The Pocket-Science Volume 7

The convergence of nanotechnology, biotechnology, information technology and cognitive science [The NBIC concept] is vital to security and economy.

Security services to access the electronic information system: some definition and the use of PKI (Public-key infrastructure).

Introduction to RNA interference for analysis of gene function and as therapeutic

The convergence of nanotechnology, biotechnology, information technology and cognitive science [The NBIC concept ] is vital to security and economy.

(i) "Scientists of very small draw disciplines together" by Barnaby J. Feder on The New York Times , February 10, 2003

Reprogramming somatic cells to obtain an artificial cytoplast [ (i) identification and analysis of the molecular processes regulating genetics reprogramming and (ii) cellular inducers of genetics reprogramming]

The enucleated oocyte is able to genetically reprogram the nucleus of a differentiated somatic cell . Nevertheless the molecular mechanisms involved in this process are unknown at present (epigenic gene regulation: DNA methylation, Methyl-CpG Binding Proteins, gene silencing, chromatin, nuclear architecture; cellular inducers (physiological / pharmacological) of genetics reprogramming.

References.: Landmines in Iraq: Present Problem, Future Disaster By Aisha El-Awady at http://www.islam-online.net/English/Science/2003/02/article09.shtml

President Bush Details Project BioShield, in the State of Union address. Project BioShield is aimed at supporting biodefense http://www.whitehouse.gov/news/releases/2003/02/print/20030203.html

Scientists fret over unprecedented increases in funding for antimicrobials and vaccines | By John Dudley Miller The Scientist Volume 17 | Issue 7 | 52 | Apr. 7, 2003 http://www.the-scientist.com/yr2003/apr/prof1_030407.html

Biowarfare Agents Producers
COMPANY	INFO
Abgenix (Fremont, CA, U.S.A.)	Human monoclonals, as protectans for a range of pathogens http://www.abgenix.com/
Acambis (Cambridge, MA, U.S.A. and Cambridge, U.K.)	http://www.acambis.com/
Accelrys (San Diego, CA, U.S.A.)	http://www.accelrys.com/ Smallpox Grid Project http://www.grid.org
Anacor Pharmaceuticals (Palo Alto, CA, U.S.A.)	Enzymes for biodefense http://www.anacor.com/
Antex Biologics (Gaithersburg, MD, U.S.A.)	Antibiotics against anthrax and anti-infectives http://www.antexbio.com/
Avant Immunotherapeutics (Needham, MA, U.S.A.)	Oral combination vaccine for anthrax, plague, cholera http://www.avantimmune.com/
Axonyx (New York, NY, U.S.A.)	acetylcholinesterase- inhibitors, butyrrylcholinesterase inhibitors against chemical warfare agents http://www.axonyx.com/
Coley Pharmaceutical Group (Wellesley, MA, U.S.A.)	Immunomodulatory oligonucleotides http://www.coleypharma.com/wt/home.php
Crucell (Amsterdam, Europe)	http://www.crucell.nl/
Cubist Pharmaceuticals (Lexington, MA, U.S.A.) (in collaboration with Syrxx (San Diego, CA, U.S.A.)	New antibiotics http://www.cubist.com/main.html
Dor BioPharm (Lake Foster, IL, U.S.A.)
DynPort Vaccine Co. (Frederick, MD, U.S.A.)	Biodefense vaccines http://www.biospace.com/b2/company_profile.cfm?Co mpanyID=3603
Evotec OAI (Hamburg, Germany)	Smallpox Grid Project ( http://www.grid.org )
GenVec (Gaithersburg, MD, U.S.A.)	http://www.genvec.com/
Hadron Advanced Biosystems (Alexandria, VA, U.S.A.)	Anthrax research (mechanisms of infection) Immune modulatory compounds http://www.bizforward.com/wdc/issues/2002-04/dealsof theyear/fedsmall.shtml
Hematech (Sioux Falls, SD, U.S.A.)	http://www.hematech.com/hematech/default.asp
Hemispherx Biopharma (Philadelphia, Penn, U.S.A.)	RNA-based oral and injectable drugs against biological pathogens in animals Double-stranded RNA products http://www.hemispherx.com/
HollisEden Pharmaceuticals (San Diego, CA, U.S.A.)	Immunestimulating hormones http://www.holliseden.com/
IBM (Armonk, NY, U.S.A.)	Smallpox Grid Project ( http://www.grid.org )
Isis Pharmaceuticals (Carlsbad, CA, U.S.A.)	RNA-based antibiotics for bioterror germs http://www.isispharm.com/index.html
Kaketsuken (Kumamoto, Japan)	Attenuated smallpox vaccine
Kirin Brewery (Tokyo, Japan)
MicroTest Laboratories (Agawam, MA, U.S.A.)	recombinant vaccines against botulinum neurotoxins http://www.microtestlabs.com/
Siga (New York, NY, U.S.A.)	http://www.siga.com/
Therion Biologics (Cambridge, MA, U.S.A.)	http://www.therionbio.com/
United Devices (Austin, Texas, U.S.A.)	Smallpox Grid Project ( http://www.grid.org )
VaxGen (Brisbane, CA, U.S.A.)	Attenuated smallpox vaccine http://www.vaxgen.com/
Vical (San Diego, CA, U.S.A.)l	Vaccines for biodefense http://www.vical.com/
Reference: Devising defenses against biowarfare agents, by Vicki Brower on Genetic Engineering News 23 (5), 1, 74. - 76, 80 (2003)

The recall involves 17,500 hip replacement units, nearly a full year's production. Patients have already undergone "revisions" - replacements of the replacement.

Security services to access the electronic information system: some definition and the use of PKI (Public-key infrastructure).

"Public-key infrastructure (PKI) is the combination of software, encryption technologies and services that enables enterprises to protect the security of their communications and business transactions on the Internet" ( http://verisign.netscape.com/security/pki/understanding.html ) PKI integrates: (i) digital certificates, (ii) public key cryptography, (iii) certificate authorities. PKI protects the information assets in several ways, as reported in the following table:

Authentication	The digital certificate establishes the validity of a transmission, message or originator
Authorization	Access privileges granted to a user, program or process. The digital certificate replaces user ID and password.
Confidentiality	Assurance that information is not disclosed to unauthorized persons, processes or devices during Internet transmission.
Data integrity	Data is unchanged from its source and has not been accidentally or maliciously modified, altered or destroyed
Non-Repudiation	Assurance the sender of data is provided with proof of delivery and the recipient is provided with proff of the sender's identity, so neither can later deny having processed the data.
References: "Security made easy with the NMCI, PKI, and the CAC" by Josephine Smidt with Rebecca Nielsen on CHIPS XXI (I), 43 - 44 (2003) PKI Law - Public key infrastrcture and the law at http://www.pkilaw.com "Ten risks of PKI: what you're not being told about public key infrastructure" by C. Ellison and B.Schneier on: Computer Security Journal 16 (1) , 1 - 7 (2000) ( http://www.counterpane.com/pki-risks.html ) "SSL Server Security Survey" by Eric Murray at http://www.meer.net/~ericm/papers/ssl_servers.html

SSL = Secure Web Servers use the Secure Sockets Layer (SSL) protocol to create an encrypted communications channel between the client and server.

The risks of PKI
Risk 1	"Who made CA trusted?"
Risk 2	"Who is using my private key?"
Risk 3	"How secure is the verifying computer?"
Risk 4	"Which John Waffer is he?" (association of a public key with a name)
Risk 5	"Is the CA an authority?" (Is it an authority on what the certificate contains?)
Risk 6	"Is the user part of the security design?"
Risk 7	"A one-part certification structure (created byCAs) or a two-part certification structure (CAs + RA)?"
Risk 8	"How did the CA identify the certificate holder?" (Identification of the applicant before issuing the certificate)
Risk 9	"How secure are the certificate practices?"
Risk 10	"Why we are uaing the CA process?" It is a business."Caveat emptor" (Let the buyer beware") (
Reference:"Ten risks of PKI: what you're not being told about public key infrastructure" by C. Ellison and B.Schneier on: Computer Security Journal 16 (1) , 1 - 7 (2000) ( http://www.counterpane.com/pki-risks.html )

Epigenetics is commonly defined as the study of heritable changes in chromosome structure or gene function, that occur without changes in DNA sequence.

"Epigenetics: Regulation Through Repression" by Alan P. Wolffe and Marjori A. Matzke in Science 286, 481 (1999)

"Genetic Medicine - A logic of disease" by Barton Childs - The Johns Hopkins University Press - Baltimore & London (1999)

"Nongenomic transmission across generations of maternal behavior and stress responses in the rat" by Darlene Francis, Josie Diorio, Dong Liu, Michael J. Meaney in: Science 286, 1155 (1999)

"Snf1 - a histone kinase that works in concert with the histone acetyltransferase Gcn5 to regulate transcription" by Wan-Sheng Lo et al. Science 293, 1142 - 1146 (2001)

"Argonaute2, a Link Between Genetic and Biochemical Analyses of RNAi" by Hammond S.M., Boettcher S., et. al. , Science 293 ,1146 - 1150 (2001)

"Tansitions in distinct histone h3 methylation patterns at the heterocromatin domain boundaries" by Ken-ichi Noma et al. Science 293, 1150 - 1155 (2001)

"Correlation between histone lysine methylation and developmental changes at the chicken beta-globin locus" by M.D. Litt et al. Science Express 293, 1007 (2001)

"RNA: guiding gene silencing" by Marjori Matzke, Antonius J.M. Matzke, Jan M. Kooter, Science 293 (5532), 1080 - 1083, (2001)

The importance of epigenetics in regulating gene activity is discussed in a special section in Science 293 (5532), 1063 - 1105 (2001)

CORDIS focus - Technology opportunities today (RTD Results Supplement) 42, 4 (May 2003)

Introduction to RNA interference for analysis of gene function and as therapeutic

The notion of "ribonomics" was introduced by Veronique Bourdeau and co-workers to describe the search for the distribution of and eventually the determination of the physiological roles of RNA genes and their structures ( "The distribution of RNA motifs in natural sequences " by Veronique Bourdeau, Gerardo Ferbeyre, Marie Pageau, Bruno Paquin, and Robert Cedergren, Nucleic Acids Res. 27: 4457 - 4467 (2000); http://www.esi.umontreal.ca/~bourdeav/Ribonomics/article.html ) . Broadly speaking, the objective of ribonomics is to delineate the relationship among RNA sequence, secondary topology, tertiarystructure and function. The approach defined previously as "ribonomics" includes also the goal to understant the role of RNA interference within the cell.

RNA interference (RNAi) represents an evolutionary conserved cellular defense mechanism for controlling the expression of alien genes in filamentous fungi, plants, and animals. It is caused by sequence-specific mRNA degradation, and is mediated by double-stranded RNA (dsRNA) homologous in sequence tothe target RNA.

"Among the 3 billion base pairs of the human genome, there are approximately 30, - 40,000 protein-coding genes, but the function of at least half of them remains unknown. A new tool - short interfering RNAs (siRNAs) - has now been developed for systematically deciphering the functions and interactions of these thousands of genes. si RNA are an intermediate of RNA interference, the process by which double-stranded RBNA silences homologous genes." (McManus, M.T. and Sharp,P.A., 2002)

RNA interference refers to the process of sequence-specific, post-transcriptional gene silencing (PTGS) initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. This phenomena, discovered in 1997 by Craig Mello (Univ. Mass. Medical School) and his collaborators at the Carnegie Institution of Washington ( http://www.hhmi.org/research/invrstigators/mello.html ), was previously recognized in plant and insect cells. More recently, Elbashir et al (2001) have shown for the first time that short

dsRNA [(21- and 22-nucleotide size), generated by ribonuclease III cleavage from longer ds RNAs] iintroduced into mammalian cells can result in sequence-specific inhibition of target mRNA without triggering an interferon response.

This finding open up the possibility of using exogenous 21-nucleotide siRNAs for analysis of gene function in human cell culture and the development of gene-specific therapeutics.

Researchers (led by Greg Hannon and Scott Lowe) at Cold Spring Harbor Laboratory have shown that RNAi can be used to set the level of a tumor suppressor gene (called p53) activity in a mouse model of lymphoma on "low", "medium", or "high"

The RNA interference (RNAi), considered as a refinement of antisense technology, has matured in 2002 and is being applied to the study of many biological phenomena, including aging. (see the articles written by Duane Hewitt).

RNA interference (RNAi) & post transcriptional gene silencing (PTGS)
The initiation step	dsRNA recognition and scanning process dsRNA cleavage through Dicer enzymes [a member of RNase IIII family of dsRNA- specific ribonucleases (endonuclease)] production of 19-21 bp duplexes (siRNAs). each with 2-nucleotide 3'overhangs. The 5'phosphate group of siRNA is maintained by a specific kinase; the free 3'hydroxyl group is essential for priming of the subsequent RdRP reaction.
	Dicer (DCR-1): DsRNA-specific endonuclease responsible (i) for processing the long targeting dsRNA (denoted the trigger) into siRNA and (ii) for demonstration of the active role played by siRNA in guiding homology-dependent destruction of the cognate mRNA A member of the Rnase III family of nucleases Evolutionarily conserved in worms, flies, plants, fungi, mammals Structure: Helicase domain dualRNase III motif RDE-4 & RDE-1 (in C. elegans ) PAZ motif (PPD proteins) ATP dependent In Caenorhabditis elegans, a model has been proposed in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to Dicer I (DCR-1) for processing. RDE-4 can bind to long dsRNA but not to siRNAs, mRNAs and microRNA precursors. RDE-4 can interact with DCR-1, RDE-1, and Dicer related helicase (DRH-1). In Caenorhabditis elegans, RDE-1, and perhaps other Argonaute proteins, might shuttle siRNSs to appropriate effector complexes (RISCs). RNAi initiators: Gene: rde-1 and rde-4 ("rde" stands for "RNAi deficient") (C.elegans)
The effector step	Association of the siRNA and associated factors in RISC-I (RNA-induced silencing complexes, I, inactive form) RISC-I activation (ATP dependent) from a latent form, containing a double-stranded siRNA to an active form, by unwinding of siRNAs. RISC-II (RISC-II, as active form) then uses the unwound siRNA as guide to substrate selection. RISC as a flexible platform upon which different regulatory modules may be superimposed Recognition of the complementary target mRNA Cleavage of the target mRNA in the center of the region complementary to the siRNA Degradation of the target mRNA and recycling of the RISC complex
	RISC - The multicomponent nuclease (RNP) contains a ribonucleoprotein complex of ~500 kilodaltons. One constituent of this complex is a member of the Argonaute family of proteins. Dicer and AGO2 might physically interact, perhaps through their shared PAZ domain. Dicer and RISC are biochemically separable, and none of the purified RISC fractions is able to process dsRNA into 22-nt fragments. The current model is that the interaction between AGO2 and Dicer facilitates the incorporation of siRNAs into RISC complexes, which ultimately dissociate from Dicer and target cognate mRNA for the distruction. Similarities between the miRNP discovered by Dreyfuss and. coll. (2002) and the RISC. Might the miniRNP and RISC be one and the same, a single RNP with multiple functions? Alternatively, the miniRNP and the RISC may be distinct complexes containing pathway-specific proteins drawn from the same protein families. Gene: rde2 and mut-7(C.elegans)
Amplification and spreading of silencing	Possible role for RNA-dependent RNA polymerase (RdRP) dsRNA-induced silencing can amplify either the dsRNA prior to cleavage or the siRNA directly.

stRNA [as microRNAs (miRNAs)] / siRNA

Properties

Small temporal RNAs [(stRNAs) referred to lin-4 and let-7 from Caenorhabditis elegans ] regulate developmental timing by mediating sequence-specific repression of mRNA translation. Recently, some laboratories succeeded in cloning additional stRNA-like RNAs from worms, flies, and human cells.These efforts added an additional 19-25nt RNAs to the original pair of stRNA. All of these tiny RNA are collectively known as miRNA. stRNA and miRNA derive from longer stem-loop precursor RNAs ( miRNA pathway ).

The 21-25 nucleotide size of miRNAs is similar to that of small interfering RNAs (siRNA) , the 21-25 nucleotide double-stranded RNAs, that mediate RNA interference ( RNAi pathway ).

Origin

stRNAs and siRNA(small interfering RNA) are excised from hairpin precursor RNAs and from long dsRNA, respectively, by Dicer, a multidomain ribonuclease III protein, thus producing RNA species of similar sizes.

siRNAs are believed to be double-stranded, whereas stRNAs are single stranded. Dicer processing of stRNA differ from that of siRNA in its asymmetry. In addition to Dicer, two members of the PPD family of proteins (ALG-1 and ALG-2) are required for the biogenesis or function of lin-4 and let-7 in worms. PPD proteins probably will be need for miRNA biogenesis in other organisms.

Action

miRNA and siRNA, obtained catalytically by the Dicer action are then incorporated into a RNP complex, the RNA-induced silencing complex ( RISC )

The binding of siRNA triggers target sequence-specific mRNA degradation, a process known as RNA interference (RNAi).siRNA must have perfect complementarity to their mRNA target.

The binding of stRNA leads to translational inhibition of their natural mRNA targets. Neither lin-4 nor let-7 show perfect complementarity to their mRNA targets.

Both the processes are mediated by the RISC. In one possible model an analogous RISC complex is formed containing either siRNA or stRNA: in the former case, cleavage is dependent upon perfect complementarity, while in the latter, cleavage does not occur, but the complex blocks ribosomal elongation ( RISC envisioned to act stoichiometrically in repressing translation, but catalytically in RNA destruction via RNAi). Alternatively, siRNAs and stRNAs may be discriminated and enter related but distinct complexes that target substrates for degradation or translational regulation, respectively.

RISC may be viewed as a flexible platform, upon which different regulatory moleculesmay be superimposed.

Amplification and spreading of silencing

In some organisms (C. elegans, Arabidopsis and Neurospora) RNAi requires a target RNA copying step, whitout which siRNA fail to reach sufficient concentration to accomplish target mRNA cleavage.Role of RNA-dependent RNA polymerases (RdRPs).

(i) A strand of siRNA can be processed into aberrant RNA by RISC , then RdRP initiates primer-independent copying at the 3' end of the aberrant RNA, converting it into dsRNA.

(ii) The siRNA can also bypass RISC processing and can be directly converted into dsRNA by RdRP which uses one of the siRNA strands as primer.

(iii) The dsRNA resulting from RdRP copying of an aberrant transcript is then converted by Dicer into secondary siRNA, which , as a part of a RISC complex, could destry additional aberrant RNA.

The pathways of dsRNA into the mammalian cells

The specific pathway

dsRNA a cleavage process short interfering RNAs [containing 21-nucleotide (nt)-long strands that overlap by 19-nt and have symmetric 2-nt overhangs at their 3' ends. The 3' overhangs can be uridines, but 2'-deoxythymidine (dT) is thought to improve nuclease resistance] RNAi with sequence specific effects (These small RNAs are incorporated into RISC and target cognate substrates for degradation) Degradation of specific mRNA Knock-out phenotype

siRNA introduced by transient transfection were found to effectively induce RNAi in mammalian cultured cells in a sequence - specific manner.

The data from Ribopharma's experiments with SIRPLEX [short (20-24 bp) dsRNA switching off genes even in human cells without initiating the acute-phase respnse] have bbe confirmed in other laboratories.

The non specific pathway

dsRNA (> 30 base pairs) non specific pathway non specific effect degradation of all mRNA global shut-down of protein synthesis Apoptosis

This suppression has been attributed to an antiviral response, which takes place through one of two pathways

In one pathway, long dsRNA activate a protein kinase (PKR), which in its active form phosphorylates and inactivate the translation initiation factor eIF2alpha leading to repression of translation; it shut down all protein synthesis

In the other pathway, long dsRNA acts on 2', 5' oligoadenylate synthetase, which synthesizes a molecule that activates RNAse L, a non specific enzyme that targets all mRNAs.(non specific RNA degradation).

Differences between
the RNAi pattern	the miRNA pattern
si RNA are not transcribed from discrete genes. siRNAs are derived from transgenes, exogenously introduced dsRNA, or viruses.	A microRNA (MIR) gene encodes a primary transcript with a stem-loop structure. The enzyme Dicer cleaves the transcript to form a microRNA (miRNA).
long dsRNA (perfect complementarity	~70nt precursors (stem-loop structures, with loops and bulges of unpaired nucleootides)
Biochemical studies have suggested that PTGS is accomplished , in part, by a mechanism similar to RNAi, in that involves a nuclease that targets RNAs for degradation; subsequently, an enzyme complex, similar to RISC, targets mRNA for translational silencing
double-stranded siRNA , with two unpaired nucleotides at each 3' end. However, there siRNAs as single stranded, like miRNAs (in C. elegans)	miRNA (one strand) (the other strand not produced or degraded ) Are miRNAs single stranded as a consequence of the catalytic activity of Dicer or are miRNAs initially double stranded like siRNAs, but then the anti-sense miRNA strand is rapidly destroyed?
Dicer activation multiple / different PPD proteins different subclassees of stRNAs / miRNAs require different PPD proteins all PPD proteins might be biochemically interchangeable, but be distinguished by distinct patterns of development or tissue-specific expression additional proteins [miRiboNucleoProtein (miRNP) complex]
perfect complementarity to their mRNA target	unperfect complementarity to their target
RISC / mini RNP RISC as the RNP that mediates RNAi (probably the same RISC is recruited by both siRNA and miRNA; in the case of perfect complementary with the target RNA, the complex mediates RNA cleavage; in case of mismatches, the complex mediates translational arrest)
destruction of a target mRNA siRNA acts as a guide restricting the ribonuclease to cleave only mRNAs complementary to one of the two siRNA strands.	Translational inhibition, without altering mRNA stability Undefined mechanism of genetic regulation
RNA interference	Transcriptional gene silencing Post transcriptional gene silencing (TGS can be separated from PTGS by assessing the effect of targeting the promoter regions of genes, regions that are not transcribed into RNA)

SELECTED COMPANIES INVOLVED
Agy therapeutics (S. San Francisco, CA, U.S.A.)	http://www.agyinc.com
Alnylam (Cambridge, MA, U.S.A.)	http://www.alnylam.com/contact.html
Amaxa Biosystems GmbH (Cologne, Germany)	http://www.amaxa.com
Ambion Inc (Austin, TX, U.S.A.)	http://www.ambion.com
Applied Biosystems (Foster City, CA, U.S.A.)	http://www.appliedbiosystems.com
Benitec Australia Limited (St. Lucia, Australia)	http://www.benitec.com.au
Cenix Bioscience (Dresden, Germany)	http://www.cenix-bioscience.com/
Compugen (Tel Aviv, Israel)	http://www.cgen.com
Dharmacon Research Inc.(Lafayette, CO, U.S.A.)	http://www.dharmacon.com
DNAengine Inc.(Seattle, WA, U.S.A.)	http://oligoengine.com
Exelixis (S. San Francisco, U.S.A.)	http://www.exelixis.com
Eurogentec (Seraing, Belgium)	http://www.eurogentec.com
Gene Therapy Systems, Inc. (San Diego, CA, U.S.A.)	http://www.genetherapysystems.com
IBA GmbH (Gottingen, Germany)	http://www.iba-go.de
Imgenex - Innovation in functional genomix (San Diego, CA, U.S.A.)	http://www.imgenex.com
InvivoGen (San Diego, CA, U.S.A.)	http://www.invivogen.com
Mirus (Madison, WI, U.S.A.)	http://www.RNAinterference.com
New England Biolabs (Beverly, MA, U.S.A.)	http://www.neb.com
Nucleonics (Malvern, PA, U.S.A.)	http://www.nucleonicsinc.com/
OligoEngine (Seattle, WA, U.S.A.)	http://www.oligoengine.com
Pierce (Rockford, IL, U.S.A.)	http://www.piercenet.com
Proligo LLC (Boulder, CO, U.S.A.)	http://www.proligo.com
Promega (Madison, WI, U.S.A.)	http://www.promega.com
Quiagen (Valencia, CA, U.S.A.)	http://www.quiagen.com
Ribopharma AG (Kulmbach, Germany)	http://www.ribopharma.de
RNA - Tec NV (Leuven, Belgium)	http://www.rna-tec.com/content0.htm
Sequitur (Natick, MA, U.S.A.)	http://www.sequiturinc.com
Upstate (Charlottesville, VA, U.S.A.)	http://www.upstate.com
Xeragon Inc.(Huntsville, ALA, U.S.A.)	http://www.xeragon.com

"RNA Interference and Gene Silencing - History and Overview" at http://www.ambion.com/hottopics/rnai and http://www.ambion.com/techlib/presentations

siRNA Wizard at the INVIVOGEN web site http://www.invivogen.com (the new web tool to find the best siRNA sequences for a specific target gene)

"The Interactive Fly - Zygotically transcrbed genes - RNAi and Posttranscriptional Gene Silencing - RNAi and PTGS functions and processes" by Thomas B. Brody at http://sdb.bio.purdue.edu/fly/aignfam/rnaistuf.htm

"Using RNA Interference to tune gene activity in stem cells - New method for the study and tratment of disease" (a series of studies by Greg Hannon at Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., U.S.A.)reported on Science Daily at http://www.sciencedaily.com/releases/2003/02/030203071028.htm (the role of a tumor suppressor gene called p53 in a mouse model of lymphoma)

"Genomewide view of gene silencing by small interfering RNAs" by Jen-Tsan Chi et al, PNAS 100 (11), 6343 - 6346 (2003)

Second Horizon Symposium, Understanding the RNAissance, are now posted on Nature 424 (6944) (2003) [ www.nature.com/horizon ]

"RNA interference: producing decaffeinated coffee plants" by Shinjiro Ogita et al, Nature 423, 823 (2003)

"Hes1 is a target of microRNA-23 during retinoic-acid-induced neuronal differentiation of NT2 cells" by Hiroaki Kawasaki and Kazunari Taira, Nature 423, 838 - 842 (2003)

"bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila" by Julius Brennecke et al, Cell 113, 25 - 36 (2003)

"Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing" by Jason W. Myers, Joshua T. Jones, Tobias Meyer & James E. Ferrell Jr. Nature Biotechnology 21, 324 - 328 (2003)

"Vertebrate MicroRNA Genes" by Lim,L.P., et al., Science 299 (5612), 1540 (2003)

"RNAi and the shape of things to come" by Jonathan B. Weitzman, Journal of Biology 2 (4), 23 (2003) http://jbiol.com/content/2/4/23

"Functional genomics of cell morphology using RNA interference: pick your style, broad or deep" by Thomas D. Pollard, Journal of Biology 2 (4), 25 (2003) http://jbiol.com/content/2/4/25

"A functional genomic analysis of cell morphology using RNA interference" by A.A. Kiger et al., Journal of Biology 2 (4), 27 (2003) http://jbiol.com/content/2/4/27

"Epigenetisches genesilencing: RNA interferenz und antisense RNA" by H. Martens & W. Nellen, BIOspektrum 4 , 351 - 354

"An expanding universe of noncoding RNAs" by Gisela Storz, Science 296, 1260 - 1263 (2002)r

"Induction and suppression of RNA silencing by an animal virus" by Hongwei Li et al., Science 296 (5571), 1319 - 1321 (2002)

"Why do miRNAs live in the miRNP?" by Dianne S. Schwarz and Phillip D. Zamore, Genes & Development 16, 1025 - 1031 (2002)

"The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and DExH-box helicase to direct RNAi in C. elegans " by Tabara H.et al, Cell 109 (7), 861 - 871 (2002)

"Nucleic -Acid Therapeutics: Basic Principles and Recent Applications" by Joanna B. Opalinska and Alan M. Gewirtz Nature Reviews (Drug Discovery) 1, 503 - 514 (2002)

"Expression of Small Interfering RNAs Targeted Against HIV-1 Rev Transcripts in Human Cells " by Lee N.S., Dohjima T., et. al., Nature Biotechnol. 20 (5), 500 - 505 (2002)

"Short interfering RNA confers intracellular immunity in human cells" by Gitlin L. et al, Nature 418 (6896), 430 - 434 (2002)

"Ancient Pathways Programmed by Small RNAs" by Zamore P.D.. Science 296: ,.1265 - 1269 (2002)

"A microRNA in a Multiple-turnover RNAi Enzyme Complex " by Gyorgy Hutvagner and Phillip Zamore Science 297 , 2056 - 2059

"Expanding small RNA interference" by Tuschl T., Nature Biotechnol 20 (5), 446 - 448 (2002).

"Gene silencing in mammals by small interfering RNAs" by McManus MT, Sharp PA, Nat. Rev. Genet 3 (10) 737 - 747 (2002)

"RNA silencing: the genome's immune system" by Roland H. A. Plasterk, Science 296, 1263 - 1265 (2002)

"RNA-dependent RNA polymerase, viruses, and RNA silencing" by Paul Ahlquist, Science 296, 1270 - 1273 (2002)

"A short primer ob RNAi: RNA-directed RNA polymerase acts as a key catalysy" by Kazuko Nishikura, Cell 107, 415 - 418 (2001)

"Post-transcriptional gene silencing by doble-stranded RNA" (Flash Animation)Nature Review Genetics 2, 110 - 119 (2001)

"On the role of RNA amplification in dsRNA-triggered gene silencing" by Sijen T. Fleenor et al, Cell 107 (4), 465 - 476 (2001)6-

"Identification of novel genes coding for small expressed RNAs" by Mariana Lagos-Quintana, Reinhard Rauhut, Winfried Lendeckel, Thomas Tuschl Science 294, 853 - 858 (2001)

"An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans " by Nelson C.Lau, Lee P. Lim, Earl G. Weinstein, David P. Bartel Science 294, 858 - 862 (2001)

"An extensive class of small RNAs in Caenorhabditis elegans " by Rosalind C. Lee and Victor Ambros Science 294, 862 - 864 (2001)

"Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells" by Sayda M. Elbashir et al. Nature 411 (6836), 494 – 498 (2001)

"RNA Interference: listening to the sound of silence" by Zamore, P.D., Nature Structural Biology 8, 746 - 750 (2001)

"Argonaute2, a Link Between Genetic and Biochemical Analyses of RNAi" by Hammond S.M., Boettcher S., et. al. , Science 293 ,1146 - 1150 (2001)

"RNA: guiding gene silencing" by Marjori Matzke, Antonius J.M. Matzke, Jan M. Kooter, Science 293 (5532), 1080 - 1083, (2001)

"Role for a bidentate ribonuclease in the initiation step of RNA interference" by Emily Bernstein, Amy A. Caudy, Scott M. Hammond and Gregory J. Hannon, Nature 409, 363 - 366 (2001)

"RNA interference: genetic wand and genetic watchdog" by Julia M. Bosher and Michael Labouesse, Nature Cell Biology, 2 (2) E31 - E36 DOI:10.1038/35000102 (2000)

"The rde-1 gene, RNA interference, and transposon silencing in C. elegans" by Tabara H., Sarkissian M., et. al., Cell 99 (2), 123 - 132 (1999)

"Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences) by Romano N., Macino G. , Mol. Microbiol. 6 (22), 3343 - 3353 (1992)

Understanding the value of technology investments : this goal can be reached by linking Management Innovation with Knowledge Management.

The definition of innovation as "success-oriented flow of knowledge in action" underlines the link between:

The management of innovation facilitates , support and integrates innovation in all aspects of the firm (organization, market, security, human resources, processes, technologies,…). It represents a "box" separated from the Management of Technology but a flow of information needs to exist between these two units for the success of the business.

"Spinning junk into gold" by Ingrid Wickelgren on Science 300, 1646 - 1649 , 2003

introns : templates for making enzymes to shoot genes into new locations ("custom cellular scissors", insertion of therapeutic genes into a chosen location)

inteins : as target for protein purification protocols [Protein property: Biorecognition - Technology: Affinity (AC) ("affinity tags", "miniinteins tags" specific to a particular ligand); peptide bond synthesis to form very large semisynthetic proteins; and for making safer transgenic plants (significant role in transgene containment in transgenic crops)].

Protein splicing is a mechanism by which an internal segment (called intein or spacer) in a protein precursor (mosaic protein) is excided and the flanking regions (called exteins) are religated to create a functional protein. In most of the cases the intein seems to be an endonuclease.

"Control of protein splicing by intein fragment reassembly" by M. W. Southworth et al., The EMBO Journal, 17 (4), 919 - 926 (1998)

"InBase, the New England Biolabs Intein Database" by Francine B. Perler, Nucleic Acid Research 27 (1), 346 - 347 (1999)

"Single-column purification and bio-characterization of recombinant human parathyroid hormone-related protein (1 - 139) " by C. Wu , P.K. Seitz, M. Falzon, Mol. Cell Endocrinol. 170 (1-2) , 163 - 174 (2000) [use of IMPACT T7 system ( (intein-mediated purificationwith an affinity chitin-binding tag); purification of the three-part fusion protein on a chitin column]

"Protein Purification II: Affinity Tags" by Aileen Constans, The Scientist 16 (4), 37 (2002)

"Protein trans-splicing in transgenic plant chloroplast: reconstraction of herbicide resistance from split genes" by Hang Gyeong Chin, et al. PNAS 100 (8), 4510 - 4515 (2003)

Topics
1. Kinetics of the chemical reactions v= dx/dt	1.1. Kinetics of irreversible chemical reactions Influence of the concentration of the interaction compounds (rate of reaction and individual molecular concentrations) - The Guldberg-Waage law: the fundamental mass action relationship valid for all chemical equilibria mA + nB + pC ==>...... v = K [A]^m[B]ⁿ[C]^p
	1.2. Molecularity and order of reaction Unimolecular, Bimolecular, First-Order, Second-order…
	1.3. Initial Rates and Rate Constants for First Order Reaction
	1.4. Zero-order reaction
	1.5. Rate constant in the bimolecular irreversible reactions Irreversible chemical reaction of second-order
	1.6. Kinetics of reversible chemical reactions
	1.7. Reaction rate and temperature. Q₁₀
2. Kinetics of the enzymatic reactions	2.1. Mechanism of action 2.1.1. Enzyme-substrate complex. - The enzyme substrate complex is formed transiently. - The decomposition of the enzyme-substrate complex
	2.2. Factors influencing the rate of the enzymatic reaction 2.2.1. Enzyme concentration 2.2.2. Substrate concentration - Michaelis-Menten Model of Enzyme Kinetics - Lineweaver-Burke form - The Eadie-Hofstee transformation is an alternative linear transformation of the Michaelis-Menten equation. Both the Lineweaver-Burk and Eadie-Hofstee transformation of the Michaelis-Menton equation are useful in the analysis of enzyme inhibition. 2.2.2.1. The Michaelis-Menten constant (K_m) 2.2.2.1.1. The significance of the Km 2.2.2.2. Thermodynamics and the complex enzyme - substrate ^{2.2.2.3. Enzymatic reactions with 2 substrates} 2.2.3. Steady-state theory 2.2.4. Further considerations on the enzymatic kinetics 2.2.5. Effect of temperature 2.2.6. Effect of the pH 2.2.7 Positive and negative effectors of the enzymatic reaction. Effector and apoenzyme; effector and coenzyme. Competitive inhibition, non-competitive inhibition, incompetitive inhibition (when an inhibitor attacks the enzyme-substrate-complex only) 2.2.7.1. Enzymatic inhibitors. 2.2.7.1.1 Competitive inhibition 2.2.7.1.1.1. Totally competitive inhibition 2.2.7.1.1.2. Partially competitive inhibition 2.2.7.1.2. Non-competitive inhibition 2.2.7.1.2.1. Totally non-competitive inhibition 2.2.7.1.2,2. Partially non competitive inhibition 2.2.7.1.3. Graphic representation of the enzymatic reaction in the presence of competitive / non-competitive inhifitors. 2.2.7.1.4. Incompetitive inhibition 2.2.7.1.5. The linkage of the inhibitor to the substrate and not to the enzyme.
	2.3 Inhibition of the enzymatic reaction by high substrate concentrations
	2.4. The substrate as activator of the enzymatic reaction
	2.5 Enzymes catalysing two reactions simultaneously
	2.6. Different enzymes acting on the same substrate
	2.7. Ternary systems 2.7.1. The bond of the enzyme to the substrate and the bond of the enzyme to the activator involve different sites of the enzymatic molecule E + S <=> ES E + A <=> EA EA + S <=> EAS ES + A <=> EAS EAS => E + P + A 2.7.2. The bond of the enzyme to the substrate is realized through the activator E + A <=> EA EA + S <=> EAS => E + P + A
	2.8 The Reversibility: the "Haldane" equation

Discussion on the paper "Mechanism of silk processing in insects and spiders" by Hyoung - Joon Jin and David L. Kaplan, Nature 424, 1057 - 1061 (28 August 2003).

Applications for the production of new materials (material engineering with desired functional properties).

Environmental protection and nanotechnology - Discussion on the decument prepared by Chris Phoenix "Projected Environmental Impacts of the Molecular Manufacturing" ( http://www.crnano.org/EPAhandout.htm ) (Center for Responsible Nanotechnology at http://www.crnano.org

The protection measures applied to intelligence sources and methods are ofyen referred to as "compartmentation" ["Sensitive Compartmented Information " ( SCI )] - (Reference: Intelforum Mailing List Archive at http://listarchives.his.com/intelforum/ )

Odour detection as a useful biometric technology (substance property / physical sensation) from the article: "Smelling out wrongdoers will put the law ahead by a nose" by Jason Burke and Peter Warren, The Observer, December 28, 2003