Effect of extended-release dexmethylphenidate and mixed amphetamine salts on sleep: a double-blind, randomized, crossover study in youth with attention-deficit hyperactivity disorder.

OBJECTIVE: We sought to determine the dose-response effects of extended-release (ER) dexmethylphenidate (d-MPH) and ER mixed amphetamine salts (MAS) on objective measures of sleep. METHODS: This was an 8-week, double-blind, placebo-controlled, randomized, two period, crossover study of youth with attention-deficit hyperactivity disorder (ADHD) as confirmed by the Kiddie Schedule for Affective Disorders for School-Age Children-Present and Lifetime version (K-SADS-PL). Children aged 10-17 years were recruited from clinical practice, colleague referrals, and flyers. Participants were randomized to initially receive either d-MPH or MAS. During each 4-week drug period, children received three dose levels (10, 20, and 25/30 mg) in ascending order, with placebo substituted for active medication in a randomized fashion during 1 week of the study. After 4 weeks, participants were switched to the alternative medication for another 4 weeks of treatment. The main outcome measure was sleep duration as measured by actigraphy. Children, parents, and researchers were blinded to drug, dose, and placebo status. RESULTS: Sixty-five participants met the inclusion criteria and were enrolled in the study. Of these, 37 participants with sufficient sleep data for analysis were included. Sleep schedule measures showed a significant effect for dose on sleep start time (F(1,36) = 6.284; p < 0.05), with a significantly later sleep start time when children were receiving 20- or 30-mg doses, compared with placebo (p < 0.05). A significant dose effect was found on actual sleep duration (F(1,36) = 8.112; p < 0.05), with significantly shorter actual sleep duration for subjects receiving 30 mg compared with those receiving placebo (p < 0.05). There were no significant differences on sleep duration or sleep schedule between the two stimulant medications. The trial is complete and closed to follow-up. CONCLUSIONS: Higher stimulant doses were associated with reduced sleep duration and later sleep start times, regardless of medication class. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00393042.

Expression of the apoptosis gene reaper in homeotic, segmentation and other mutants in Drosophila.

Apoptosis is an essential process required for development and morphogenesis in metazoan organisms. The apoptosis pathway and cell death machinery have been extensively studied, but little is known how apoptosis genes are regulated in the course of development . In this study, we analyzed the transcriptional regulation of the pro-apoptotic gene reaper (rpr) by performing whole-mount in situ hybridization in embryos mutant for a number of transcription factor genes in Drosophila melanogaster. In sum, our data show that all factors studied have very specific temporal and spatial effects on rpr transcription . Thus, our results reinforce the concept that apoptosis is an essential process for morphogenesis and that apoptosis related genes very tight developmental factors identified in sculpting the morphology of various embryonic structures by modulating the apoptosis pathway.

Atherosclerotic mice exhibit systemic inflammation in periadventitial and visceral adipose tissue, liver, and pancreatic islets.

OBJECTIVE: Atherosclerosis is a chronic inflammatory disease of major conduit arteries. Similarly, obesity and type 2 diabetes mellitus are associated with accumulation of macrophages in visceral white adipose tissue and pancreatic islets. Our goal was to characterize systemic inflammation in atherosclerosis with hypercholesterolemia, but without obesity. METHODS AND RESULTS: We compared 22-week-old apolipoprotein E knockout (ApoE(-/-)) with wild-type mice kept for 14 weeks on a high cholesterol (1.25%) diet (CD, n=8) and 8-week-old ApoE(-/-) with wild-type mice kept on a normal diet (ND, n=8). Hypercholesterolemic, atherosclerotic ApoE(-/-) mice on CD exhibited increased macrophages and T-cells in plaques and periadventitial adipose tissue that revealed elevated expression of MIP-1alpha, IL-1beta, IL-1 receptor, and IL-6. Mesenteric adipose tissue and pancreatic islets in ApoE(-/-) mice showed increased macrophages. Expression of IL-1beta was enhanced in mesenteric adipose tissue of ApoE(-/-) mice on CD. Furthermore, these mice exhibited steatohepatitis with macrophage and T-cell infiltrations as well as increased MIP-1alpha and IL-1 receptor expression. Blood glucose, insulin and total body weight did not differ between the groups. CONCLUSIONS: In hypercholesterolemic lean ApoE(-/-) mice, inflammation extends beyond atherosclerotic plaques to the periadventitial and visceral adipose tissue, liver, and pancreatic islets without affecting glucose homeostasis.

Multifactorial regulation of a hox target gene.

Hox proteins play fundamental roles in controlling morphogenetic diversity along the anterior-posterior body axis of animals by regulating distinct sets of target genes. Within their rather broad expression domains, individual Hox proteins control cell diversification and pattern formation and consequently target gene expression in a highly localized manner, sometimes even only in a single cell. To achieve this high-regulatory specificity, it has been postulated that Hox proteins co-operate with other transcription factors to activate or repress their target genes in a highly context-specific manner in vivo. However, only a few of these factors have been identified. Here, we analyze the regulation of the cell death gene reaper (rpr) by the Hox protein Deformed (Dfd) and suggest that local activation of rpr expression in the anterior part of the maxillary segment is achieved through a combinatorial interaction of Dfd with at least eight functionally diverse transcriptional regulators on a minimal enhancer. It follows that context-dependent combinations of Hox proteins and other transcription factors on small, modular Hox response elements (HREs) could be responsible for the proper spatio-temporal expression of Hox targets. Thus, a large number of transcription factors are likely to be directly involved in Hox target gene regulation in vivo.

Children, sleep, and behavior: a complex association.

Pediatric sleep physiology begins with development of the sleep/wake cycle, and the origins of active versus quiet sleep. The 24-hour circadian cycle becomes established at 3-6 months. Sleep disorders during infancy commonly include mild, usually self-limited conditions such as sleep-onset association disorder, excessive nighttime feedings, and poor limit-setting. These require behavioral management to avoid long-term deleterious sleep habits. In contrast, other sleep disorders are more ominous, including SIDS, central congenital hypoventilation syndrome, and sleep apnea. Childhood is generally considered the golden age of sleep, with brief latency to sleep onset, high efficiency, and easy awakening. Yet parasomnias, psychological factors, and sleep disturbances associated with common disorders such as ADHD disrupt the idealistic notion of childhood being a period of unfettered sleep. Adolescents have sleep requirements similar to adults, posing a challenge for them to adapt to school schedules and increasingly demanding lifestyles. Narcolepsy, usually diagnosed in adolescence or early adulthood, is a lifelong sleep disorder and has led to the identification of the hypocretin/orexin neurotransmitter system. Research advances in the complex interrelationships between developmental neurobiology, sleep disorders and behavior will lead to an enhanced understanding of the pathophysiology of sleep problems and lead to novel therapeutic strategies for sleep disturbances in children.

Medical mimics. Medical and neurological conditions simulating ADHD.

The medical and neurological conditions that simulate ADHD are reviewed, as well as those disorders frequently presenting as comorbidities with ADHD. The localization of ADHD has invoked multiple areas, including frontal lobes, nondominant parietal lobe, and basal ganglia, and the neural network theory of cortical-subcortical-cortical loops has been implicated in the pathogenesis of ADHD. The medical evaluation of patients presenting with ADHD should be comprehensive, with an emphasis on demonstrating chronic and permeating symptoms since early childhood without a better medical explanation. Associated thyroid disorders are reviewed, including the syndrome of resistance to thyroid hormone. Suggested laboratory studies are provided, depending on the clinical circumstances.

Sleep and behavior problems in school-aged children.

OBJECTIVES: The primary purposes of the present study were to survey the prevalence of sleep problems in school-aged children and to examine these associations with parental perception of sleep problems, medical history, and childhood psychopathology. METHODS: Sleep and medical history questionnaires and the Child Behavior Checklist were administered to the parents of 472 children between ages 4 and 12 years receiving routine pediatric care from urban, rural, and suburban pediatric practices. RESULTS: Although sleep problems were reported for 10.8% of the sample during the past 6 months, less than one half of the parents who identified sleep problems reported that they had discussed sleep with their child's pediatrician. The best predictor of current sleep problems was a history of sleep problems before age 2 years. Sleep problems such as snoring, tiredness during the day, and taking excessive time to fall asleep were very common, occurring at least 1 night per week in over 20% of the total sample. Factor analysis of the sleep problems questionnaire resulted in 5 sleep problem factors that accounted for 58.7% of the variance. Specific sleep problem factors include: parasomnias, enuresis/gags, tiredness, noisy sleep, and insomnia. Sleep problem factor scores were differentially associated with medical history variables and measures of childhood psychopathology. Children rated highly on parasomnias were more likely to have frequent falls and to display pica. Parasomnias and noisy sleep were inversely associated with socioeconomic status (SES). Children from lower SES families were rated higher on these factors than children from higher SES families. Enuresis/gags was the only sleep problem factor associated with age. Younger children scored higher on this factor. Duration of naps was highly correlated with age and with bed times during the week and weekends. As expected, younger children were more likely to nap for longer periods and to have earlier bed times. In addition, higher tiredness factor scores were associated with napping and with later bed times during the week and weekend. Boys were much more likely than were girls to have higher scores on enuresis/gags, and higher enuresis/gags scores were associated with an increased prevalence of trauma and falls. Bed times were not associated with any other sleep problem factor score. Children rated highly on tiredness were more likely to have a history of hospitalizations. Tiredness factor scores were strongly associated with the sleep practice of sharing a bed but not with sharing a room. Sharing a room was not associated with any sleep problem factor score. High scores on noisy sleep were associated with allergies, falls frequently, and with sharing a bed. Children with high scores on the insomnias were also more likely to display an increased prevalence of allergies. CONCLUSIONS: Parental perception of global sleep problems was surprisingly common in school-aged children receiving routine pediatric care. Parental reports of their children's sleep problems may be a red flag for specific sleep problems and psychiatric, social, or medical problems. Sleep problems should be queried about during pediatric visits for school-aged children.

Indoor environmental quality in six commercial office buildings in the midwest United States.

The aims of this study were to characterize physical, mechanical, and environmental factors influencing indoor environmental quality (IEQ) in commercial office buildings; document occupant perceptions and psychosocial attributes; and evaluate relationships among these parameters. Six large office buildings in metropolitan areas were selected in Iowa, Minnesota, and Nebraska. Comprehensive sampling was conducted over one week in each building, during all four seasons. This paper presents the study methods and selected results from the first round of sampling (November 1996 to April 1997). Air flow and recirculation rates were quite variable, with the proportion of outdoor air provided to occupants ranging from 10 to 79 CFM/person. Carbon dioxide, carbon monoxide, and temperature were within ranges anticipated for nonproblem buildings. Relative humidity was low, ranging from 11.7 to 24.0 percent. Indoor geometric mean concentrations of total volatile organic compounds (TVOCs) ranged from 73 to 235 microg/m3. The most prevalent compounds included xylene, toluene, 2-propanol, limonene, and heptane. Geometric mean formaldehyde concentrations ranged from 1.7 to 13.3 microg/m3, and mean acetaldehyde levels ranged from <3.0 to 7.5 microg/m3. Airborne concentrations of culturable bacteria and fungi were low, with no samples exceeding 150 CFU/m3. Total (direct count) bioaerosols were more variable, ranging from 5010 to 10,700 organisms/m3. Geometric mean endotoxin concentrations ranged from 0.5 to 3.0 EU/m3. Respirable particulates (PM10) were low (14 to 36 microg/m3). Noise levels ranged from 48 to 56 dBA, with mean light values ranging from 200 to 420 lux. Environmental parameters were significantly correlated with each other. The prevalence of upper respiratory symptoms (dry eyes, runny nose), central nervous system symptoms (headache, irritability), and musculoskeletal symptoms (pain/stiffness in shoulders/neck) were elevated compared to other studies using similar questionnaires. Importantly, psychosocial factors were significantly related to increased symptoms in females, while environmental factors were more closely correlated with symptoms in males. Endotoxin concentrations were associated with symptoms in both males and females. These data will help to identify and quantify the relative role of factors that contribute to sick building syndrome. The data collected in this study may also be used to evaluate the effectiveness of current building operation practices, and can be used to prioritize allocations of resources for reduction of risk associated with IEQ complaints.

A fluorescence microscopy based genetic screen to identify mutants altered for interactions with host cells.

The study of microbial intracellular pathogenesis has benefited from the application of immunofluorescence microscopy to characterize interactions of the pathogen with host cells. Unfortunately, immunofluorescence microscopy is impractical for screening the large number of bacterial mutants necessary to represent the entire genome of the pathogen. Screening has been limited due to the lack of materials suitable for high-throughput processing (e.g. 96-well plates) that also possess the optical features needed for high resolution fluorescence microscopy. Recently marketed 96-well Special Optics (SO) plates provide both the 96-well template ideal for high-throughput analysis and optical features suitable for fluorescence microscopy. Until this work, mutants needed for the study of a fluorescence-based virulence phenotype could not be obtained by direct screening approaches. In this study, SO plates were used to examine 11520 individual Salmonella typhimurium MudJ mutants for the loss of the ability to disrupt host cell endocytic compartments. The direct application of the fluorescence phenotype for screening allowed us to obtain a set of mutants to characterize the formation of lysosomal membrane glycoprotein (lgp) containing tubules upon Salmonella infection of HeLa epithelial cells. This approach will facilitate the characterization of a wide range of microbial phenotypes detectable by fluorescence microscopy.

Aggregation of host endosomes by Salmonella requires SPI2 translocation of SseFG and involves SpvR and the fms-aroE intragenic region.

Salmonella-induced aggregation of host endosomal compartments into tubules, termed lgp-tubules, requires sifA and ompR. Lgp-tubules result from Salmonella-directed alteration of the endocytic system and typify the unique intracellular locale where Salmonella replicate. A high-throughput method devised to screen 11 520 MudJ mutants for loss of lgp-tubule formation identified one auxotrophic and nine prototrophic mutants. Molecular characterization identified four new loci required to alter epithelial endocytic structure. Salmonella pathogenicity island 2 (SPI2) is the locus central to the phenotype. A subset of SPI2 effectors is essential: SpiC and SseFG are required, but not SseE. A subset of apparatus proteins is also implicated: SsaJ, L, M, V and P are required. SPI2 was implicated further, as SifA shows similarity with known SPI2 translocation targets, and OmpR regulates SPI2. Another locus lies within the smf-aroE intragenic region. Lgp-tubule formation also involves a locus on the virulence plasmid pSLT. The pSLT-encoded SpvR negatively regulates an unknown repressor of the phenotype located on pSLT. Finally, disruption of carB leads to multiple auxotrophy that prevents lgp-tubule formation. This study demonstrates that lgp-tubule formation is a virulence mechanism that underlies the selective disruption of host endocytic trafficking and is associated with the formation of a replication-permissive locale.

Unravelling sleep problems in treated and untreated children with ADHD.

This study investigated parental perception of sleep problems in stimulant treated and untreated children with Attention Deficit Hyperactivity Disorder (ADHD). Parents of 135 psychiatric clinic referred children and 83 pediatric outpatients completed a sleep questionnaire and the Child Behavior Checklist. Moderate to severe "sleep problems" reportedly occurred at least once a week in 19.3% of children with ADHD, 13.3% of the psychiatric controls, and 6.2% of the pediatric controls. Children with ADHD treated with stimulants were reported to display a higher prevalence of nightly "severe" sleep problems than did untreated children with ADHD. Almost a third (29%) of stimulant treated ADHD children were reported to display increased sleep latency or insomnia every night versus 10% of untreated children with ADHD. Despite the high prevalence of sleep related problems in ADHD, the significance of the association between delayed sleep onset and ADHD with regard to etiology and management of ADHD is still poorly understood.

The third factor of the WISC-III: it's (probably) not freedom from distractibility.

OBJECTIVE: This study examined the ecological validity, construct validity, and diagnostic utility of the third factor of the WISC-III, heuristically labeled "Freedom From Distractibility" (FFD). METHOD: A sample of 200 children, aged 6 to 11 years, with attention-deficit hyperactivity disorder (ADHD) completed the WISC-III, the Wide Range Achievement Test-Revised, and the Test of Variables of Attention. Objective parent and teacher report measures of attention and hyperactivity were completed. RESULTS: Mean FFD scores were significantly lower than other WISC-III factor scores. The diagnostic utility of FFD is limited, however, as the majority of these children did not show a significant relative weakness on this index. Correlational analyses failed to support the concurrent, ecological, or construct validity of the FFD. FFD scores were not correlated with a measure of sustained visual attention. Findings suggest that among children with ADHD, a low FFD score may be associated with the presence of a learning disability or poor academic performance. This finding was maintained after level of general intelligence was statistically controlled. CONCLUSIONS: Clinicians and researchers should not view FFD as a reliable or valid index of attention or as a diagnostic screening measure for identifying children with ADHD.

Validity of DSM-IV attention-deficit/hyperactivity disorder for younger children.

OBJECTIVE: Little is known about the validity of the diagnosis of attention-deficit/hyperactivity disorder (ADHD) in young children. Moreover, the results of the DSM-IV field trials raised concerns that inclusion of the new predominantly hyperactive-impulsive type of ADHD in DSM-IV might increase the likelihood of the diagnosis being given to active but unimpaired preschool and primary school children. METHOD: The validity of DSM-IV criteria for each subtype of ADHD was evaluated in 126 children, aged 4 through 6 years, and 126 matched comparison children. Probands and controls were classified by using structured diagnostic interviews of the parent and a DSM-IV checklist completed by the teacher. RESULTS: Children who met DSM-IV criteria for each subtype of ADHD according to parent and teacher reports differed consistently from controls on a wide range of measures of social and academic impairment, even when other types of psychopathology and other potential confounds were controlled. CONCLUSIONS: When diagnosed by means of a structured diagnostic protocol, all three DSM-IV subtypes of ADHD are valid for 4- through 6-year-old children in the sense of identifying children with lower mean scores on measures of adaptive functioning that are independently associated with ADHD.

Trafficking of porin-deficient Salmonella typhimurium mutants inside HeLa cells: ompR and envZ mutants are defective for the formation of Salmonella-induced filaments.

Outer membrane porin genes of Salmonella typhimurium, including ompC, ompF, and tppB, are regulated by the products of ompB, a two-component regulatory locus encoding OmpR and EnvZ. S. typhimurium ompR mutants are attenuated in mice, but to date no one has studied the intracellular trafficking of S. typhimurium porin-deficient mutants. In this study, isogenic transposon mutants of S. typhimurium with insertions in ompR, envZ, ompF, ompC, ompD, osmZ, and tppB were compared with wild-type SL1344 for trafficking in the human epithelial cell line HeLa. We found that ompR and envZ mutants were reduced or completely inhibited for the formation of Salmonella-induced filaments (Sifs). This result was confirmed with an ompB deletion mutant. Sifs are tubular structures containing lysosomal glycoprotein which are induced specifically by intracellular Salmonella. Genetic analysis showed that the ompR mutation could be complemented in trans by cloned ompR to restore its ability to induce Sifs. In contrast, mutations in the known ompR-regulated genes ompF, ompC, and tppB (as well as the ompR-independent porin gene, ompD) had no effect on Sif formation relative to that of wild-type SL1344, thus indicating that OmpR does not exert its role on these genes to induce Sif formation. The omp mutants studied were able to invade and replicate in HeLa cells at levels comparable to those in wild-type SL1344. We conclude that OmpR and EnvZ appear to regulate Sif formation triggered by intracellular S. typhimurium.

Cloning, nucleotide sequence, and overexpression of smoS, a component of a novel operon encoding an ABC transporter and polyol dehydrogenases of Rhodobacter sphaeroides Si4.

The gene coding for sorbitol dehydrogenase (SDH) of Rhodobacter sphaeroides Si4 was located 55 nucleotides upstream of the mannitol dehydrogenase gene (mtlK) within a previously unrecognized polyol operon. This operon probably consists of all the proteins necessary for transport and metabolization of various polyols. The gene encoding SDH (smoS) was cloned and sequenced. Analysis of the deduced amino acid sequence revealed homology to enzymes of the short-chain dehydrogenase/reductase protein family. For structure analysis of this unique bacterial enzyme, smoS was subcloned into the overexpression vector pET-24a(+) and then overproduced in Escherichia coli BL21(DE3), which yielded a specific activity of 24.8 U/mg of protein and a volumetric yield of 38,000 U/liter. Compared to values derived with the native host, R. sphaeroides, these values reflected a 270-fold increase in expression of SDH and a 971-fold increase in the volumetric yield. SDH was purified to homogeneity, with a recovery of 49%, on the basis of a three-step procedure. Upstream from smoS, another gene (smoK), which encoded a putative ATP-binding protein of an ABC transporter, was identified.

Mannitol dehydrogenase from Rhodobacter sphaeroides Si4: subcloning, overexpression in Escherichia coli and characterization of the recombinant enzyme.

By polymerase chain reaction mutagenesis techniques, an NdeI restriction site was introduced at the initiation codon of the mannitol dehydrogenase (MDH) gene (mtlK) of Rhodobacter sphaeroides Si4. The mtlK gene was then subcloned from plasmid pAK74 into the NdeI site of the overexpression vector pET24a+ to give plasmid pASFG1. Plasmid pASFG1 was introduced into Escherichia coli BL21(DE3), which was grown in a 1.5-1 bioreactor at 37 degrees C and pH 7.0. Overexpression of MDH in Escherichia coli BL21(DE3) [pASFG1] was determined by enzymatic analysis and sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis. Under standard growth conditions, E. coli produced considerable amounts of a polypeptide that correlated with MDH in SDS gels, but the activity yield was low. Decreasing the growth temperature to 27 degrees C and omitting pH regulation resulted in a significant increase in the formation of soluble and enzymatically active MDH up to a specific activity of 12.4 U/mg protein and a yield of 26,000 U/l, which corresponds to 0.38 g/l MDH. This was an 87-fold overexpression of MDH compared to that of the natural host R. sphaeroides Si4, and a 236-fold improvement of the volumetric yield. MDH was purified from E. coli BL21(DE3) [pASFG1] with 67% recovery, using ammonium sulfate precipitation, hydrophobic interaction chromatography, and gel filtration. Partial characterization of the recombinant MDH revealed no significant differences to the wild-type enzyme.

Behavioral effects of liothyronine (L-T3) in children with attention deficit hyperactivity disorder in the presence and absence of resistance to thyroid hormone.

Evidence that the thyroid may play a role in the pathogenesis of attention deficit hyperactivity disorder (ADHD) comes from observations that 48% to 73% of children with the syndrome of resistance to thyroid hormone (RTH) have ADHD. Casual observations in subjects with RTH have suggested that treatment with thyroid hormone may improve the symptoms of ADHD. The aim of this study was to determine whether thyroid hormone has a beneficial effect on the behavior of children with RTH. A prospective, randomized, double-blinded, placebo-controlled, cross-over study was conducted to evaluate the effect of the rapid acting thyroid hormone, liothyronine (L-T3), on the behavior of 8 children with ADHD + RTH, and 9 children with ADHD and normal thyroid function (ADHD Only). Parent and teacher ratings of hyperactivity (Conners scale) and a computerized continuous performance test (CPT) were used as objective measures of hyperactivity, attention and impulsivity. L-T3 had no effect on Conners Hyperactivity Index in 7 of 9 children with ADHD Only; it caused improvement and deterioration in 1 subject each. In contrast, the rating in 5 of 8 subjects with ADHD + RTH showed improvement, whereas 3 of 8 subjects remained unchanged. L-T3 was associated with increased commission errors in 5 of 8 children with ADHD Only and decreased commission errors in 4 of 7 with ADHD + RTH. In children with RTH and ADHD, particularly those that exhibit hyperactivity, L-T3 in supraphysiological doses may be beneficial in reducing hyperactivity and impulsivity. In the majority of children with ADHD who do not have RTH, L-T3 treatment has no effect or may be detrimental.