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This review highlights the most important discovery in the reticular activating system (RAS) in the last 10 years, the manifestation of gamma (γ) band activity in cells of the RAS, especially in the pedunculopontine nucleus (PPN), which is in charge of the high frequency states of waking and rapid eye movement sleep. This discovery is critical to understanding the modulation of movement by the RAS and how it sets the background over which we generate voluntary and triggered movements. The presence of γ band activity in the RAS is proposed to participate in the process of preconscious awareness, and provide the essential stream of information for the formulation of many of our actions. Early findings using stimulation of this region to induce arousal, and also to elicit stepping, are placed in this context. This finding also helps explain the novel use of PPN deep brain stimulation for the treatment of Parkinson’s disease, although considerable work remains to be done.

The seventh iteration of the reference genome assembly for Rattus norvegicus-mRatBN7.2-corrects numerous misplaced segments and reduces base-level errors by approximately 9-fold and increases contiguity by 290-fold compared with its predecessor. Gene annotations are now more complete, improving the mapping precision of genomic, transcriptomic, and proteomics datasets. We jointly analyzed 163 short-read whole-genome sequencing datasets representing 120 laboratory rat strains and substrains using mRatBN7.2. We defined ∼20.0 million sequence variations, of which 18,700 are predicted to potentially impact the function of 6,677 genes. We also generated a new rat genetic map from 1,893 heterogeneous stock rats and annotated transcription start sites and alternative polyadenylation sites. The mRatBN7.2 assembly, along with the extensive analysis of genomic variations among rat strains, enhances our understanding of the rat genome, providing researchers with an expanded resource for studies involving rats., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The Hybrid Rat Diversity Panel (HRDP) is a stable and well-characterized set of more than 90 inbred rat strains that can be leveraged for systems genetics approaches to understanding the genetic and genomic variation associated with complex disease. The HRDP exhibits substantial between-strain diversity while retaining substantial within-strain isogenicity, allowing for the precise mapping of genetic variation associated with complex phenotypes and providing statistical power to identify associated variants. In order to robustly identify associated genetic variants, it is important to account for the population structure induced by inbreeding. To this end, we investigate the performance of four plausible approaches towards modeling quantitative traits in the HRDP and quantify their operating characteristics. In particular, we investigate three approaches based on genome-wide mixed model analysis, and one approach based on ordinary least squares linear regression. Towards facilitating study planning and design, we conduct extensive simulations to investigate the power of genetic association analyses in the HRDP, and characterize the impressive attained power. In simulation of eQTL data in the HRDP, we find that a mixed model approach that leverages leave-one-chromosome-out kinship estimation attains the highest power while controlling type I error., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pattee, Vanderlinden, Mahaffey, Hoffman, Tabakoff and Saba.)

Post transcriptional modifications of RNA are powerful mechanisms by which eukaryotes expand their genetic diversity. For instance, researchers estimate that most transcripts in humans undergo alternative splicing and alternative polyadenylation. These splicing events produce distinct RNA molecules, which in turn yield distinct protein isoforms and/or influence RNA stability, translation, nuclear export, and RNA/protein cellular localization. Due to their pervasiveness and impact, we hypothesized that alternative splicing and alternative polyadenylation in brain can contribute to a predisposition for voluntary alcohol consumption. Using the HXB/BXH recombinant inbred rat panel (a subset of the Hybrid Rat Diversity Panel), we generated over one terabyte of brain RNA sequencing data (total RNA) and identified novel splice variants ( via StringTie) and alternative polyadenylation sites ( via aptardi) to determine the transcriptional landscape in the brains of these animals. After establishing an analysis pipeline to ascertain high quality transcripts, we quantitated transcripts and integrated genotype data to identify candidate transcript coexpression networks and individual candidate transcripts associated with predisposition to voluntary alcohol consumption in the two-bottle choice paradigm. For genes that were previously associated with this trait (e.g., Lrap , Ift81 , and P2rx4 ) (Saba et al., Febs. J., 282, 3556-3578, Saba et al., Genes. Brain. Behav., 20, e12698), we were able to distinguish between transcript variants to provide further information about the specific isoforms related to the trait. We also identified additional candidate transcripts associated with the trait of voluntary alcohol consumption (i.e., isoforms of Mapkapk5 , Aldh1a7 , and Map3k7 ). Consistent with our previous work, our results indicate that transcripts and networks related to inflammation and the immune system in brain can be linked to voluntary alcohol consumption. Overall, we have established a pipeline for including the quantitation of alternative splicing and alternative polyadenylation variants in the transcriptome in the analysis of the relationship between the transcriptome and complex traits., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lusk, Hoffman, Mahaffey, Rosean, Smith, Silhavy, Pravenec, Tabakoff and Saba.)

The National Institute on Drug Abuse and Joint Institute for Biological Sciences at the Oak Ridge National Laboratory hosted a meeting attended by a diverse group of scientists with expertise in substance use disorders (SUDs), computational biology, and FAIR (Findability, Accessibility, Interoperability, and Reusability) data sharing. The meeting's objective was to discuss and evaluate better strategies to integrate genetic, epigenetic, and 'omics data across human and model organisms to achieve deeper mechanistic insight into SUDs. Specific topics were to (a) evaluate the current state of substance use genetics and genomics research and fundamental gaps, (b) identify opportunities and challenges of integration and sharing across species and data types, (c) identify current tools and resources for integration of genetic, epigenetic, and phenotypic data, (d) discuss steps and impediment related to data integration, and (e) outline future steps to support more effective collaboration-particularly between animal model research communities and human genetics and clinical research teams. This review summarizes key facets of this catalytic discussion with a focus on new opportunities and gaps in resources and knowledge on SUDs., (© 2021 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.)

LncRNAs are important regulators of quantitative and qualitative features of the transcriptome. We have used QTL and other statistical analyses to identify a gene coexpression module associated with alcohol consumption. The "hub gene" of this module, Lrap (Long non-coding RNA for alcohol preference), was an unannotated transcript resembling a lncRNA. We used partial correlation analyses to establish that Lrap is a major contributor to the integrity of the coexpression module. Using CRISPR/Cas9 technology, we disrupted an exon of Lrap in Wistar rats. Measures of alcohol consumption in wild type, heterozygous and knockout rats showed that disruption of Lrap produced increases in alcohol consumption/alcohol preference. The disruption of Lrap also produced changes in expression of over 700 other transcripts. Furthermore, it became apparent that Lrap may have a function in alternative splicing of the affected transcripts. The GO category of "Response to Ethanol" emerged as one of the top candidates in an enrichment analysis of the differentially expressed transcripts. We validate the role of Lrap as a mediator of alcohol consumption by rats, and also implicate Lrap as a modifier of the expression and splicing of a large number of brain transcripts. A defined subset of these transcripts significantly impacts alcohol consumption by rats (and possibly humans). Our work shows the pleiotropic nature of non-coding elements of the genome, the power of network analysis in identifying the critical elements influencing phenotypes, and the fact that not all changes produced by genetic editing are critical for the concomitant changes in phenotype., (© 2020 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.)

Background: Access to primary care is an important determinant of health, and data are sparse on primary care utilization for people who experience imprisonment. We aimed to describe primary care utilization for persons released from prison, and to compare utilization with the general population., Methods: We linked correctional data for all persons released from provincial prison in Ontario, Canada in 2010 with health administrative data. We matched each person by age and sex with four general population controls. We compared primary care utilization rates using generalized estimating equations. We adjusted rate ratios for aggregated diagnosis groups, to explore this association independent of comorbidity. We examined the proportion of people using primary care using chi squared tests and time to first primary care visit post-release using the Kaplan-Meier method., Results: Compared to the general population controls, the prison release group had significantly increased relative rates of primary care utilization: at 6.1 (95% CI 5.9-6.2) in prison, 3.7 (95% CI 3.6-3.8) in the week post-release and between 2.4 and 2.6 in the two years after prison release. All rate ratios remained significantly increased after adjusting for comorbidity. In the month after release, however, 66.3% of women and 75.5% of men did not access primary care., Conclusions: Primary care utilization is high in prison and post-release for people who experience imprisonment in Ontario, Canada. Increased use is only partly explained by comorbidity. The majority of people do not access primary care in the month after prison release. Future research should identify reasons for increased use and interventions to improve care access for persons who are not accessing care post-release.

Epigenetic mechanisms (i.e., histone post-translational modification and DNA methylation) play a role in regulation of gene expression. The pedunculopontine nucleus (PPN), part of the reticular activating system, manifests intrinsic gamma oscillations generated by voltage-dependent, high threshold N- and P/Q-type Ca 2+ channels. We studied whether PPN intrinsic gamma oscillations are affected by inhibition of histone deacetylation. We showed that, a) acute in vitro exposure to the histone deacetylation Class I and II inhibitor trichostatin A (TSA, 1 μM) eliminated oscillations in the gamma range, but not lower frequencies, b) pre-incubation with TSA (1 μM, 90-120 min) also decreased gamma oscillations, c) Ca 2+ currents (I Ca ) were reduced by TSA, especially on cells with P/Q-type channels, d) a HDAC Class I inhibitor MS275 (500 nM), and a Class IIb inhibitor Tubastatin A (150-500 nM), failed to affect gamma oscillations, e) MC1568, a HDAC Class IIa inhibitor (1 μM), blocked gamma oscillations, and f) the effects of both TSA and MC1568 were blunted by blockade of CaMKII with KN-93 (1 μM). These results suggest a cell type specific effect on gamma oscillations when histone deacetylation is blocked, suggesting that gamma oscillations through P/Q-type channels modulated by CaMKII may be linked to processes related to gene transcription.

Bipolar disorder is characterized by a host of sleep-wake abnormalities that suggests that the reticular activating system (RAS) is involved in these symptoms. One of the signs of the disease is a decrease in high frequency gamma band activity, which accounts for a number of additional deficits. Bipolar disorder has also been found to overexpress neuronal calcium sensor protein 1 (NCS-1). Recent studies showed that elements in the RAS generate gamma band activity that is mediated by high threshold calcium (Ca 2+ ) channels. This mini-review provides a description of recent findings on the role of Ca 2+ and Ca 2+ channels in bipolar disorder, emphasizing the involvement of arousal-related systems in the manifestation of many of the disease symptoms. This will hopefully bring attention to a much-needed area of research and provide novel avenues for therapeutic development., Competing Interests: Conflict of Interest The authors certify that they have NO affiliations with or involvement in any organization or entity with any financial interest, or non-financial interest in the subject matter or materials discussed in this manuscript.

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the centrolateral/parafascicular; Cl/Pf nucleus). The activation of either the PPN or Cl/Pf nuclei in vivo has been described to induce the arousal of the animal and an increment in gamma band activity in the cortical electroencephalogram (EEG). The cellular mechanisms for the generation of gamma band oscillations in Reticular Activating System (RAS) neurons are the same as those found to generate gamma band oscillations in other brains nuclei. During current-clamp recordings of PPN neurons (from parasagittal slices from 9 - 25 day-old rats), the use of depolarizing square steps rapidly activated voltage-dependent potassium channels that prevented PPN neurons from being depolarized beyond -25 mV. Injecting 1 - 2 sec long depolarizing current ramps gradually depolarized PPN membrane potential resting values towards 0 mV. However, injecting depolarizing square pulses generated gamma-band oscillations of membrane potential that showed to be smaller in amplitude compared to the oscillations generated by ramps. All experiments were performed in the presence of voltage-gated sodium channels and fast synaptic receptors blockers. It has been shown that the activation of high-threshold voltage-dependent calcium channels underlie gamma-band oscillatory activity in PPN neurons. Specific methodological and pharmacological interventions are described here, providing the necessary tools to induce and sustain PPN subthreshold gamma band oscillation in vitro.

A 10 Hz rhythm is present in the occipital cortex when the eyes are closed (alpha waves), in the precentral cortex at rest ( mu rhythm), in the superior and middle temporal lobe ( tau rhythm), in the inferior olive (projection to cerebellar cortex), and in physiological tremor (underlying all voluntary movement). These are all considered resting rhythms in the waking brain which are "replaced" by higher frequency activity with sensorimotor stimulation. That is, the 10 Hz frequency fulcrum is replaced on the one hand by lower frequencies during sleep, or on the other hand by higher frequencies during volition and cognition. The 10 Hz frequency fulcrum is proposed as the natural frequency of the brain during quiet waking, but is replaced by higher frequencies capable of permitting more complex functions, or by lower frequencies during sleep and inactivity. At the center of the transition shifts to and from the resting rhythm is the reticular activating system, a phylogenetically preserved area of the brain essential for preconscious awareness.

Gamma rhythms have been proposed to promote the feed forward or "bottom-up" flow of information from lower to higher regions in the brain during perception. On the other hand, beta rhythms have been proposed to represent feed back or "top-down" influence from higher regions to lower. The pedunculopontine nucleus (PPN) has been implicated in sleep-wake control and arousal, and is part of the reticular activating system (RAS). This review describes the properties of the cells in this nucleus. These properties are unique, and perhaps it is the particular characteristics of these cells that allow the PPN to be involved in a host of functions and disorders. The fact that all PPN neurons fire maximally at gamma band frequency regardless of electrophysiological or transmitter type, make this an unusual cell group. In other regions, for example in the cortex, cells with such a property represent only a sub-population. More importantly, the fact that this cell group's functions are related to the capacity to generate coherent activity at a preferred natural frequency, gamma band, speaks volumes about how the PPN functions. We propose that "bottom-up" gamma band influence arises in the RAS and contributes to the build-up of the background of activity necessary for preconscious awareness and gamma activity at cortical levels.

Recent discoveries on the nature of the activity generated by the reticular activating system (RAS) suggest that arousal is much more involved in perception and movement than previously thought. The RAS is not simply an amorphous, unspecific region but rather a distinct group of nuclei with specific cell and transmitter types that control waking and modulate such processes as perception and movement. Thus, disturbances in the RAS will affect a number of neurological disorders. The discovery of gamma band activity in the RAS determined that high threshold calcium channels are responsible for generating gamma band activity in the RAS. Results showing that waking is mediated by CaMKII modulation of P/Q-type channels and REM sleep is modulated by cAMP/PK modulation of N-type channels points to different intracellular pathways influencing each state. Few studies address these important breakthroughs. Novel findings also show that the same primate RAS neurons exhibiting activity in relation to arousal are also involved in locomotion. Moreover, deep brain stimulation of this region, specifically the pedunculopontine nucleus (PPN DBS), in Parkinson's disease has salutary effects on movement, sleep, and cognition. Gamma oscillations appear to participate in sensory perception, problem solving, and memory, and coherence at these frequencies may occur at cortical or thalamocortical levels. However, rather than participating in the temporal binding of sensory events, gamma band activity generated in the RAS may help stabilize coherence related to arousal, providing a stable activation state during waking, and relay such activation to the cortex. Continuous sensory input will thus induce gamma band activity in the RAS to participate in the processes of preconscious awareness, and provide the essential stream of information for the formulation of many of our perceptions and actions. Such a role has received little attention but promises to help understand and treat a number of neurological disorders.

This review highlights the most important discovery in the reticular activating system in the last 10 years, the manifestation of gamma band activity in cells of the reticular activating system (RAS), especially in the pedunculopontine nucleus, which is in charge of waking and rapid eye movement (REM) sleep. The identification of different cell groups manifesting P/Q-type Ca(2+) channels that control waking vs. those that manifest N-type channels that control REM sleep provides novel avenues for the differential control of waking vs. REM sleep. Recent discoveries on the development of this system can help explain the developmental decrease in REM sleep and the basic rest-activity cycle.

This review describes the wake/sleep symptoms present in Parkinson׳s disease, and the role of the pedunculopontine nucleus in these symptoms. The physiology of PPN cells is important not only because it is a major element of the reticular activating system, but also because it is a novel target for deep brain stimulation in the treatment of gait and postural deficits in Parkinson׳s disease. A greater understanding of the physiology of the target nuclei within the brainstem and basal ganglia, amassed over the past decades, has enabled increasingly better patient outcomes from deep brain stimulation for movement disorders.

This review describes the interactions between the pedunculopontine nucleus (PPN), the ventral tegmental area (VTA), and the thalamocortical system. Experiments using modulators of cholinergic receptors in the PPN clarified its role on psychostimulant-induced locomotion. PPN activation was found to be involved in the animal's voluntary search for psychostimulants. Every PPN neuron is known to generate gamma band oscillations. Voltage-gated calcium channels are key elements in the generation and maintenance of gamma band activity of PPN neurons. Calcium channels are also key elements mediating psychostimulant-induced alterations in the thalamic targets of PPN output. Thus, the PPN is a key substrate for maintaining arousal and REM sleep, but also in modulating psychostimulant self-administration.

Schizophrenia is characterized by major sleep/wake disturbances including increased vigilance and arousal, decreased slow wave sleep, and increased REM sleep drive. Other arousal-related symptoms include sensory gating deficits as exemplified by decreased habituation of the blink reflex. There is also dysregulation of gamma band activity, suggestive of disturbances in a host of arousal-related mechanisms. This review examines the role of the reticular activating system, especially the pedunculopontine nucleus, in the symptoms of the disease. Recent discoveries on the physiology of the pedunculopontine nucleus help explain many of these disorders of arousal in, and point to novel therapeutic avenues for, schizophrenia.

We consider insomnia a disorder of waking rather than a disorder of sleep. This review examines the role of the reticular activating system, especially the pedunculopontine nucleus, in the symptoms of insomnia, mainly representing an overactive waking drive. We determined that high frequency activity during waking and REM sleep is controlled by two different intracellular pathways and channel types in PPN cells. We found three different PPN cell types that have one or both channels and may be active during waking only, REM sleep only, or both. These discoveries point to a specific mechanism and novel therapeutic avenues for insomnia.

Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital aspect of metabolomics is sample preparation whereby inconsistent techniques generate unreliable results. This technique encompasses protein precipitation, liquid-liquid extraction, and solid-phase extraction as a means of fractionating metabolites into four distinct classes. Improved enrichment of low abundance molecules with a resulting increase in sensitivity is obtained, and ultimately results in more confident identification of molecules. This technique has been applied to plasma, bronchoalveolar lavage fluid, and cerebrospinal fluid samples with volumes as low as 50 µl.  Samples can be used for multiple downstream applications; for example, the pellet resulting from protein precipitation can be stored for later analysis. The supernatant from that step undergoes liquid-liquid extraction using water and strong organic solvent to separate the hydrophilic and hydrophobic compounds. Once fractionated, the hydrophilic layer can be processed for later analysis or discarded if not needed. The hydrophobic fraction is further treated with a series of solvents during three solid-phase extraction steps to separate it into fatty acids, neutral lipids, and phospholipids. This allows the technician the flexibility to choose which class of compounds is preferred for analysis. It also aids in more reliable metabolite identification since some knowledge of chemical class exists.

Cigarette smoke exposure is linked to the development of a variety of chronic lung and systemic diseases in susceptible individuals. Metabolomics approaches may aid in defining disease phenotypes, may help predict responses to treatment, and could identify biomarkers of risk for developing disease. Using a mouse model of chronic cigarette smoke exposure sufficient to cause mild emphysema, we investigated whether cigarette smoke induces distinct metabolic profiles and determined their persistence following smoking cessation. Metabolites were extracted from plasma and fractionated based on chemical class using liquid-liquid and solid-phase extraction prior to performing liquid chromatography mass spectrometry-based metabolomics. Metabolites were evaluated for statistically significant differences among group means (p-value≤0.05) and fold change ≥1.5). Cigarette smoke exposure was associated with significant differences in amino acid, purine, lipid, fatty acid, and steroid metabolite levels compared to air exposed animals. Whereas 60% of the metabolite changes were reversible, 40% of metabolites remained persistently altered even following 2 months of smoking cessation, including nicotine metabolites. Validation of metabolite species and translation of these findings to human plasma metabolite signatures induced by cigarette smoking may lead to the discovery of biomarkers or pathogenic pathways of smoking-induced disease.

microRNAs (miRNAs) regulate expression by promoting degradation or repressing translation of target transcripts. miRNA target sites have been catalogued in databases based on experimental validation and computational prediction using various algorithms. Several online resources provide collections of multiple databases but need to be imported into other software, such as R, for processing, tabulation, graphing and computation. Currently available miRNA target site packages in R are limited in the number of databases, types of databases and flexibility. We present multiMiR, a new miRNA-target interaction R package and database, which includes several novel features not available in existing R packages: (i) compilation of nearly 50 million records in human and mouse from 14 different databases, more than any other collection; (ii) expansion of databases to those based on disease annotation and drug microRNAresponse, in addition to many experimental and computational databases; and (iii) user-defined cutoffs for predicted binding strength to provide the most confident selection. Case studies are reported on various biomedical applications including mouse models of alcohol consumption, studies of chronic obstructive pulmonary disease in human subjects, and human cell line models of bladder cancer metastasis. We also demonstrate how multiMiR was used to generate testable hypotheses that were pursued experimentally., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The pedunculopontine nucleus (PPN), the cholinergic arm of the reticular activating system, regulates waking and rapid eye movement sleep. Here, we demonstrate immunohistochemical labeling of the leptin receptor signaling isoform in PPN neurons, and investigated the effects of G-protein modulation and the leptin triple antagonist (TA) on the action of leptin in the PPN. Whole-cell patch clamp recordings were performed in rat brainstem slices from 9 to 17 day old pups. Previous results showed that leptin caused a partial blockade of sodium (I(Na)) and h-current (I(H)) in PPN neurons. TA (100 nM) reduced the blockade of I(Na) (~ 50% reduction) and I(H) (~ 93% reduction) caused by leptin. Intracellular guanosine 5'-[β-thio]diphosphate trilithium salt (a G-protein inhibitor) significantly reduced the effect of leptin on I(Na) (~ 60% reduction) but not on I(H) (~ 25% reduction). Intracellular GTPγS (a G-protein activator) reduced the effect of leptin on both I(Na) (~ 80% reduction) and I(H) (~ 90% reduction). These results suggest that the effects of leptin on the intrinsic properties of PPN neurons are leptin receptor- and G-protein dependent. We also found that leptin enhanced NMDA receptor-mediated responses in single neurons and in the PPN population as a whole, an effect blocked by TA. These experiments further strengthen the association between leptin dysregulation and sleep disturbances. Beck et al. investigated the effects of leptin on the intrinsic properties of neurons from the pedunculopontine nucleus (PPN). Leptin reduced the amplitude of voltage-gated sodium (I(Na)) and hyperpolarization-activated cyclic nucleotide-gated HCN (I(H)) channels. These effects were antagonized by a leptin receptor (OB-R) antagonist and by the G-protein antagonist GDPβ., (© 2013 International Society for Neurochemistry.)

Sample preparation remains a challenge in untargeted metabolomics studies and no method currently results in complete extraction of all metabolite classes in human plasma. Because a large variety of molecules, with vast differences in dynamic range, could be involved in human disease, there is an urgent need to develop analytical techniques that result in comprehensive coverage of metabolites. Furthermore, analysis of more focused molecular classes could be necessary to more fully interrogate markers of human disease. However, such techniques, which generally involve multiple steps, often result in high variability. We have optimized a combined liquid-liquid and solid phase extraction method for plasma and have compared that to traditional methanol precipitation using spiked internal standards as controls. This method, based largely on previously published methods, results in 5 separate fractions enriched for aqueous species, phospholipids, fatty acids, neutral lipids, and hydrophobic lipids. Using liquid chromatography mass spectrometry as the analytical method, we detect over 3806 metabolites using the new method versus 1851 metabolites using methanol alone. Qualitative analysis and quantitative analysis of both internal standards (ISTDs) and endogenous metabolites demonstrate excellent reproducibility with CV's below 15% for the combined method compared to 30% using the methanol method. While both methods have applications for clinical metabolomics, fractionation resulted in greater overall coverage and can be used for initial classification of molecular species., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The role of Group IVA cytosolic phospholipase A2 (cPLA2α) activation in regulating macrophage transcriptional responses to Candida albicans infection was investigated. cPLA2α releases arachidonic acid for the production of eicosanoids. In mouse resident peritoneal macrophages, prostacyclin, prostaglandin E2 and leukotriene C4 were produced within minutes of C. albicans addition before cyclooxygenase 2 expression. The production of TNFα was lower in C. albicans-stimulated cPLA2α(+/+) than cPLA2α(-/-) macrophages due to an autocrine effect of prostaglandins that increased cAMP to a greater extent in cPLA2α(+/+) than cPLA2α(-/-) macrophages. For global insight, differential gene expression in C. albicans-stimulated cPLA2α(+/+) and cPLA2α(-/-) macrophages (3 h) was compared by microarray. cPLA2α(+/+) macrophages expressed 86 genes at lower levels and 181 genes at higher levels than cPLA2α(-/-) macrophages (≥2-fold, p<0.05). Several pro-inflammatory genes were expressed at lower levels (Tnfα, Cx3cl1, Cd40, Ccl5, Csf1, Edn1, CxCr7, Irf1, Irf4, Akna, Ifnγ, several IFNγ-inducible GTPases). Genes that dampen inflammation (Socs3, Il10, Crem, Stat3, Thbd, Thbs1, Abca1) and genes involved in host defense (Gja1, Csf3, Trem1, Hdc) were expressed at higher levels in cPLA2α(+/+) macrophages. Representative genes expressed lower in cPLA2α(+/+) macrophages (Tnfα, Csf1) were increased by treatment with a prostacyclin receptor antagonist and protein kinase A inhibitor, whereas genes expressed at higher levels (Crem, Nr4a2, Il10, Csf3) were suppressed. The results suggest that C. albicans stimulates an autocrine loop in macrophages involving cPLA2α, cyclooxygenase 1-derived prostaglandins and increased cAMP that globally effects expression of genes involved in host defense and inflammation.

Galectin-9 is a pleiotropic immune modulator affecting numerous cell types of innate and adaptive immunity. Patients with chronic infection with either hepatitis C virus (HCV) or HIV have elevated circulating levels. Limited data exist on the regulation of natural killer (NK) cell function through interaction with galectin-9. We found that galectin-9 ligation downregulates multiple immune-activating genes, including eight involved in the NK cell-mediated cytotoxicity pathway, impairs lymphokine-activated killing, and decreases the proportion of gamma interferon (IFN-γ)-producing NK cells that had been stimulated with interleukin-12 (IL-12)/IL-15. We demonstrate that the transcriptional and functional changes induced by galectin-9 are independent of Tim-3. Consistent with these results for humans, we find that the genetic absence of galectin-9 in mice is associated with greater IFN-γ production by NK cells and enhanced degranulation. We also show that in the setting of a short-term (4-day) murine cytomegalovirus infection, terminally differentiated NKs accumulate in the livers of galectin-9 knockout mice, and that hepatic NKs spontaneously produce significantly more IFN-γ in this setting. Taken together, our results indicate that galectin-9 engagement impairs the function of NK cells, including cytotoxicity and cytokine production.

Background: Atopic dermatitis (AD) is the most common inflammatory skin disorder in the general population worldwide, and the majority of patients are colonized with Staphylococcus aureus. Eczema herpeticum is a disseminated herpes simplex virus infection that occurs in a small subset of patients., Objectives: The goal was to conduct proteomic profiling of patients with AD based on S. aureus colonization status and history of eczema herpeticum. We hoped to identify new biomarkers for improved diagnosis and prediction of eczema herpeticum and S. aureus susceptibility and to generate new hypotheses regarding disease pathogenesis., Methods: Skin taping was performed on nonlesional skin of nonatopic control subjects and on lesional and nonlesional skin of patients with AD. Subjects were classified according to the history of eczema herpeticum and S. aureus colonization. Proteins were analyzed by using mass spectrometry; diagnostic groups were compared for statistically significant differences in protein expression., Results: Proteins related to the skin barrier (filaggrin-2, corneodesmosin, desmoglein-1, desmocollin-1, and transglutaminase-3) and generation of natural moisturizing factor (arginase-1, caspase-14, and gamma-glutamyl cyclotransferase) were expressed at significantly lower levels in lesional versus nonlesional sites of patients with AD with and without history of eczema herpeticum; epidermal fatty acid-binding protein was expressed at significantly higher levels in patients with methicillin-resistant S. aureus., Conclusion: This noninvasive, semiquantitative profiling method has revealed novel proteins likely involved in the pathogenesis of AD. The lower expression of skin barrier proteins and enzymes involved in the generation of the natural moisturizing factor could further exacerbate barrier defects and perpetuate water loss from the skin. The greater expression of epidermal fatty acid-binding protein, especially in patients colonized with methicillin-resistant S. aureus, might perpetuate the inflammatory response through eicosanoid signaling., (Copyright © 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: New and innovative approaches must be used to rationally allocate scarce resources such as operating room time while simultaneously optimizing the associated financial return. In this article we use the technique of linear programming to optimize allocation of OR time among a group of surgeons based on professional fee generation., Study Design: For the period of December 1, 2000, to July 31, 2002, the following individualized data were obtained for the Division of General Surgery at Duke University Medical Center: allocated OR time (hours), case mix as determined by CPT codes, total OR time used, and normalized professional charges and receipts. Inpatient, outpatient, and emergency cases were included. The Solver linear programming routine in Microsoft Excel (Microsoft Corp.) was used to determine the optimal mix of surgical OR time allocation to maximize professional receipts., Results: Our model of optimized OR allocation would maximize weekly professional revenues at 237,523 US dollars, a potential increase of 15% over the historical value of 207,700 US dollars or an annualized increase of approximately 1.5 million US dollars., Conclusions: Our results suggest that mathematical modeling techniques used in operations research, management science, or decision science may rationally optimize OR allocation to maximize revenue or to minimize costs. These techniques may optimize allocation of scarce resources in the context of the goals specific to individual academic departments of surgery.

Background: The DiGeorge syndrome is a congenital disorder that affects the heart, parathyroid glands, and thymus. In complete DiGeorge syndrome, patients have severely reduced T-cell function., Methods: We treated five infants (age, one to four months) with complete DiGeorge syndrome by transplantation of cultured postnatal thymus tissue. Follow-up evaluations included immune phenotyping and proliferative studies of peripheral-blood mononuclear cells plus biopsy of the thymus allograft. Thymic production of new T cells was assessed in peripheral blood by tests for T-cell-receptor recombination excision circles, which are formed from excised DNA during the rearrangement of T-cell-receptor genes., Results: After the transplantation of thymus tissue, T-cell proliferative responses to mitogens developed in four of the five patients. Two of the patients survived with restoration of immune function; three patients died from infection or abnormalities unrelated to transplantation. Biopsies of grafted thymus in the surviving patients showed normal morphologic features and active T-cell production. In three patients, donor T cells could be detected about four weeks after transplantation, although there was no evidence of graft-versus-host disease on biopsy or at autopsy. In one patient, the T-cell development within the graft was demonstrated to accompany the appearance of recently developed T cells in the periphery and coincided with the onset of normal T-cell function. In one patient, there was evidence of thymus function and CD45RA+CD62L+ T cells more than five years after transplantation., Conclusions: In some infants with profound immunodeficiency and complete DiGeorge syndrome, the transplantation of thymus tissue can restore normal immune function. Early thymus transplantation - before the development of infectious complications - may promote successful immune reconstitution.

Objectives: To answer the following questions: a) Does jugular venous ligation (simulating venovenous extracorporeal life support) alter proximal jugular venous pressure, intracranial pressure, hemispheric cerebral blood flow, or cerebral metabolism? b) Does release of ligation reverse these effects? and c) What are the comparative effects of venous ligation alone vs. venous ligation in combination with arterial ligation?, Design: Prospective, randomized, laboratory investigation., Setting: Multidisciplinary laboratory setting., Subjects: Sixteen swine, weighing 8.1 to 12.1 kg, 3 to 4 wks of age., Interventions: Sixteen swine were randomly assigned to two groups, utilizing a random sequence of vessel ligation. Nine swine underwent occlusion of the right internal and external jugular veins alone (venovenous ligation) followed by release of the occlusion and then occlusion of the right common carotid artery and the right internal and external jugular veins together (venoarterial ligation). The remaining seven swine underwent venoarterial ligation, followed by release of the occlusion and then venovenous ligation. In the experimental group in which venovenous ligation was performed first, the 5, and 30-min release periods after ligation were taken to represent the effects of draining the right jugular vein during venovenous extracorporeal life support., Measurements and Main Results: Data were obtained at baseline, 5, and 30 mins after each ligation/release period. Intracranial pressure, right and left internal jugular pressures/flow rates, and cerebral sinus lactate concentrations were measured. Cerebral blood flow was determined using 133Xe clearance methodology, and the cerebral metabolic rate was calculated. There were no significant differences between the ipsilateral internal jugular pressure or extracorporeal life support at 5 or 30 mins after venovenous or venoarterial ligation compared with baseline values or compared with the release of the ligation at 5 or 30 mins. There was a significant increase in right-side (44.7 +/- 2.0 vs. 38.8 +/- 2.4 mL/kg/min; p < .05) and left-side (42.9 +/- 2.3 vs. 38.7 +/- 1.9 mL/kg/min; p < .05) cerebral blood flow 5 mins after venovenous ligation when compared with baseline values. Similarly, after venoarterial ligation, there was a significant increase in right-side (44.6 +/- 2.2 vs. 38.8 +/- 2.4 mL/kg/min; p < .05) and left-side (43.9 +/- 1.5 vs. 38.7 +/- 1.9 mL/kg/min; p < .05) and cerebral blood flow. Cerebral oxygen consumption was significantly increased after venovenous (2.7 +/- 0.2 to 3.2 +/- 0.2 mL/kg/min; p < .05) and venoarterial (2.7 +/- 0.2 to 3.1 +/- 0.2 mL/kg/min; p < .05) ligation at 5 mins after ligation. This increase persisted at the 30-min period and after release of ligation., Conclusions: Ligation of the right jugular veins alone (venovenous ligation) or jugular veins and right carotid artery (venoarterial ligation) does not increase jugular venous pressures or intracranial pressure. However, this procedure does increase cerebral blood flow and cerebral oxygen consumption. These findings demonstrate that there is adequate decompression of the venous system by the cerebrovascular system and retrograde decompression during extracorporeal life support appears unwarranted.

Significant alterations of liver function have been identified in experimental sepsis including changes in protein and glucose production. The specific changes which are evident in vivo appear to depend upon the specific experimental model and probably represent the relative contribution of hepatocellular function and extrahepatic influences as well as the time course of the septic process. Relatively few studies have focused on function of the whole organ. In an effort to study intrinsic hepatic function during chronic sepsis, control and septic animals (intraabdominal abscess) were studied using the isolated perfused liver model. Basal hepatic oxygen utilization was mildly elevated compared to that in control livers and the oxygen consumption response to a metabolic load was found to be essentially identical to that in control and septic livers. Glucose and albumin production were not substantially different in these two groups. These findings suggest that alterations in liver function following the induction of sepsis may result from extra hepatic factors, since intrinsic liver function appears to be normal.

The consumer-driven Heart Smart Restaurant Program was developed by the Heart and Stroke Foundation of Saskatchewan and the Saskatoon Community Health Unit, with the aim of improving the nutritional quality of food consumed in table-service restaurants. To participate, restaurateurs must agree to provide smoke-free seating and specific more healthful food choices upon request of the customer. The program was evaluated through telephone interviews with 999 individuals in randomly selected households in Saskatoon and Regina. While public awareness of the program was satisfactory, over half of those who knew of the program misunderstood its function, believing that more healthful choices are indicated on the menu. When choosing a restaurant, individuals are not greatly influenced by whether it is Heart Smart, but in Saskatoon, they are more likely to request a more healthful alternative in a Heart Smart restaurant than in others. Implications of these and other findings for future program development and research are discussed.

Enterocolitis is the most common cause of significant morbidity and death in Hirschsprung's disease. Although most cases respond to nasogastric decompression, antibiotics, and colonic evacuation, some children have an unusually fulminant or protracted clinical course. Four cases are reported of pseudomembranous colitis (PMC) that developed 1 to 18 months (mean, 8 months) after definitive surgery for Hirschsprung's disease (Soave endorectal pull-though, 2; Duhamel procedure, 2). While all children presented with fever, abdominal distention, and diarrhea, indistinguishable from typical Hirschsprung's enterocolitis, the clinical course was fulminant in two cases, both of whom died of septic shock. Postmortem examination in both showed extensive colonic pseudomembranes despite identification of Clostridium difficile toxin and subsequent vancomycin therapy (initiated late in the clinical course). Two children in the series had protracted hospitalizations and eventually required diverting enterostomy despite recognition of C difficile toxin and treatment with enteral vancomycin, in one child necessitating multiple courses of antibiotic therapy. Awareness of the virulence of PMC associated with Hirschsprung's disease (even after definitive resection) should prompt submission of stool specimens from any child who presents with enterocolitis for both C difficile culture and toxin levels. On the basis of our experience it is our policy to initiate a prompt course of vancomycin by rectal lavage or nasogastric tube in all children with Hirschsprung's enterocolitis, pending culture results, in view of the significant morbidity and mortality exemplified by cases in this review.

A 3-year-old child was referred with a tentative diagnosis of Hirschsprung's disease because of life-long constipation and "megacolon" demonstrated radiographically. Our rectal biopsy revealed hyperganglionosis suggestive of multiple endocrine neoplasia (MEN) type II B. This, in addition to an elevated serum calcitonin level, prompted surgical removal of her thyroid, which appeared grossly normal but on sectioning, contained a medullary carcinoma in each lobe. She remains disease-free 5 years later. Gastrointestinal symptoms are a significant component of the MEN type II B syndrome, and often antedate the full phenotypic expression of the syndrome and the development of potentially lethal endocrine neoplasms. On the basis of this experience, it is recommended that MEN II B be included in the differential diagnosis of chronic constipation.

The influence of alternate forms of nutritional support on primary tumor growth rate, tumor DNA synthesis rate, and number of lung metastases was examined in Swiss mice bearing subcutaneously implanted Lewis lung carcinoma (LLC). From Day 14 through 22 postimplant, mice were fed by continuous intravenous infusion of dextrose/amino acid (TPN), were offered the same solution from a feeding bottle (PO), were offered a casein-based, solid diet (CASEIN), or were infused with an electrolyte (ELECT) solution while energy and nitrogen were provided from the casein diet. Tumor weight and doubling time were decreased in the PO group compared to CASEIN; however, host weight decreased by 22% in the PO group. Tumor weight and DNA synthesis were decreased in the TPN group compared to CASEIN, and host weight increased by 4.6%. The decreased rate of tumor growth in the PO group was not reflected in a decrease in DNA synthesis, perhaps a result of the circadian pattern of DNA synthesis as previously reported for LLC. The number of metastatic lung nodules was significantly decreased in both the TPN and ELECT groups compared to PO and CASEIN, suggesting that intravenous fluid load rather than nutrient intake was the causative factor. In this host-tumor system, parenteral feeding was associated with a decrease in primary tumor weight and DNA synthesis rate, maintenance of host weight, and a decrease in pulmonary metastatic disease compared to mice fed a conventional diet.

The effect of N-free energy substrate manipulation on tumor growth and metastasis, host maintenance, and intermediary metabolism was studied in parenterally fed Swiss mice bearing subcutaneously implanted Lewis lung carcinoma. Non-N energy was provided from dextrose (CHO), lipid emulsion (FAT), or a 75:25 balanced (BAL) solution, infused from day 14 through day 22 postimplant. Control mice were offered equivalent energy and N from a balanced, casein-based solid diet (CAS). Tumor-doubling time was significantly prolonged in the CHO group compared to FAT and CAS. Pulmonary metastatic nodules were decreased in number in all parenterally fed mice compared to CAS, suggesting that the route of administration altered pulmonary physiology in such a way that the transmissability and/or growth of the tumor cells was inhibited. Tumor-free body weight was maintained in the CHO (+ 1.3%) and BAL (+ 0.3%) groups. However, significant weight loss occurred, despite equal intake, in the FAT (-4.7%) and CAS (-7.5%) groups. The energy appeared to be channeled into nonoxidative pathways, reflected by an increase in hepatic and adipose tissue lipogenesis and hepatic glycogen content. During the period studied, parenteral dextrose/amino acid infusion in this host-tumor system resulted in a decrease in primary tumor growth and optimal host maintenance compared to fat-based TPN and enteral feeding of a balanced, solid diet. Tumor metastasis was decreased in all parenterally fed mice, a phenomenon related to the route of administration and apparently independent of energy substrate.

Filters used in i.v. administration sets were tested for their ability to retain bacterial endotoxins for up to 96 hours of continuous infusion. Inline filters composed of cellulose ester, polyacrylate, polypropylene, polyethylene, or Posidyne Nylon 66 were used during continuous infusion of 5% dextrose injection at 83 mL/hr. One milliliter of inoculum containing 10(8) Escherichia coli was injected through a port upstream from the filter. A bacterial filter was used to monitor the sterility of effluent from the inline filters. The effluent was tested with limulus amebocyte lysate (LAL) that could detect endotoxin concentrations greater than 50 pg/mL. A control solution was monitored for viability of the bacteria throughout the course of the study, and positive endotoxin controls were used to confirm the sensitivity of the LAL. Samples of effluent were tested at 0, 4, 19, 24, 48, 72, and 96 hours. Effluent from all filters was sterile throughout the study. LAL assay indicated that only the effluent from filters containing Posidyne Nylon 66 was free of endotoxins for 96 hours. Effluent from the other filters contained endotoxins immediately after injection of the E. coli. Of the inline filters tested, only the one composed of Posidyne Nylon 66 was able to retain E. coli endotoxin for 96 hours. Further study is needed with E. coli and other microorganisms that are likely contaminants of i.v. infusions.