DNA methylation is a key epigenetic mark when occurring in the promoter and enhancer regions regulates the accessibility of the binding protein and gene transcription. DNA methylation is inheritable and can be de novo-synthesized, erased and reinstated, making it arguably one of the most dynamic upstream regulators for gene expression and the most influential pacer for development. Recent progress has demonstrated that two forms of cytosine methylation and two pathways for demethylation constitute ample complexity for an instructional program for orchestrated gene expression and development. The forum of the current discussion and review are whether there is such a program, if so what the DNA methylation program entails, and what environment can change the DNA methylation program. The translational implication of the DNA methylation program is also proposed.
epigenetics; neural development; 5-hydroxymethylcytosine; epigenome; environmental factors; DNA demethylation
During hippocampal development, the Cornus Ammonis (CA) and the dentate gyrus (DG) undergo waves of neurogenesis and neuronal migration and maturation independently. This stage is widely known to be vulnerable to environmental stresses, but its underlying mechanism is unclear. Alcohol exposure has been shown to alter the expression of genes that regulate the fate, survival, migration and differentiation of pyramidal and granule cells. Undermining this process might compromise hippocampal development underlying the learning and memory deficits known in Fetal Alcohol Spectrum Disorders (FASD). We have previously demonstrated that DNA methylation was programmed along with neural tube development. Here, we demonstrated that DNA methylation program (DMP) proceeded along with hippocampal neuronal differentiation and maturation, and how this DMP was affected by fetal alcohol exposure. C57BL/6 mice were treated with 4% v/v ethanol through a liquid diet along with pair-fed and chow-fed controls from gestation day (E) 7 to E16. We found that a characteristic DMP, including 5-methylcytidine (5mC), 5-hydroxylmethylcytidine (5hmC) and their binding proteins, led the hippocampal neuronal differentiation and maturation spatiotemporally as indicated by their phenotypic marks in the CA and DG pre- and post-natally. Alcohol hindered the acquisition and progression of methylation marks, and altered the chromatin translocation of these marks in the nucleus, which was correlated with developmental retardation.
Alcohol exposure during development can result in variable growth retardation and facial dysmorphology known as fetal alcohol spectrum disorders. Although the mechanisms underlying the disorder are not fully understood, recent progress has been made that alcohol induces aberrant changes in gene expression and in the epigenome of embryos. To inform the gene and epigenetic changes in alcohol-induced teratology, we used whole-embryo culture to identify the alcohol-signature protein profile of neurulating C6 mice. Alcohol-treated and control cultures were homogenized, isoelectrically focused, and loaded for 2D gel electrophoresis. Stained gels were cross matched with analytical software. We identified 40 differentially expressed protein spots (P < 0.01), and 9 spots were selected for LC/MS-MS identification. Misregulated proteins include serotransferrin, triosephosphate isomerase and ubiquitin-conjugating enzyme E2 N. Misregulation of serotransferrin and triosephosphate isomerase was confirmed with immunologic analysis. Alteration of proteins with roles in cellular function, cell cycle, and the ubiquitin-proteasome pathway was induced by alcohol. Several misregulated proteins interact with effectors of the NF-κB and Myc transcription factor cascades. Using a whole-embryo culture, we have identified misregulated proteins known to be involved in nervous system development and function.
WNT signalling plays a central role in mammalian sex determination by promoting ovarian development and repressing aspects of testis development in the early gonad. Dickkopf homolog 1 (DKK1) is a WNT signalling antagonist that plays critical roles in multiple developmental systems by modulating WNT activity. Here, we examined the role of DKK1 in mouse sex determination and early gonadal development. Dkk1 mRNA was upregulated sex-specifically during testis differentiation, suggesting that DKK1 could repress WNT signalling in the developing testis. However, we observed overtly normal testis development in Dkk1-null XY gonads, and found no significant upregulation of Axin2 or Sp5 that would indicate increased canonical WNT signalling. Nor did we find significant differences in expression of key markers of testis and ovarian development. We propose that DKK1 may play a protective role that is not unmasked by loss-of-function in the absence of other stressors.
Dkk1; Rspo1; Testis development; Wnt4
Potential epigenetic mechanisms underlying fetal alcohol syndrome (FAS) include alcohol-induced alterations of methyl metabolism, resulting in aberrant patterns of DNA methylation and gene expression during development. Having previously demonstrated an essential role for epigenetics in neural stem cell (NSC) development and that inhibiting DNA methylation prevents NSC differentiation, here we investigated the effect of alcohol exposure on genome-wide DNA methylation patterns and NSC differentiation.
NSCs in culture were treated with or without a 6-hr 88mM (“binge-like”) alcohol exposure and examined at 48 hrs, for migration, growth, and genome-wide DNA methylation. The DNA methylation was examined using DNA-methylation immunoprecipitation (MeDIP) followed by microarray analysis. Further validation was performed using Independent Sequenom analysis.
NSC differentiated in 24 to 48 hrs with migration, neuronal expression, and morphological transformation. Alcohol exposure retarded the migration, neuronal formation, and growth processes of NSC, similar to treatment with the methylation inhibitor 5-aza-cytidine. When NSC departed from the quiescent state, a genome-wide diversification of DNA methylation was observed—that is, many moderately methylated genes altered methylation levels and became hyper- and hypomethylated. Alcohol prevented many genes from such diversification, including genes related to neural development, neuronal receptors, and olfaction, while retarding differentiation. Validation of specific genes by Sequenom analysis demonstrated that alcohol exposure prevented methylation of specific genes associated with neural development [cutl2 (cut-like 2), Igf1 (insulin-like growth factor 1), Efemp1 (epidermal growth factor-containing fibulin-like extracellular matrix protein 1), and Sox 7 (SRY-box containing gene 7)]; eye development, Lim 2 (lens intrinsic membrane protein 2); the epigenetic mark Smarca2 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2); and developmental disorder [Dgcr2 (DiGeorge syndrome critical region gene 2)]. Specific sites altered by DNA methylation also correlated with transcription factor binding sites known to be critical for regulating neural development.
The data indicate that alcohol prevents normal DNA methylation programming of key neural stem cell genes and retards NSC differentiation. Thus, the role of DNA methylation in FAS warrants further investigation.
Epigenetics; Epigenomics; MeDIP-Chip; Neural development; Fetal alcohol syndrome
Alcohol consumption during pregnancy causes Fetal Alcohol Spectrum Disorder (FASD), which includes a range of developmental deficits. Fetal alcohol syndrome (FAS) is the most severe form of FASD and can be diagnosed with pathognomonic facial features (smooth philtrum, short palpebral fissure, and thin upper vermilion). However, many children with developmental damage due to prenatal alcohol exposure exhibit no, or only a subset, of the above features, making diagnosis difficult. This study explored novel analyses to quantify the effect of a known dose of alcohol on specific facial measurements in sub-strains C57BL/B6J (B6J) and C57BL/6NHsd (B6N) mice. Mouse dams were provided alcohol (Alc) consisting of 4.8% (v/v) alcohol in a liquid diet for 16 days pre-pregnancy, chow and water diet during mating, and the alcohol liquid diet reinstated on gestational days 7(E7) to E17. Treatment controls included a pair-fed (PF) group given matched volumes of an alcohol-free liquid diet made isocalorically, and a group given ad lib access to lab chow and water (Chow). Maternal diet intake (Alc and PF), blood alcohol concentrations (BACs), embryo weights, and 15 morphometric facial measurements for E17 embryos were analyzed. B6N dams drank more alcohol during pregnancy and generated higher BAC than B6J dams. Both the Alc and PF treatments induced significant reductions in embryo weights relative to Chow in both sub-strains. Alcohol treatments produced significant changes, relative to controls, in four of the 15 facial measures for the B6N sub-strain, but only in two measures for the B6J sub-strain. Discriminant analysis demonstrated successful classification of the B6N alcohol-exposed versus non-alcohol exposed embryos with high sensitivity (86%), and specificity (80%), and overall classification (total correct 83%), while, B6J mice yielded sensitivity of 80%, specificity 78%, and overall correct classification in 79%. In addition, B6N mice showed significantly more effects of pair feeding on these facial measures than did B6J, suggesting that the B6N sub-strain may be more vulnerable to nutritional stress during pregnancy. Overall, these data indicate that B6N and B6J mice were both vulnerable to alcohol but show differences in the severity and location of alcohol-induced dysmorphic facial features and may parallel findings from human studies comparing different ethnic groups. Furthermore these findings suggest that discriminant analysis may be useful in predicting alcohol exposure in either mouse sub-strain.
facial development; genetic variance; morphometrics; anthropometry; diagnosis; craniofacial; translational study
We have previously demonstrated that alcohol exposure at early neurulation induces growth retardation, neural tube abnormalities, and alteration of DNA methylation. To explore the global gene expression changes which may underline these developmental defects, microarray analyses were performed in a whole embryo mouse culture model that allows control over alcohol and embryonic variables.
Alcohol caused teratogenesis in brain, heart, forelimb, and optic vesicle; a subset of the embryos also showed cranial neural tube defects. In microarray analysis (accession number GSM9545), adopting hypothesis-driven Gene Set Enrichment Analysis (GSEA) informatics and intersection analysis of two independent experiments, we found that there was a collective reduction in expression of neural specification genes (neurogenin, Sox5, Bhlhe22), neural growth factor genes [Igf1, Efemp1, Klf10 (Tieg), and Edil3], and alteration of genes involved in cell growth, apoptosis, histone variants, eye and heart development. There was also a reduction of retinol binding protein 1 (Rbp1), and de novo expression of aldehyde dehydrogenase 1B1 (Aldh1B1). Remarkably, four key hematopoiesis genes (glycophorin A, adducin 2, beta-2 microglobulin, and ceruloplasmin) were absent after alcohol treatment, and histone variant genes were reduced. The down-regulation of the neurospecification and the neurotrophic genes were further confirmed by quantitative RT-PCR. Furthermore, the gene expression profile demonstrated distinct subgroups which corresponded with two distinct alcohol-related neural tube phenotypes: an open (ALC-NTO) and a closed neural tube (ALC-NTC). Further, the epidermal growth factor signaling pathway and histone variants were specifically altered in ALC-NTO, and a greater number of neurotrophic/growth factor genes were down-regulated in the ALC-NTO than in the ALC-NTC embryos.
This study revealed a set of genes vulnerable to alcohol exposure and genes that were associated with neural tube defects during early neurulation.
The annual meeting of the Fetal Alcohol Spectrum Disorders Study Group (FASDSG) was held on June 28, 2008 in Washington DC, as a satellite to the Research Society on Alcoholism meeting. The FASDSG membership includes clinical, basic and social scientists, who meet to discuss recent advances and issues in FASD research. The main theme of the meeting was “Factors that Influence Brain and Behavioral Development: Implications for Prevention and Intervention.” Two keynote speakers, Dr. Stephen Suomi and Dr. Carl Keen addressed how early environment and nutrition may influence outcome following prenatal alcohol exposure. The final keynote speaker, Kathy Mitchell, addressed issues regarding the relationship between scientists and the families with children with FASD. Members of the FASDSG provided updates on new findings through brief (FASt) data reports, and national agency representative provided updates of activities and funding priorities. Presentations were also made by recipients of the Student Research Merit award and Rosett award.
fetal alcohol syndrome; fetal alcohol spectrum disorders; teratology; ethanol; prenatal
Emerging information indicates that epigenetic modification (i.e. histone code and DNA methylation) may be integral to the maintenance and differentiation of neural stem cells (NSC), but their actual involvements have not yet been illustrated. In this study, we demonstrated the dynamic nature of epigenetic marks during the differentiation of quiescent adult rat NSCs in neurospheres. A subpopulation of OCT4+ NSCs in the neurosphere contained Histone marks, trimethylated Histone 3 on lysine 27 (3me-H3K27), 2me-H3K4, and acetylated H4 (Ac-H4). A major decrease of these marks was found prior to or during differentiation, and was further diminished or reprogrammed in diverse subpopulations of migrated NSCs expressing nestin or β-III-tubulin. The DNA methylation mark 5-methyl-cytosine (5-MeC) and the DNA methyltransferase (DNMT) 1 and 3a expression also correlated to the state of differentiation; they were highly present in undifferentiated NSCs but down-regulated in migrated populations. In contrast, the DNA methyl-CpG-binding protein (MBD1) was low in undifferentiated NSCs in neurospheres, but highly appeared in differentiating NSCs. Furthermore, we found a outward translocation of DNA-methylation marks 5-MeC, DNMT1, DNMT3a, and MBD1 in NSCs as differentiation began and proceeded; the 5-MeC from homogeneous nucleus to peripheral-nucleus, and DMNT1a and 3a from nuclear to cytoplasm, indicating chromatin remodeling. Treatment with DNA a methylation inhibitor, 5-aza-cytidine, altered DNA methylation and disrupted migration as indicated by a reduction of migrated neurons and differentiation. These results indicate that chromatin is dynamically remodeled when NSCs transform from the quiescent state to active growth, and that DNA methylation modification is essential for neural stem cell differentiation.
Neural progenitor cells; DNA methylation; Histone code; DNMT; MBD1; 5-azacytidine
Alcohol exposure during development can cause variable neurofacial deficit and growth retardation known as fetal alcohol spectrum disorders (FASD). The mechanism underlying FASD is not fully understood. However, alcohol, which is known to affect methyl donor metabolism, may induce aberrant epigenetic changes contributing to FASD. Using a tightly controlled whole-embryo culture, we investigated the effect of alcohol exposure (88 mM) at early embryonic neurulation on genome-wide DNA methylation and gene expression in the C57BL/6 mouse. The DNA methylation landscape around promoter CpG islands at early mouse development was analyzed using MeDIP (methylated DNA immunoprecipitation) coupled with microarray (MeDIP-chip). At early neurulation, genes associated with high CpG promoters (HCP) had a lower ratio of methylation but a greater ratio of expression. Alcohol-induced alterations in DNA methylation were observed, particularly in genes on chromosomes 7, 10 and X; remarkably, a >10 fold increase in the number of genes with increased methylation on chromosomes 10 and X was observed in alcohol-exposed embryos with a neural tube defect phenotype compared to embryos without a neural tube defect. Significant changes in methylation were seen in imprinted genes, genes known to play roles in cell cycle, growth, apoptosis, cancer, and in a large number of genes associated with olfaction. Altered methylation was associated with significant (p < 0.01) changes in expression for 84 genes. Sequenom EpiTYPER DNA methylation analysis was used for validation of the MeDIP-chip data. Increased methylation of genes known to play a role in metabolism (Cyp4f13) and decreased methylation of genes associated with development (Nlgn3, Elavl2, Sox21 and Sim1), imprinting (Igf2r) and chromatin (Hist1h3d) was confirmed. In a mouse model for FASD, we show for the first time that alcohol exposure during early neurulation can induce aberrant changes in DNA methylation patterns with associated changes in gene expression, which together may contribute to the observed abnormal fetal development.
fetal alcohol syndrome; epigenetics; MeDIP-chip; sequenom mass array; microarray; neural tube defect
Background: European Organization for Research and Treatment quality of life (QOL) questionnaire (QLQ-C30) has been used frequently and many language versions have been developed, including the simplified Chinese version. It is important to study psychometric properties of the simplified Chinese version from the clinical standpoint.
Patients and methods: The simplified Chinese version of the QLQ-C30 was used in a longitudinal study of 600 patients with five types of cancer: lung, breast, head and neck, colorectal, and stomach. The psychometric properties of the scale were evaluated by indicators of validity and reliability coefficients such as Cronbach's α and Pearson's correlation coefficient r, standardized response mean (SRM), correlational analysis, t-tests, and structural equation models.
Results: Correlation and structural equation model analyses confirmed good construct validity with root mean square error of approximation 0.054, standardized root mean square residual 0.037, non-normed fit index 0.972, and comparative fit index 0.980. The α coefficients for all domains are >0.7 except for cognitive functioning (0.49). The test–retest reliability coefficients for most domains are >0.80 except for appetite loss (0.77) and diarrhea (0.75). The QOL score changes after treatments were of statistical significance with higher or moderate SRM in most domains.
Conclusion: The simplified Chinese version of QLQ-C30 has good validity, reliability, and responsiveness and can be used to measure QOL for Chinese cancer patients.
quality of life; QLICP-GM; structural equation model; standardized response mean; psychometric properties
Prenatal alcohol exposure via maternal liquid diet consumption by C57BL/6 (B6) mice causes conspicuous midline neural tube deficit (dysraphia) and disruption of genesis and development of serotonin (5-HT) neurons in the raphe nuclei, together with brain growth retardation. The current study tested the hypothesis that concurrent treatment with either an activity-dependent neurotrophic factor (ADNF) agonist peptide [SALLRSIPA, (SAL)] or an activity-dependent neurotrophic protein (ADNP) agonist peptide [NAPVSIPQ, (NAP)] would protect against these alcohol-induced deficits in brain development.
Timed-pregnant B6 dams consumed alcohol from embryonic day 7 (E7, before the onset of neurulation) until E15. Fetuses were obtained on E15 and brain sections processed for 5-HT immunocytochemistry, for evaluation of morphologic development of the brainstem raphe and its 5-HT neurons. Additional groups were treated either with SAL or NAP daily from E7 to E15 to assess the potential protective effects of these peptides. Measures of incomplete occlusion of the ventral canal and the frequency and extent of the openings in the rhombencephalon were obtained to assess fetal dysraphia. Counts of 5-HT-immunostained neurons were also obtained in the rostral and caudal raphe.
Prenatal alcohol exposure resulted in abnormal openings along the midline and delayed closure of ventral canal in the brainstem. This dysraphia was associated with reductions in the number of 5-HT neurons both in the rostral raphe nuclei (that gives rise to ascending 5-HT projections) and in the caudal raphe (that gives rise to the descending 5-HT projections). Concurrent treatment of the alcohol-consuming dams with SAL prevented dysraphia and protected against the alcohol-induced reductions in 5-HT neurons in both the rostral and caudal raphe. NAP was less effective in protecting against dysraphia and did not protect against 5-HT loss in the rostral raphe, but did protect against loss in the caudal raphe.
These findings further support the potential usefulness of these peptides for therapeutic interventions in pregnancies at risk for alcohol-induced developmental deficits. Notably, the ascending 5-HT projections of the rostral raphe have profound effects in regulating forebrain development and function, and the descending 5-HT projections of the caudal raphe are critical for regulating respiration. Protection of the rostral 5-HT-system may help prevent structural and functional deficits linked to abnormal forebrain development, and protection of the caudal systems may also reduce the increased risk for sudden infant death syndrome associated with prenatal alcohol exposure.
Fetal Alcohol Syndrome; Dysraphia; Neurotrophic Factor; Neural Tube Defect; Activity-Dependent Neuroprotective Peptide
Preservation of neural stem cells (NSCs) in the adult peripheral nervous system (PNS) has recently been confirmed. However, it is not clear whether peripheral NSCs possess predestined, bona fide phenotypes or a response to innate developmental cues. In this study, we first demonstrated the longevity, multipotency, and high fidelity of sensory features of postmigrating adult dorsal root ganglia (aDRG) stem cells. Derived from aDRG and after 4–5 years in culture without dissociating, the aDRG NSCs were found capable of proliferation, expressing neuroepithelial, neuronal, and glial markers. Remarkably, these aDRG NSCs expressed sensory neuronal markers vesicular glutamate transporter2 (VGluT2—glutamate terminals), transient receptor potential vanilloid1 (TrpV1—capsaicin sensitive), phosphorylated 200 kDa neurofilaments (pNF200—capsaicin insensitive, myelinated), and the serotonin transporter (5-HTT), which normally is transiently expressed in developing DRG. Furthermore, in response to neurotrophins, the aDRG NSCs enhanced TrpV1 expression upon exposure to nerve growth factor (NGF), but not to brain-derived neurotrophic factor (BDNF). On the contrary, BDNF increased the expression of NeuN. Third, the characterization of aDRG NSCs was demonstrated by transplantation of red fluorescent-expressing aDRG NSCs into injured spinal cord. These cells expressed nestin, Hu, and β-III-tubulin (immature neuronal markers), GFAP (astrocyte marker) as well as sensory neural marker TrpV1 (capsaicin sensitive) and pNF200 (mature, capsaicin insensitive, myelinated). Our results demonstrated that the postmigrating neural crest adult DRG stem cells not only preserved their multipotency but also were retentive in sensory potency despite the age and long-term ex vivo status.
Long-term potency; Neural progenitor cells; Spinal cord injury; Sensory neurons; Brain-derived neurotrophic factor (BDNF); Nerve growth factor (NGF)
Summary: PIQA is a quality analysis pipeline designed to examine genomic reads produced by Next Generation Sequencing technology (Illumina G1 Genome Analyzer). A short statistical summary, as well as tile-by-tile and cycle-by-cycle graphical representation of clusters density, quality scores and nucleotide frequencies allow easy identification of various technical problems including defective tiles, mistakes in sample/library preparations and abnormalities in the frequencies of appearance of sequenced genomic reads. PIQA is written in the R statistical programming language and is compatible with bustard, fastq and scarf Illumina G1 Genome Analyzer data formats.
Availability: The PIQA pipeline, installation instructions and examples are available at the supplementary web site (http://bioinfo.uh.edu/PIQA).
Background: Fetal alcohol exposure causes growth deficits, microencephaly, and neurological abnormalities. Although the effects of alcohol on developmental delay and growth-related deficits have been hypothesized, little is understood about how alcohol alters, in particular, the cyclin pathway within the cell cycle, which is critical to proliferation and apoptotic control. In this study, we examined cell cycle proteins pertinent to the G1–S phase transition and apoptosis, to determine if cell cycle misregulation can be attributed to apoptotic induction and growth defects. Methods: We examined cell cycle regulation during G1 and S-phase, and DNA fragmentation damage, using E14 dorsal root ganglia neural stem cells (DRG-NC), and cultured mouse embryos exposed to 200 and 400 mg/dl ethanol. Results: Alcohol-exposed DRG-NC demonstrated a dose-dependent increase in cells expressing increased cyclin D1 protein, and increased DNA fragmentation. Western blot analysis, using embryos, demonstrated an overexpression of cyclin D1, D2, and E2F1, key G1 to S-phase cell cycle regulatory components, and increases in p53, linking the cell cycle and apoptotic pathways. Bromodeoxyuridine incorporation indicated reduced DNA synthesis and growth in several embryonic regions. Propidium iodide staining demonstrated decreases in DNA content and increases in DNA fragmentation in several embryonic tissues. Conclusions: This study indicated that retarded growth of DRG-NC and embryos, induced by alcohol, is associated with altered expression of cell cycle and apoptotic proteins and concurrent inhibition of proliferation and increased DNA fragmentation. We suggest that alcohol induces an increase in cyclin D1 expression, premature S-phase entry, and disjointed DNA synthesis with increased apoptosis.
A CadDX system that confers resistance to Cd2+ and Zn2+ was identified in Streptococcus salivarius 57.I. Unlike with other CadDX systems, the expression of the cad promoter was negatively regulated by CadX, and the repression was inducible by Cd2+ and Zn2+, similar to what was found for CadCA systems. The lower G+C content of the S. salivarius cadDX genes suggests acquisition by horizontal gene transfer.
This is a first report, using our MotifModeler informatics program, to simultaneously identify transcription factor (TF) and microRNA (miRNA) binding sites from gene expression microarray data. Based on the assumption that gene expression is controlled by combinatorial effects of transcription factors binding in the 5'-upstream regulatory region and miRNAs binding in the 3'-untranslated region (3'-UTR), we developed a model for (1) predicting the most influential cis-acting elements under a given biological condition, and (2) estimating the effects of those elements on gene expression levels. The regulatory regions, TF and miRNA, which mediate the differential genes expression in fetal alcohol syndrome were unknown; microarray data from alcohol exposure paradigm was used. The model predicted strong inhibitory effects of 5' cis-acting elements and stimulatory effects of 3'-UTR under alcohol treatment. Current predictive model derived a key hypothesis for the first time a novel role of miRNAs in gene expression changes associated with abnormal mouse embryo development after alcohol exposure. This suggests that disturbance of miRNA functions may contribute to the alcohol-induced developmental deficiencies.
Alcohol is known to affect glutamate transmission. However, how chronic alcohol affects the synaptic structure mediating glutamate transmission is unknown. Repeated alcohol exposure in a subject with familial alcoholic history often leads to alcohol addiction. The current study adopts alcohol-preferring rats, which are known to develop high drinking. Two-photon microscopy analysis indicates that chronic alcohol of 14 weeks either, under continuous alcohol (C-Alc) or with repeated deprivation (RD-Alc), causes dysmorphology–– thickened, beaded, and disoriented dendrites that are reminiscent of reactive astrocytes –– in a subpopulation of medium spiny neurons. The density of dendritic spines was found differentially lower in the nucleus accumbens of RD-Alc and C-Alc groups as compared with those of Water groups. Large-sized spines and multiple-headed spines were increased in the RD-Alc group. The NMDA receptor subunit NR1 proteins, as analyzed with western blot, were upregulated in C-Alc, but not in RD-Alc. The upregulated NMDA receptor subunits of NR1 however, are predominantly a splice variant isoform with truncated exon 21, which is required for membrane-bound trafficking or anchoring into a spine synaptic site. These maladaptations may contribute to the transformation of spines. The changes, in density and head-size of spines and the corresponding NMDA receptors, demonstrated an alteration of microcircuitry for glutamate reception. The current study demonstrates for the first time that chronic alcohol exposure causes structural alteration of dendrites and their spines in the key reward brain region in animals that have a genetic background leading to alcohol addiction.
Glutamate; NMDA; Extended amygdala; repeated deprivation; medium spiny neurons; Two-photon laser microscopy; postsynaptic; GABA; addiction; genetics; P rats
Bacteria-derived glucosyltransferases (Gtf) (EC 18.104.22.168), through synthesizing glucan polymers from sucrose and starch hydrolysates, play an essential role in the etiology and pathogenesis of caries. We attempted to correlate the levels of Gtf in whole saliva with the prevalence of carious lesions in young children. We examined saliva from children who were either free of overt carious lesions, or had severe early childhood caries (mean dmfs = 18.72 ± 9.0 SD), for Gtf by direct enzyme assay. The levels of GtfB, GtfC and GtfD from Streptococcus mutans in the saliva using monoclonal/specific antibodies in an enzyme-linked immunosorbent assay were determined. Multiple logistic regression analyses with model selection showed that GtfB levels correlated with dmfs values of the subjects (p = 0.006). There was no correlation between total Gtf activity as measured by direct enzyme assay and dmfs values. There was a strong correlation between mutans streptococci populations in saliva and caries activity. Collectively, these data show that GtfB levels in saliva correlate strongly with presence of clinical caries and with number of carious lesions in young children. It is also possible to measure different Gtfs, separately, in whole saliva. These observations may have important clinical implications, may lead to development of a chair side caries activity test and support the importance of GtfB in the pathogenesis of dental caries.
Dental caries; Glucosyltransferase B; Saliva; Streptococcus mutans
Alterations in cardiac autonomic control, assessed by changes in heart
rate variability (HRV), provide one plausible mechanistic explanation
for consistent associations between exposure to airborne particulate matter (PM) and
increased risks of cardiovascular mortality. Decreased
HRV has been linked with exposures to PM10 (PM with aerodynamic diameter ≤ 10 μm) and with fine particles (PM
with aerodynamic diameter ≤ 2.5 μm) originating
primarily from combustion sources. However, little is known about
the relationship between HRV and coarse particles [PM with
aerodynamic diameter 10–2.5 μm (PM10–2.5)], which typically result from entrainment of dust and soil or
from mechanical abrasive processes in industry and transportation. We
measured several HRV variables in 19 nonsmoking older adults with coronary
artery disease residing in the Coachella Valley, California, a desert
resort and retirement area in which ambient PM10 consists predominantly of PM10–2.5. Study subjects wore Holter monitors for 24 hr once per week for up to 12 weeks
during spring 2000. Pollutant concentrations were assessed at
nearby fixed-site monitors. We used mixed models that controlled for
individual-specific effects to examine relationships between air pollutants
and several HRV metrics. Decrements in several measures of HRV
were consistently associated with both PM10 and PM10–2.5; however, there was little relationship of HRV variables with PM2.5 concentrations. The magnitude of the associations (~ 1–4% decrease
in HRV per 10-μg/m3 increase in PM10 or PM10–2.5) was comparable with those observed in several other studies of PM. Elevated
levels of ambient PM10–2.5 may adversely affect HRV in older subjects with coronary artery disease.
cardiovascular; coarse particles; epidemiology; heart rate variability; particulate matter; PM; PM2.5; PM10; PM10–2.5
OBJECTIVE—Primary brain tumours may be associated
with coagulation disorders which can pose intraoperative and
postoperative management difficulties. The aim was to evaluate the
coagulation profile of patients with brain tumours undergoing surgery
using thromboelastography (TEG) in combination with simple laboratory tests.
METHODS—Fifty adult patients with primary brain
tumours larger than 4 cm in maximum diameter and no history of
coagulation disorders were studied in a prospective, observational
manner over a one year period. Preoperative, intraoperative, and
postoperative measurements included haemoglobin concentration, platelet
count, prothrombin and partial thromboplastin times, fibrin(ogen)
degradation product concentration, D-dimer concentration, and TEG.
RESULTS—Eleven patients (22%) had abnormal
intraoperative TEGs, of whom six (12%) subsequently developed
haematomas requiring surgical evacuation. The coagulopathy seemed to be
hyperfibrinolysis in two cases (4%) and disseminated intravascular
coagulation in four (8%). There was no preoperative difference in
reaction time (R time) for clot formation between the non-haematoma and
haematoma groups(mean 11.44 (SD 3.42) v 12.33 (2.50) min,
P= 0.46). However, when other preoperative indices were compared, in
the non-haematoma group, K time (time to reach a clot amplitude of 20 mm) was shorter (6.72 (2.15) v 10.56 (3.50) min, P=0.001),
rate of clot growth (å) was faster (43.67°(7.53) v
27.11° (5.42) , P<0.0001) and maximum amplitude of clot strength
(MA) was greater (52.64 (7.85) v 40.33(6.59) mm, P<
0.001). Intraoperatively, R time was significantly shortened in the
non-haematoma group, (7.67 (1.78) min, P<0.0001) unlike the haematoma
group (10.67 (1.58) minutes, P=0.11).
CONCLUSIONS—Although these results indicate a
general hypercoagulability during brain tumour surgery, in certain
cases, a predisposition towards hypocoagulability may exist even before
surgery, detectable only when the physical characteristics of clot
formation are studied by TEG. Judicious replacement of clotting
factors, platelets, and antifibrinolytic agents should be considered
intraoperatively if the TEG is abnormal, without waiting for laboratory
The control of leprosy will be facilitated by the identification of major Mycobacterium leprae-specific antigens which mirror the immune response to the organism across the leprosy spectrum. We have investigated the host response to a 35-kDa protein of M. leprae. Recombinant 35-kDa protein purified from Mycobacterium smegmatis resembled the native antigen in the formation of multimeric complexes and binding by monoclonal antibodies and sera from leprosy patients. These properties were not shared by two forms of 35-kDa protein purified from Escherichia coli. The M. smegmatis-derived 35-kDa protein stimulated a gamma interferon-secreting T-cell proliferative response in the majority of paucibacillary leprosy patients and healthy contacts of leprosy patients tested. Cellular responses to the protein in patients with multibacillary leprosy were weak or absent, consistent with hyporesponsiveness to M. leprae characteristic of this form of the disease. Almost all leprosy patients and contacts recognized the 35-kDa protein by either a T-cell proliferative or an immunoglobulin G antibody response, whereas few tuberculosis patients recognized the antigen. This specificity was confirmed in guinea pigs, with the 35-kDa protein eliciting strong delayed-type hypersensitivity in M. leprae-sensitized animals but not in those sensitized with Mycobacterium tuberculosis or Mycobacterium bovis BCG. Therefore, the M. leprae 35-kDa protein appears to be a major and relatively specific target of the human immune response to M. leprae and is a potential component of a diagnostic test to detect exposure to leprosy or a vaccine to combat the disease.
Hepatitis B virus (HBV) variants with precore mutation(s) resulting in the absence of HBeAg production have been associated with the occurrence of fulminant hepatitis in Japan, Israel, and southern Europe, where the prevalence of this HBV strain appears common. In areas such as United States, where HBV infection is not endemic, the role of this mutant virus in fulminant hepatitis is unknown. We developed an amplification refractory mutation detection system to detect specifically the presence of the G to A mutation at nucleotide position 1898, which is the most frequently observed mutation resulting in a precore stop codon. In addition, this method provided a quantitative measurement of the relative ratio of one strain to the other. Using this system, we tested HBV strains for the presence of the stop codon mutation in sera from 40 cases of fulminant hepatitis B occurring in the United States. Serum HBV DNAs from 28 patients were analyzed successfully. A mixture of wild-type and mutant strains in various ratios were observed in 15 patients, wild type exclusively in 11, and mutant exclusively in 2. Four of these patients had undergone liver transplantation for HBV-associated cirrhosis and developed fulminant HBV-associated hepatitis after transplantation. Pre- and posttransplant serum samples from one patient were analyzed: a mixture of wild-type and mutant HBV strains was detected in both samples. Our study demonstrated that both wild-type and mutant HBV strains are associated with fulminant hepatitis, and that in some patients in the United States, factors other than precore mutations contribute to the development of fulminant hepatitis.
During a five and a half year period, exchange transfusions were performed through the peripheral vessels in 201 of the 214 infants (94%) who required either double volume or partial plasma exchange transfusions. Peripheral vessel exchange transfusion is simple, practicable, and safe with few complications. Technical difficulties in catheterizing the peripheral artery and vein may be overcome by using a 24 gauge catheter, which causes no more catheter induced haemolysis than standard umbilical catheters.