Functional genes and gene array analysis as tools for monitoring hydrocarbon biodegradation

Bioremediation, which is the exploitation of the intrinsic ability of environmental microbes to degrade and remove harmful compounds from nature, is considered to be an environmentally sustainable and cost-effective means for environmental clean-up. However, a comprehensive understanding of the biodegradation potential of microbial communities and their response to decontamination measures is required for the effective management of bioremediation processes. In this thesis, the potential to use hydrocarbon-degradative genes as indicators of aerobic hydrocarbon biodegradation was investigated. Small-scale functional gene macro- and microarrays targeting aliphatic, monoaromatic and low molecular weight polyaromatic hydrocarbon biodegradation were developed in order to simultaneously monitor the biodegradation of mixtures of hydrocarbons. The validity of the array analysis in monitoring hydrocarbon biodegradation was evaluated in microcosm studies and field-scale bioremediation processes by comparing the hybridization signal intensities to hydrocarbon mineralization, real-time polymerase chain reaction (PCR), dot blot hybridization and both chemical and microbiological monitoring data. The results obtained by real-time PCR, dot blot hybridization and gene array analysis were in good agreement with hydrocarbon biodegradation in laboratory-scale microcosms. Mineralization of several hydrocarbons could be monitored simultaneously using gene array analysis. In the field-scale bioremediation processes, the detection and enumeration of hydrocarbon-degradative genes provided important additional information for process optimization and design. In creosote-contaminated groundwater, gene array analysis demonstrated that the aerobic biodegradation potential that was present at the site, but restrained under the oxygen-limited conditions, could be successfully stimulated with aeration and nutrient infiltration. During ex situ bioremediation of diesel oil- and lubrication oil-contaminated soil, the functional gene array analysis revealed inefficient hydrocarbon biodegradation, caused by poor aeration during composting. The functional gene array specifically detected upper and lower biodegradation pathways required for complete mineralization of hydrocarbons. Bacteria representing 1 % of the microbial community could be detected without prior PCR amplification. Molecular biological monitoring methods based on functional genes provide powerful tools for the development of more efficient remediation processes. The parallel detection of several functional genes using functional gene array analysis is an especially promising tool for monitoring the biodegradation of mixtures of hydrocarbons.

The trishanku gene and terminal morphogenesis in Dictyostelium discoideum

P>Multicellular development in the social amoeba Dictyostelium discoideum is triggered by starvation. It involves a series of morphogenetic movements, among them being the rising of the spore mass to the tip of the stalk. The process requires precise coordination between two distinct cell types-presumptive (pre-) spore cells and presumptive (pre-) stalk cells. Trishanku (triA) is a gene expressed in prespore cells that is required for normal morphogenesis. The triA- mutant shows pleiotropic effects that include an inability of the spore mass to go all the way to the top. We have examined the cellular behavior required for the normal ascent of the spore mass. Grafting and mixing experiments carried out with tissue fragments and cells show that the upper cup, a tissue that derives from prestalk cells and anterior-like cells (ALCs), does not develop properly in a triA- background. A mutant upper cup is unable to lift the spore mass to the top of the fruiting body, likely due to defective intercellular adhesion. If wild-type upper cup function is provided by prestalk and ALCs, trishanku spores ascend all the way. Conversely, Ax2 spores fail to do so in chimeras in which the upper cup is largely made up of mutant cells. Besides proving that under these conditions the wild-type phenotype of the upper cup is necessary and sufficient for terminal morphogenesis in D. discoideum, this study provides novel insights into developmental and evolutionary aspects of morphogenesis in general. Genes that are active exclusively in one cell type can elicit behavior in a second cell type that enhances the reproductive fitness of the first cell type, thereby showing that morphogenesis is a cooperative process.

Prediction of protein-protein interactions between Helicobacter pylori and a human host

A lack of information on protein-protein interactions at the host-pathogen interface is impeding the understanding of the pathogenesis process. A recently developed, homology search-based method to predict protein-protein interactions is applied to the gastric pathogen, Helicobacter pylori to predict the interactions between proteins of H. pylori and human proteins in vitro. Many of the predicted interactions could potentially occur between the pathogen and its human host during pathogenesis as we focused mainly on the H. pylori proteins that have a transmembrane region or are encoded in the pathogenic island and those which are known to be secreted into the human host. By applying the homology search approach to protein-protein interaction databases DIP and iPfam, we could predict in vitro interactions for a total of 623 H. pylori proteins with 6559 human proteins. The predicted interactions include 549 hypothetical proteins of as yet unknown function encoded in the H. pylori genome and 13 experimentally verified secreted proteins. We have recognized 833 interactions involving the extracellular domains of transmembrane proteins of H. pylori. Structural analysis of some of the examples reveals that the interaction predicted by us is consistent with the structural compatibility of binding partners. Examples of interactions with discernible biological relevance are discussed.

An extragenic suppressor of prp24-1 defines genetic interaction between PRP24 and PRP21 gene products of Saccharomyces cerevisiae

The temperature-sensitive prp24-1 mutation defines a gene product required for the first step in pre-mRNA splicing. PRP24 is probably a component of the U6 snRNP particle. We have applied genetic reversion analysis to identify proteins that interact with PRP24. Spontaneous revertants of the temperature-sensitive (ts) prp24-1 phenotype were analyzed for those that are due to extragenic suppression. We then extended our analysis to screen for suppressors that confer a distinct conditional phenotype. We have identified a temperature-sensitive extragenic suppressor, which was shown by genetic complementation analysis to be allelic to prp21-1. This suppressor, prp21-2, accumulates pre-mRNA at the non-permissive temperature, a phenotype similar to that of prp21-1. prp21-2 completely suppresses the splicing defect and restores in vivo levels of the U6 snRNA in the prp24-1 strain. Genetic analysis of the suppressor showed that prp21-2 is not a bypass suppressor of prp24-1. The suppression of prp24-1 by prp21-2 is gene specific and also allele specific with respect to both the loci. Genetic interactions with other components of the pre-spliceosome have also been studied. Our results indicate an interaction between PRP21, a component of the U2 snRNP, and PRP24, a component of the U6 snRNP. These results substantiate other data showing U2-U6 snRNA interactions.

Next-generation sequencing: A frameshift in skeletal dysplasia gene discovery

In the last decade, huge breakthroughs in genetics - driven by new technology and different statistical approaches - have resulted in a plethora of new disease genes identified for both common and rare diseases. Massive parallel sequencing, commonly known as next-generation sequencing, is the latest advance in genetics, and has already facilitated the discovery of the molecular cause of many monogenic disorders. This article describes this new technology and reviews how this approach has been used successfully in patients with skeletal dysplasias. Moreover, this article illustrates how the study of rare diseases can inform understanding and therapeutic developments for common diseases such as osteoporosis. © International Osteoporosis Foundation and National Osteoporosis Foundation 2013.

Regulation of Osteoblast Differentiation and Gene Expression by Phosphodiesterases and Bioactive Peptides

Bone is a mineralized tissue that enables multiple mechanical and metabolic functions to be carried out in the skeleton. Bone contains distinct cell types: osteoblasts (bone-forming cells), osteocytes (mature osteoblast that embedded in mineralized bone matrix) and the osteoclasts (bone-resorbing cells). Remodelling of bone begins early in foetal life, and once the skeleton is fully formed in young adults, almost all of the metabolic activity is in this form. Bone is constantly destroyed or resorbed by osteoclasts and then replaced by osteoblasts. Many bone diseases, i.e. osteoporosis, also known as bone loss, typically reflect an imbalance in skeletal turnover. The cyclic adenosine monophosphate (cAMP) and the cyclic guanosine monophosphate (cGMP) are second messengers involved in a variety of cellular responses to such extracellular agents as hormones and neurotransmitters. In the hormonal regulation of bone metabolism, i.e. via parathyroid hormone (PTH), parathyroid hormone-related peptide (PTHrp) and prostaglandin E2 signal via cAMP. cAMP and cGMP are formed by adenylate and guanylate cyclases and are degraded by phosphodiesterases (PDEs). PDEs determine the amplitudes of cyclic nucleotide-mediated hormonal responses and modulate the duration of the signal. The activities of the PDEs are regulated by multiple inputs from other signalling systems and are crucial points of cross-talk between the pathways. Food-derived bioactive peptides are reported to express a variety of functions in vivo. The angiotensin-converting enzymes (ACEs) are involved in the regulation of the specific maturation or degradation of a number of mammalian bioactive peptides. The bioactive peptides offer also a nutriceutical and a nutrigenomic aspect to bone cell biology. The aim of this study was to investigate the influence of PDEs and bioactive peptides on the activation and the differentiation of human osteoblast cells. The profile of PDEs in human osteoblast-like cells and the effect of glucocorticoids on the function of cAMP PDEs, were investigated at the mRNA and enzyme levels. The effects of PDEs on bone formation and osteoblast gene expression were determined with chemical inhibitors and siRNAs (short interfering RNAs). The influence of bioactive peptides on osteoblast gene expression and proliferation was studied at the mRNA and cellular levels. This work provides information on how PDEs are involved in the function and the differentiation of osteoblasts. The findings illustrate that gene-specific silencing with an RNA interference (RNAi) method is useful in inhibiting, the gene expression of specific PDEs and further, PDE7 inhibition upregulates several osteogenic genes and increases bALP activity and mineralization in human mesenchymal stem cells-derived osteoblasts. PDEs appear to be involved in a mechanism by which glucocorticoids affect cAMP signaling. This may provide a potential route in the formation of glucocorticoid-induced bone loss, involving the down-regulation of cAMP-PDE. PDEs may play an important role in the regulation of osteoblastic differentiation. Isoleucine-proline-proline (IPP), a bioactive peptide, possesses the potential to increase osteoblast proliferation, differentiation and signalling.

Neuronal K-Cl Cotransporter : Transcriptional Mechanisms of KCC2 Gene Regulation

K-Cl cotransporter 2 (KCC2) maintains a low intracellular Cl concentration required for fast hyperpolarizing responses of neurons to classical inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. Decreased Cl extrusion observed in genetically modified KCC2-deficient mice leads to depolarizing GABA responses, impaired brain inhibition, and as a consequence to epileptic seizures. Identification of mechanisms regulating activity of the SLC12A5 gene, which encodes the KCC2 cotransporter, in normal and pathological conditions is, thus, of extreme importance. Multiple reports have previously elucidated in details a spatio-temporal pattern of KCC2 expression. Among the characteristic features are an exclusive neuronal specificity, a dramatic upregulation during embryonic and early postnatal development, and a significant downregulation by neuronal trauma. Numerous studies confirmed these expressional features, however transcriptional mechanisms predetermining the SLC12A5 gene behaviour are still unknown. The aim of the presented thesis is to recognize such transcriptional mechanisms and, on their basis, to create a transcriptional model that would explain the established SLC12A5 gene behaviour. Up to recently, only one KCC2 transcript has been thought to exist. A particular novelty of the presented work is the identification of two SLC12A5 gene promoters (SLC12A5-1a and SLC12A5-1b) that produce at least two KCC2 isoforms (KCC2a and KCC2b) differing by their N-terminal parts. Even though a functional 86Rb+ assay reveals no significant difference between transport activities of the isoforms, consensus sites for several protein kinases, found in KCC2a but not in KCC2b, imply a distinct kinetic regulation. As a logical continuation, the current work presents a detailed analysis of the KCC2a and KCC2b expression patterns. This analysis shows an exclusively neuron-specific pattern and similar expression levels for both isoforms during embryonic and neonatal development in rodents. During subsequent postnatal development, the KCC2b expression dramatically increases, while KCC2a expression, depending on central nervous system (CNS) area, either remains at the same level or moderately decreases. In an attempt to explain both the neuronal specificity and the distinct expressional kinetics of the KCC2a and KCC2b isoforms during postnatal development, the corresponding SLC12A5-1a and SLC12A5-1b promoters have been subjected to a comprehensive bioinformatical analysis. Binding sites of several transcription factors (TFs), conserved in the mammalian SLC12A5 gene orthologs, have been identified that might shed light on the observed behaviour of the SLC12A5 gene. Possible roles of these TFs in the regulating of the SLC12A5 gene expression have been elucidated in subsequent experiments and are discussed in the current thesis.

Pheo-type: A diagnostic gene-expression assay for the classification of pheochromocytoma and paraganglioma

Context: Pheochromocytomas and paragangliomas (PPGLs) are heritable neoplasms that can be classified into gene-expression subtypes corresponding to their underlying specific genetic drivers. Objective: This study aimed to develop a diagnostic and research tool (Pheo-type) capable of classifying PPGL tumors into gene-expression subtypes that could be used to guide and interpret genetic testing, determine surveillance programs, and aid in elucidation of PPGL biology. Design: A compendium of published microarray data representing 205 PPGL tumors was used for the selection of subtype-specific genes that were then translated to the Nanostring gene-expression platform. A support vector machine was trained on the microarray dataset and then tested on an independent Nanostring dataset representing 38 familial and sporadic cases of PPGL of known genotype (RET, NF1, TMEM127, MAX, HRAS, VHL, and SDHx). Different classifier models involving between three and six subtypes were compared for their discrimination potential. Results: A gene set of 46 genes and six endogenous controls was selected representing six known PPGL subtypes; RTK1–3 (RET, NF1, TMEM127, and HRAS), MAX-like, VHL, and SDHx. Of 38 test cases, 34 (90%) were correctly predicted to six subtypes based on the known genotype to gene-expression subtype association. Removal of the RTK2 subtype from training, characterized by an admixture of tumor and normal adrenal cortex, improved the classification accuracy (35/38). Consolidation of RTK and pseudohypoxic PPGL subtypes to four- and then three-class architectures improved the classification accuracy for clinical application. Conclusions: The Pheo-type gene-expression assay is a reliable method for predicting PPGL genotype using routine diagnostic tumor samples.

Positional cloning and pathway analysis of the asthma susceptibility gene, NPSR1

In the present study, we identified a novel asthma susceptibility gene, NPSR1 (neuropeptide S receptor 1) on chromosome 7p14.3 by the positional cloning strategy. An earlier significant linkage mapping result among Finnish Kainuu asthma families was confirmed in two independent cohorts: in asthma families from Quebec, Canada and in allergy families from North Karelia, Finland. The linkage region was narrowed down to a 133-kb segment by a hierarchial genotyping method. The observed 77-kb haplotype block showed 7 haplotypes and a similar risk and nonrisk pattern in all three populations studied. All seven haplotypes occur in all three populations at frequences > 2%. Significant elevated relative risks were detected for elevated total IgE (immunoglobulin E) or asthma. Risk effects of the gene variants varied from 1.4 to 2.5. NPSR1 belongs to the G protein-coupled receptor (GPCR) family with a topology of seven transmembrane domains. NPSR1 has 9 exons, with the two main transcripts, A and B, encoding proteins of 371 and 377 amino acids, respectively. We detected a low but ubiquitous expression level of NPSR1-B in various tissues and endogenous cell lines while NPSR1-A has a more restricted expression pattern. Both isoforms were expressed in the lung epithelium. We observed aberrant expression levels of NPSR1-B in smooth muscle in asthmatic bronchi as compared to healthy. In an experimental mouse model, the induced lung inflammation resulted in elevated Npsr1 levels. Furthermore, we demonstrated that the activation of NPSR1 with its endogenous agonist, neuropeptide S (NPS), resulted in a significant inhibition of the growth of NPSR1-A overexpressing stable cell lines (NPSR1-A cells). To determine which target genes were regulated by the NPS-NPSR1 pathway, NPSR1-A cells were stimulated with NPS, and differentially expressed genes were identified using the Affymetrix HGU133Plus2 GeneChip. A total of 104 genes were found significantly up-regulated and 42 down-regulated 6 h after NPS administration. The up-regulated genes included many neuronal genes and some putative susceptibility genes for respiratory disorders. By Gene Ontology enrichment analysis, the biological process terms, cell proliferation, morphogenesis and immune response were among the most altered. The expression of four up-regulated genes, matrix metallopeptidase 10 (MMP10), INHBA (activin A), interleukin 8 (IL8) and EPH receptor A2 (EPHA2), were verified and confirmed by quantitative reverse-transcriptase-PCR. In conclusion, we identified a novel asthma susceptibility gene, NPSR1, on chromosome 7p14.3. NPS-NPSR1 represents a novel pathway that regulates cell proliferation and immune responses, and thus may have functional relevance in the pathogenesis of asthma.

Genetic and epigenetic variants in the MTHFR gene are not associated with non-Hodgkin lymphoma

The methylenetetrahydrofolate reductase (MTHFR) gene codes for the MTHFR enzyme which plays a key role in the pathway of folate and methionine metabolism. Polymorphisms of genes in this pathway affect its regulation and have been linked to lymphoma. In this study we examined whether we could detect an association between two common non-synonomous MTHFR polymorphisms, 677C>T (rs1801133) and 1298A>C (rs1801131), and susceptibility to non-Hodgkin lymphoma (NHL) in an Australian case-control cohort. We found no significant differences between genotype or allele frequencies for either polymorphisms between lymphoma cases and controls. We also explored whether epigenetic modification of MTHFR, specifically DNA methylation of a CpG island in the MTHFR promoter region, is associated with NHL using blood samples from patients. No difference in methylation levels was detected between the case and control samples suggesting that although hypermethylation of MTHFR has been reported in tumour tissues, particularly in the diffuse large B-cell lymphoma subtype of NHL, methylation of this MTHFR promoter CpG island is not a suitable epigenetic biomarker for NHL diagnosis or prognosis in peripheral blood samples. Further studies into epigenetic variants could focus on genes that are robustly associated with NHL susceptibility.

Determinants of outcome in critically ill patients

Assessment of the outcome of critical illness is complex. Severity scoring systems and organ dysfunction scores are traditional tools in mortality and morbidity prediction in intensive care. Their ability to explain risk of death is impressive for large cohorts of patients, but insufficient for an individual patient. Although events before intensive care unit (ICU) admission are prognostically important, the prediction models utilize data collected at and just after ICU admission. In addition, several biomarkers have been evaluated to predict mortality, but none has proven entirely useful in clinical practice. Therefore, new prognostic markers of critical illness are vital when evaluating the intensive care outcome. The aim of this dissertation was to investigate new measures and biological markers of critical illness and to evaluate their predictive value and association with mortality and disease severity. The impact of delay in emergency department (ED) on intensive care outcome, measured as hospital mortality and health-related quality of life (HRQoL) at 6 months, was assessed in 1537 consecutive patients admitted to medical ICU. Two new biological markers were investigated in two separate patient populations: in 231 ICU patients and 255 patients with severe sepsis or septic shock. Cell-free plasma DNA is a surrogate marker of apoptosis. Its association with disease severity and mortality rate was evaluated in ICU patients. Next, the predictive value of plasma DNA regarding mortality and its association with the degree of organ dysfunction and disease severity was evaluated in severe sepsis or septic shock. Heme oxygenase-1 (HO-1) is a potential regulator of apoptosis. Finally, HO-1 plasma concentrations and HO-1 gene polymorphisms and their association with outcome were evaluated in ICU patients. The length of ED stay was not associated with outcome of intensive care. The hospital mortality rate was significantly lower in patients admitted to the medical ICU from the ED than from the non-ED, and the HRQoL in the critically ill at 6 months was significantly lower than in the age- and sex-matched general population. In the ICU patient population, the maximum plasma DNA concentration measured during the first 96 hours in intensive care correlated significantly with disease severity and degree of organ failure and was independently associated with hospital mortality. In patients with severe sepsis or septic shock, the cell-free plasma DNA concentrations were significantly higher in ICU and hospital nonsurvivors than in survivors and showed a moderate discriminative power regarding ICU mortality. Plasma DNA was an independent predictor for ICU mortality, but not for hospital mortality. The degree of organ dysfunction correlated independently with plasma DNA concentration in severe sepsis and plasma HO-1 concentration in ICU patients. The HO-1 -413T/GT(L)/+99C haplotype was associated with HO-1 plasma levels and frequency of multiple organ dysfunction. Plasma DNA and HO-1 concentrations may support the assessment of outcome or organ failure development in critically ill patients, although their value is limited and requires further evaluation.

Evaluation of a 7-gene genetic profile for athletic endurance phenotype in Ironman championship triathletes

Polygenic profiling has been proposed for elite endurance performance, using an additive model determining the proportion of optimal alleles in endurance athletes. To investigate this model’s utility for elite triathletes, we genotyped seven polymorphisms previously associated with an endurance polygenic profile (ACE Ins/Del, ACTN3 Arg577Ter, AMPD1 Gln12Ter, CKMM 1170bp/985+185bp, HFE His63Asp, GDF8 Lys153Arg and PPARGC1A Gly482Ser) in a cohort of 196 elite athletes who participated in the 2008 Kona Ironman championship triathlon. Mean performance time (PT) was not significantly different in individual marker analysis. Age, sex, and continent of origin had a significant influence on PT and were adjusted for. Only the AMPD1 endurance-optimal Gln allele was found to be significantly associated with an improvement in PT (model p=5.79 x 10-17, AMPD1 genotype p=0.01). Individual genotypes were combined into a total genotype score (TGS); TGS distribution ranged from 28.6 to 92.9, concordant with prior studies in endurance athletes (mean±SD: 60.75±12.95). TGS distribution was shifted toward higher TGS in the top 10% of athletes, though the mean TGS was not significantly different (p=0.164) and not significantly associated with PT even when adjusted for age, sex, and origin. Receiver operating characteristic curve analysis determined that TGS alone could not significantly predict athlete finishing time with discriminating sensitivity and specificity for three outcomes (less than median PT, less than mean PT, or in the top 10%), though models with the age, sex, continent of origin, and either TGS or AMPD1 genotype could. These results suggest three things: that more sophisticated genetic models may be necessary to accurately predict athlete finishing time in endurance events; that non-genetic factors such as training are hugely influential and should be included in genetic analyses to prevent confounding; and that large collaborations may be necessary to obtain sufficient sample sizes for powerful and complex analyses of endurance performance.

The influence of OLR1 and PCSK9 gene polymorphisms on ischemic stroke: Evidence from a meta-analysis

It has been reported that both OLR1 and PCSK9 genes are related to various vascular diseases such as atherosclerosis, cardiovascular disease, peripheral artery disease and stroke, in particular ischemic stroke. The prevalence of PCSK9 rs505151 and OLR1 rs11053646 variants in ischemic stroke were 0.005 and 0.116, respectively. However, to date, association between OLR1 rs11053646 and PCSK9 rs505151 polymorphisms and the risk of ischemic stroke remains unclear and inconclusive. Therefore, this first meta-analysis was carried out to clarify the presumed influence of genetic polymorphisms on ischemic stroke, by analyzing the complete coverage of all relevant studies. All eligible case-control and cohort studies that met the search term were retrieved in multiple scientific databases. Data of interest such as demographic data and genotyping methods were extracted from each study, and the meta-analysis was performed using RevMan 5.3 and Metafor R 3.2.1. The pooled odd ratios (ORs) and 95% confidence intervals (CIs) were calculated using both fixed- and random-effect models. A total of seven case-control studies encompassing 1897 ischemic stroke cases and 2119 healthy controls were critically evaluated. Pooled results from the genetic models indicated that OLR1 rs11053646 dominant (OR=1.33. 95%CI:1.11-1.58) and co-dominant models (OR=1.24, 95%CI:1.02-1.51) were significantly associated with ischemic stroke. For PCSK9 rs505151 polymorphism, the OR of co-dominant model (OR=1.36, 95%CI:1.01-1.58) was found to be higher among ischemic stroke patients. In conclusion, the current meta-analysis highlighted that variant allele of OLR1 rs11053646 G>C and PCSK9 rs505151 A>G may contribute to the susceptibility risk of ischemic stroke.