Structural Insights into the Recovery of Aldolase Activity in N -Acetylneuraminic Acid Lyase by Replacement of the Catalytically Active Lysine with γ-Thialysine by Using a Chemical Mutagenesis Strategy

Chemical modification has been used to introduce the unnatural amino acid γ‐thialysine in place of the catalytically important Lys165 in the enzyme N‐acetylneuraminic acid lyase (NAL). The Staphylococcus aureus nanA gene, encoding NAL, was cloned and expressed in E. coli. The protein, purified in high yield, has all the properties expected of a class I NAL. The S. aureus NAL which contains no natural cysteine residues was subjected to site‐directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site. Subsequently chemical mutagenesis completely converted the cysteine into γ‐thialysine through dehydroalanine (Dha) as demonstrated by ESI‐MS. Initial kinetic characterisation showed that the protein containing γ‐thialysine regained 17 % of the wild‐type activity. To understand the reason for this lower activity, we solved X‐ray crystal structures of the wild‐type S. aureus NAL, both in the absence of, and in complex with, pyruvate. We also report the structures of the K165C variant, and the K165‐γ‐thialysine enzyme in the presence, or absence, of pyruvate. These structures reveal that γ‐thialysine in NAL is an excellent structural mimic of lysine. Measurement of the pH‐activity profile of the thialysine modified enzyme revealed that its pH optimum is shifted from 7.4 to 6.8. At its optimum pH, the thialysine‐containing enzyme showed almost 30 % of the activity of the wild‐type enzyme at its pH optimum. The lowered activity and altered pH profile of the unnatural amino acid‐containing enzyme can be rationalised by imbalances of the ionisation states of residues within the active site when the pKa of the residue at position 165 is perturbed by replacement with γ‐thialysine. The results reveal the utility of chemical mutagenesis for the modification of enzyme active sites and the exquisite sensitivity of catalysis to the local structural and electrostatic environment in NAL

Adolescent Binge Drinking Leads to Changes in Alcohol Drinking, Anxiety, and Amygdalar Corticotropin Releasing Factor Cells in Adulthood in Male Rats

Heavy episodic drinking early in adolescence is associated with increased risk of addiction and other stress-related disorders later in life. This suggests that adolescent alcohol abuse is an early marker of innate vulnerability and/or binge exposure impacts the developing brain to increase vulnerability to these disorders in adulthood. Animal models are ideal for clarifying the relationship between adolescent and adult alcohol abuse, but we show that methods of involuntary alcohol exposure are not effective. We describe an operant model that uses multiple bouts of intermittent access to sweetened alcohol to elicit voluntary binge alcohol drinking early in adolescence (∼postnatal days 28–42) in genetically heterogeneous male Wistar rats. We next examined the effects of adolescent binge drinking on alcohol drinking and anxiety-like behavior in dependent and non-dependent adult rats, and counted corticotropin-releasing factor (CRF) cell in the lateral portion of the central amygdala (CeA), a region that contributes to regulation of anxiety- and alcohol-related behaviors. Adolescent binge drinking did not alter alcohol drinking under baseline drinking conditions in adulthood. However, alcohol-dependent and non-dependent adult rats with a history of adolescent alcohol binge drinking did exhibit increased alcohol drinking when access to alcohol was intermittent. Adult rats that binged alcohol during adolescence exhibited increased exploration on the open arms of the elevated plus maze (possibly indicating either decreased anxiety or increased impulsivity), an effect that was reversed by a history of alcohol dependence during adulthood. Finally, CRF cell counts were reduced in the lateral CeA of rats with adolescent alcohol binge history, suggesting semi-permanent changes in the limbic stress peptide system with this treatment. These data suggest that voluntary binge drinking during early adolescence produces long-lasting neural and behavioral effects with implications for anxiety and alcohol use disorders

The oxytocin analogue carbetocin prevents emotional impairment and stress-induced reinstatement of opioid-seeking in morphine-abstinent mice.

The main challenge in treating opioid addicts is to maintain abstinence due to the affective consequences associated with withdrawal which may trigger relapse. Emerging evidence suggests a role of the neurohypophysial peptide oxytocin (OT) in the modulation of mood disorders as well as drug addiction. However, its involvement in the emotional consequences of drug abstinence remains unclear. We investigated the effect of 7-day opioid abstinence on the oxytocinergic system and assessed the effect of the OT analogue carbetocin (CBT) on the emotional consequences of opioid abstinence, as well as relapse. Male C57BL/6J mice were treated with a chronic escalating-dose morphine regimen (20-100 mg/kg/day, i.p.). Seven days withdrawal from this administration paradigm induced a decrease of hypothalamic OT levels and a concomitant increase of oxytocin receptor (OTR) binding in the lateral septum and amygdala. Although no physical withdrawal symptoms or alterations in the plasma corticosterone levels were observed after 7 days of abstinence, mice exhibited increased anxiety-like and depressive-like behaviors and impaired sociability. CBT (6.4 mg/kg, i.p.) attenuated the observed negative emotional consequences of opioid withdrawal. Furthermore, in the conditioned place preference paradigm with 10 mg/kg morphine conditioning, CBT (6.4 mg/kg, i.p.) was able to prevent the stress-induced reinstatement to morphine-seeking following extinction. Overall, our results suggest that alterations of the oxytocinergic system contribute to the mechanisms underlying anxiety, depression, and social deficits observed during opioid abstinence. This study also highlights the oxytocinergic system as a target for developing pharmacotherapy for the treatment of emotional impairment associated with abstinence and thereby prevention of relapse

Is obesity associated with reduced health-related quality of life in Latino, black and white children in the community?

OBJECTIVE: Few studies have examined the impact of obesity on health-related quality of life (HRQOL) in non-clinical community samples of children, and methodological limitations have hindered drawing firm conclusions, especially whether the impact is similar across racial/ethnic groups. The present aims were to examine at what levels of non-normal weight, school-aged children experience lower HRQOL and whether this differs among racial/ethnic groups, when controlling for socioeconomic status (SES) differences. DESIGN: Cross-sectional community cohort survey. SUBJECTS AND METHODS: Data are from the Healthy Passages, reporting on 4824 Latino, black and white 5th graders in a population-based survey conducted in three United States metropolitan areas. Children's weight status was classified from measured weight and height into underweight (1%), normal weight (52%), overweight (19%), obese (13%) and extremely obese (14%). Children reported their own HRQOL using the Pediatric Quality of Life Inventory and additional scales addressing global self-worth, physical appearance and body satisfaction. Parents reported children's overall health status. RESULTS: Each increment in higher non-healthy weight class—overweight to obese to extremely obese—was associated with significantly lower scores in more domains of psychosocial HRQOL compared with that in normal weight. However, only extremely obese children reported significantly lower physical HRQOL. Differences among weight classes remained when adjusting for SES and were independent of race/ethnicity. Underweight children generally reported HRQOL that was not significantly different from normal weight children. CONCLUSIONS: Overweight, obese and extremely obese 5th graders on average experience worse HRQOL than normal weight children, especially in psychosocial domains including self-worth and peer relationships, regardless of race/ethnicity. If messages can be conveyed in a sensitive and supportive manner, the desire to improve HRQOL could provide additional motivation for children and their parents in addressing unhealthy weight

Transplantation of Specific Human Astrocytes Promotes Functional Recovery after Spinal Cord Injury

Repairing trauma to the central nervous system by replacement of glial support cells is an increasingly attractive therapeutic strategy. We have focused on the less-studied replacement of astrocytes, the major support cell in the central nervous system, by generating astrocytes from embryonic human glial precursor cells using two different astrocyte differentiation inducing factors. The resulting astrocytes differed in expression of multiple proteins thought to either promote or inhibit central nervous system homeostasis and regeneration. When transplanted into acute transection injuries of the adult rat spinal cord, astrocytes generated by exposing human glial precursor cells to bone morphogenetic protein promoted significant recovery of volitional foot placement, axonal growth and notably robust increases in neuronal survival in multiple spinal cord laminae. In marked contrast, human glial precursor cells and astrocytes generated from these cells by exposure to ciliary neurotrophic factor both failed to promote significant behavioral recovery or similarly robust neuronal survival and support of axon growth at sites of injury. Our studies thus demonstrate functional differences between human astrocyte populations and suggest that pre-differentiation of precursor cells into a specific astrocyte subtype is required to optimize astrocyte replacement therapies. To our knowledge, this study is the first to show functional differences in ability to promote repair of the injured adult central nervous system between two distinct subtypes of human astrocytes derived from a common fetal glial precursor population. These findings are consistent with our previous studies of transplanting specific subtypes of rodent glial precursor derived astrocytes into sites of spinal cord injury, and indicate a remarkable conservation from rat to human of functional differences between astrocyte subtypes. In addition, our studies provide a specific population of human astrocytes that appears to be particularly suitable for further development towards clinical application in treating the traumatically injured or diseased human central nervous system

A systematic review of interactive multimedia interventions to promote children's communication with health professionals: implications for communicating with overweight children

Background: Interactive multimedia is an emerging technology that is being used to facilitate interactions between patients and health professionals. The purpose of this review was to identify and evaluate the impact of multimedia interventions (MIs), delivered in the context of paediatric healthcare, in order to inform the development of a MI to promote the communication of dietetic messages with overweight preadolescent children. Of particular interest were the effects of these MIs on child engagement and participation in treatment, and the subsequent effect on health-related treatment outcomes. Methods: An extensive search of 12 bibliographic databases was conducted in April 2012. Studies were included if: one or more child-participant was 7 to 11 years-of-age; a MI was used to improve health-related behaviour; child-participants were diagnosed with a health condition and were receiving treatment for that condition at the time of the study. Data describing study characteristics and intervention effects on communication, satisfaction, knowledge acquisition, changes in self-efficacy, healthcare utilisation, and health outcomes were extracted and summarised using qualitative and quantitative methods. Results: A total of 14 controlled trials, published between 1997 and 2006 met the selection criteria. Several MIs had the capacity to facilitate engagement between the child and a clinician, but only one sought to utilise the MI to improve communication between the child and health professional. In spite of concerns over the quality of some studies and small study populations, MIs were found useful in educating children about their health, and they demonstrated potential to improve children’s health- related self-efficacy, which could make them more able partners in face-to-face communications with health professionals. Conclusions: The findings of this review suggest that MIs have the capacity to support preadolescent child-clinician communication, but further research in this field is needed. Particular attention should be given to designing appropriate MIs that are clinically relevant

Fluid Intelligence and Psychosocial Outcome: From Logical Problem Solving to Social Adaptation

While fluid intelligence has proved to be central to executive functioning, logical reasoning and other frontal functions, the role of this ability in psychosocial adaptation has not been well characterized.Lower fluid intelligence scores were associated with physical violence, both in the role of victim and victimizer. Drug intake, especially cannabis, cocaine and inhalants and lower self-esteem were also associated with lower fluid intelligence. Finally, scores on the perceived mental health assessment were better when fluid intelligence scores were higher.Our results show evidence of a strong association between psychosocial adaptation and fluid intelligence, suggesting that the latter is not only central to executive functioning but also forms part of a more general capacity for adaptation to social contexts

Gender role orientation is associated with health-related quality of life differently among African-American, Hispanic, and White youth

PurposeThis study examined the association between gender role orientation (GRO) and health-related quality of life (HRQOL) in youth, and how this relationship may differ between males and females as well as among African-American, White, and Hispanic individuals. GRO has been reported to influence serious health outcomes including cancer, heart disease, mental illness, and mortality rates. However, few studies have examined the link between GRO and health outcomes for children, even though gender identity is formed in childhood.MethodsData were examined from 4824 participants in the Healthy Passages™ project, a population-based survey of fifth-grade children in three US metropolitan areas. Children reported their own HRQOL using the PedsQL and degree of female, male, and androgynous GRO using the Children's Sex Role Inventory.ResultsBased on structural equations analysis, male GRO was positively associated with HRQOL for all racial/ethnic groups, regardless of sex, whereas female GRO was associated with better HRQOL for Hispanic and White females and poorer HRQOL for Hispanic males. Androgynous GRO was associated with better HRQOL among Hispanic and White females, but not males nor African-Americans of either sex.ConclusionsRacial/ethnic differences emerged for female and androgynous, but not male, GROs. Hispanic males are the only group for which GRO (female) was associated with poorer HRQOL. Future research should find ways to help youth overcome negative effects on health from gender beliefs and behavior patterns with sensitivity to racial/ethnic membership