The Epigenetics of Psychosis: A Structured Review with Representative Loci.

The evidence for an environmental component in chronic psychotic disorders is strong and research on the epigenetic manifestations of these environmental impacts has commenced in earnest. In reviewing this research, the focus is on three genes as models for differential methylation, MCHR1, AKT1 and TDO2, each of which have been investigated for genetic association with psychotic disorders. Environmental factors associated with psychotic disorders, and which interact with these model genes, are explored in depth. The location of transcription factor motifs relative to key methylation sites is evaluated for predicted gene expression results, and for other sites, evidence is presented for methylation directing alternative splicing. Experimental results from key studies show differential methylation: for MCHR1, in psychosis cases versus controls; for AKT1, as a pre-existing methylation pattern influencing brain activation following acute administration of a psychosis-eliciting environmental stimulus; and for TDO2, in a pattern associated with a developmental factor of risk for psychosis, in all cases the predicted expression impact being highly dependent on location. Methylation induced by smoking, a confounding variable, exhibits an intriguing pattern for all three genes. Finally, how differential methylation meshes with Darwinian principles is examined, in particular as it relates to the "flexible stem" theory of evolution.

A Career in Nursing: Calling or Choice?

Is nursing a choice, a vocation, a calling? What is the significance of these terms? Caring remains the underlying foundation of nursing practice that can transform the choice of nursing as a career into a higher calling. Nurses often find life satisfaction in fulfilling God's call to care.

Analysis of DNA and RNA Binding Properties of Borrelia burgdorferi Regulatory Proteins.

Bioinformatic approaches and a large volume of prokaryotic genome sequences have enabled rapid identification of regulatory proteins with features to bind DNA or RNA in a given prokaryote. However, biological relevance of these regulatory proteins requires methods to rapidly purify and determine their binding properties within the physiological context or life style of the organism. Here, we describe the experimental approaches to determine the nucleic acid binding properties of regulatory proteins of Borrelia burgdorferi using Borrelia host-adaptation Re.3gulator (BadR-a DNA binding protein) and Carbon storage regulators A of B. b urgdorferi (CsrABb-an RNA binding protein) as examples. Best laboratory practices associated with overexpression/purification of recombinant borrelial proteins, synthesis of target nucleic acid sequences, and electrophoretic mobility assays to assess the protein/nucleic acid interactions are described. The methods described are intended to facilitate empirical assessment of the binding affinity, co-factor requirements, quality of the interacting partners, and readily modifiable assay conditions to assess the binding properties to define known and unknown regulatory properties of nucleic acid binding proteins of B. burgdorferi.

Homer1a protein expression in schizophrenia, bipolar disorder, and major depression.

In recent years, there was growing interest in postsynaptic density proteins in the central nervous system. Of the most important candidates of this specialized region are proteins belonging to the Homer protein family. This family of scaffolding proteins is suspected to participate in the pathogenesis of a variety of diseases. The present study aims to compare Homer1a expression in the hippocampus and cingulate gyrus of patients with major psychiatric disorders including schizophrenia, bipolar disorder and major depression. Immunohistochemistry was used to analyze changes of Homer1a protein expression in the hippocampal formation and the cingulate gyrus from the respective disease groups. Glial cells of the cingulate gyrus gray matter showed decreased Homer1a levels in bipolar disorder when compared to controls. The same results were seen when comparing cingulate gyrus gray matter glial cells in bipolar disorder with major depression. Stratum oriens glial cells of the hippocampus showed decreased Homer1a levels in bipolar disorder when compared to controls and major depression. Stratum lacunosum glial cells showed decreased Homer1a levels in bipolar disorder when compared to major depression. In stratum oriens interneurons Homer1a levels were increased in all disease groups when compared to controls. Stratum lucidum axons showed decreased Homer1a levels in bipolar disorder when compared to controls. Our data demonstrate altered Homer1a levels in specific brain regions and cell types of patients suffering from schizophrenia, bipolar disorder and major depression. These findings support the role of Homer proteins as interesting candidates in neuropsychiatric pathophysiology and treatment.

Spermine and Spermidine Alter Gene Expression and Antigenic Profile of Borrelia burgdorferi.

Borrelia burgdorferi, the agent of Lyme disease, responds to numerous host-derived signals to alter adaptive capabilities during its enzootic cycle in an arthropod vector and mammalian host. Molecular mechanisms that enable B. burgdorferi to detect, channel, and respond to these signals have become an intense area of study for developing strategies to limit transmission/infection. Bioinformatic analysis of the borrelial genome revealed the presence of polyamine transport components (PotA, PotB, PotC, and PotD), while homologs for polyamine biosynthesis were conspicuously absent. Although potABCD is cotranscribed, the level of PotA was elevated under in vitro growth conditions mimicking unfed ticks compared to the level in fed ticks, while the levels of PotD were similar under the aforementioned conditions in B. burgdorferi Among several polyamines and polyamine precursors, supplementation of spermine or spermidine in the borrelial growth medium induced synthesis of major regulators of gene expression in B. burgdorferi, such as RpoS and BosR, with a concomitant increase in proteins that contribute to colonization and survival of B. burgdorferi in the mammalian host. Short transcripts of rpoS were elevated in response to spermidine, which was correlated with increased protein levels of RpoS. Transcriptional analysis of rpoZ and B. burgdorferirel (relBbu ; bb0198) in the presence of spermidine revealed the interplay of multiple regulatory factors in B. burgdorferi gene expression. The effect of spermidine on the levels of select borrelial proteins was also influenced by serum factors. These studies suggest that multiple host-derived signals/nutrients and their transport systems contribute to B. burgdorferi adaptation during the vector and vertebrate host phases of infection.

Global transcriptome responses including small RNAs during mixed-species interactions with methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

Pseudomonas aeruginosa and Staphylococcus aureus mixed-species biofilm infections are more resilient to biocide attacks compared to their single-species counterparts. Therefore, this study used an in vitro model recapitulating bacterial burdens seen in in vivo infections to investigate the interactions of P. aeruginosa and S. aureus in biofilms. RNA sequencing (RNA-seq) was utilized to identify the entire genomic response, both open reading frames (ORFs) and small RNAs (sRNAs), of each species. Using competitive indexes, transposon mutants validated uncharacterized PA1595 of P. aeruginosa and Panton-Valentine leukocidin ORFs of S. aureus are required for competitive success. Assessing spent media on biofilm development determined that the effects of these ORFs are not solely mediated by mechanisms of secretion. Unlike PA1595, leukocidin (lukS-PV) mutants of S. aureus lack a competitive advantage through contact-mediated mechanisms demonstrated by cross-hatch assays. RNA-seq results suggested that during planktonic mixed-species growth there is a robust genomic response or active combat from both pathogens until a state of equilibrium is reached during the maturation of a biofilm. In mixed-species biofilms, P. aeruginosa differentially expressed only 0.3% of its genome, with most ORFs necessary for growth and biofilm development, whereas S. aureus modulated approximately 5% of its genome, with ORFs suggestive of a phenotype of increased virulence and metabolic quiescence. Specific expression of characterized sRNAs aligned with the genomic response to presumably coordinate the adaptive changes necessary for this homeostatic mixed-species biofilm and sRNAs may provide viable foci for the design of future therapeutics.

RsmW, Pseudomonas aeruginosa small non-coding RsmA-binding RNA upregulated in biofilm versus planktonic growth conditions.

BACKGROUND: Biofilm development, specifically the fundamentally adaptive switch from acute to chronic infection phenotypes, requires global regulators and small non-coding regulatory RNAs (sRNAs). This work utilized RNA-sequencing (RNA-seq) to detect sRNAs differentially expressed in Pseudomonas aeruginosa biofilm versus planktonic state. RESULTS: A computational algorithm was devised to detect and categorize sRNAs into 5 types: intergenic, intragenic, 5'-UTR, 3'-UTR, and antisense. Here we report a novel RsmY/RsmZ-type sRNA, termed RsmW, in P. aeruginosa up-transcribed in biofilm versus planktonic growth. RNA-Seq, 5'-RACE and Mfold predictions suggest RsmW has a secondary structure with 3 of 7 GGA motifs located on outer stem loops. Northern blot revealed two RsmW binding bands of 400 and 120 bases, suggesting RsmW is derived from the 3'-UTR of the upstream hypothetical gene, PA4570. RsmW expression is elevated in late stationary versus logarithmic growth phase in PB minimal media, at higher temperatures (37 °C versus 28 °C), and in both gacA and rhlR transposon mutants versus wild-type. RsmW specifically binds to RsmA protein in vitro and restores biofilm production and reduces swarming in an rsmY/rsmZ double mutant. PA4570 weakly resembles an RsmA/RsmN homolog having 49 % and 51 % similarity, and 16 % and 17 % identity to RsmA and RsmN amino acid sequences, respectively. PA4570 was unable to restore biofilm and swarming phenotypes in ΔrsmA deficient strains. CONCLUSION: Collectively, our study reveals an interesting theme regarding another sRNA regulator of the Rsm system and further unravels the complexities regulating adaptive responses for Pseudomonas species.

Statins reduce spirochetal burden and modulate immune responses in the C3H/HeN mouse model of Lyme disease.

Lyme disease (LD) is a systemic disorder caused by Borrelia burgdorferi. Lyme spirochetes encode for a functional 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR EC 1.1.1.88) serving as a rate limiting enzyme of the mevalonate pathway that contribute to components critical for cell wall biogenesis. Statins have been shown to inhibit B. burgdorferi in vitro. Using a mouse model of Lyme disease, we found that statins contribute to reducing bacterial burden and altering the murine immune response to favor clearance of spirochetes.

Genome Sequence of Highly Virulent Pseudomonas aeruginosa Strain VA-134, Isolated from a Burn Patient.

Infection with Pseudomonas aeruginosa leads to impairment of healing and many deaths in severe burn patients. The phenotypic diversity of P. aeruginosa strains makes it difficult to define a therapeutic strategy. Here we report the genome sequence of a highly virulent strain of P. aeruginosa, VA-134, isolated from a burn patient.

Increased expression of retinoic acid-induced gene 1 in the dorsolateral prefrontal cortex in schizophrenia, bipolar disorder, and major depression.

BACKGROUND: Retinoids regulate gene expression in different cells and tissues at the transcriptional level. Retinoic acid transcriptionally regulates downstream regulatory molecules, including enzymes, transcription factors, cytokines, and cytokine receptors. Animal models indicate an involvement of retinoid signaling pathways in the regulation of synaptic plasticity and learning, especially in the hippocampus. Retinoic acid-inducible or induced gene 1 (RAI-1) is induced during neuronal differentiation, and was associated with the severity of the phenotype and response to medication in schizophrenic patients. METHODS: In the present study, we used immunohistochemistry to investigate the expression of RAI-1 in 60 brains from the Stanley Neuropathology Consortium (15 cases each from controls and from patients with schizophrenia, bipolar disorder, and major depression). Rating scores for density and intensity were determined in the dorsolateral prefrontal cortex. RESULTS: All four groups showed high interindividual variation. RAI-1-positive cells were identified as neurons and astrocytes. Significantly increased intensities in cortical neurons were noted in all three major psychiatric groups compared with controls. The density of RAI-1-positive neurons was increased (P=0.06) in schizophrenia and bipolar disorder. In bipolar disorder, RAI-1-positive astrocytes in gray matter showed a significantly increased intensity and compound value. Thus, a significant increase in the parameters measured was found in schizophrenia, bipolar disorder, and major depression. CONCLUSION: Our study shows a significant increase in expression of RAI-1 in the brains from patients with schizophrenia, bipolar disorder, or major depression. The increased expression might reflect altered signaling pathways, like that for retinoic acid. The underlying mechanisms leading to the increased expression and its functional consequences are so far unknown, and remain to be investigated in future studies.

The chemical interaction between adrenochrome, three different classes of antipsychotic drugs and metabolites of the kynurenine pathway.

Two kynurenine metabolites, 3-hydroxykynurenine and 3-hydroxyanthranilic acid, are known to inhibit melanin polymer formation in in vitro reactions catalyzed by tyrosinase. The present study expands that finding to include inhibition of chlorpromazine-stimulated melanin formation from the endogenous melanin precursor adrenochrome. Several kynurenine pathway metabolites tested had no measurable effect on the reaction: tryptophan, kynurenine, kynurenic acid, quinolinic acid and nicotinic acid. However, at a concentration of 0.5mM in a pH 7.4 reaction mix, 3-hydroxykynurenine exerted~72% inhibition on product formation and the same concentration of 3-hydroxyanthranilic acid caused complete inhibition. Two other classes of antipsychotic drugs were evaluated in this paradigm, represented by olanzapine and minocycline. Although the adrenochrome reaction of both drugs was strongly inhibited by 3-hydroxyanthranilic acid, 3-hydroxykynurenine inhibited product formation from only the minocycline reaction. The results are discussed in terms of the well-studied kynurenine pathway upregulation in psychotic disorders and how such upregulation may either influence the efficacy of antipsychotic drug treatment or relate to the mechanism of action of these drugs.

Evidence for phenotypic plasticity in response to photic cues and the connection with genes of risk in schizophrenia.

Numerous environmental factors have been identified as influential in the development of schizophrenia. Some are byproducts of modern life, yet others were present in our evolutionary past and persist to a lesser degree in the current era. The present study brings together published epidemiological data for schizophrenia and data on variables related to photic input for places of residence across geographical regions, using rainfall as an inverse, proxy measure for light levels. Data were gathered from the literature for two countries, the former Yugoslavia and Ireland, during a time in the early 20th century when mobility was relatively limited. The data for Yugoslavia showed a strong correlation between hospital census rates for schizophrenia (by place of birth) and annual rain (r = 0.96, p = 0.008). In Ireland, the hospital census rates and first admissions for schizophrenia (by place of permanent residence) showed a trend for correlation with annual rain, reaching significance for 1st admissions when the rainfall data was weighted by the underlying population distribution (r = 0.71, p = 0.047). In addition, across the years 1921-1945, birth-year variations in a spring quarter season-of-birth effect for schizophrenia in Ireland showed a trend for correlation with January-March rainfall (r = 0.80, p ≤ 0.10). The data are discussed in terms of the effect of photoperiod on the gestation and behavior of offspring in animals, and the premise is put forth that vestigial phenotypic plasticity for such photic cues still exists in humans. Moreover, genetic polymorphisms of risk identified for psychotic disorders include genes modulated by photoperiod and sunlight intensity. Such a relationship between phenotypic plasticity in response to a particular environmental regime and subsequent natural selection for fixed changes in the environmentally responsive genes, has been well studied in animals and should not be discounted when considering human disease.

Contributions of environmental signals and conserved residues to the functions of carbon storage regulator A of Borrelia burgdorferi.

Carbon storage regulator A of Borrelia burgdorferi (CsrABb) contributes to vertebrate host-specific adaptation by modulating activation of the Rrp2-RpoN-RpoS pathway and is critical for infectivity. We hypothesized that the functions of CsrABb are dependent on environmental signals and on select residues. We analyzed the phenotype of csrABb deletion and site-specific mutants to determine the conserved and pathogen-specific attributes of CsrABb. Levels of phosphate acetyltransferase (Pta) involved in conversion of acetyl phosphate to acetyl-coenzyme A (acetyl-CoA) and posttranscriptionally regulated by CsrABb in the csrABb mutant were reduced from or similar to those in the control strains under unfed- or fed-tick conditions, respectively. Increased levels of supplemental acetate restored vertebrate host-responsive determinants in the csrABb mutant to parental levels, indicating that both the levels of CsrABb and the acetyl phosphate and acetyl-CoA balance contribute to the activation of the Rrp2-RpoN-RpoS pathway. Site-specific replacement of 8 key residues of CsrABb (8S) with alanines resulted in increased levels of CsrABb and reduced levels of Pta and acetyl-CoA, while levels of RpoS, BosR, and other members of rpoS regulon were elevated. Truncation of 7 amino acids at the C terminus of CsrABb (7D) resulted in reduced csrABb transcripts and posttranscriptionally reduced levels of FliW located upstream of CsrABb. Electrophoretic mobility shift assays revealed increased binding of 8S mutant protein to the CsrA binding box upstream of pta compared to the parental and 7D truncated protein. Two CsrABb binding sites were also identified upstream of fliW within the flgK coding sequence. These observations reveal conserved and unique functions of CsrABb that regulate adaptive gene expression in B. burgdorferi.

Borrelia host adaptation Regulator (BadR) regulates rpoS to modulate host adaptation and virulence factors in Borrelia burgdorferi.

The RpoS transcription factor of Borrelia burgdorferi is a 'gatekeeper' because it activates genes required for spirochaetes to transition from tick to vertebrate hosts. However, it remains unknown how RpoS becomes repressed to allow the spirochaetes to transition back from the vertebrate host to the tick vector. Here we show that a putative carbohydrate-responsive regulatory protein, designated BadR (Borrelia host adaptation Regulator), is a transcriptional repressor of rpoS. BadR levels are elevated in B. burgdorferi cultures grown under in vitro conditions mimicking unfed-ticks and badR-deficient strains are defective for growth under these same conditions. Microarray and immunoblot analyses of badR-deficient strains showed upregulation of rpoS and other factors important for virulence in vertebrate hosts, as well as downregulation of putative tick-specific determinants (e.g. linear plasmid 28-4 genes). DNA-binding assays revealed BadR binds to upstream regions of rpoS. Site-directed mutations in BadR and the presence of phosphorylated sugars affected BadR's binding to the rpoS promoters. badR-deficient B. burgdorferi were unable to colonize mice. Several putative tick-specific targets have been identified. Our study identified a novel regulator, BadR, and provides a link between nutritional environmental cues utilized by spirochaetes to adaptation to disparate conditions found in the tick and vertebrate hosts.

On the mechanism of action of antipsychotic drugs: a chemical reaction not receptor blockade.

Over forty years ago, biochemist Lauro Galzigna conducted an in-vitro experiment showing that the antipsychotic chlorpromazine reacted with the putative psychotogen adrenochrome to form a polymer resembling melanin. The field of psychopharmacology has essentially ignored that simple but illustrative experiment in the intervening time. The present study reproduces principle elements of Galzigna's experiment and expands the scope to include the antipsychotic medications olanzapine and minocycline. The rate of reaction was slow, with maximal yield of black polymer being achieved by 4, 10 and 7 days with chlorpromazine, olanzapine and minocycline, respectively. Changing the pH was most informative for chlorpromazine and minocycline reactions, where yield increased sharply between pH 6.1 and 6.9, and decreased slightly between pH 6.9 and 7.8, consistent with reaction profiles expected for aromatic substitution. Preincubation of olanzapine with iodine doubled the polymer yield, facilitated by the addition of iodine to the aromatic ring and presumably followed by its departure as a "leaving group". Increasing the salt concentration 1.5-fold depressed yields for all three drugs, most likely via ionic shielding of charged functional groups, diminishing reactivity. The results are discussed in regards to the mechanism of action of antipsychotic medications, casting doubt on commonly held theories. The time course of the chemical reactions presented here and the concentrations required, are much more consistent with clinical results than are models concerning receptor-mediated mechanisms. Furthermore, minocycline was effective in this model, but does not appear to have affinity for the primary receptor families thought by many to mediate antipsychotic efficacy.

A novel gammaherpesvirus found in oral squamous cell carcinomas in sun bears (Helarctos malayanus).

A novel herpesvirus was detected in sun bears (Helarctos malayanus) with oral squamous cell carcinoma. Five captive sun bears from 4 institutions in the United States presented with oral lesions ranging from erythema and mild erosions to nodular, ulcerated masses. All 5 were diagnosed with squamous cell carcinoma. The tumors were treated with surgical resection but recurrence, local extension, or appearance of new lesions was noted in all cases. Intralesional chemotherapy was administered in 2 cases, and the nonsteroidal anti-inflammatory drug piroxicam was administered in 3 cases. Virus was detected in 4 of the 5 bears' tissue samples using a consensus herpesvirus polymerase chain reaction. Nucleotide sequencing and phylogenetic analysis showed that this herpesvirus is in the subfamily Gammaherpesvirinae and distinct from other known herpesviruses. The association between the herpesvirus and squamous cell carcinoma is unknown. The current study presents a novel gammaherpesvirus within the order Ursidae, with the name Ursid herpesvirus 1 proposed.

Effect of levels of acetate on the mevalonate pathway of Borrelia burgdorferi.

Borrelia burgdorferi, the agent of Lyme disease, is a spirochetal pathogen with limited metabolic capabilities that survives under highly disparate host-specific conditions. However, the borrelial genome encodes several proteins of the mevalonate pathway (MP) that utilizes acetyl-CoA as a substrate leading to intermediate metabolites critical for biogenesis of peptidoglycan and post-translational modifications of proteins. In this study, we analyzed the MP and contributions of acetate in modulation of adaptive responses in B. burgdorferi. Reverse-transcription PCR revealed that components of the MP are transcribed as individual open reading frames. Immunoblot analysis using monospecific sera confirmed synthesis of members of the MP in B. burgdorferi. The rate-limiting step of the MP is mediated by HMG-CoA reductase (HMGR) via conversion of HMG-CoA to mevalonate. Recombinant borrelial HMGR exhibited a K(m) value of 132 µM with a V(max) of 1.94 µmol NADPH oxidized minute(-1) (mg protein)(-1) and was inhibited by statins. Total protein lysates from two different infectious, clonal isolates of B. burgdorferi grown under conditions that mimicked fed-ticks (pH 6.8/37°C) exhibited increased levels of HMGR while other members of the MP were elevated under unfed-tick (pH 7.6/23°C) conditions. Increased extra-cellular acetate gave rise to elevated levels of MP proteins along with RpoS, CsrA(Bb) and their respective regulons responsible for mediating vertebrate host-specific adaptation. Both lactone and acid forms of two different statins inhibited growth of B. burgdorferi strain B31, while overexpression of HMGR was able to partially overcome that inhibition. In summary, these studies on MP and contributions of acetate to host-specific adaptation have helped identify potential metabolic targets that can be manipulated to reduce the incidence of Lyme disease.