Functional importance of conserved nucleotides at the histone RNA 3' processing site.

Histone pre-mRNA 3' processing is controlled by a hairpin element preceding the processing site that interacts with a hairpin-binding protein (HBP) and a downstream spacer element that serves as anchoring site for the U7 snRNP. In addition, the nucleotides following the hairpin and surrounding the processing site (ACCCA'CA) are conserved among vertebrate histone genes. Single to triple nucleotide mutations of this sequence were tested for their ability to be processed in nuclear extract from animal cells. Changing the first four nucleotides had no qualitative and little if any quantitative effects on histone RNA 3' processing in mouse K21 cell extract, where processing of this gene is virtually independent of the HBP. A gel mobility shift assay revealing HBP interactions and a processing assay in HeLa cell extract (where the contribution of HBP to efficient processing is more important) showed that only one of these mutations, predicted to extend the hairpin by one base pair, affected the interaction with HBP. Mutations in the next three nucleotides affected both the cleavage efficiency and the choice of processing sites. Analysis of these novel sites indicated a preference for the nucleotide 5' of the cleavage site in the order A > C > U > G. Moreover, a guanosine in the 3' position inhibited cleavage. The preference for an A is shared with the cleavage/polyadenylation reaction, but the preference order for the other nucleotides is different [Chen F, MacDonald CC, Wilusz J, 1995, Nucleic Acids Res 23:2614-2620].

The functional significance of EEG microstates-Associations with modalities of thinking

The momentary, global functional state of the brain is reflected by its electric field configuration. Cluster analytical approaches consistently extracted four head-surface brain electric field configurations that optimally explain the variance of their changes across time in spontaneous EEG recordings. These four configurations are referred to as EEG microstate classes A, B, C, and D and have been associated with verbal/phonological, visual, attention reorientation, and subjective interoceptive-autonomic processing, respectively. The present study tested these associations via an intra-individual and inter-individual analysis approach. The intra-individual approach tested the effect of task-induced increased modality-specific processing on EEG microstate parameters. The inter-individual approach tested the effect of personal modality-specific parameters on EEG microstate parameters. We obtained multichannel EEG from 61 healthy, right-handed, male students during four eyes-closed conditions: object-visualization, spatial-visualization, verbalization (6 runs each), and resting (7 runs). After each run, we assessed participants' degrees of object-visual, spatial-visual, and verbal thinking using subjective reports. Before and after the recording, we assessed modality-specific cognitive abilities and styles using nine cognitive tests and two questionnaires. The EEG of all participants, conditions, and runs was clustered into four classes of EEG microstates (A, B, C, and D). RMANOVAs, ANOVAs and post-hoc paired t-tests compared microstate parameters between conditions. TANOVAs compared microstate class topographies between conditions. Differences were localized using eLORETA. Pearson correlations assessed interrelationships between personal modality-specific parameters and EEG microstate parameters during no-task resting. As hypothesized, verbal as opposed to visual conditions consistently affected the duration, occurrence, and coverage of microstate classes A and B. Contrary to associations suggested by previous reports, parameters were increased for class A during visualization, and class B during verbalization. In line with previous reports, microstate D parameters were increased during no-task resting compared to the three internal, goal-directed tasks. Topographic differences between conditions concerned particular sub-regions of components of the metabolic default mode network. Modality-specific personal parameters did not consistently correlate with microstate parameters except verbal cognitive style which correlated negatively with microstate class A duration and positively with class C occurrence. This is the first study that aimed to induce EEG microstate class parameter changes based on their hypothesized functional significance. Beyond, the associations of microstate classes A and B with visual and verbal processing, respectively and microstate class D with interoceptive-autonomic processing, our results suggest that a finely-tuned interplay between all four EEG microstate classes is necessary for the continuous formation of visual and verbal thoughts, as well as interoceptive-autonomic processing. Our results point to the possibility that the EEG microstate classes may represent the head-surface measured activity of intra-cortical sources primarily exhibiting inhibitory functions. However, additional studies are needed to verify and elaborate on this hypothesis.

Chow–Liu trees are sufficient predictive models for reproducing key features of functional networks of periictal EEG time-series

Seizure freedom in patients suffering from pharmacoresistant epilepsies is still not achieved in 20–30% of all cases. Hence, current therapies need to be improved, based on a more complete understanding of ictogenesis. In this respect, the analysis of functional networks derived from intracranial electroencephalographic (iEEG) data has recently become a standard tool. Functional networks however are purely descriptive models and thus are conceptually unable to predict fundamental features of iEEG time-series, e.g., in the context of therapeutical brain stimulation. In this paper we present some first steps towards overcoming the limitations of functional network analysis, by showing that its results are implied by a simple predictive model of time-sliced iEEG time-series. More specifically, we learn distinct graphical models (so called Chow–Liu (CL) trees) as models for the spatial dependencies between iEEG signals. Bayesian inference is then applied to the CL trees, allowing for an analytic derivation/prediction of functional networks, based on thresholding of the absolute value Pearson correlation coefficient (CC) matrix. Using various measures, the thus obtained networks are then compared to those which were derived in the classical way from the empirical CC-matrix. In the high threshold limit we find (a) an excellent agreement between the two networks and (b) key features of periictal networks as they have previously been reported in the literature. Apart from functional networks, both matrices are also compared element-wise, showing that the CL approach leads to a sparse representation, by setting small correlations to values close to zero while preserving the larger ones. Overall, this paper shows the validity of CL-trees as simple, spatially predictive models for periictal iEEG data. Moreover, we suggest straightforward generalizations of the CL-approach for modeling also the temporal features of iEEG signals.

Functional interplay of SP family members and nuclear factor Y is essential for transcriptional activation of the human Calreticulin gene

Calreticulin (CALR) is a highly conserved, multifunctional protein involved in a variety of cellular processes including the maintenance of intracellular calcium homeostasis, proper protein folding, differentiation and immunogenic cell death. More recently, a crucial role for CALR in the pathogenesis of certain hematologic malignancies was discovered: in clinical subgroups of acute myeloid leukemia, CALR overexpression mediates a block in differentiation, while somatic mutations have been found in the majority of patients with myeloproliferative neoplasms with nonmutated Janus kinase 2 gene (JAK2) or thrombopoietin receptor gene (MPL). However, the mechanisms underlying CALR promoter activation have insufficiently been investigated so far. By dissecting the core promoter region, we could identify a functional TATA-box relevant for transcriptional activation. In addition, we characterized two evolutionary highly conserved cis-regulatory modules (CRMs) within the proximal promoter each composed of one binding site for the transcription factors SP1 and SP3 as well as for the nuclear transcription factor Y (NFY) and we verified binding of these factors to their cognate sites in vitro and in vivo.

A novel CYP17A1 deletion causes a functional knockout of the steroid enzyme 17-hydroxylase and 17,20-lyase in a Turkish family and illustrates the precise role of the CYP17A1 gene

A novel homozygous long-range deletion of the CYP17A1 gene abolished protein expression and caused the severest form of 17-hydroxylase deficiency in one kindred of a Turkish family. The affected subjects presented with 46,XY sex reversal and 46,XX lack of pubertal development as well as severe hypertension.

A legacy of human-induced ecosystem changes: spatial processes drive the taxonomic and functional diversities of testate amoebae in Sphagnum peatlands of the Galápagos

Aim Our aims were to compare the composition of testate amoeba (TA) communities from Santa Cruz Island, Galápagos Archipelago, which are likely in existence only as a result of anthropogenic habitat transformation, with similar naturally occurring communities from northern and southern continental peatlands. Additionally, we aimed at assessing the importance of niche-based and dispersal-based processes in determining community composition and taxonomic and functional diversity. Location The humid highlands of the central island of Santa Cruz, Galápagos Archipelago. Methods We survey the alpha, beta and gamma taxonomic and functional diversities of TA, and the changes in functional traits along a gradient of wet to dry habitats. We compare the TA community composition, abundance and frequency recorded in the insular peatlands with that recorded in continental peatlands of Northern and Southern Hemispheres. We use generalized linear models to determine how environmental conditions influence taxonomic and functional diversity as well as the mean values of functional traits within communities. We finally apply variance partitioning to assess the relative importance of niche- and dispersal-based processes in determining community composition. Results TA communities in Santa Cruz Island were different from their Northern Hemisphere and South American counterparts with most genera considered as characteristic for Northern Hemisphere and South American Sphagnum peatlands missing or very rare in the Galápagos. Functional traits were most correlated with elevation and site topography and alpha functional diversity to the type of material sampled and site topography. Community composition was more strongly correlated with spatial variables than with environmental ones. Main conclusions TA communities of the Sphagnum peatlands of Santa Cruz Island and the mechanisms shaping these communities contrast with Northern Hemisphere and South American peatlands. Soil moisture was not a strong predictor of community composition most likely because rainfall and clouds provide sufficient moisture. Dispersal limitation was more important than environmental filtering because of the isolation of the insular peatlands from continental ones and the young ecological history of these ecosystems.

Different-sized grazers have distinctive effects on plant functional composition of an African savannah

Grazing ungulates play a key role in many ecosystems worldwide and can form diverse assemblages, such as in African savannahs. In many of these ecosystems, present-day ungulate communities are impoverished subsets of once diverse assemblages. While we know that excluding all ungulates from grasslands can exert major effects on both the structure and composition of the vegetation, how different individual ungulate species may have contrasting effects on grassland communities remains poorly understood. Here, we performed a long-term ‘Russian doll’ grazing exclosure experiment in an African savannah to test for the effects of different size classes of grazers on grassland structure and composition. At five sites, grazer species of decreasing size class (ranging from white rhino to scrub hare) were excluded using four fence types, to experimentally create different realized grazer assemblages. The vegetation structure and the grass functional community composition were characterized in 6 different years over a 10-year period. Additionally, animal footprints were counted to quantify the abundance of different ungulate species in each treatment. We found that while vegetation height was mostly driven by total grazing pressure of all species together, ungulate community composition best explained the functional community composition of grasses. In the short term, smaller ungulate species (‘mesoherbivores’) had strongest effects on vegetation composition, by shifting communities towards dominance by species with low specific leaf area and low nutritional value. In the long term, large grazers had stronger but similar effects on the functional composition of the system. Surprisingly, the largest ‘mega-herbivore’, the white rhinoceros, did not have strong effects on the vegetation structure or composition. Synthesis. Our results support the idea that different size classes of grazers have varying effects on the functional composition of grassland plant communities. Therefore, the worldwide decline in the diversity of ungulate species is expected to have (had) major impacts on community composition and functioning of grassland ecosystems, even if total grazing pressure has remained constant, for example, due to replacement by livestock.

Functional requirements of histone deacetylases in Tup1-Ssn6 repression

The corepressor complex Tup1-Ssn6 regulates many classes of genes in yeast including cell type specific, glucose repressible, and DNA damage inducible. Tup1 and Ssn6 are recruited to target promoters through their interactions with specific DNA binding proteins such as α2, Mig1, and Crt1. Most promoters that are repressed by this corepressor complex exhibit a high degree of nucleosomal organization. This chromatin domain occludes transcription factor access to the promoter element resulting in gene repression. Previous work indicated that Tup1 interacts with underacetylated isoforms of H3 and H4, and that mutation of these histones synergistically compromises repression. These studies predict that Tup1-hypoacetyalted histone interaction is important to the repression mechanism, and in vivo hyperacetylation might compromise the corepressors ability to repress target genes. ^ One way to alter histone acetylation levels in vivo is to alter the balance between histone acetyltransferases and histone deacetylases. To date five histone deacetylases (HDACs) have been identified in yeast Rpd3, Hos1, Hos2, Hos3 and Hda1. Deletion of single or double HDAC genes had little to no effect on Tup1-Ssn6 repression, but simultaneous deletion of three specific activities Rpd3, Hos1, and Hos2 abolished repression in vivo. Promoter regions of Tup1-Ssn6 target genes in these triple deacetylase mutant cells are dramatically hyperacetylated in both H3 and H4. Examination of bulk histone acetylation levels showed that this specific HDAC triple mutant combination (rpd3 hos1 hos2) caused a dramatic and concomitant hyperacetylation of both H3 and H4. The loss of repression in the rpd3 hos1 hos2 cells, but not in other mutants, is consistent with previous observations, which indicate that histones provide redundant functions in the repression mechanism and that high levels of acetylation are required to prevent Tup1 binding. Investigation into a potential direct interaction between the Tup1-Ssn6 corepressor complex and one or more HDAC activities showed that both Rpd3 and Hos2 interact with the corepressor complex in vivo. These findings indicate that Tup1-Ssn6 repression involves the recruitment of histone deacetylase activities to target promoters, where they locally deacetylate histone residues promoting Tup1-histone tail interaction to initiate and/or maintain the repressed state. ^

Physical and functional mapping of a novel tumor suppressor locus for prostate cancer within chromosome 10p12.31-q11

Prostate cancer remains the second leading cause of male cancer deaths in the United States, yet the molecular mechanisms underlying this disease remain largely unknown. Cytogenetic and molecular analyses of prostate tumors suggest a consistent association with the loss of chromosome 10. Previously, we have defined a novel tumor suppressor locus PAC-1 within chromosome 10pter-q11. Introduction of the short arm of chromosome 10 into a prostatic adenocarcinoma cell line PC-3H resulted in dramatic tumor suppression and restoration of a programmed cell death pathway. Using a combined approach of comparative genomic hybridization and microsatellite analysis of PC-3H, I have identified a region of hemizygosity within 10p12-p15. This region has been shown to be involved in frequent loss of heterozygosity in gliomas and melanoma. To functionally dissect the region within chromosome 10p containing PAC-1, we developed a strategy of serial microcell fusion, a technique that allows the transfer of defined fragments of chromosome 10p into PC-3H. Serial microcell fusion was used to transfer defined 10p fragments into a mouse A9 fibrosarcoma cell line. Once characterized by FISH and microsatellite analyses, the 10p fragments were subsequently transferred into PC-3H to generate a panel of microcell hybrid clones containing overlapping deletions of chromosome 10p. In vivo and microsatellite analyses of these PC hybrids identified a small chromosome 10p fragment (an estimated 31 Mb in size inclusive of the centromere) that when transferred into the PC-3H background, resulted in significant tumor suppression and limited a region of functional tumor suppressor activity to chromosome 10p12.31-q11. This region coincides with a region of LOH demonstrated in prostate cancer. These studies demonstrate the utility of this approach as a powerful tool to limit regions of functional tumor suppressor activity. Furthermore, these data used in conjunction with data generated by the Human Genome Project lent a focused approach to identify candidate tumor suppressor genes involved in prostate cancer. ^

Functional analysis of BMP4 signaling in mouse neural development

During early mouse neural development, bone morphogenetic protein (BMP) signaling patterns the dorsal neural tube and defines distinct neural progenitor cell domains along the dorsoventral axis. Unlike the ventral signaling molecule Sonic hedgehog, which has long-range activity by establishing a concentration gradient in the ventral neural tube, these dorsally expressed BMPs appear to have a limited domain of action. This raises questions as to how BMP activity is restricted locally and how restricted BMP signaling directs dorsal neural patterning and differentiation. I hypothesize that BMPs are restricted in the dorsal neural tube for correct dorsoventral patterning. ^ Previous studies have shown that the positively charged basic amino acids located at the N-terminus of several BMPs are essential for heparin binding and diffusion. This provides a novel tool to address these questions. Here I adapted a UAS/GAL4 bigenic mouse system to control the ectopic expression of BMP4 and a mutant form of BMP4 that lacks a subset of the N-terminal basic amino acids. The target genes, UAS-Bmp4 and UAS-mBmp4 , were introduced into the Hprt locus by gene targeting in mouse embryonic stem cells. The expression of the GAL4 transactivator was driven by a roof plate specific Wnt1 promoter. ^ The bigenic mouse embryos exhibit phenotype variations, ranging from mid/hindbrain defects, hemorrhage, and eye abnormalities to vasculture formation. Embryonic death starts around E11.5 because of severe hemorrhage. The different expression levels of the activated transgene may account for the phenotype variation. Further marker analysis reveals that mutant BMP4 induces ectopic expression of the dorsal markers MSX1/2 and PAX7 in the ventral neural tube. In addition, the expression of the ventral neural marker NKX2.2 is affected by the expanded BMP4 activity, indicating that ectopic BMP signaling can antagonize ventral signaling. Comparison of the phenotypes of the Wnt1/ Bmp4 and Wnt1/mBmp4 bigenic embryos that express transgenes at the same level, respectively, shows that mutant BMP4 causes the expansion of dorsal neural fates ventrally while wild type BMP4 does not, suggesting that mutant BMP4 acts farther than wild type BMP4. Together, these data suggest that the N-terminus basic amino acid core controls BMP4 long-range activity in neural development, and that BMP signaling patterns the dorsal neural tube through a secondary signaling pathway that involves homeodomain transcription factors MSX1/2 and PAX7. ^

Functional studies of insulin-like growth factor binding protein 2 pathway in glioma progression

Comparison of gene expressing profiles between gliomas with different grades revealed frequent overexpression of insulin-like growth factor binding protein 2 (IGFBP2) in glioblastomas (GBM), in which uncontrolled cell proliferation, angiogenesis, invasion and anti-apoptosis are hallmarks. Using the glia-specific gene transfer transgenic mouse and the stable LN229(BP2) GBM cell lines, we found that IGFBP2 by itself cannot transform cells in vitro and in vivo. IGFBP2 had growth inhibitory effects on mouse primary neural progenitors, but overexpression of IGFBP2 had no effect on GBM cells. ^ Although IGFBP2 does not initiate gliomagenesis, using tissue array technology, we observed strong correlation between IGFBP2 overexpression and VEGF up-regulation in human diffuse gliomas. Furthermore, overexpression of IGFBP2 in GBM cells not only enhanced VEGF expression but also increased the malignant potential of U87 MG cells in our angiogenesis xenograft animal model. ^ In parallel to these studies, using established stable SNB19 GBM cells that overexpress IGFBP2, we found that IGFBP2 significantly increased invasion by induction of matrix metalloproteinase-2 (MMP-2) as well as other invasion related genes, providing evidence that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and in turn tumor cell invasion. ^ Finally, we found that primary filial cells infected with an anti-sense IGFBP2 construct have markedly increased sensitivity to γ irradiation and reduced Akt activation. On the other hand, SNB19(BP2) stable lines have consistently increased levels of Akt and NFkB activation, suggesting that one possible mechanism for anti-apoptosic function of IGFBP2 is through the activation of Akt and NFkB. Beside this, what is especially interesting is the finding that Akt protein was cleaved and inactivated during apoptosis by caspases, and IGFBP2 can prevent Akt cleavage, revealing another possible mechanism through it IGFBP2 exhibit strong antiapoptotic effects. Our data showed that IGFBP2 is a specific substrate for caspase-3, raising the possibility that IGFBP2 may inhibit apoptosis by a suicide mechanism. ^ In summary, using cellular, genomics, and molecular approaches, this thesis documented the potential roles of IGFBP2 in glioma progression. Our findings shed light on an important biological aspect of glioma progression and may provide new insights useful for the design of novel mechanism-based therapies for GBM. ^

Interrelationships between the structural and functional aspects of social relationships, comorbidity and health related quality of life in breast cancer survivors

It is widely acknowledged in theoretical and empirical literature that social relationships, comprising of structural measures (social networks) and functional measures (perceived social support) have an undeniable effect on health outcomes. However, the actual mechanism of this effect has yet to be clearly understood or explicated. In addition, comorbidity is found to adversely affect social relationships and health related quality of life (a valued outcome measure in cancer patients and survivors). ^ This cross sectional study uses selected baseline data (N=3088) from the Women's Healthy Eating and Living (WHEL) study. Lisrel 8.72 was used for the latent variable structural equation modeling. Due to the ordinal nature of the data, Weighted Least Squares (WLS) method of estimation using Asymptotic Distribution Free covariance matrices was chosen for this analysis. The primary exogenous predictor variables are Social Networks and Comorbidity; Perceived Social Support is the endogenous predictor variable. Three dimensions of HRQoL, physical, mental and satisfaction with current quality of life were the outcome variables. ^ This study hypothesizes and tests the mechanism and pathways between comorbidity, social relationships and HRQoL using latent variable structural equation modeling. After testing the measurement models of social networks and perceived social support, a structural model hypothesizing associations between the latent exogenous and endogenous variables was tested. The results of the study after listwise deletion (N=2131) mostly confirmed the hypothesized relationships (TLI, CFI >0.95, RMSEA = 0.05, p=0.15). Comorbidity was adversely associated with all three HRQoL outcomes. Strong ties were negatively associated with perceived social support; social network had a strong positive association with perceived social support, which served as a mediator between social networks and HRQoL. Mental health quality of life was the most adversely affected by the predictor variables. ^ This study is a preliminary look at the integration of structural and functional measures of social relationships, comorbidity and three HRQoL indicators using LVSEM. Developing stronger social networks and forming supportive relationships is beneficial for health outcomes such as HRQoL of cancer survivors. Thus, the medical community treating cancer survivors as well as the survivor's social networks need to be informed and cognizant of these possible relationships. ^

Comparison of Bispectral Index (BIS) and Patient State Index (PSI) as objective measures of sedation and analgesia in critically ill/injured patients

Critically ill and injured patients require pain relief and sedation to reduce the body's stress response and to facilitate painful diagnostic and therapeutic procedures. Presently, the level of sedation and analgesia is guided by the use of clinical scores which can be unreliable. There is therefore, a need for an objective measure of sedation and analgesia. The Bispectral Index (BIS) and Patient State Index (PSI) were recently introduced into clinical practice as objective measures of the depth of analgesia and sedation. ^ Aim. To compare the different measures of sedation and analgesia (BIS and PSI) to the standard and commonly used modified Ramsay Score (MRS) and determine if the monitors can be used interchangeably. ^ Methods. MRS, BIS and PSI values were obtained in 50 postoperative cardiac surgery patients requiring analgesia and sedation from June to December 2004. The MRS, BIS and PSI values were assessed hourly for up to 6-h by a single observer. ^ The relationship between BIS and PSI values were explored using scatter plots and correlation between MRS, BIS and PSI was determined using Spearman's correlation coefficient. Intra-class correlation (ICC) was used to determine the inter-rater reliability of MRS, BIS and PSI. Kappa statistics was used to further evaluate the agreement between BIS and PSI at light, moderate and deep levels of sedation. ^ Results. There was a positive correlation between BIS and PSI values (Rho = 0.731, p<0.001). Intra-class correlation between BIS and PSI was 0.58, MRS and BIS 0.43 and MRS and PSI 0.27. Using Kappa statistics, agreement between MRS and BIS was 0.35 (95% CI: 0.27–0.43) and for MRS and PSI was 0.21 (95% CI: 0.15–0.28). The kappa statistic for BIS and PSI was 0.45 (95% CI: 0.37–0.52). Receiver operating characteristics (ROC) curves constructed to detect undersedation indicated an area under the curve (AUC) of 0.91 (95% CI = 0.87 to 0.94) for the BIS and 0.84 (95% CI = 0.79 to 0.88) for the PSI. For detection of oversedation, AUC for the BIS was 0.89 (95% CI = 0.84 to 0.92) and 0.80 (95% CI = 0.75 to 0.85) for the PSI. ^ Conclusions. There is a statistically significant positive correlation between the BIS and PSI but poor correlation and poor test agreement between the MRS and BIS as well as MRS and PSI. Both the BIS and PSI demonstrated a high level of prediction for undersedation and oversedation; however, the BIS and PSI can not be considered interchangeable monitors of sedation. ^