reasonable choice for patients struggling with significant amounts of hypoglycemia.

Astrocyte-mediated purinergic signaling is upregulated in a mouse model of Fragile X syndrome.

Fragile X syndrome (FXS) is the leading monogenic cause of intellectual disability and autism spectrum disorders. With increasing investigation into the molecular mechanisms underlying FXS, there is growing evidence that perturbations in glial signaling are widely associated with neurological pathology. Purinergic signaling, which utilizes nucleoside triphosphates as signaling molecules, provides one of the most ubiquitous signaling systems for glial-neuronal and glial-glial crosstalk. Here, we sought to identify whether purinergic signaling is dysregulated within the FXS mouse cortex, and whether this dysregulation contributes to aberrant intercellular communication. In primary astrocyte cultures derived from the Fmr1 knockout (KO) mouse model of FXS, we found that application of exogenous ATP and UTP evoked elevated intracellular calcium responses compared to wildtype levels. Accordingly, purinergic P2Y2 and P2Y6 receptor expression was increased in Fmr1 KO astrocytes both in vitro and in acutely dissociated tissue, while P2Y antagonism via suramin prevented intracellular calcium elevations, suggesting a role for these receptors in aberrant FXS astrocyte activation. To investigate the impact of elevated purinergic signaling on astrocyte-mediated synaptogenesis, we quantified synaptogenic protein TSP-1, known to be regulated by P2Y activation. TSP-1 secretion and expression were both heightened in Fmr1 KO vs wildtype astrocytes following UTP application, while naïve TSP-1 cortical expression was also transiently elevated in vivo, indicating increased potential for excitatory TSP-1-mediated synaptogenesis in the FXS cortex. Together, our results demonstrate novel and significant purinergic signaling elevations in Fmr1 KO astrocytes, which may serve as a potential therapeutic target to mitigate the signaling aberrations observed in FXS.

Fructose metabolism in Chromohalobacter salexigens: interplay between the Embden-Meyerhof-Parnas and Entner-Doudoroff pathways.

BACKGROUND: The halophilic bacterium Chromohalobacter salexigens metabolizes glucose exclusively through the Entner-Doudoroff (ED) pathway, an adaptation which results in inefficient growth, with significant carbon overflow, especially at low salinity. Preliminary analysis of C. salexigens genome suggests that fructose metabolism could proceed through the Entner-Doudoroff and Embden-Meyerhof-Parnas (EMP) pathways. In order to thrive at high salinity, this bacterium relies on the biosynthesis and accumulation of ectoines as major compatible solutes. This metabolic pathway imposes a high metabolic burden due to the consumption of a relevant proportion of cellular resources, including both energy molecules (NADPH and ATP) and carbon building blocks. Therefore, the existence of more than one glycolytic pathway with different stoichiometries may be an advantage for C. salexigens. The aim of this work is to experimentally characterize the metabolism of fructose in C. salexigens. RESULTS: Fructose metabolism was analyzed using in silico genome analysis, RT-PCR, isotopic labeling, and genetic approaches. During growth on fructose as the sole carbon source, carbon overflow was not observed in a wide range of salt concentrations, and higher biomass yields were reached. We unveiled the initial steps of the two pathways for fructose incorporation and their links to central metabolism. While glucose is metabolized exclusively through the Entner-Doudoroff (ED) pathway, fructose is also partially metabolized by the Embden-Meyerhof-Parnas (EMP) route. Tracking isotopic label from [1-13C] fructose to ectoines revealed that 81% and 19% of the fructose were metabolized through ED and EMP-like routes, respectively. Activities of enzymes from both routes were demonstrated in vitro by 31P-NMR. Genes encoding predicted fructokinase and 1-phosphofructokinase were cloned and the activities of their protein products were confirmed. Importantly, the protein encoded by csal1534 gene functions as fructose bisphosphatase, although it had been annotated previously as pyrophosphate-dependent phosphofructokinase. The gluconeogenic rather than glycolytic role of this enzyme in vivo is in agreement with the lack of 6-phosphofructokinase activity previously described. CONCLUSIONS: Overall, this study shows that C. salexigens possesses a greater metabolic flexibility for fructose catabolism, the ED and EMP pathways contributing to a fine balancing of energy and biosynthetic demands and, subsequently, to a more efficient metabolism.

Percutaneous device embolization of an aneurysmal pulmonary artery in an infant weighing <3 kilograms.

Pulmonary artery aneurysm (PAA) and pulmonary artery pseudoaneurysm (PAP) are rare diagnoses in the pediatric population and carry a high risk of mortality if rupture occurs. There is currently no standard therapeutic approach to PAAs and PAPs. Reports of surgical intervention describe high mortality. We present a case of an infant with a PAP that was successfully treated with a percutaneous device closure. Our approach included deployment of a 6-mm Amplatzer Vascular Plug 2 (Abbott, St. Paul, MN) in the right lower pulmonary artery segmental branch just proximal to the origin of the pseudoaneurysm. Subsequent imaging 1-month post-procedure demonstrated a >50% reduction in the size of the PAP when compared to original imaging studies and near-complete resolution 14 months following the intervention. Percutaneous device placement to occlude the vessel supplying peripheral PAAs and PAPs may be a reasonable alternative to open surgical resection when treating patients with this rare, but potentially life-threatening vascular anomaly. To our knowledge, this is the first case describing a successful device closure of a PAP in an infant weighing <3 kg.

Stereotypic behaviour in standard non-enriched cages is an alternative to depression-like responses in C57BL/6 mice.

Depressive-like forms of waking inactivity have been recently observed in laboratory primates and horses. We tested the hypotheses that being awake but motionless within the home-cage is a depression-like symptom in mice, and that in impoverished housing, it represents an alternative response to stereotypic behaviour. We raised C57BL/6 ('C57') and DBA/2 ('DBA') females to adulthood in non-enriched (n=62 mice) or enriched (n=60 mice) cages, observing home-cage behaviour during the active (dark) phases. We predicted that being still but awake would be reduced by environmental enrichment; more pronounced in C57s, as the strain most prone to learned helplessness; negatively related to stereotypic behaviour; and positively related to immobility in Forced Swim Tests (FST). Compared to enriched mice, non-enriched subjects did spend more time spent being inactive but awake, especially if they displayed relatively little stereotypic behaviour. C57 mice also spent more time awake but motionless than DBAs. Furthermore, even after statistically controlling for housing type and strain, this behaviour very strongly tended to predict increased immobility in the FST, while high levels of stereotypic behaviours in contrast predicted low immobility in the FST. Being awake but motionless is thus a reaction to non-enriched housing that seems to be an alternative to stereotypic behaviour, and could reflect depression-like states.

Cage-induced stereotypic behaviour in laboratory mice covaries with nucleus accumbens FosB/ΔFosB expression.

Stereotypic behaviour (SB) occurs in certain human disorders (e.g. autism), and animals treated with stimulants or raised in impoverished conditions, including laboratory mice in standard cages. Dysfunctional cortico-basal ganglia pathways have been implicated in these examples, but for cage-induced forms of SB, the relative roles of ventral versus dorsal striatum had not been fully ascertained. Here, we used immunohistochemical staining of FosB and ΔFosB to assess long-term activation within the nucleus accumbens and caudate-putamen of C57BL/6 mice. Housed in typical laboratory cages, these mice spontaneously developed different degrees of route-tracing, bar-mouthing and other forms of SB (spending 0% to over 50% of their active time budgets in this behaviour). The most highly stereotypic mice showed the most elevated FosB/ΔFosB activity in the nucleus accumbens. No such patterns occurred in the caudate-putamen. The cage-induced SB common in standard-housed mice thus involves elevated activity within the ventral striatum, suggesting an aetiology closer to compulsive gambling, eating and drug-seeking than to classic amphetamine stereotypies and other behaviours induced by motor loop over-activation.

Rapid increases in immature synapses parallel estrogen-induced hippocampal learning enhancements.

Dramatic increases in hippocampal spine synapse density are known to occur within minutes of estrogen exposure. Until now, it has been assumed that enhanced spinogenesis increased excitatory input received by the CA1 pyramidal neurons, but how this facilitated learning and memory was unclear. Delivery of 17ß-estradiol or an estrogen receptor (ER)-α (but not ER-ß) agonist into the dorsal hippocampus rapidly improved general discrimination learning in female mice. The same treatments increased CA1 dendritic spines in hippocampal sections over a time course consistent with the learning acquisition phase. Surprisingly, estrogen-activated spinogenesis was associated with a decrease in CA1 hippocampal excitatory input, rapidly and transiently reducing CA1 AMPA activity via a mechanism likely reflecting AMPA receptor internalization and creation of silent or immature synapses. We propose that estrogens promote hippocampally mediated learning via a mechanism resembling some of the broad features of normal development, an initial overproduction of functionally immature connections being subsequently "pruned" by experience.

The school age child with congenital heart disease.

Currently, in the United States, there are approximately 1 in 150 adults living with congenital heart disease (CHD) (). Infant and childhood mortality related to CHD decreased by 31% between 1987 and 2005 (). This survival trend is predicted to increase each year due to advancements in treatment and management of CHD. This significant shift in the epidemiology of CHD requires nurses to take action in preparing children with CHD and their families for their teenage years and young adulthood. The school-age child is the ideal age to begin teaching the child about their healthcare needs and how to care for themselves in preparation for the future. The school-age child with CHD has specific physical, intellectual, emotional, and developmental needs that must be considered and managed using a multidisciplinary approach. Pediatric nurses must be aware of these needs as they help the child and their family seamlessly and successfully transition into young adulthood as a happy and healthy CHD survivor.

Promoting cultural understanding through pediatric clinical dyads: an education research project.

This project explored the experiences of six undergraduate nursing students, three American nursing students and three nursing students from the Sultan of Oman, who participated in a faculty initiated education research project as part of their pediatric clinical practicum. Students were placed in dyads, with one American-born student and one Omani student in each dyad. Omani students also were paired with American nurse preceptors. A transcript-based content analysis was used to analyze data generated from qualitative focus group student interviews and student journals. The analysis generated three themes that described how myths were dispelled, cultural barriers were broken down and knowledge gained from another cultural perspective. The nurse preceptors were surveyed at the conclusion of the program. The survey findings suggest that preceptors gained a different cultural perspective of nursing care and they were better informed of the Omani students' learning needs. There was, however, an additional investment of preceptor time in meeting the learning needs of international students. Additional faculty time was also required for preparation and time during clinical conferencing to address differences in nursing practice between U.S. and Oman while meeting course learning objectives. Overall, the educational program provided evidence of enhancing American and Omani student cultural competence and Omani student adaptation to the United States. Coupling a domestic student with an international student to form dyads from the beginning of international students' experience could be a significant enhancement to both groups of students' learning experience.

Customized nutritional enhancement for pregnant women appears to lower incidence of certain common maternal and neonatal complications: an observational study.

A retrospective chart review analyzed the effect of customized nutrition on the incidence of pregnancy-induced hypertension (PIH), gestational diabetes (GDM), and small- and large-for-gestational-age (SGA, LGA) neonates, examining consecutive deliveries between January 1, 2011, and Decem ber 31, 2012, at a low-risk community hospital. The population was divided into 3 groups: (1) study group (SG), (2) private practice (PP), and (3) community healthcare clinic (CHCC). All groups received standard perinatal management, but additionally the study group was analyzed for serum zinc, carnitine, total 25-hydroxy cholecalciferol (25 OH-D), methylene tetrahydrofolate reductase, and catechol-O-methyl transferase polymorphisms in the first trimester prior to intervention, with subsequent second trimester and postpartum assessment of zinc, carnitine, and 25 OH-D after intervention. Intervention consisted of trimesterby-trimester nutrition and lifestyle education, supplementation of L-methyl folate, magnesium, essential fatty acids, and probiotics for all SG patients, with targeted supplementation of zinc, carnitine, and 25 OH-D. Because of small case occurrence rates of individual conditions in the study group, unreportable reductions were found, except GDM (SG vs CHCC, P value .046 with 95.38% confidence interval [CI]), and PIH (SG vs PP, P value .0505 with 94.95% CIl). The aggregated occurrence rate of the four conditions, however, was significantly lower in the study population than in either comparison population (PP P value .0154 with 98.46% CI, and CHCC P value .0265 with 97.35% CI). Customized nutritional intervention appears to have significantly reduced adverse perinatal outcomes. Prospective study within larger, at-risk populations is needed to determine whether customized nutrition improves conditions individually.

En una revisión retrospectiva de historias clínicas se analizó el efecto de la nutrición personalizada sobre la incidencia de hipertensión inducida por el embarazo (HIE), diabetes gestacional (DG) y los neonatos pequeños o grandes para su edad gestacional (PEG, GED), examinando partos consecutivos entre el 1 de enero de 2011 y el 31 de diciembre de 2012 en un hospital general de bajo riesgo. La población se dividió en 3 grupos: (1) grupo del estudio (GE), (2) consulta privada (CP), y (3) clínica de atención médica general (CAMG). Todos los grupos recibieron una gestión perinatal estándar, pero en el grupo del estudio se realizaron, además, análisis del zinc en suero, carnitina, 25-hidroxicolecalciferol (25 OH-D) total, metilentetrahidrofolato reductasa y polimorfismos en catecol-o-metil-transferasa en el primer trimestre, antes de la intervención, con las posteriores valoraciones en el segundo y tercer trimestre y postparto de zinc, carnitina y 25 OH-D tras la intervención. La intervención consistió en un proceso educativo trimestral sobre nutrición y estilo de vida, suplementos de L-metilfolato, magnesio, ácidos grasos esenciales y probióticos, para todas las pacientes del GE, con suplementos dirigidos con zinc, carnitina y 25 OH-D. Debido a las bajas tasas de incidencia de las afecciones individuales en el grupo del estudio, se encontraron reducciones que no deben comunicarse, exceptuando la DG (GE frente a CAMG, valor de P 0,046 con un nivel de confianza del 95,38 %) y la HIE (GE frente a CP, valor de P 0,0505 con un nivel de confianza del 94,95 %). Sin embargo, la tasa de incidencia agregada de las cuatro afecciones fue significativamente más baja en la población del estudio que en cualquiera de las poblaciones de comparación (CP, valor de P 0,0154 con un nivel de confianza del 98,46 %, y CAMG, valor de P 0,0265 con un nivel de confianza del 97,35 %). La intervención nutricional personalizada parece tener resultados perinatales adversos significativamente reducidos. Es necesario un estudio prospectivo con poblaciones más grandes y de riesgo, para determinar si la nutrición personalizada mejora las afecciones de forma individual.

Effect of short-term iPRO continuous glucose monitoring on hemoglobin A1c levels in clinical practice.

OBJECTIVE: This study determined if short-term (professional) blinded continuous glucose monitoring (CGM) improves hemoglobin A1c levels in a mixed group of patients with type 1 and type 2 diabetes in the clinical setting of an office practice. RESEARCH DESIGN AND METHODS: This was a retrospective analysis of 102 consecutive patients with diabetes over the course of 10 months in a subspecialty practice undergoing 3-day blinded CGM using the iPRO(™) device (Medtronic, Northridge, CA). Hemoglobin A1c levels were measured prior to and up to 7 months after the CGM procedure. RESULTS: Before blinded CGM the average hemoglobin A1c level was 7.7±1.0%, and after it was 7.8±1.1%. These values are not statistically different. A subgroup analysis of subjects using continuous subcutaneous insulin infusion treatment also failed to show a significant hemoglobin A1c difference pre- and post-CGM. CONCLUSIONS: Using the iPro device for short-term (professional) blinded CGM in an office setting to improve hemoglobin A1c levels may not be a feasible goal for patients with type 1 and type 2 diabetes. The expectations of benefit, choice of patient, and choice of technology for short-term CGM are factors requiring careful consideration before testing takes place.

Copper delivery by the copper chaperone for chloroplast and cytosolic copper/zinc-superoxide dismutases: regulation and unexpected phenotypes in an Arabidopsis mutant.

Copper (Cu) is an important mineral nutrient found in chloroplasts as a cofactor associated with plastocyanin and Cu/Zn superoxide dismutase (Cu/ZnSOD). Superoxide dismutases are metallo-enzymes found in most oxygenic organisms with proposed roles in reducing oxidative stress. Several recent studies in Arabidopsis have shown that microRNAs and a SQUAMOSA promoter binding protein-like7 (SPL7) transcription factor function to down-regulate the expression of many Cu-proteins, including Cu/ZnSOD in both plastids and the cytosol, during growth on low Cu. Plants contain the Cu Chaperone for SOD (CCS) that delivers Cu to Cu/ZnSODs, and, in Arabidopsis, both cytosolic and plastidic CCS versions are encoded by one gene. In this study, we demonstrate that Arabidopsis CCS transcript levels are regulated by Cu, mediated by microRNA 398 that was not previously predicted to target CCS. The microRNA target site is conserved in CCS of Oryza sativa. The data suggest that Cu-regulated microRNAs may have more mRNA targets than was previously predicted. A CCS null mutant has no measurable SOD activity in the chloroplast and cytosol, indicating an absolute requirement for CCS. When the CCS null mutant was grown on high Cu media, it lacked both Fe superoxide dismutase (FeSOD) and Cu/ZnSOD activity. However, this did not lead to a visual phenotype and no photosynthetic deficiencies were detected, even after high light stress. These results indicate that Cu/ZnSOD is not a pivotal component of the photosynthetic anti-oxidant system during growth in laboratory conditions.

Copper homeostasis.

Copper (Cu) is a cofactor in proteins that are involved in electron transfer reactions and is an essential micronutrient for plants. Copper delivery is accomplished by the concerted action of a set of evolutionarily conserved transporters and metallochaperones. As a result of regulation of transporters in the root and the rarity of natural soils with high Cu levels, very few plants in nature will experience Cu in toxic excess in their tissues. However, low Cu bioavailability can limit plant productivity and plants have an interesting response to impending Cu deficiency, which is regulated by an evolutionarily conserved master switch. When Cu supply is insufficient, systems to increase uptake are activated and the available Cu is utilized with economy. A number of Cu-regulated small RNA molecules, the Cu-microRNAs, are used to downregulate Cu proteins that are seemingly not essential. On low Cu, the Cu-microRNAs are upregulated by the master Cu-responsive transcription factor SPL7, which also activates expression of genes involved in Cu assimilation. This regulation allows the most important proteins, which are required for photo-autotrophic growth, to remain active over a wide range of Cu concentrations and this should broaden the range where plants can thrive.