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Lipopolysaccharide induces the expression of T-like receptor 9
Posted on September 2nd, 2010 No commentsThe human bronchial epithelial cells have been recently used to investigate the expression of Toll like receptor 9, after the induction by lipopolysaccharide. This receptor has a transmembrane structure and signals though MAPK and IKK pathway, at last instance. It is involved in the innate immune response: indeed, it recognizes the molecules shared by pathogen, as different from the host ones and generates a response. Firstly, IL2 and IL18 are produced in antigen presenting cells, and these cytokines induce the maturation of naïve T cells. Then, pathogens are captured and internalized into the cells through phagocytosis or endocytosis. Lipopolysaccharide is usually expressed on bacterial surface and it’s reasonable to hypothesize that the interaction between LPS and human cells induces the expression of proteins involved in the innate immune response. The work published in the last number of Nature Preceding demonstrates and confirms this hypothesis. Scientists at the Department of Chemistry of Anna University (India) showed by RT-PCR and western blot analysis the induction of T- like receptor 9 after stimulation by LPS. This novel and innovative approach may be applied to study the regulation of other proteins, in order to better understand the mechanism that controls the innate response.
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Pfam database
Posted on August 30th, 2010 No commentsPfam is a novel database that contains conserved proteins and domains, usually employed in proteome analyses or sequencing classification. In this database, proteins are classified into two groups: Pfam-A families are manually annotated and inserted into the database after overcoming strictly threshold, while Pfam-B families are automatically created and clustered through similar sequence regions not matched with Pfam-A. A further rearrangement divides Pfam-A families in clans in respect to a hierarchical classification. The proteome coverage of Pfam database is different among species: information about bacteria are the most complete. Coverage is defined as sequence or amino acid coverage. In the first case, the proportion of sequence with a match with at least one of Pfam-A family describes the grade of completeness of the information. In the second case, is the proportion of amino acid belonging to the Pfam-A family that corresponds to the coverage. In order to grow and increase the information contained in the database, authors affiliated to the Wellcome Trust Sanger Institute, require the contribution of all scientists working on proteome. They are looking for new alignments, annotations or references for novel families, and updates for the existing ones.
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The basal cell carcinoma
Posted on August 27th, 2010 No commentsBasal cell carcinoma (BCC) is the most diffused tumor among the Caucasian population. Its more frequent in men than in women and usually arises in the fifth decade. BCC involves keratinocytes and keratinocyte stem cells that are present in the epidermis and in the follicular hair. These cells acquire some genetic mutations, generating the transforming phenotype. Based on the current data available in literature, it is not totally understood which genomic alterations are crucial for BCC development. By contrast, multiple genes seem altered in this kind of cancer. Firstly, the hedgehog signaling pathway is considered as the most frequently mutated pathway. In particular ptch1 gene that normally acts as a tumor suppressor is inactivated. Therefore, HH signaling could be a promising target for a pharmacological therapy. Inhibitors are already in clinical trials and seem to have good results and positive perspectives.
Another class of gene that is frequently altered in BCC encodes for the damaged DNA repairing proteins. Of course, as well as in other skin cancer, some mutations in p53 gene have been reported also in the case of the basal cell carcinoma. It is not surprising that BCC could arise in children affected by Xeroderma pigmentosous during the childhood. The polymorphism of the gene of the receptor of the melanocyte stimulating hormone is a risk factor. Indeed, the occurrence of BCC is higher in presence of a certain single nucleotide polymorphism that is responsible for the red or fair phenotype. In fact, in individuals with red or fair hair and pale skin, the pheomelanin is less protective against the UV damage than the eumelanin (the black or brown type) and these persons are more prone to skin cancer, in general. Nevertheless, it has not completely clarified what is the role of UVR damage in the genesis of the basal cell carcinoma.
Data reported in literature are quite contradictory. It seems that there is a correlation between sun exposure and BCC arising, but the systematic use of UV screens doesn’t change the rate or the likelihood to have this kind of cancer. Again, it seems that is the sporadic sun exposure –one or twice a week during jogging or cycling- the most dangerous. Further studies must be done to better clarify this point and give some suggestions to the general population in order to prevent the BCC. Indeed, in terms of economic and social cost the BCC is really expensive and even if normally is not lethal, as the other skin cancer, is aggressive and disfiguring. Surgeon and radiotherapy are the current treatment for the BCC. The great advances of medicine in this field has given satisfactory results either from a clinical point of view and quality of life. Further studies will optimize these techniques and improve the cosmetic outcome. -
DNA and RNA editing survey
Posted on August 25th, 2010 No commentsA recent survey of millions of sequencing products demonstrated that the occurrence of RNA and DNA editing in human and mouse genome is higher than previously documented. Indeed, while the RNA editing has been identified in several model organisms as well as in human, the DNA editing has been reported only in viruses and mice –through retrotransposons- but not in human genome. Scientists of the Harvard University collected the raw sequences from the National Center for Biotechnology Information Trace Archive and aligned them. They identified the mismatch motifs caused by sequencing errors by using a selective and stringent quality threshold. Then, the remaining thousands of mismatches were considered as genuine editing events that occurred in this case also in human genome. Furthermore, this study, recently published on Plos Journal, revealed the existence of numerous other RNA editing positions both in human and mouse genomes and an extensive editing process in Xenopus tropicalis genome. This technique must be applied in the next future to data obtained from next- generation sequencing methods with at least two important advantages: the first is the rapid proofreading system to avoid sequencing errors and the second is the immediate identification of editing sites. Indeed, RNA and DNA editing are mechanism of expression regulation that have to be further deepened.
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The PINEM technology
Posted on August 23rd, 2010 No commentsThe imaging techniques have done important advances and fine cell details have been resolved with high definition. Anyway, the study of certain processes or surfaces is sometimes limited because of the requirement of gold coat or permeabilization treatment that disrupt cells and organelles. At the California Institute of Technology a novel kind of microscopy has been set up. This techniques, namely PINEM (photon induced, near field electron microscopy), uses the combination of photons and electrons beam to resolve structure of nanometer within few femtoseconds. The capture an image only when a photon and an electron beam coincide on the cell or structure or membrane that have to be studied. The first work has been published on protein vesicles in Escherichia Coli cells: the high resolution allows to distinguish the space between the inner and the outer membranes. The polarization of laser and the orientation of cellular layers are two factors that can be regulated in order to achieve image of a single structure. The PINEM technology is being marketed by an electron microscopy company and it belong available to other labs. At the moment the power of resolution is at nanometer level, but the California Institute’s scientists are confident of further improvements.
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The binary expression system Q
Posted on August 19th, 2010 No commentsThe binary expression system is a strategy to finely tune the tragene expression in a model organism. One example of this kind of system is the GAL4- UAS in which the GAL4 gene is under control of the promoter of interest. GAL 4 activates a second exogenous gene bearing the upstream activating sequence. A possible alternative to the GAL 4- UAS system is the Q system. In this case the transcriptional activator QF binds to a specific sequence Q- UAS, as well as its repressor QS. QS in turn can be regulated by the addition of quinic acid into the medium and a further level of control can be added. A common application for this kind of systems is the generation of mosaic animals through the MARCM, the mosaic analysis with a repressible cell marker. In MARCM cell division is coupled to the repression of the gene of interest in one of daughter cells, allowing to study at the same time the effects of knock down in comparison to the normal cells. The Q system works in mammal cells but further improvements are necessary to use this tool for generating transgenic mice or for some application to zebrafish and worm, rather than flies.
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Novel application of live-cell imaging
Posted on August 17th, 2010 No commentsThe reprogramming of somatic cell is a difficult process and the very low rate of success is the challenge that scientists must overcome. Researchers at the Harvard Stem Cell Institute set up a live cell imaging system to follow the differentiation process step by step. They used mouse fibroblast as a model. These cells had been previously transduced with viruses carrying the expression of Oct4, Sox2, c-Myc and Nanog under control of doxycycline. In order to recognize specific lineage during reprogramming, scientists introduced also a doxycycline- inducible fluorescent protein. After induction, they took pictures every 6-12 hours to identify the pluripotent stem cell colonies. They traced many colonies back to a group of cells that quickly proliferated and had peculiar characteristics in terms of size and shape. This observation is in contrast with previous ones because it seems that cells follow an early decision in the ability to reprogramm, rather than a stoichastic event. Nevertheless, this kind of experiment didn’t investigate the presence of a stoichastic event at the beginning of the process, so this hypothesis cannot be ruled out without further studies. Anyway, the live imaging gave important information and other advances in this technique can help to deepen the knowledge of cell dynamics.
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Secret of the effective communication
Posted on August 13th, 2010 No commentsA team of psychologists of the University of Princeton demonstrated that speaker and listener activate the same neuronal areas during a conversation. The brain area involved in speaking is the Broca’s area, while a speech is completely understood in the Wernicke’s area. Thus, in principle the areas of brain involved in a conversation may be different between speaker and listener. Nevertheless, some recent theories hypothesizes the presence of mirror neurons which fire when one person observes the action or listens the speech of his partner. Scientists wanted to understand whether this mirror property was effectively present in our behaviour. Thus, they analyzed the brain activity of a speaker by magnetic resonance imaging, they recorded the story and played it to some listeners. Again, scientists measured the brain activity by the same technique and observed a synchronization of the brain areas activated in the speaker and in the listeners. Furthermore, they found a correlation between the strength of the coupling and the ability to recall the story listened. This study may be the biological explanation of the effective communication: a good speaker is able to induce attention and neural mirror in the listeners.
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The siRNA principle and some suggestions to design good siRNA
Posted on August 11th, 2010 No commentssiRNA is a gene-regulatory mechanism triggered by dsRNAs. siRNAs which consist of duplexes of 21-23nt RNAs that are base paired with 2-nt 3’ overhangs and is complementary to target mRNA, mimic intermediates of the natural processing of longer double-stranded RNA triggers by RNA-polymerase III. RNA-polymerase III and other component of RNAi machinery specifically recognize an siRNA duplex and selectively incorporate one of the siRNA strands into different RISCs (RNA-induced silencing complex), including the catalytic endonuclease-containing complex, which is responsible for the strong gene-knock down effect. The strand antisense to the targeted mRNA is often referred to as guide strand and its base-paired sense strand is named passenger strand, which is destroyed upon the incorporation of the guide strand into RISC. The catalytic RISC recognizes mRNAs containing perfect or near perfect complementary sequence and cleaves the mRNA at the site precisely 10nt upstream of the nucleotide opposite the 5’-most nucleotide of the guide strand. The mRNA fragments are subsequently degraded by cellular nucleases, resulting in knockdown of the expression of the corresponding gene.
The selection of siRNA against a gene of interest starts with an annotated target mRNA sequence, including its 5’-3’ untranslated regions (UTRs), splice, polymorphic and allelic variants; nevertheless the coding sequence is most commonly targeted. Several algorithms have been developed in recent years that rely on intrinsic sequence and stability features of functional siRNAs; after selection, each candidate siRNA is examined for similarity to all other mRNA transcripts that might unintentionally be targeted at a genome-wide level. It has been demonstrated that structurally symmetric but primary sequence-asymmetric siRNAs from which the target mRNA complementary guide strand has great propensity to be assembled into RISC than the passenger strand, show improved efficacy and specificity. Most of the functional siRNAs have a low to medium G+C content ranging between 30-55%. Indeed, too low G+C content may destabilize siRNA duplexes and reduce the affinity for target mRNA binding, while too high G+C content may avoid RISC loading or cleavage-product release. Additionally, stable duplexes avoid of internal repeats or palindromes are the better silencers.
Single nucleotide positional preferences into siRNA sequence have been identified: U or A at position 1, C or G at position 19, A+U richness between position 1 and 7, these features correlate with the rule of thermodynamic asymmetry and the preferred nucleotides on indicated positions may contribute to the bias for selection of antisense strand; A or U at position 10 may promote catalytic RISC mediated passenger strand and substrate cleavage.
Each strand of the siRNA duplex , once assembled to RISC, can guide recognition of fully or partially complementary target mRNAs, named as on and off targets respectively. Off targets can share contiguous and centrally located sequence complementarity over more than half of the siRNA sequence somewhere within the mRNA sequence, as well as show solely 6 or 7 nucleotides of perfect match preferentially in the 3’UTRs with position 2-7 and 2-8 of the guide siRNA. To enforce the specificity, the current strategy is to select siRNAs in which the strand entering in RISC has some mismatches to all undesired target mRNAs. -
Fatty food addiction?
Posted on August 9th, 2010 No commentsIt’s largely known that drugs are addictive because they alter the expression of dopamine receptors on the brain. The same mechanism seems to involve the pleasure centre of the brain to generate addiction to fatty food. The study has been recently published in Nature Neuroscience journal. Scientists from the Scripps Research Institute studied the behaviour of three groups of rats. The first group was fed with normal food, the second was fed with fatty food, such as cheesecake, bacon, sausage, frosting, for one hour a day, while the third group was fed with this junky food for 23 hours a day. Animals of the third group became obese and began eat compulsively: in other words they developed resistance to the pleasure gained with the food. In few day these rats focused their attention only on the food and even if they were stimulated by the electroshock, they didn’t go away from the food. Scientists analysed the brain and noticed that the level of dopamine receptor decreased in obese rats. This preliminary work on animals must be confirmed in human: indeed, the transposition of this animal behaviour on human could be tricky. Further studies are necessary to deepen this interesting topic.
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