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Astronomia Astrofizyka

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Metodologia nauk, Matematyka, Filozofia, Miary i wagi, Pomiary

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Przewidywania Kosmologia Religie Ideologia Polityka

Geologia, geofizyka, geochemia, środowisko przyrodnicze

Biologia, biologia molekularna i genetyka

Technologia cyberprzestrzeni, cyberkultura, media i komunikacja

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Budownictwo, energetyka, transport, wytwarzanie, technologie informacyjne

Journal of Nucleic Acids Investigation

In previous work, we have shown that the entropy of a folded RNA molecule can be divided into local and global contributions using the cross-linking entropy (CLE) model, where, in the case of RNA, the cross- links are the base-pair stacking interactions. The local contribution to the CLE is revealed in the Kuhn length (a measure of the stiffness of the RNA). The Kuhn length acts as a scaling parameter. When the size of the system is rescaled, the relationship between local and global free energy must be renormalized to reflect this rescaling. In this renormalization process, the Kuhn length increases, the local entropy also increases due to freezing out of the local conformational degrees of freedom. At the same time, as the number of degrees of freedom decrease, there is a significant reduction in the global entropy. Here we present a method, based on the concepts of renormalization theory, to quantitatively estimate the size of the contribution from the local entropy as a function of the Kuhn length. The local entropy correction is used to predict the current empirically derived constant in the Jacobson-Stockmayer equation. The variation in the Kuhn length is shown to be largely influenced by the length of the double-stranded RNA stems formed in the secondary structure of folded RNA. This result is used to test the resulting entropy under a variable Kuhn length in stem-loop structures. Comparisons between a variable Kuhn length and a static Kuhn length on a short stem-loop of RNA are also examined. The model is quite general and is also directly applicable to protein structure and folding problems. 2014/03/07 - 21:05

The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, Schizosaccharomyces pombe Pot1 and Saccharomyces cerevisiae Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization. 2013/09/21 - 07:53

Searching for new cancer biomarkers, circulating cell-free DNA (cfDNA) has become an appealing target of interest as an elevated level of cfDNA has been detected in the circulation of cancer patients in comparison with healthy controls. Since cfDNA can be isolated from the circulation and other body fluids of patients without harming their physical condition, cfDNA is becoming a promising candidate as a novel non-invasive biomarker for cancer. The challenge in the diagnostic analysis of cfDNA is its very low presence in human plasma/serum and its partially strong fragmentation. Here we evaluated a modified phenol/chloroform extraction method for the isolation of cfDNA and compared it with published standard methods for cfDNA isolation. 2013/03/11 - 18:53

The concept of a free energy (FE) landscape, in which the surface spirals down like a funnel, has long been viewed as the reason why a complex protein structure forms so rapidly compared to the number of conformations available to it. On the other hand, this landscape picture is less clear with RNA due to the multiplicity of conformations and the uncertainties in the current thermodynamics. It is therefore sometimes proposed that the ensemble average is the main factor deciding the structure. However, calculations of the free energy of observed structures often suggest that this ensemble is far from the minimum FE, particularly in the case of long sequences. If so, then such a FE surface is unlikely to be funnel shaped. We have been developing a version of vsfold that can evaluate the suboptimal structures of the FE surface (vs_subopt). Here we show that the ensemble for a number of known RNA structures can actually be both close to the minimum FE and also be the dominant observed structure when a proper Kuhn length is selected. Two state aptamers known as riboswitches can show neighboring FE states in the suboptimal structures that match the observed structures and their relative difference in FE is well within the range of the binding free energy of the metabolite. For the riboswitches and other short RNA sequences (less than 250 nt), the flow of the suboptimal structures (including pseudoknots) tended to resemble a rock rolling down a hill along the reaction coordinate axis. 2012/07/25 - 22:23

In previous work, we have shown that the entropy of a folded RNA molecule can be divided into local and global contributions using the cross-linking entropy (CLE) model, where, in the case of RNA, the cross-links are the base-pair stacking interactions. The local contribution to the CLE is revealed in the Kuhn length (a measure of the stiffness of the RNA). As the Kuhn length increases, the local entropy also increases due to freezing out of the local conformational degrees of freedom. At the same time, as the number of degrees of freedom decrease, there is a significant reduction in the global entropy. Here we present a method, based on the concepts of renormalization theory, to quantitatively estimate the size of the contribution from the local entropy as a function of the Kuhn length. The local entropy correction is used to predict the current empirically derived constant in the Jacobson-Stockmayer equation. The model is quite general and is also directly applicable to protein structure and folding problems. Comparisons between a variable Kuhn length and a static Kuhn length on a short stem loop of RNA are also examined. 2012/07/25 - 22:23

The Jacobson-Stockmayer (JS) model is used in a number of standard programs for calculating the conformational entropy of RNA (and proteins). However, it is shown in this study that, in certain limiting cases, the current form of this model can lead to highly unphysical conclusions. The origin of this behavior can be traced to misunderstandings that occurred during the development of the model as applied to folded, single-stranded RNA. Here we show that an alternative model known as the cross linking entropy (CLE) model can overcome these issues. The principal object that causes entropy loss on a global scale in the CLE model is the stem, the primary measure of structural order in such coarse-grained calculations. The principal objects in the JS-model are various types of loops, and, with the exception of the hairpin loop, they are topologically local in character. To extract experimentally measurable variables, a simplified version of the CLE model is developed that resembles many features of the contact order model used in RNA and protein folding. These modifications are then applied to single molecule force-extension experiments (molecular tweezers) to extract quantitative information. It is further shown that a crude derivative of the CLE model itself can be derived directly from the JS-model when the misunderstandings are examined and corrected. 2012/07/25 - 01:17

Flax is an important agronomic crop grown for its fiber (linen) and oil (linseed oil). In spite of many thousands of years of breeding some fiber varieties have been shown to rapidly respond to environmental stress with heritable changes to its genome. Many miRNAs appear to be induced by abiotic or biotic conditions experienced through the plant life cycle. Computational miRNA analysis of the flax genome provides a foundation for subsequent research on miRNA function in Linum usitatissimum and may also provide novel insight into any regulatory role the RNAi pathway may play in generating adaptive structural variation in response to environmental stress. Here a bioinformatics approach is used to screen for miRNAs previously identified in other plant species, as well as to predict putative miRNAs unique to a particular species which may not have been identified as they are less abundant or dependent upon a specific set of environmental conditions. Twelve miRNA genes were identified in flax on the basis of unique pre-miRNA positions with structural homology to plant pre-miRNAs and complete sequence homology to published plant miRNAs. These miRNAs were found to belong to 7 miRNA families, with an additional 2 matches corresponding to as yet unnamed poplar miRNAs and a parologous miRNA with partial sequence homology to mtr-miR4414b. An additional 649 novel and distinct flax miRNA genes were identified to form from canonical hairpin structures and to have putative targets among the ~30,000 flax Unigenes. 2012/06/05 - 20:13

The High Mobility Group A (HMGA) proteins, a family of DNA architectural factors, by interacting with different proteins play crucial roles in neoplastic transformation of a wide range of tissues. Therefore, the search for HMGA-interacting partners was carried out by several laboratories in order to investigate the mechanisms underlying HMGA-dependent tumorigenesis. Three of the several HMGA-binding proteins are discussed in this review. These are the Chromobox family protein (chromobox protein homolog 7, CBX7), the homeodomain interacting protein kinase 2 (HIPK2) and the POZ/domain and Kruppel zinc finger family member, PATZ. All of them play a critical role in tumorigenesis, and may also be independent markers of cancer. Their activities are linked to cell cycle, apoptosis and senescence. In this review, we discuss the properties of each protein, including their effect on HMGA1 functions, and propose a model accounting for how their activities might be coordinated. 2012/03/17 - 03:32

Antisense oligonucleotides (AON) delivered via inhalation are in drug development for respiratory diseases. In rodents and monkeys, repeated exposure to high doses of inhaled phosphorothioate (PS) AON can lead to microscopic changes in the lungs, including accumulation of alveolar macrophages in the lower airway that have a foamy appearance. The functional consequences that result from this morphological change are unclear as there is controversy whether the vacuoles/inclusion bodies reflect normal clearance of the inhaled AON or are early indicators of lung toxicity. The morphological and functional responses of macrophage to PS AON were characterized in vitro using the comparator drug amiodarone, as a known inducer of foamy macrophages. Morphological changes of increased vacuolization with the presence of lamellated structures were observed in macrophages in response to both amiodarone and AON treatment. Functional responses to the drugs clearly differed with amiodarone treatment leading to apoptosis of cells and cell death, release of proinflammatory mediators IL-1RA, MIP-1α and TNFα, decrease in IP-10, a cytokine shown to be involved in protection against pulmonary fibrosis and altered phagocytosis capacity of the cells. In contrast, AON in concentrations up to 30 μM, had no effect on cell viability or apoptosis, had minimal effects on pro-inflammatory cytokines, increased IP-10 levels and did not alter the phagocytic capacity of the cells. Exposure of macrophages to AON in vitro, led to morphological changes of increased vacuolization, but did not lead to functional consequences which were observed with another vacuolization-inducing drug, suggesting that the in vivo phenotypic changes observed following inhalation of AON may be consistent with a clearance mechanism and not an activation or impairment of macrophages. 2011/11/19 - 17:23

The MLC1 gene is involved in an autosomal recessive neurological disorder, megalencephalic leucoencephalopathy with subcortical cysts (MLC), which is characterized by macrocephaly during the first year of life and swollen white matter (leucoencephaly). Variants of MLC1 have also been associated with psychiatric disorders such as schizophrenia, major depression and bipolar disorder. Currently, little is known about the encoded protein (MLC1). Judging from its similarity to other known proteins, it may serve as a trans-membrane transporter. However, the function of the encoded protein and its gene regulation has not been investigated successfully so far. We investigated the 5’ region of the murine Mlc1 with respect to regulatory elements for gene expression. A promoter search and an in silico analysis were conducted. Luciferase reporter gene constructs with potential promoter regions were created to study promoter activity in vitro. We found two alternative first exons for the murine Mlc1 but were not able to detect any promoter activity for the investigated reporter gene constructs in different cell lines, thus pointing to the presence of essential cis-acting elements far outside of the region. In silico analysis indicated an uncommon promoter structure for Mlc1, with CCAAT-boxes representing the only noticeable elements. 2011/10/23 - 00:31

Cervical cancer is the second most common form of death by cancer in women worldwide and has special attention for the development of new treatment strategies. Human Papilloma Virus (HPV) persistent infection is the main etiological agent of this neoplasia, and the main cellular transformation mechanism is by disruption of p53 and pRb function by interaction with HPV E6 and E7 oncoproteins. This generates alterations in cellular differentiation and cellular death inhibition. Thus, HPV E6 and E7 oncogenes represent suitable targets for the development of gene therapy strategies against cervical cancer. An attractive technology platform is developing for post-transcriptional selective silencing of gene expression, using small interference RNA. Therefore, in the present study, we used SiHa cells (HPV16+) transiently transfected with specific siRNA expression plasmids for HPV16 E6 and E7 oncogenes. In this model we detected repression of E6 and E7 oncogene and oncoprotein expression, an increase in p53 and hypophosphorylated pRb isoform protein expression, and autophagy and apoptosis morphology features. These findings suggest that selective silencing of HPV16 E6 and E7 oncogenes by siRNAs, has significant biological effects on the survival of human cancer cells and is a potential gene therapy strategy against cervical cancer. 2011/08/30 - 11:37

There has been considerable progress in characterising chronic lymphocytic leukemia with respect to the underlying biology and the corresponding clinical presentation. Most patients with CLL live for years without the need for therapy. This does not hold true for patients that exhibit inactivation of the tumour suppressor p53 or, possibly, its pathway members. Deletions of chromosome 17p and mutations of TP53 are the main characteristic of aggressive and refractory disease. In this review we will outline the current understanding of mechanisms known to contribute to dysfunctional p53 and its connection to microRNAs. 2011/07/12 - 22:31

The HER2 gene amplification occurs in 20-30% of breast cancer and is correlated with a poorer prognosis compared to HER2-negative disease due to increased proliferation and metastatic potential. Two major types of receptor inhibitors have been developed for therapy and one for each categories is currently used in clinic: i) the humanized monoclonal antibody trastuzumab, directed against the HER2 extracellular domain; and ii) the EGFR/HER2 dual tyrosine kinase inhibitor lapatinib. However, patients may develop resistance to drugs and show disease progression. Several resistant mechanisms have been explored and are still under investigation. Here, we focus our attention on the role played by the alternative splicing forms of HER2 in mediating HER2 oncogenic activity and in conditioning the response to HER2 therapies. Three HER2 splice variants have been described so far; the p100 and the herstatin gave raised to two secreted proteins of 100 kd and 68 kd, respectively that act as cell growth inhibitors. Herstatin has been described for its ability to interrupt the constitutive HER2 activation, but also for its capacity to hamper HER2 dimerization with the others HER receptors. Interestingly, herstatin, present as mRNA and protein in non cancerous tissue in areas adjacent to breast carcinoma, is absent as protein in 75% of mammary tumors, which indicates that cancer cells are protected by some intrinsic mechanism against the putative growth-inhibitory effects of this naturally occurring molecule. The third splice form of HER2 gene is the Δ16HER2, encoding for a receptor lacking exon16, whose absence determines a constitutive active dimers with transforming activity in vitro and in vivo. The Δ16HER2 binds to trastuzumab to a less extend, due to conformational changes of the extracellular domain. The Δ16HER2 accounts for almost 9% of the total HER2 transcripts in human breast cancers and, additionally, Δ16HER2 levels are supposed to increase proportionally at the increasing of the HER2 wild-type copy numbers in human primary breast cancers. The availability of a specific assay to determine and quantify the expression levels of this splicing form and the availability of Δ16HER2 transgenic mice models made this variant as the most promising for the development of biodrugs. Finally, HER2 carboxy-terminal fragments (CTFs), generated by alternative initiation of translation, were observed in breast cancer patients. In particular, 611-CTF was described to activate multiple signaling pathways since it is expressed as a constitutively active homodimer. Expression of 611-CTF led to development of aggressive and invasive mammary tumors and it was suggested to be a potent oncogene capable of promoting mammary tumor progression and metastasis. 2011/07/06 - 15:23

Species of the genus Campylobacter are responsible for the largest number of bacterial food-borne illness cases occurring yearly in the developed world. The majority of disease is caused by three of the thermotolerant Campylobacter species: Campylobacter jejuni, Campylobacter coli, and Campylobacter lari. An inability to differentiate these three species using the commonly employed 16S rRNA sequencing procedure has led to the development of alternative methods to identify these bacteria. Some of these methods include the utilization of the gyrB gene. A reliable method was developed for the differentiation of C. jejuni, C. coli, and C. lari employing the gyrB gene. It involves amplification and sequencing of a species-variable region of the gene with a single pair of DNA primers. The method works well for the separation and organization of the three Campylobacter strains as well as satisfying the suggested guidelines for sequence based identification for most strains investigated. 2011/05/05 - 15:14

Accumulating experimental evidence indicates that microRNAs play important roles in various biological processes, such as cell differentiation, proliferation, metabolism and apoptosis. In addition, several reports concluded that altered expression of specific microRNA genes contributes to the initiation and progression of cancer. Here, we summarize the current knowledge about aberrant expression of various microRNAs in human solid cancers (e.g., lung, breast, and gastric cancers), their target proteins, and the relationship between their expression and response to chemotherapies. We also review the potential for using microRNAs as biomarkers for the diagnosis and cancer therapy. The development of treatment strategies against human solid cancers based on the profile and/or certain features of microRNAs is promising. 2011/04/19 - 19:31

MiRNAs are probable regulators of cell events such as differentiation, propagation and apoptosis. These cellular phenomena are also associated with benign and malignant tumor cells, therefore, it is presumed that miRNAs act as natural oncogenes or tumor suppressor genes. Whether a particular miRNA serves as either could almost be moot when the additional problems of SNPs enter the fray. A miRNA involved with SNPs (miR-SNPs) on any regulatory level, whether naturally cancerinducing or not, could easily undergo an oncogenic transformation. This work reviews targets of miRNAs and the miRNAs themselves frequently containing SNPs reflecting different risks and markers of cancer with emphasis on familial groups and populations of shared heredity. 2011/04/11 - 11:40

Non-coding RNAs were previously thought to have little importance because they are not directly translated into a protein like their coding counterparts.  However, it was recently found that non-coding RNAs do in fact have a much bigger role than previously thought.  They are involved in cancer predisposition, development and progression.  MicroRNAs, very short non-coding RNAs, are abnormally expressed in cancer and some harbor more mutations that affect expression levels.  MicroRNA alterations have been observed in all forms of cancer that have been researched to the current date.  MicroRNAs are also located in cancer-associated genomic regions, which have been previously shown to affect gene expression leading to the activation or inhibition of cancer growth.  Single-nucleotide polymorphisms within microRNAs can predispose someone to cancer.  MicroRNAs have been shown to target both tumor suppressors, inhibiting cancer development, as well as oncogenes, stimulating cancer development.  Some microRNAs can switch between these two functions and behave as a tumor suppressor at one time and an oncogene at another time.  MicroRNAs can be used for diagnostic purposes as well as prognostic evaluations.  Outside of microRNAs, ultraconserved genes, another group of non-coding RNAs, also express differently in cancer patients.  Large intervening non-coding RNAs, specifically one termed HOTAIR, have been quantified in very high levels in cancer cells and have been implicated in metastasis.  Further research into non-coding RNAs may allow for the development of therapies that will target non-coding RNAs creating better treatment options for cancer patients, improving their prognosis.  This review discusses the most current discoveries about non-coding RNAs, revealing their associations with cancer. 2011/03/11 - 22:54

The use of online tools for bioinformatics analyses is becoming increasingly widespread. Resources specific to the field of microRNAs are available, varying in scope and usability. Online tools are the most useful for casual as well as power users since they need no installation, are hardware independent and are used mostly through graphic user interfaces and links to external sources. Here, we present an overview of useful online resources that have to do with microRNA genomics, gene finding, target prediction and functional analysis. 2011/02/25 - 11:05

MicroRNAs (miRNAs) are a class of highly evolutionarily conserved non-coding RNAs (ncRNAs) that modulate gene expression. Several studies have shown that the expression of miRNAs is deregulated in human malignancies. For ncRNAs and miRNAs, such gene-profiling studies in tumorigenic tissues have identified significant signatures that are of both diagnostic and prognostic value. Addressing the functions of ncRNAs not only give insights into the molecular mechanisms that underlie complex genetic processes, but may also elucidate novel mechanisms that contribute to early stages of tumor development, progression and metastasis. MiRNA-based novel approaches target the ncRNAome, including, for instance, miRNA expression levels and improved designs of miRNA-mimics or more precise target-predictions, prevent off-target effects of novel drugs and make miRNAs become a highly efficient class of therapeutics. For miRNA-based therapeutic studies two direct strategies are currently under investigation, viz. (i) the overexpression of given miRNAs to inhibit the expression of protein-coding genes or (ii) the inhibition of target miRNAs with antisense constructs like antagomiRs. Indirect strategies include the use of novel drugs that modulate miRNA expression levels by directly targeting their processing or transcription. Further, miRNA-based biomarkers have a significant impact on the development of both therapeutic and diagnostic agents, a concept known as theranostics and are highly relevant for drug development and personalized medicine. 2011/02/18 - 01:11

Accepted tools for early cancer detection run the gamut from Pap staining to detect cervical cancer detection to colonoscopy and biopsy for colorectal cancer detection to imaging (mammogram and, in high risk women, magnetic resonance imaging) and biopsy for breast cancer detection. These modalities use standard cytopathologic assessment to determine if disease is present. There are few biologic (DNA, RNA, protein or carbohydrate) markers (biomarkers) that are in general use for the detection of any cancer, or to identify individuals at increased cancer risk. Biomarkers have been identified that provide information to physicians on disease prognosis. Panels of biomarkers are being developed to predict response to treatment in individuals with known cancer, and some are currently in use. Nonethless, there is a great need to identify accurate biomarkers for the early detection of a new cancer, to identify individuals at increased cancer risk, and among those with cancer, to determine their likelihood of responding to a given treatment, risk of disease relapse and death. Micro (mi)RNAs hold promise as biomarkers for determining at risk individuals, for early cancer detection, and among those with cancer, to assess how likely a person is to respond a given treatment, their risk of disease recurrence and death. 2011/02/18 - 01:11