Integrative analysis of RUNX1 downstream pathways and target genes

BACKGROUND: The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia. RESULTS: Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFbeta, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFbeta. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes. CONCLUSION: This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications.

Functional analysis of anionic phospholipids in cell metabolism in {\it Escherichia coli\/}

The major goal of this work was to understand the function of anionic phospholipid in E. coli cell metabolism. One important finding from this work is the requirement of anionic phospholipid for the DnaA protein-dependent initiation of DNA replication. An rnhA mutation, which bypasses the need for the DnaA protein through induction of constitutive stable DNA replication, suppressed the growth arrest phenotype of a $pgsA$ mutant in which the synthesis of anionic phospholipid was blocked. The maintenance of plasmids dependent on an $oriC$ site for replication, and therefore DnaA protein, was also compromised under conditions of limiting anionic phospholipid synthesis. These results provide support for the involvement of anionic phospholipids in normal initiation of DNA replication at oriC in vivo by the DnaA protein. In addition, structural and functional requirements of two major anionic phospholipids, phosphatidylglycerol and cardiolipin, were examined. Introduction into cells of the ability to make phosphatidylinositol did not suppress the need for the naturally occurring phosphatidylglycerol. The requirement for phosphatidylglycerol was concluded to be more than maintenance of the proper membrane surface charge. Examination of the role of cardiolipin revealed its ability to replace the zwitterionic phospholipid, phosphatidylethanolamine, in maintaining an optimal membrane lipid organization. This work also reported the DNA sequence of the cls gene, which encodes the CL synthase responsible for the synthesis of cardiolipin. ^

A 4 Mb high resolution BAC contig on bovine chromosome 1q12 and comparative analysis with human chromosome 21q22

The bovine RPCI-42 BAC library was screened to construct a sequence-ready ~4 Mb single contig of 92 BAC clones on BTA 1q12. The contig covers the region between the genes KRTAP8P1 and CLIC6. This genomic segment in cattle is of special interest as it contains the dominant gene responsible for the hornless or polled phenotype in cattle. The construction of the BAC contig was initiated by screening the bovine BAC library with heterologous cDNA probes derived from 12 human genes of the syntenic region on HSA 21q22. Contig building was facilitated by BAC end sequencing and chromosome walking. During the construction of the contig, 165 BAC end sequences and 109 single-copy STS markers were generated. For comparative mapping of 25 HSA 21q22 genes, genomic PCR primers were designed from bovine EST sequences and the gene-associated STSs mapped on the contig. Furthermore, bovine BAC end sequence comparisons against the human genome sequence revealed significant matches to HSA 21q22 and allowed the in silico mapping of two new genes in cattle. In total, 31 orthologues of human genes located on HSA 21q22 were directly mapped within the bovine BAC contig, of which 16 genes have been cloned and mapped for the first time in cattle. In contrast to the existing comparative bovine-human RH maps of this region, these results provide a better alignment and reveal a completely conserved gene order in this 4 Mb segment between cattle, human and mouse. The mapping of known polled linked BTA 1q12 microsatellite markers allowed the integration of the physical contig map with existing linkage maps of this region and also determined the exact order of these markers for the first time. Our physical map and transcript map may be useful for positional cloning of the putative polled gene in cattle.

There is a place for radical cystectomy and urinary diversion, including orthotopic bladder substitution, in patients aged 75 and older: Results of a retrospective observational analysis from a high-volume center

INTRODUCTION The incidence of cancer increases with age and owing to the changing demographics we are increasingly confronted with treating bladder cancer in old patients. We report our results in patients>75 years of age who underwent open radical cystectomy (RC) and urinary diversion. MATERIAL AND METHODS From January 2000 to March 2013, a consecutive series of 224 old patients with complete follow-up who underwent RC and urinary diversion (ileal orthotopic bladder substitute [OBS], ileal conduit [IC], and ureterocutaneostomy [UCST]) were included in this retrospective single-center study. End points were the 90-day complication rates (Clavien-Dindo classification), 90-day mortality rates, overall and cancer-specific survival rates, and continence rates (OBS). RESULTS Median age was 79.2 years (range: 75.1-91.6); 35 of the 224 patients (17%) received an OBS, 178 of the 224 patients (78%) an IC, and 11 of the 224 patients (5%) an UCST. The 90-day complication rate was 54.3% in the OBS (major: Clavien grade 3-5: 22.9%, minor: Clavien Grade 1-2: 31.4%), 56.7% in the IC (major: 27%, minor: 29.8%), and 63.6% in the UCST group (major: 36.4%, minor: 27.3%); P = 0.001. The 90-day mortality was 0% in the OBS group, 13% in the IC group, and 10% in the UCST group (P = 0.077). The Glasgow prognostic score was an independent predictor of all survival parameters assessed, including 90-day mortality. Median follow-up was 22 months. Overall and cancer-specific survivals were 90 and 98, 47 and 91, and 11 and 12 months for OBS, IC, and UCST, respectively. In OBS patients, daytime continence was considered as dry in 66% and humid in 20% of patients. Nighttime continence was dry in 46% and humid 26% of patients. CONCLUSION With careful patient selection, oncological and functional outcome after RC can be good in old patients. Old age as the sole criterion should not preclude the indication for RC or the option of OBS. In old patients undergoing OBS, satisfactory continence results can be achieved.

DNA-based analysis of protein variants reveals different genetic variability of the paralogous equine ß-lactoglobulin genes LGB1 and LGB2

The genetic variability of milk protein genes may influence the nutritive value or processing and functional properties of the milk. While numerous protein variants are known in ruminants, knowledge about milk protein variability in horses is still limited. Mare's milk is, however, produced for human consumption in many countries. Beta-lactoglobulin belonging to the protein family of lipocalins, which are known as common food- and airborne allergens, is a major whey protein. It is absent from human milk and thus a key agent in provoking cow's milk protein allergy. Mare's milk is, however, usually better tolerated by most affected people. Several functions of β-lactoglobulin have been discussed, but its ultimate physiological role remains unclear. In the current study, the open reading frames of the two equine β-lactoglobulin paralogues LGB1 and LGB2 were re-sequenced in 249 horses belonging to 14 different breeds in order to predict the existence of protein variants at the DNA-level. Thereby, only a single signal peptide variant of LGB1, but 10 different putative protein variants of LGB2 were identified. In horses, both genes are expressed and in such this is a striking previously unknown difference in genetic variability between the two genes. It can be assumed that LGB1 is the ancestral paralogue, which has an essential function causing a high selection pressure. As horses have very low milk fat content this unknown function might well be related to vitamin-uptake. Further studies are, however, needed, to elucidate the properties of the different gene products.

Functional data analysis approaches for genotype-phenotype association studies from next-generation sequencing

Next-generation DNA sequencing platforms can effectively detect the entire spectrum of genomic variation and is emerging to be a major tool for systematic exploration of the universe of variants and interactions in the entire genome. However, the data produced by next-generation sequencing technologies will suffer from three basic problems: sequence errors, assembly errors, and missing data. Current statistical methods for genetic analysis are well suited for detecting the association of common variants, but are less suitable to rare variants. This raises great challenge for sequence-based genetic studies of complex diseases.^ This research dissertation utilized genome continuum model as a general principle, and stochastic calculus and functional data analysis as tools for developing novel and powerful statistical methods for next generation of association studies of both qualitative and quantitative traits in the context of sequencing data, which finally lead to shifting the paradigm of association analysis from the current locus-by-locus analysis to collectively analyzing genome regions.^ In this project, the functional principal component (FPC) methods coupled with high-dimensional data reduction techniques will be used to develop novel and powerful methods for testing the associations of the entire spectrum of genetic variation within a segment of genome or a gene regardless of whether the variants are common or rare.^ The classical quantitative genetics suffer from high type I error rates and low power for rare variants. To overcome these limitations for resequencing data, this project used functional linear models with scalar response to develop statistics for identifying quantitative trait loci (QTLs) for both common and rare variants. To illustrate their applications, the functional linear models were applied to five quantitative traits in Framingham heart studies. ^ This project proposed a novel concept of gene-gene co-association in which a gene or a genomic region is taken as a unit of association analysis and used stochastic calculus to develop a unified framework for testing the association of multiple genes or genomic regions for both common and rare alleles. The proposed methods were applied to gene-gene co-association analysis of psoriasis in two independent GWAS datasets which led to discovery of networks significantly associated with psoriasis.^

Cloning and molecular analysis of paraxis: A {\it trans\/}-acting factor required for somite maturation

During vertebrate embryogenesis, cells from the paraxial mesoderm coalesce in a rostral-to-caudal progression to form the somites. Subsequent compartmentalization of the somites yields the sclerotome, myotome and dermatome, which give rise to the axial skeleton, axial musculature, and dermis, respectively. Recently, we cloned a novel basic-Helix-Loop-Helix (bHLH) protein, called scleraxis, which is expressed in the sclerotome, in mesenchymal precursors of bone and cartilage, and in connective tissues. This dissertation focuses on the cloning, expression and functional analysis of a bHLH protein termed paraxis, which is nearly identical to scleraxis within the bHLH region but diverges in both its amino and carboxyl termini. During the process of mouse embryogenesis, paraxis transcripts are first detected at about day 7.5 post coitum within the primitive mesoderm lying posterior to the head and heart primordia. Subsequently, paraxis expression progresses caudally through the paraxial mesoderm, immediately preceding somite formation. Paraxis is expressed at high levels in newly formed somites before the first detectable expression of the myogenic bHLH genes, and as the somite becomes compartmentalized, paraxis becomes downregulated within the myotome.^ To determine the function of paraxis during mammalian embryogenesis, mice were generated with a null mutation in the paraxis locus. Paraxis null mice survived until birth, but exhibited severe foreshortening along the anteroposterior axis due to the absence of vertebrae caudal to the midthoracic region. The phenotype also included axial skeletal defects, particularly shortened bifurcated ribs which were detached from the vertebral column, fused vertebrae and extensive truncation and disorganization caudal to the hindlimbs. Mutant neonates also lacked normal levels of trunk muscle and exhibited defects in the dermis as well as the stratification of the epidermis. Analysis of paraxis -/- mutant embryos has revealed a failure of the somites to both properly epithelialize and compartmentalize, resulting in defects in somite-derived cell lineages. These results suggest that paraxis is an essential component of the genetic pathway regulating somitogenesis. ^

Identification and analysis of the plant peroxisomal targeting signal 1 receptor NtPEX5

Protein translocation into peroxisomes takes place via recognition of a peroxisomal targeting signal present at either the extreme C termini (PTS1) or N termini (PTS2) of matrix proteins. In mammals and yeast, the peroxisomal targeting signal receptor, Pex5p, recognizes the PTS1 consisting of -SKL or variants thereof. Although many plant peroxisomal matrix proteins are transported through the PTS1 pathway, little is known about the PTS1 receptor or any other peroxisome assembly protein from plants. We cloned tobacco (Nicotiana tabacum) cDNAs encoding Pex5p (NtPEX5) based on the protein’s interaction with a PTS1-containing protein in the yeast two-hybrid system. Nucleotide sequence analysis revealed that the tobacco Pex5p contains seven tetratricopeptide repeats and that NtPEX5 shares greater sequence similarity with its homolog from humans than from yeast. Expression of NtPEX5 fusion proteins, consisting of the N-terminal part of yeast Pex5p and the C-terminal region of NtPEX5, in a Saccharomyces cerevisiae pex5 mutant restored protein translocation into peroxisomes. These experiments confirmed the identity of the tobacco protein as a PTS1 receptor and indicated that components of the peroxisomal translocation apparatus are conserved functionally. Two-hybrid assays showed that NtPEX5 interacts with a wide range of PTS1 variants that also interact with the human Pex5p. Interestingly, the C-terminal residues of some of these peptides deviated from the established plant PTS1 consensus sequence. We conclude that there are significant sequence and functional similarities between the plant and human Pex5ps.

Crystallization, preliminary X-ray analysis and molecular-replacement solution of haemoglobin-II from the fish matrinxa (Brycon cephalus)

Haemoglobins constitute a set of proteins with interesting structural and functional properties, especially when the two large animal groups reptiles and fishes are focused on. Here, the crystallization and preliminary X-ray analysis of haemoglobin-II from the South American fish matrinxa (Brycon cephalus) is reported. X-ray diffraction data have been collected to 3.0 Angstrom resolution using synchrotron radiation (LNLS). Crystals were determined to belong to space group P2(1) and preliminary structural analysis revealed the presence of two tetramers in the asymmetric unit. The structure was determined using the standard molecular-replacement technique.

Cluster Analysis To Identify Elderly People's Profiles: A Healthcare Strategy Based On Frailty Characteristics.

The new social panorama resulting from aging of the Brazilian population is leading to significant transformations within healthcare. Through the cluster analysis strategy, it was sought to describe the specific care demands of the elderly population, using frailty components. Cross-sectional study based on reviewing medical records, conducted in the geriatric outpatient clinic, Hospital de Clínicas, Universidade Estadual de Campinas (Unicamp). Ninety-eight elderly users of this clinic were evaluated using cluster analysis and instruments for assessing their overall geriatric status and frailty characteristics. The variables that most strongly influenced the formation of clusters were age, functional capacities, cognitive capacity, presence of comorbidities and number of medications used. Three main groups of elderly people could be identified: one with good cognitive and functional performance but with high prevalence of comorbidities (mean age 77.9 years, cognitive impairment in 28.6% and mean of 7.4 comorbidities); a second with more advanced age, greater cognitive impairment and greater dependence (mean age 88.5 years old, cognitive impairment in 84.6% and mean of 7.1 comorbidities); and a third younger group with poor cognitive performance and greater number of comorbidities but functionally independent (mean age 78.5 years old, cognitive impairment in 89.6% and mean of 7.4 comorbidities). These data characterize the profile of this population and can be used as the basis for developing efficient strategies aimed at diminishing functional dependence, poor self-rated health and impaired quality of life.

Screening for Alzheimer`s Disease Among Illiterate Elderly: Accuracy Analysis for Multiple Instruments

One of the challenges in screening for dementia in developing countries is related to performance differences due to educational and cultural factors. This study evaluated the accuracy of single screening tests as well as combined protocols including the Mini-Mental State Examination (MMSE), Verbal Fluency animal category (VF), Clock Drawing test (CDT), and Pfeffer Functional Activities Questionnaire (PFAQ) to discriminate illiterate elderly with and without Alzheimer`s disease (AD) in a clinical sample. Cross-sectional study with 66 illiterate outpatients diagnosed with mild and moderate AD and 40 illiterate normal controls. Diagnosis of AD was based on NINCDS-ADRDA. All patients were submitted to a diagnostic protocol including a clinical interview based on the CAMDEX sections. ROC curves area analyses were carried out to compare sensitivity and specificity for the cognitive tests to differentiate the two groups (each test separately and in two by two combinations). Scores for all cognitive (MMSE, CDT, VF) and functional assessments (PFAQ) were significantly different between the two groups (p < 0.001). The best screening instruments for this sample of illiterate elderly were the MMSE and the PFAQ. The cut-off scores for the MMSE, VF, CDT, and PFAQ were 17.5, 7.5, 2.5, and 11.5, respectively. The most sensitive combination came from the MMSE and PFAQ (94.1%), and the best specificity was observed with the combination of the MMSE and CDT (89%). Illiterate patients can be successfully screened for AD using well-known screening instruments, especially in combined protocols.

Representational Difference Analysis (RDA) reveals differential expression of conserved as well as novel genes during caste-specific development of the honey bee (Apis mellifera L.) ovary

In highly eusocial insects, such as the honey bee, Apis mellifera, the reproductive bias has become embedded in morphological caste differences. These are most expressively denoted in ovary size, with adult queens having large ovaries consisting of 150-200 ovarioles each, while workers typically have only 1-20 ovarioles per ovary. This morphological differentiation is a result of hormonal signals triggered by the diet change in the third larval instar, which eventually generate caste-specific gene expression patterns. To reveal these we produced differential gene expression libraries by Representational Difference Analysis (RDA) for queen and worker ovaries in a developmental stage when cell death is a prominent feature in the ovarioles of workers, whereas all ovarioles are maintained and extend in length in queens. In the queen library, 48% of the gene set represented homologs of known Drosophila genes, whereas in the worker ovary, the largest set (59%) were ESTs evidencing novel genes, not even computationally predicted in the honey bee genome. Differential expression was confirmed by quantitative RT-PCR for a selected gene set, denoting major differences for two queen and two worker library genes. These included two unpredicted genes located in chromosome 11 (Group11.35 and Group11.31, respectively) possibly representing long non-coding RNAs. Being candidates as modulators of ovary development, their expression and functional analysis should be a focal point for future studies. (C) 2011 Elsevier Ltd. All rights reserved.

Multi-dimensional scaling applied to histogram-based DNA analysis

This paper aims to study the relationships between chromosomal DNA sequences of twenty species. We propose a methodology combining DNA-based word frequency histograms, correlation methods, and an MDS technique to visualize structural information underlying chromosomes (CRs) and species. Four statistical measures are tested (Minkowski, Cosine, Pearson product-moment, and Kendall τ rank correlations) to analyze the information content of 421 nuclear CRs from twenty species. The proposed methodology is built on mathematical tools and allows the analysis and visualization of very large amounts of stream data, like DNA sequences, with almost no assumptions other than the predefined DNA “word length.” This methodology is able to produce comprehensible three-dimensional visualizations of CR clustering and related spatial and structural patterns. The results of the four test correlation scenarios show that the high-level information clusterings produced by the MDS tool are qualitatively similar, with small variations due to each correlation method characteristics, and that the clusterings are a consequence of the input data and not method’s artifacts.

Functional and therapeutical implications of ligand recognition by the scavenger-like lymphocyte receptors CD5 and CD6

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Light-distortion analysis as a possible indicator of visual quality after refractive lens exchange with diffractive multifocal intraocular lenses

METHODS: Refractive lens exchange was performed with implantation of an AT Lisa 839M (trifocal) or 909MP (bifocal toric) IOL, the latter if corneal astigmatism was more than 0.75 diopter (D). The postoperative visual and refractive outcomes were evaluated. A prototype light-distortion analyzer was used to quantify the postoperative light-distortion indices. A control group of eyes in which a Tecnis ZCB00 1-piece monofocal IOL was implanted had the same examinations. RESULTS: A trifocal or bifocal toric IOL was implanted in 66 eyes. The control IOL was implanted in 18 eyes. All 3 groups obtained a significant improvement in uncorrected distance visual acuity (UDVA) (P < .001) and corrected distance visual acuity (CDVA) (P Z .001). The mean uncorrected near visual acuity (UNVA) was 0.123 logMAR with the trifocal IOL and 0.130 logMAR with the bifocal toric IOL. The residual refractive cylinder was less than 1.00 D in 86.7% of cases with the toric IOL. The mean light-distortion index was significantly higher in the multifocal IOL groups than in the monofocal group (P < .001), although no correlation was found between the light-distortion index and CDVA. CONCLUSIONS: The multifocal IOLs provided excellent UDVA and functional UNVA despite increased light-distortion indices. The light-distortion analyzer reliably quantified a subjective component of vision distinct from visual acuity; it may become a useful adjunct in the evaluation of visual quality obtained with multifocal IOLs.