The human immunodeficiency virus type 1 (HIV-1) is the primary cause of the acquired immunodeficiency syndrome (AIDS), which is a slow, progressive and degenerative disease of the human immune system. The pathogenesis of HIV-1 is complex and characterized by the interplay of both viral and host factors. An intense global research effort into understanding the individual steps of the viral replication cycle and the dynamics during an infection has inspired researchers in the development of a wide spectrum of antiviral strategies. Practically every stage in the viral life cycle and every viral gene product is a potential target. In addition, several strategies are targeting host proteins that play an essential role in the viral life cycle. This review summarizes the main genetic approaches taken in such antiviral strategies. The infection spreads to the lymphatic tissue that contains follicular dendritic cells that may act as a storage place for latent viruses. Over time, virus replication leads to a slow and progressive destruction of the immune system. The development of possible methods that can delay progression of the infection or block replication of HIV-1 in infected individuals has been the subject of dedicated research efforts over the past decades. One important issue is that HIV-1 makes use of the replication machinery of the host cell, which minimizes the number of potential viral targets. On the other hand, the close host-virus relationship limits the evolutionary freedom for the viral components that interact with the host molecules.HIV-1 is a lentivirus belonging to the retrovirus family. The virus is diploid and contains two plus-stranded RNA copies of its genome. The approximately 9 kb RNA genome encodes at least 9 proteins, Gag, Pol, Env, Tat, Rev, Nef, Vif, Vpu and Vpr of which only the former five are essential for viral replication in vitro. HIV-1 primarily infects CD4The aim of this review is to take a comprehensive look at the molecular, intracellularly based antiviral strategies that have been reported in literature, and to discuss their potential for development into clinical protocols. We will not discuss vaccine-based strategies that recently have been reviewed in  and 2]..2].The inhibition strategies can be divided into two groups:The RNA-based strategies including anti-sense RNA , RNA decoys (sense RNA), ribozymes, RNA aptamers, small interfering RNA (siRNA), microRNAs (miRNAs) and the protein-based strategies including transdominant negative proteins (TNPs), chimeric proteins (fusion proteins), nucleases, anti-infective cellular proteins, intracellular single-chain antibodies (sFvs) and monoclonal antibodies (Mabs). In addition, other strategies based on suicide genes, protease inhibitors and nucleoside or non-nucleoside analogues have shown to possess the ability to reduce HIV-1 replication.The HIV-1 life cycle including the inhibiting strategies targeted against the various steps in the viral life cycle is summarized in Fig. + T-lymphocytes and the TM protein mediates the fusion between the viral membrane and the host cell membrane [HIV-1 infection is initiated by binding of the virion gp120 surface subunit (SU protein) to the CD4 receptor. The SU protein is attached to the virus by a non-covalent binding to the gp41 transmembrane subunit (TM protein). Both SU and TM are proteolytically cleaved from the Envelope (Env) precursor protein by a cellular convertase, furin, within the endoplasmatic reticulum (ER). Both remain noncovalently attached and are targeted to the host plasma membrane by vesicular transport. The SU protein is responsible for receptor recognition on CD4membrane .Binding to CD4 induces a structural alteration in SU that exposes the binding site for a co-receptor of the chemokine family. The major co-receptors required for entry of HIV-1 are the chemokine receptor molecules CCR-5 (R5 HIV-1 isolates) and CXCR-4 (X4 HIV-1 isolates), which are used by monocytes/macrophage-tropic and T-cell tropic HIV-1 viruses, respectively . When thCurrent strategies are targeting particularly the CD4-SU interaction, the SU-chemokine co-receptor interaction, and the TM-mediated virus-cell membrane fusion process.CCR-5 and CXCR-4 co-receptors have specific chemokine ligands/antagonists that possess the ability to block the virus infection. The molecules that bind to the co-receptors can be divided into four categories: naturally occurring chemokines and their derivatives, peptides and small molecules (< 1 kDa), and Mabs, which recognize epitopes on for instance the extracellular domains of certain receptors.α-(n-nonanoyl)-des-Ser1 [L-thioproline2, L-α-cyclohexyl-glycine3] RANTES (PSC-RANTES) [in vitro antiviral activity than AOP-RANTES. Furthermore, it has successfully protected rhesus macaques from intravaginal exposure to a chimeric simian/human immunodeficiency virus containing an R5-tropic envelope of HIV-1 [Examples of chemokine ligands (beta-chemokines) that inhibit infection of R5 isolates include RANTES, a physiological ligand for the HIV-1 co-receptors CCR3 and CCR5. RANTES is actively secreted by normal T-cells. Derivatives of this peptide have been used, including aminooxypentane (AOP)-RANTES , and fro-RANTES) . RANTES of HIV-1 .In addition to RANTES and its derivatives, the chemokine ligands macrophage inflammatory proteins 1alpha/beta  also show an inhibiting effect by mediating a receptor blockade ,11-13. EThe CCR5 amino-terminal domain is thought to play an important role in virus fusion and entry. This knowledge has been utilized in the development of anti-CCR5 Mabs whose epitopes include residues in the amino-terminal domain. Mabs of this kind strongly inhibit SU binding to CCR5 but only moderately inhibit HIV-1 fusion and entry . AnotherPeptides, resembling the CCR5 transmembrane helices, inhibit HIV-1 replication and chemokine signaling by disrupting helix-helix interactions, which may influence the CCR5 structure . T22 is Toxoplasma gondii [Recently, a new kind of CCR5 antagonist has been discovered in a protozoan parasite, a gondii . This prSmall organic molecules, such as AMD3100, potently inhibit HIV-1 replication by an interaction with residues present on one of the CXCR4 extracellular loops, ECL2, and residues within a transmembrane helix, TM4. Upon binding to these residues AMD3100 spans the main ligand-binding cavity of CXCR4, which probably constrains the co-receptor in an inactive conformation .Individuals with a homozygous deletion in the gene encoding CCR5 are healthy and protected against HIV-1 transmission, which suggests that down regulation may not pose any clinical side effects. This knowledge has led to the development of strategies that directly target the mRNA encoding CCR5 or CXC4, either by ribozymes ,18, antiSoluble CD4 (sCD4) is an anti-HIV-1 protein, which can be expressed and secreted from genetically engineered cells. It is a truncated form of the CD4 receptor, composed of the ectodomain that inhibits laboratory-adapted strains of HIV-1. sCD4 probably prevents the binding of the virus to the cell, by binding directly to Env, or indirectly by inducing or repressing cellular factors that influence the viral gene expression ,19.- cells expressing the co-receptor [When sCD4 binds to SU it acts by extending the distance to TM, which inhibits the fusion. But when used against primary isolates, sCD4 was much less successful because of a lower affinity for sCD4. Surprisingly, some isolates became more infectious upon sCD4 treatment. An explanation for this may be that an interaction between the SU protein and sCD4 induces changes in SU, allowing it to bind the co-receptor with higher affinity or increased kinetics. In addition this interaction can eventually facilitate the fusion of HIV-1 with CD4receptor . This hareceptor ,13.+ for a relatively long period of time [siRNA-directed silencing of CD4 mRNA expression has been shown to specifically inhibit HIV-1 entry and thus HIV-1 replication ,24. Howe of time .Strategies based on the intracellular expression of antibodies specific for the HIV-1 envelope (anti-SU) have also been shown to inhibit virus replication. This strategy is based on the usage of sFvs, containing the smallest structural domain that still possesses complete antigen and binding-site specificity of the parental antibody. They are secreted into the medium where they probably act as inhibitors by direct interaction with the viral proteins  to neutrCyanovirin (CV-N), an 11 kDa protein originally isolated from cyanobacteria, potently inactivates diverse strains of HIV-1. It has a high affinity for the SU protein, and when bound it inhibits the SU-CD4 interaction. CV-N possesses the advantage that even high concentrations are non-toxic and it is an extremely stable protein. CV-N has also been coupled to a cytotoxin (Pseudomonas exotoxin), thereby selectively killing HIV-1 infected SU-expressing cells .As the SU protein binds to CD4, it initiates conformational changes in SU, making the interaction between the SU protein and the co-receptors more favorable. After attachment to the co-receptor further conformational changes occur in both the SU and TM proteins, thus weakening their interaction. During this process a transitory pre-hairpin intermediate of the TM protein is created, freeing the previously buried fusion peptide to interact with the host-cell membrane. This exposes the N-peptide and the C-peptide regions on the pre-hairpin intermediate that have been targets for several inhibiting strategies including synthetic C-peptides, N-peptides and sFvs.C-peptides are based on the C terminal end of the fusion peptide, and mimics this part of the fusion peptide when it has its correct fusogenic conformation. T-20, a 36-amino acid C-peptide, is a potent inhibitor of HIV-1 infection. It acts through a dominant negative mechanism and interacts by binding to a conserved domain on the N-peptide present in the pre-hairpin intermediate. The function of this domain is to mediate a structural change, which allows the pre-hairpin intermediate to form a fusogenic hairpin state. Binding of T-20 inhibits this process and thereby impedes fusion. Disadvantages of the C-peptide strategy are the cost of C-peptide synthesis and the relatively large amounts necessary for an antiviral effect. In addition, their size makes them non-amenable to oral routes of entry and they must be injected instead ,27,28.The 5-Helix is a 25 amino acid N-peptide consisting of five of the six helixes constituting the C-peptide. The peptide is presumed to inhibit fusion, through binding with high affinity to the C-peptide. However because N-peptides have a strong tendency to aggregate the inhibition could also be due to their intercalation into the TM amino-terminal coiled coil ,28.A third kind of peptides named D-peptides have also proven effective. These peptides are small 16–18 D-amino acids residues that specifically bind to three hydrophobic pockets present at the end of the N-peptide. Since such peptides are unnatural, they are resistant to proteolytic degradation, which makes them attractive for clinical use ,27.Recently, a non-neutralizing antibody directed against epitopes exposed on the fusion peptide has been reported to possess antiviral effect . This an3lys. The latter acts as primer and initiates negative strand DNA synthesis by binding to the primer binding site (PBS) region, located immediately 3' to the U5 region [After fusion the viral core enters the cytoplasm and the viral RNA is copied into double-stranded cDNA. This process is mediated by the viral reverse transcriptase (RT) enzyme in a complex consisting of RT, the viral genome, and a cellular tRNA5 region ,4. RT po5 region ,4.Because RT is essential for viral replication it has been one of the most popular targets. This has led to the following antiviral strategies.Inhibiting strategies against RT involve the utilization of nucleosides and non-nucleosides. The nucleoside analogues lack the 3'-hydroxyl group, prevent the continued polymerization of the DNA chain, and are usually named nucleoside reverse transcriptase inhibitors (NRTIs). Clinically approved examples include Zidovudine (AZT), Didanosine (ddI), Zalcitabine (ddC), Lamivudine (3TC), Abacavir succinate and Stavudine (d4T) .The non-nucleoside analogues, often referred to as non-nucleoside reverse transcriptase inhibitors (NNRTIs), act at the same step in the viral life cycle as the nucleoside analogous, but by a significantly different mechanism. Instead of acting as false nucleosides, the NNRTIs bind directly and non-competitively to RT in a way that inhibits the enzyme's activity. Examples of clinically approved NNRTIs include Nevirapine, Delaviridine and Efavirenz .NRTIs bind to the deoxynucleoside triphosphate-binding pocket, which is formed partly by the template-primer nucleic acid and partly by the protein surfaces. NNRTIs bind to a hydrophobic pocket exclusively present in the RT enzyme of M subtype HIV-1. When used in combination they have a more pronounced antiviral effect.3Lys mutant containing an 11 nucleotide 3'-end complementary to the HIV-1 TAR region, shows an inhibiting effect. This mutant competes with cellular tRNA3Lys for the binding to RT and primes reverse transcription from the TAR region instead of the PBS region [The RNA decoy strategy aimed at RT involves the expression of RNAs lacking the PBS region, thus preventing it from acting as template for reverse transcription. The RNA competes with HIV-1 RNA when RT makes the first jump during the first strand transfer . AnotherS region .Other strategies against the RT enzyme involve the usage of small peptides, about 15–19 amino acids long, that inhibit RT activity by interfering with the dimerization process of the RT enzyme. The amino acid sequence corresponds to the so-called connection domain of RT, in particular a tryptophan-rich 19-mer sequence corresponding to residues 389–407, which efficiently inhibits viral replication . LikewisAnti-sense RNAs designed to be complementary to the Psi-gag and the U3-5'UTR-gag-env regions have been shown to inhibit RT in new virion particles. They are co-packaged together with the genomic RNA into the virus progeny, and inhibit reverse transcription by hybridizing to the genomic RNA .siRNAs directed against several regions of the HIV-1 genome, including the viral long terminal repeat (LTR) and the accessory genes vif and nef have provided evidence that the viral genomic RNA, as it exists within the virion as a nucleoprotein reverse transcription complex, is amenable to siRNA-mediated degradation . In addiVal-U5-ribozyme has shown promising results and is currently being tested in clinical trials. Moreover, hairpin and hammerhead ribozymes targeted against the HIV-1 pol region also show promising results [Hammerhead ribozymes targeted against the HIV-1 gag region will cleave the viral RNA before reverse transcription is completed . Hairpin results ,36. In t3Lys is being packaged into new virus particles, this molecule is often used when ribozymes have to be co-packaged. An example is the tRNA3Lys-hammerhead ribozymes targeted against the PBS region. Besides cleaving the HIV-1 RNA, the tRNA3Lys-ribozyme inhibits reverse transcription by competing with host tRNA3Lys for RT binding and/or for the binding to the PBS sequence. Also, when bound to the PBS, the tRNA3Lys-ribozyme is unable to prime reverse transcription [Since host tRNAcription .In a study closely related to the earlier mentioned CCR5 antagonist, C-18, human cyclophilin A (CyPA) has been shown to be incorporated into HIV-1 during virion assembly through interaction with an exposed proline-rich loop within the capsid domain of Gag ,38. CyPAThe next step is the translocation of the cDNA containing capsid into the nucleus. This process is mediated by independent pathways involving either the Vpr accessory protein, the matrix protein (MA) or the integrase (IN) protein. Vpr is thought to mediate the nuclear import of the preintegration complex through the nuclear pore complex (NPC) in non-dividing cells by interacting directly with proteins in the NPC. This transfer of viral DNA is mediated by a nuclear localization signal present in the Vpr protein. Furthermore, it has been shown that Vpr is involved in arresting HIV-infected cells in the G2 phase of the cell cycle, where the virus production has been shown to reach a maximum level -7. IntegSeveral strategies aiming at the IN function have been reported:IN has no known functional analogue in human cells and is therefore an appealing target for inhibiting strategies, which generally involves the usage of oligonucleotides, dinucleotides and different kinds of chemical agents, such as dicaffeoylquinic acids (DCQAs)  and 2,4-The inhibiting effect of a dinucleotide, named pdCpIsodU, is due to its ability to interact with the catalytic core domain . This moDCQAs are non-competitive inhibitors that act by irreversible binding to the catalytic core domain. The exact chemical mechanism for this anti-IN activity is unknown, but it is thought to be caused by a simple redox-process. Two examples are 1-Methoxy-3,5-dicaffeoylquinic acid and 3,4-Dicaffeoylquinic acid. Both are relative non-toxic . FinallysFvs interacting with different domains on IN have been isolated, and by fusion with a nuclease, a fusion protein is created that can interact with IN in the pre-integration complex, leading to cleavage of proviral DNA. Likewise IN-specific sFvs have been shown to be inhibitory to HIV-1 replication ,18.Finally, siRNAs targeted against the capsid protein, p24-siRNA, is thought to interact with the gag gene in the unspliced viral RNA when present in the cytoplasm. Thereby, the viral RNA genome is cleaved before integration occurs ,24,42.Transcriptional regulation of HIV-1 gene expression is controlled by co-operative and cell-specific interactions between several host cells transcription factors, including AP-1, NF-κB, NF-AT, NF-IL-6, CREB, IRF, Sp1, LEF-1/TCF-1α, Ets-1 and USF, and the viral Tat protein ,7,43. ThThe Tat/TAR interaction is essential for activation of HIV-1 transcription and is therefore a popular target for inhibiting strategies. Another reason for choosing strategies directed against this step is that the Tat-TAR interaction is highly conserved. Thus the chance for development of escape mutants is very low, due to the fact that mutations in either Tat or TAR will cause an impaired interaction between them and thereby abolish HIV-1 replication.One strategy is to express a Tat protein that displays a transdominant negative phenotype, which can inhibit the replication of HIV-1. These proteins act as competitors for Tat binding to an essential substrate or co-factor, or alternatively by associating with wild-type monomers to form an inactive mixed multimer. Examples include Tat proteins containing mutations in the activating domain, the protein-binding domain, or in the TAR binding domain ,18,45. ASeveral studies have shown promising results in blocking the interaction of cellular TAR RNA-binding proteins and viral Tat protein to TAR RNA. For instance these include a study in which a compound termed TR87 directly competes with Tat for binding to TAR , and a sAn inhibiting effect has also been observed using anti-sense-, nuclease-, or siRNA-based strategies directed against tat mRNA ,35,45,49A Tat-nuclease fusion protein has been engineered by fusing the HIV-1 TAR RNA binding domain of HIV-1 Tat with the RNase H domain of HIV-1 RT. Since TAR is present at the 5' and 3' ends of all HIV RNAs, this Tat-nuclease can recognize and cleave all HIV-1 RNA transcripts. The Tat protein cycles in and out of the nucleus and the cleavage of HIV-1 transcripts should therefore take place both in the nucleus and in the cytoplasm [TAR decoys represent another example of a suitable strategy. These sense RNAs act by interacting with Tat-containing RNA polymerase II transcription complexes that assemble on the HIV-1 promoter . In addiBesides targeting the Tat encoding regions, siRNAs have been directed against the Gag ,24,42 anRecently it has been reported that minocycline (MC), a second-generation non-toxic tetracycline, possesses the ability to inhibit NF-κB transcriptional activation and thereby viral replication in microglia . These rViral gene expression can be divided into an early and late phase, which is Rev-independent and Rev-dependent, respectively. In the early phase the newly transcribed mRNA is spliced by the cellular splicing machinery into multiply spliced transcripts, which mainly produces the Tat, Rev and Nef proteins. When Rev has accumulated to a critical level the mRNA production shifts from multiply spliced to the singly spliced and unspliced transcripts, characteristic of the late phase of gene expression. Rev contains an RNA binding motif that directly interacts with stem-loop IIB located within an RNA multi-stem-loop secondary structure, termed the rev response element (RRE), which is present in the env gene of all incompletely spliced viral mRNAs. The RRE can accommodate the binding of at least 8 Rev molecules, and at a certain threshold concentration of Rev protein in the nucleus, functional Rev/RRE complexes are formed, which greatly stimulate the export of unspliced and singly spliced RNA to the cytoplasm where translation can proceed. Nuclear export is mediated by cellular co-factors termed exportin 1 and Ran-GTP, which interacts cooperatively with the Rev nuclear export signal (NES) sequence . The nucMany of the used strategies are based on inhibiting the Rev/RRE interaction. Examples include Rev TNPs, RRE RNA decoys, anti-sense Rev/RRE RNAs, siRNAs, sFvs, nucleases, ribozymes, aptamers, chimeric proteins, and small inhibiting molecules.The most well characterized Rev TNP is the RevM10 protein, which contains amino acid substitutions within the NES region. The ability of RevM10 to form multimeric structures and to interact with RRE is not hindered by these mutations, but the interaction with cellular co-factors is prevented leading to inhibition of Rev function ,25,57-59RRE RNA decoys act by recruiting the Rev molecules and thereby hindering their interaction with the viral transcript. Potent inhibition has been achieved by overexpression of a 45 nucleotide chimeric tRNA-RRE transcript . This tysFvs targeted against Rev potently recruit Rev proteins in the cytoplasm, which accelerates the degradation of Rev ,18,25,57Additional strategies for inhibition of Rev function include, 1) RNA aptamers, possessing a higher affinity for Rev than the RRE sequence, and which recognize other epitopes than the natural RNA binding site on Rev , 2) peptAs with inhibition of the Tat/TAR interaction, the anti-sense and ribozyme strategies can also be targeted towards the Rev/RRE interaction. Anti-sense directed against RRE will inhibit Rev binding, whereas anti-sense directed against the Rev encoding region will hinder the expression of Rev protein . The antBy fusing the Rev protein with a nuclease it is possible to create a nuclease with affinity for the RRE and which therefore has the potential ability to specifically cleave all HIV-1 RNAs carrying the RRE sequence . Also, sFinally, strategies based on small molecules that either interact with host cellular proteins or bind to RRE, have also been applied to inhibit Rev function. Due to the ability to interact with cellular proteins, these molecules are often cytotoxic and therefore not usable for eventual clinical trials. Nevertheless they are included here because less toxic derivatives could be developed in the future. An example is Leptomycin (LMB), an antibiotic agent that interacts with the cellular protein CRM1, blocking the binding to the Rev NES domain. A clear disadvantage of LMB is, besides long-term toxicity, its ability to block the transport of other proteins with a NES domain ,56,57. AAminoglycoside antibiotics, such as neomycin B and derivatives have shown antiviral effect by binding to RRE and thereby hindering the Rev/RRE interaction. The binding of aminoglycoside antibiotics to RNA is very unspecific, and together with a low selectivity, this drug is unfortunately highly toxic for humans ,57.The intercalating dye, pyronin Y, completely blocks the formation of the Rev/RRE complex in vitro. But since pyronin Y also intercalates in DNA, the result is an elevated level of cell death. Other intercalating agents, derivatives of diphenylfuran, have been reported to inhibit the Rev/RRE interaction by causing a conformational change in RRE. These compounds possess the same disadvantages as pyronin Y .As illustrated in fig. Designed anti-sense RNAs directed against sequences located in the 5' UTR of all HIV-1 mRNAs, would be able to hinder the ribosome-complex in completing the translation process and thereby inhibit the protein synthesis . HairpinA study involving nef shRNAs corresponding to nef-derived miRNAs, which recently have been demonstrated to be produced in HIV-1-infected cells , shows tThe virus particle is assembled at the plasma membrane. In this process the Gag and Gag-Pol polyproteins interact with each other by protein-protein interaction, most probably via the capsid (CA) protein domain . The virAfter virus budding from the cell surface, maturation of the virus particle proceeds. At this stage in the virion life cycle the Gag and Gag-Pol polyprotein are proteolytically cleaved by the protease domain of Gag-Pol precursors. Cleavage of Gag results in the MA, CA, NC and CEL viral proteins. The MA proteins forms a matrix under the viral envelope, the CA proteins condenses to form a conical core surrounding the NC-coated RNA genome and the CEL protein is thought to associate with the Env protein and, in addition to mediating packaging of Vpr.Cleavage of the Gag-Pol polyproteins, which is made by ribosomal frameshifting during translation of unspliced mRNA, results in the enzymatic proteins IN, RT and Pro. Both the Gag and Gag-Pol precursors associate with the Env protein by protein-protein interactions between the MA domain and the TM protein. After maturation the virus is ready for another round of infection . StrategThe packaging signal Psi is a highly conserved RNA sequence and is therefore an obvious target for inhibition strategies. The Psi region contains four stem-loops located near the 5' major splice donor and the start of the gag open reading frame and is essential and sufficient for RNA packaging .RNA containing a 1.43 kb region anti-sense to the Psi-gag region has been reported to inhibit packaging of genomic RNA and thus HIV-1 replication with a higher efficiency than the RevM10 mutant ,7,45. AnSince the Psi element is recognized by the NC domain, one strategy is to design a NC-nuclease fusion protein, which will recognize and cleave all unspliced HIV-1 RNAs. The cleavage process occurs in the cytoplasm and this will also inhibit the production of the Gag and Gag-Pol fusion proteins . ExpressThe ability of the NC domains to recognize the genomic RNA can be blocked with RNA decoys containing the Psi sequence. These decoys may form dimers with HIV-1 RNA and thereby compete with HIV-1 RNA dimers for packaging into new virus particles ,45.The structural proteins, Gag and Env, form multimeric complexes during viral assembly. By means of different kinds of Gag TNPs it has been possible to inhibit this step. Besides inhibiting viral assembly, these Gag TNPs also interfere with the viral release process, the uncoating of the viral genome and the reverse transcription . A limitGiven that one function of the Vif protein is to block early processing of the Gag protein, it has inspired the development of a Vif TNP that is missing the blocking function. Applying this protein means that the virus assembly is impaired, but a clear disadvantage is that HIV-1 has the potential to evolve escape mutants . SimilarRecently, a specific inhibition of virus budding was demonstrated by overexpression of an amino-terminal fragment of tumour susceptibility gene 101 (Tsg101) . The rolex vivo or in mice in vivo.By targeting an intracellular sFv specifically against the CD4 binding region of the SU protein, it has been possible to make cells temporarily resistant to HIV-1 infection. This sFv, named sFv105, acts by binding to the Env protein, and traps Env in the ER. This prevents the maturation process in which Env is cleaved into the SU-TM proteins. As a result, Env is prevented from reaching the cell surface ,18. AnotFurthermore, anti-sense RNA approaches directed against the Env message also belong to the applied strategies .Multitarget ribozymes that target different sites in the SU sequence also exert antiviral effects. Some of these designed ribozymes bind and cleave up to nine different conserved regions in the SU sequence .A Gag-nuclease fusion protein can be packed into new virions and thereafter, in the proceeding rounds of infection, efficiently cleave the viral genomic RNA. Since the Gag protein has many essential functions it is unlikely that HIV-1 will develop escape mutants when using the Gag-nuclease. Vpr- and Nef-nuclease fusion proteins also seem to cleave viral RNAs, either during or after viral assembly ,7.A Nef TNP has shown an antiviral effect by inhibiting down-regulation of e.g. the CD4 cell surface protein. It is thought that CD4 may interact with the Env protein present on new viral particles, thus hampering viral release from the cell surface . LikewisBy overexpressing different kinds of CD4 variants in the infected cell, it is possible to inhibit virus budding. The reason is most likely that the CD4 variants possess the ability to trap and restrain new virions in the cell .The HIV-1 protease plays an important role in virus maturation. As mentioned earlier, the HIV-1 protease cleaves the Gag and Gag-Pol polyproteins to form the structural and enzymatic proteins. Consequently, the protease is a potent target for inhibiting strategies. The current strategies involve protease inhibitors that bind to the active site of the HIV-1 protease and thereby inhibit processing of the Gag and Gag-Pol polyprotein precursors. This results in immature and noninfectious viral particles. The HIV-1 protease is an aspartyl protease and inhibitors have been designed that optimally bind to the catalytic aspartate residues and additionally to the water molecule that is critical for enzymatic action. The inhibitors are transition state analogues that bind the enzyme much more tightly than the natural substrate, making them competitive enzyme inhibitors. Examples of approved protease inhibitors include Saquinavir, Indinavir, Ritonavir, Nelfinavir and Amprenavir ,11,73.Other strategies that target Pro involve the application of Pro TNP , and theBy combining the different antiviral strategies, the effectiveness can be increased and the chances of generating escape mutants will be minimized. Examples of combination therapies include ribozymes combined with decoy or anti-sense RNAs ,75, and The RT and Pro inhibitors are preferentially not used alone to avoid the risk of generating viral escape mutants. By combining the different kinds of inhibitors  significant inhibition of HIV-1 is achieved. This combination strategy is also known as HAART . Because nucleoside and non-nucleoside analogues act on two different positions on the RT enzyme they will not compete for binding and when used in combination they exhibit a more potent effect. The disadvantage concerning this strategy is the relative strong toxic effects related to these RT drugs. Another problem arises if the prescribed treatment is not exactly followed and resistant viral mutants emerge .Val-RRE-SLII-U5 hairpin ribozyme, in which SLII (stem loop II) contains the Rev protein-binding site that acts as a decoy [When combining strategies involving ribozymes and RNA decoys one can obtain better results than by using one of the strategies. This is clearly illustrated by the tRNA a decoy .In an attempt to interfere with two essential HIV-1 activities at the same time, a double transdominant negative Tat-Rev fusion protein, Trev, has been designed. This fusion protein inhibits both Tat transactivation and Rev mediated nuclear transport . A similThe combination of an anti-Rev sFv, which targets the Rev activation domain, and a ribozyme that targets RRE, or an RRE RNA decoy, which recruits the Rev molecules, has also shown good results . PromisiConditional expression of suicide genes in cells infected with HIV-1, e.g. by expression from a Tat dependent promoter or under Rev dependent control, has been designed in different versions. Examples of suicide gene approaches include engineering cells with a diphtheria toxin A-chain (DT-A) gene, a cytosine deaminase gene, a herpes simplex virus (HSV) thymidine kinase (tk) gene, and a herpes simplex shutoff (vhs) gene.DT-A is a very effective cellular toxin that kills cells by blocking the protein synthesis via the ADP-ribosylating elongation factor 2 . CytosinAnother approach involves the design of a hybrid molecule consisting of the human CD4 and a modified version of the Pseudomonas exotoxin A (CD4-PE40). This molecule binds to infected cells by a CD4-SU interaction at the cell surface and, after uptake, the exotoxin inhibits protein synthesis, thus leading to cell death .Finally, the last example involves a modified apoptosis-promoting caspase-3 protein, Tat-Casp3, which acts by cleavage of the inhibitor of caspase-activated DNase, resulting in the activation of caspase-activated DNase and, ultimately, cell death. In this design the endogenous cleavage sites have been substituted with the HIV proteolytic cleavage sites, and the Pro domain of the modified Casp3 protein was removed and substituted with the Tat transduction domain. Hence, the fusion protein is only activated by the HIV protease in infected cells, resulting in apoptosis, whereas in uninfected cells it remains in the inactive zymogen form. Tat-Casp3 proteins may also be packaged inside the virion and kill the virion after it buds from the cell and/or initiate apoptosis immediately after subsequent infection of a cell .+ T-cells and thereby a progression of the disease [In spite of the astonishing diversity of methods developed as antivirals against HIV-1, still many problems remain. Perhaps the most difficult problem to solve is the remarkable ability of HIV-1 to evade the different inhibiting strategies. The selection pressure enforced by the treatment may result in the selection of escape mutants that are more pathogenic than the original virus. For instance: Blocking the interaction with CCR5, which is the primarily used co-receptor, could result in usage of both the CCR5 and the CXCR4 co-receptors or CXCR4 alone. The outcome will be an accelerated reduction of CD4 disease . The risRNAi is a very promising novel approach that in principle will provide a large number of new targets that may be combined, but unfortunately one of the biggest hurdles is the in vivo delivery problem, which needs to be addressed. A gene therapy approach may be used to make hematopoietic stem cells resistant to HIV-1, which could eventually lead to  restoration of the immune system.In spite of the advanced technology used in the different virus intervention strategies and the rapidly growing knowledge about the molecular biology of HIV-1, it has not yet been accomplished to block HIV-1 replication completely. Hopefully, scientists will succeed to push the balance of the virus-host battle in the right direction so that the immune system will be able to handle the remaining viruses, or vaccine strategies may be added to clear the virus. Independently of the path taken, it will most likely require that the latent reservoirs of virus are activated to make them more vulnerable to treatment. A combination of these directions may eventually lead to a complete eradication of the virus in infected patients.Aids; acquired immunodeficiency syndromeHIV-1; human immunodeficiency virus type 1shRNA; short hairpin RNAsiRNA; small interfering RNARNAi; RNA interferencemiRNA; microRNAdsRNA; double stranded RNAGag; group specific antigenCA; capsid proteinMA; matrix proteinNC; nucleocapsid proteinCEL; core-envelope-link proteinGag-Pol; group specific antigen-polymeraseRT; reverse transcriptaseIN; integrasePro; proteaseEnv; envelopeSU; surface proteinTM; transmembrane proteinTat; trans-activator of transcriptionRev; regulator of expression of virion proteinsNef; negative effectorVif; virion infectivity factorVpr; viral protein rVpu; viral protein uTAR; trans-activation responsive elementRRE; rev response elementPsi; packaging signalDLS; dimer linkage structurePBS; primer binding siteLTR; long terminal repeatR; repeatU5; unique 5'U3; unique 3'ER; endoplasmatic reticulumNES; nuclear export signalNLS; nuclear localization signalTNP; transdominant negative proteinsFv; intracellular single-chain antibodyMab; monoclonal antibodyTRTI; template analogue RT inhibitorNRTI; nucleoside reverse transcriptase inhibitorNNRTI; non-nucleoside reverse transcriptase inhibitorsHAART; highly active antiretroviral therapySELEX; systematic evolution of ligands by exponential enrichmentDCQA; dicaffeoylquinic acidLMB; leptomycinTsg101; tumour susceptibility gene 101MVB; multivesicular bodyDT-A; diphtheria toxin A-chaintk; thymidine kinaseRANTES; regulated upon activation, normal T-cell expressed and secreted beta-chemokinePBMC; human peripheral blood mononuclear cellCV-N; CyanovirinCyPA; human cyclophilin AThe author(s) declare that they have no competing interests.


Familial Juvenile Hyperuricemic Nephropathy is an autosomal dominant nephropathy, characterized by decreased urate excretion and progressive interstitial nephritis. Mutations in the uromodulin coding UMOD gene have been found responsible for the disease in some families.novel heterozygous p.K307T mutation in an affected female with hyperuricemia, renal cysts and renal failure. The proband's only son is also affected and the mutation was found to segregate with the disease.We here describe a This mutation is the fourth reported in exon 5. Initial studies identified a mutation clustering in exon 4 and it has been recommended that sequencing this exon alone should be the first diagnostic test in patients with chronic interstitial nephritis with gout or hyperuricemia. However, regarding the increasing number of mutations being reported in exon 5, we now suggest that sequencing exon 5 should also be performed. Renal impairment usually appears between 15 and 40 years of age, leading to end-stage renal disease (ESRD) within 10 to 20 years 62000 is . The can disease  are resp disease  and NPHP disease  account  disease ,12.novel heterozygous missense mutation in affected individuals from a Portuguese FJHN family that also displays corticomedullary cysts on ultrasound examination. This mutation, c.920A→C, resides in exon 5 and is the fourth reported outside exon 4.We here describe a The proband is an affected female who was first evaluated at the age of 24, when she presented with a gout attack and hyperuricemia. At age 27 she was told having renal failure. However, a renal biopsy was not performed. At age 44, serum creatinine was 2.8 mg/dl and ultrasound imaging detected numerous renal cysts. Renal disease slowly progressed and the patient reached ESRD when she was 49 years old. Her father died at the age of 55 from ESRD and suffered from hyperuricemia and gout. The proband's only son is also affected. At the age of 18 years he had a gout attack. On the initial evaluation, serum uric acid was 15 mg/dl and serum creatinine 1.6 mg/dl. Renal cysts were also detected on ultrasound examination.Informed consent was obtained from tested individuals. Genomic DNA was isolated from peripheral blood leucocytes and the coding region of the UMOD gene was screened for mutations by direct sequencing of PCR products. We used a set of primers previously described  except inovel heterozygous missense mutation, c.920A→C, was detected  anchored protein that accounts for the primary structure of the 85-kD Tamm-Horsfall glycoprotein (THP). THP is the most abundant protein in the urine and, in normal kidney, uromodulin expression is restricted to the thick ascending limb (TAL) and distal convoluted tubule. Urinary excretion occurs by proteolytic cleavage of the GPI counterpart at the luminal surface of TAL. It has been suggested that one of THP major role is of an urinary anti-adherence factor preventing type 1 fimbriated E coli from binding to the urothelial receptors .missense mutations affecting cysteine residues  declare that they have no competing interests.JC was responsible for the study design and drafted the manuscript. JC and CC carried out the molecular analysis. AG collected the clinical data. JR participated in the study design.The pre-publication history for this paper can be accessed here:


Cell migration is a complex phenomenon that requires the coordination of numerous cellular processes. Investigation of cell migration and its underlying biology is of interest to basic scientists and those in search of therapeutics. Current migration assays for screening small molecules, siRNAs, or other perturbations are difficult to perform in parallel at the scale required to screen large libraries.We have adapted the commonly used scratch wound healing assay of tissue-culture cell monolayers to a 384 well plate format. By mechanically scratching the cell substrate with a pin array, we are able to create characteristically sized wounds in all wells of a 384 well plate. Imaging of the healing wounds with an automated fluorescence microscope allows us to distinguish perturbations that affect cell migration, morphology, and division. Readout requires ~1 hr per plate but is high in information content i.e. high content. We compare readouts using different imaging technologies, automated microscopy, scanners and a fluorescence macroscope, and evaluate the trade-off between information content and data acquisition rate.The adaptation of a wound healing assay to a 384 well format facilitates the study of aspects of cell migration, tissue reorganization, cell division, and other processes that underlie wound healing. This assay allows greater than 10,000 perturbations to be screened per day with a quantitative, high-content readout, and can also be used to characterize small numbers of perturbations in detail. When wounded or scratched, cell monolayers respond to the disruption of cell-cell contacts and an increased concentration of growth factors at the wound margin by healing the wound through a combination of proliferation and migration -3; theseWound healing assays are a classic and commonly used method for studying cell migration and the biology underlying it . They haIn order to perform high throughput screening of cell migration, we developed a wound healing assay in a 384 well plate format that does not require expensive reagents, provides consistently shaped wounds, can provide detailed information on numerous processes involved in cell migration, and provides a quantitative, information-rich readout. We use multiple imaging technologies to assay the results and compare their relative merits.For the development of a high-throughput wound healing assay we chose to use BS-C-1 cells, a cell type with a classic wound healing response on glass coverslips Figure . BS-C-1 To adapt this assay for parallel screening, we needed a method for introducing uniformly sized wounds in the same position of each well. We used a 96 well floating-pin transfer device – a tool primarily used for the transfer of solutions between plates. A floating pin array, with foam padding placed between the top plate and the pins, provides an adaptive stop to pin height and overcomes problems with plate planarity. We have adapted a 24-channel aspirator in the same manner for small-scale work.To wound all 384 wells in a plate, the 96 well pin array is placed in the corner of a well, pushed down to engage all pins with the surface of the plate, and then moved laterally to produce the wound. This is then repeated in three neighboring wells to cover the plate and produce uniform wounds throughout Figure . After tWe used four different imaging technologies to analyze the results of this assay, each with distinct advantages and drawbacks [summarized in For highest resolution imaging of the assay we chose an end point of 7 hrs, when migration can be clearly seen, and used an automated fluorescence microscope to image individual wells after fixing and staining for filamentous actin and DNA Figure . The micUsing automated microscopy, the image resolution is relatively high and the time required to image an entire plate is relatively long. Imaging one 384-well plate takes ~1 hr at 4× magnification. At 10× magnification, ~1.5 hrs are required per plate because at higher magnification two images per well must be taken to ensure that the wound edge is captured. We found it more informative to observe the entirety of the wound at 4× rather than parts of it at 10×, despite the higher resolution in the latter case.A fluorescence scanner can be used to determine the extent of cell migration at the 7 hr timepoint. By staining filamentous actin and using a fluorescence scanner with a 42 μm/pixel resolution setting, we can observe consistent differences between normal and inhibited migration. Control wells show a veil of less-densely stained, migrating cells that extend into the wound with a concomitant decrease in wound width Figure . TitratiA simple transmitted-light scanner, normally used for scanning documents and costing less than $1,200, can also be used to monitor wound healing at 7 hrs and only requires that cells are stained with a dye. Figure In both cases, scanners do not provide specific information on cell morphology or other subtle effects in the 7 hr assay; however, acquisition time is greatly decreased. For the fluorescence scanner, acquisition time is 26 minutes at the resolution and image quality shown . For the conventional scanner, acquisition time is 8.5 minutes (at 10 μm/pixel, 2400 dpi) and cell staining with Coomassie Brilliant Blue takes only 10 minutes.The lowest resolution imaging technology that we tested, a fluorescence macroscope had a resolution of ~100 μm/pixel. Detecting wound healing at this resolution required an incubation time of 24 hours after wounding as differences are not readily seen with this method at the 7 hr timepoint. Cells are wounded and allowed to recover for 24 hours before fixing, staining for filamentous actin, and imaging. The macroscope captures an image of the entire plate. At this magnification and resolution, an unhealed wound is seen as a non-staining, black streak within the monolayer Figure . In contThe time required for acquisition of images using this technique, is limited only by the fluorescence signal and was typically ~5 seconds per plate.With all of the imaging techniques discussed here, we initially scored the assay by visual inspection. Visual inspection is fast, information-rich, and can distinguish subtle effects. This method proved useful during adaptation and optimization of the assay in high-throughput format. However, screening large numbers of perturbations by visual inspection is limited by subjectivity, operator fatigue, and the lack of quantifiable metrics. Thus, we developed an automated image analysis routine to provide a rapid and quantitative measurement of the wound healing assay with images captured using the automated microscope . Using images captured by automated microscopy with a 4× objective, we are able delineate measurable characteristics of the wound. After 7 hrs recovery, actin staining defines the extent of healing as well as the morphology of the cells and, because of cell polarization, DNA staining defines the approximate starting point of the migrating cells Figure . ApplyinAutomated analysis and visual inspection are complementary approaches. To illustrate this, we compare the two approaches using the results from one 384 well plate of a screen for small molecules that affect wound healing Figure . The autWe have used methods described in this paper (notably, automated microscopy) to screen ~1,000 bioactive and ~20,000 random drug-like compounds. We were able to reproducibly identify compounds that affected wound healing with different effects including: inhibiting migration, affecting morphology, blocking completion of mitosis, and disrupting the cell monolayer. The details of these results will be presented elsewhere.We present a cell migration assay in a 384 well plate format through the adaptation of tissue culture cell wound healing. We have also compared the readouts provided by four distinct imaging techniques technologies – automated microscopy, fluorescence and transmitted-light scanners, and a fluorescence macroscope – for their relative acquisition speed, image resolution, and information content.High-content image-based screening is often performed at high magnification, however, we have found that low magnification images are information-rich and can be sufficient for observation of detailed phenomena. New screens utilizing imaging technologies are often explicitly developed to image plates at the highest resolution possible given time constraints (10× or higher), rather than at the lowest resolution required to discern differences between wells by visual inspection or automated analysis. Increased magnification further requires acquisition of a greater number of images in order to guard against sampling error. The more sophisticated imaging technologies also carry a hefty price tag. We show here that images taken at magnifications of 4× and lower still allow us to easily discern wells in which wound healing is inhibited and morphology affected. Low magnification imaging is an underutilized method and may be particularly effective for screens that monitor gross changes in protein localization such as nuclear transport, or protein transport from Golgi to plasma membrane; processes in which changes in localization might manifest as changes in the image texture of the stained cell monolayer.While the choice of imaging technologies represents a tradeoff between resolution and information, we think that they should be used together rather than suggesting that they are mutually exclusive. One can iteratively image a given assay plate and take advantage of the best aspects of each technique. For example, a screen for inhibitors of cell migration could initially be performed using a lower resolution technique, taking advantage of the speed to identify interesting wells before imaging just those wells at higher resolution. Related to this, one can re-stain assay plates with different molecular markers. Plates initially screened with a marker that most easily defines the phenotype of interest can be subsequently re-stained with markers that provide different information. In this way, one can consider an assay plate a resource to which one can return to ask new questions.Similar to the tradeoffs seen with different imaging technologies, different image processing techniques like automated image analysis and visual inspection have their distinct advantages but also provide complementary approaches. Our scheme for automated analysis of wound healing allows us to rapidly and easily identify wells in which wound healing is significantly inhibited or morphology clearly affected. However, the automated analysis missed a number of interesting wells that were identified by visual inspection. In part this is because the analysis is based on only a few measures – lamellar width, area, and smoothness. Any automated analysis based on measurable parameters will be limited to phenotypes that manifest along those parameters and might miss more subtle or complex phenotypes that "jump off the screen" when viewed by eye. Automated analysis can also be confounded by artifacts within the plate, dust particles, precipitates, or fluorescent small molecules. Instead of being thought of as a complete answer, automated analysis can enrich for wells that differ from the norm. We don't address automated analysis with the other imaging techniques, but from what can be seen by eye, automated approaches that measure the intensity of staining at the wound edge, or the steepness to which the intensity at the wound edge drops off should provide a good measure of wound healing at very low magnification and resolution.As with any complex phenotypic assay, cell migration during wound healing can be inhibited by effects on global cellular processes – e.g. inhibition of protein translation, disruption of metabolism, disruption of ion homeostasis, etc. – and validation of the specificity of new perturbations is required. Even if the molecular target of the perturbation is unknown, this assay can be adapted or used to distinguish compounds that specifically inhibit or potentiate cell migration. For example, partial inhibition of migration with low concentrations of cytochalasin D could create a sensitized screen for cell migration and used to find suppressors or enhancers. Other complementary approaches include comparative screens across multiple cell lines that migrate differently and the use of specific trophic factors and cognate cell lines such as VEGF and endothelial cells. In our studies, we used still another approach: a series of counter screens that eliminated from our pool of hits, toxic compounds and compounds that do not inhibit processes of interest . This approach has worked well and results will be reported elsewhere.The adaptation of a wound healing assay to a 384 well format facilitates the study of aspects of cell migration, tissue reorganization, cell division, and other processes that underlie wound healing. This assay allows greater than 10,000 perturbations to be screened per day with a quantitative, information-rich readout, and can also be used to characterize small numbers of perturbations in detail.2) and wounding was preformed 12 hours later.BS-C-1 (ATCC CCL-26) cells were grown in DMEM, 10% FCS, and antibiotics. Cells were plated in black 384 well plates with clear bottoms (Corning Costar 3712) at a density of 8500 cells/well in a volume of 50 μl using a liquid dispenser (Labsystems Multidrop). Plates were spun briefly at 500 rpm for ~30 s to in a tabletop centrifuge . Cells were incubated overnight . Foam backing was inserted between the plates of the pin array to provide a resistive stop and the external guide pins were bent to allow greater movement in the z-axis. The pin array was placed in the top corner of a well, pushed down into the plate to engage all pins, and then pulled toward the user. This was repeated in the three neighboring wells to cover all 384. Plates were returned to the tissue culture incubator for 7 or 24 hours before fixation.2, 0.2% Triton X-100, 3.7% Formaldehyde) was added as 30 μl and incubated for 15 min. Wells were aspirated and washed 2× with TBS with 0.1% Triton-X 100 (TBS-Tx) and stained.Cells were fixed after removal of the media with a wand aspirator (VP scientific VP-186L) used along with the Labsystems Multidrop for all liquid handling. Fixation solution  0.5 μg/ml and Hoechst (Sigma B2261) 0.1 μg/ml as 15 μl per well for 15 minutes. Wells were washed 2× with TBS-Tx and imaged. For experiments using the fluorescence plate scanner, after fixation, cells were incubated in TBS-Tx with 2% BSA (AbDil) for 30 minutes, incubated with mouse anti-actin antibody (Chemicon MAB1501) at 1:10,000 in AbDil for 45 minutes, washed 2× with TBS-Tx, incubated with secondary antibodies appropriate for the plate scanner (Molecular Probes A-21057), washed 2× with TBS-Tx and imaged. For experiments using the transmitted-light scanner, after fixation, cells were incubated with SDS-Page gel staining solution (0.25% Coomassie Brilliant Blue R-250, 50% methanol, 10% acetic acid) for 10 minutes, washed 2× with TBS and imaged.We used a NikonTE300 inverted fluorescence microscope with filter wheel (Sutter Lamda10-2), x-y stage (Prior H107N300), and piezoelectric-motorized objective holder (Physik Instrumente P-723.10). Images were captured on a CCD camera (Hammamatsu OrcaER). Metamorph software  running the "Screen Acquisition" drop-in allowed coordination of software-based auto-focusing, movement between wells, imaging, and image evaluation. Images were acquired using a 4× or 10× objective with 2 × 2 binning. Exposure times were ~300 ms for TRITC-phalloidin and ~10 ms for Hoechst. An individual plate took ~1 hr to image at 4× and ~1.5 hrs at 10×. Individual black and white actin images (.tif) were compiled as a .stk file and scrolled through using keystrokes to visually annotate. Visual inspection of 1 plate of images (384) took 10 minutes.An Epson 1680 scanner was used with the positive film setting. A scanning resolution of 1200 dpi  gave an acceptable image with a read time for one plate of 3.5 minute. The image shown in Figure An Odyssey scanner (LiCor) was tested at all combinations of scanning resolution and image quality. Scanning at a resolution setting of 42 um with medium image quality was found to be the optimal balance between speed and image quality. The scanner was used as per the manual, with no modifications.A Tundra macroscope, (Imaging Research – now available as the Leadseeker from Amersham ) a 12 cmWound healing images were analyzed using software written using Visual Basic 6.0 (Microsoft) and Halcon 6.0.1 (MVTec Software) but could be implemented with most basic image analysis software. This software iterates the analysis over images specified by the Metamorph .HTS file and returns values to an Excel spreadsheet.Hoechst images are convolved with a Laplacian-of-a-Gaussian (σ = 2 pixels) kernel. The resulting image is thresholded for pixels of value 0 and binarized. After a binary morphological opening (an erosion followed by a dilation) with a disc of radius 2.5 pixels, the largest contiguous region that does not touch the image edge is defined, and holes within this region are filled.A Kirsch edge detection filter is applied to actin images and the resulting image is thresholded at a manually set value (changed when needed to account for variation in staining intensity). The largest contiguous region that does not touch the image edge is defined and holes within this binary region are filled.lamellar area: area of annulus defined by the difference between the non-actin staining area and the non-DNA staining area; lamellar width: (lamellar area)/(perimeter length of the non-DNA staining region); lamellar smoothness  Wounds generated with the 96 well floating-pin array healed for 0, 3, 7, 12, or 24 hours, were processed as above, and wound regions were measured using automated analysis. Normalized average values and standard deviations of both the area and median width of the non-actin staining region and the non-DNA staining region (see Methods for definitions) are shown. The non-DNA staining region at the 24 hr timepoint was automatically not measured (asterisks) because the non-actin staining region was 0. At least 24 wells of each condition were measured.Click here for fileSample images illustrating automated analysis of wound healing images. (A) Processing of DAPI image to generate non-DNA staining region. (B) Processing of actin image to generate non-actin staining image. (C) Comparison of lamellar wound region with its morphological closing allows assessment of lamellar smoothness.Click here for file


Increases in urokinase-like plasminogen activator (uPA) activity are reported to be amongst the earliest events occurring in remnant liver following partial hepatectomy in rats, and have been proposed as a key component of the regenerative response. Remodelling of the extracellular matrix, conversion of single chain hepatocyte growth factor to the active two-chain form and a possible activation of a mitogenic signalling pathway have all been ascribed to the increased uPA activity. The present study aimed to determine whether similar early increases in uPA activity could be detected in the remnant liver following resection of metastatic tumours in surgical patients.Eighteen patients undergoing partial hepatectomy for the removal of hepatic metastases secondary to primary colonic tumours were studied. Increased plasminogen activator activity was found in the final liver samples for the group of patients in whom the resection size was at least 50%. For smaller resections, the increased activity was not observed. The increased activity did not correlate with the age of the patient or with the time between the start of resection and the end of the operation. There was, however, a negative correlation between plasminogen activator activity and the time for which blood supply to the liver was clamped.Our findings are in accordance with those from experimental animal models and show, for the first time, that rapid increases in plasminogen activator activity can occur following similarly large liver resection in humans. Thus, increases in plasminogen activator activity are an early event in the remnant liver following major liver resection in man. Our observations provide support for the contention that increases in plasminogen activators play a key role in the initiation of hepatic regeneration in man. In vitro uPA and tissue-like plasminogen activator (tPA) have been shown to convert single chain inactive HGF into the active two chain form [et al. [Urokinase-like plasminogen activator (uPA), initially recognised by its ability to convert plasminogen to plasmin and to participate in the fibrinolytic cascade, is now considered to have a wider role, which encompasses metastatic invasion by tumour cells and liver regeneration. In regeneration of the liver following partial hepatectomy, uPA has a number of potential roles. These include initiating the remodelling of the extracellular matrix to allow cell division, activation of extra-cellular pro-metalloproteases and the release of the bound single-chain form of hepatocyte growth factor (HGF) from the extracellular matrix (ECM). ain form  in cultuain form . Followiain form . This imain form . As disc [et al. , uPA mayIncreases in uPA activity are amongst the earliest documented changes following partial hepatectomy in rats . Raised 3H]-thymidine into DNA and mitotic index were reduced by almost half at 44 h post-hepatectomy (the peak time for control mice), suggesting a slower hepatocyte growth response [Additional support for a role for uPA in the hepatic regenerative process comes from studies of uPA-deficient (uPA-/-) mice. In these animals, uptake of [response . In a seresponse . GeneratMore recently, studies with uPA or plasminogen deficient mice confirmed the requirement for plasminogen activation in liver regeneration and also showed a need for plasminogen in regeneration-associated hepatic angiogenesis . CollectThough not necessarily identical, it is clear from the literature that regeneration in humans and rodents share similar mechanisms. Many of the cytokines and growth factors essential for regeneration in rodents [reviewed in ] are alsHowever, clear differences between species do exist; a notable example being the differences in the time at which DNA synthesis peaks in the remnant liver. In rats, this is at about 24 h; in mice, at about 40 h; and in man, at 180–200 h following hepatectomy. In the case of the human studies, this may partially reflect the relatively greater age of the patients since the rate of regeneration slows with age. Such age related effects are less likely in the rodent studies where the timing of hepatocyte entry into DNA synthesis following partial hepatectomy has been shown to be an intrinsic, cell-autonomous, feature . Thus, aThe unique sensitivity of the human hepatocyte to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand)  likewiseThe vast majority of the literature concerns regeneration in rats and mice and much less information is available from human studies since the opportunity to study liver regeneration in humans is generally limited to units specialising in liver surgery and is necessarily constrained by ethical considerations. Surgical removal of liver metastases affords the opportunity to obtain small samples of liver at the start, time of resection and time of wound closure approximating to the early sampling times in the animal studies. In this vein, the aim of the present study was to determine whether very early increases in uPA activity occur in the remnant liver following resection in man.p = 0.7), nor there was any difference between the values for male patients (mean = 9.14 nmol/min/mg, SD = 3.84, n = 7) and female patients (mean = 10.45, SD = 5.83, n = 11) with a non-significant unpaired Student's t test (p = 0.61).The basal uPA activity associated with the membrane preparations showed a wide patient to patient variation ranging from 4 to 24 nmol/min/mg protein with a mean of 9.94 nmol/min/mg protein (n = 18, SD = 5.06). This variation in basal activity correlated neither with the age of the patient – linear regression analysis gave a slope of -0.03 and correlation analysis gave a Spearman coefficient of -0.097 .Standard operative procedures were followed. The liver was mobilised and the resection delineated with diathermy. The portal inflow was clamped while resection with an ultrasonic dissector was carried out. Typically, the portal inflow was released every 15 minutes for 5 minutes intervals to prevent ischaemic damage and the total clamping time was recorded. Resection margins were sent separately for histopathology. The magnitude of the resection was estimated as percentage of the total liver volume, by the surgeon.The following samples were taken from tumour-free regions of the liver during the operation. A sample was obtained before the resection was started (this was labelled 'Start'). Immediately following resection samples were taken from the remnant liver (labelled 'Rem 0' for remnant liver at time 0) and from the resected liver as far away from the tumour as possible (labelled 'Res 0' for resected liver at time 0). The samples were placed in cryovials and immediately frozen in liquid nitrogen in the operating theatre. A second sample of the resected liver (labelled 'Res end') was kept at room temperature until the end of the operative procedure and was only transferred to liquid nitrogen when the final sample from the remnant liver was taken. The final sample ('Rem end' for remnant end) was taken from the remnant liver as late into the operation as possible and frozen immediately. The 'Res end' sample was also frozen at this time. The interval between the time of sampling 'Rem 0' and 'Rem end' ranged from 7 to 90 minutes. The median was 20.5 minutes and 15 of the 18 intervals were between 10 and 33 minutes. Samples were stored in liquid nitrogen in the laboratory and only thawed immediately prior to analysis.Casein, plasminogen, uPA (high and low molecular weight forms) and tPA were purchased from Calbiochem (CN Biosciences Ltd., UK). The fluorometric substrates 7-amino-4-methylcoumarin (AMC), Z-Gly-Gly-Arg-AMC and EGR-CMK (Glu-Gly-Arg-Chloromethylketone) were from Bachem Ltd. (UK). Electrophoresis reagents were from BioRad and Geneflow Ltd. Other reagents were from Sigma-Aldrich Co Ltd. (Poole, UK).Liver samples were homogenised in a ten-fold volume of homogenisation buffer: 250 mM sucrose / 10 mM MOPS pH 7.4 containing the protease inhibitors E-64 (20 μM), Pepstatin A (20 μM), and EDTA (0.2 mM). Inhibitors against the serine proteases, which include uPA, were not included.A membrane preparation was made by differential centrifugation of the homogenate in a TLS 55 swinging bucket rotor in a Beckman TL-100 bench top ultracentrifuge (Beckman Coulter Ltd., High Wycombe, UK). The homogenate was initially centrifuged at 40,000 g, for 20 minutes, to pellet large cell organelles such as nuclei and mitochondria. After centrifugation the fat at the top of each tube was removed with a piece of tissue and the supernatants transferred to clean tubes and recentrifuged at 105,000 g, for 1 hour. The membranous pellets were then washed twice by resuspending in homogenisation buffer and recentrifuged at 105,000 g, for 1 hour. All centrifugations were carried out at 4°C.The protein content of the homogenates and membrane preparations was determined by the BCA (bicinchoninic acid) method  using a uPA activity was determined by a fluorimetric continuous rate assay of Z-Gly-Gly-Arg-AMC hydrolysis using a Perkin Elmer LS50B fluorimeter linked to an IBM compatible computer running the FLUSYS software . CleavagAt the end of the assay, EGR-CMK (Glu-Gly-Arg-Chloromethylketone), a chemical inhibitor of uPA, was added to check that this compound inhibited the measured activity. Any activity still persisting was taken as not uPA-mediated and subtracted from the rate measured in the absence of EGR-CMK.Since the biologically relevant fraction of uPA is generally considered to be associated with its receptor uPAR and therefore localised to the cell membrane, uPA activity measurements were performed with washed membrane preparations rather than with total liver homogenates. Preliminary experiments demonstrated the necessary linear response between the measured activity and the volume of membrane preparation assayed (data not shown).All samples were assayed in triplicate at two sample volumes to ensure linearity of activity with amount of extract. The measured rates were then adjusted for the protein concentration of each sample to give a rate in nmoles/min/mg of protein.et al. [Zymography was carried out with 7.5% SDS PAGE two-substrate gels essentially as described by Bryson et al. . The cont tests, simple linear regression, Spearman correlations) were performed using the software in the cited package.All Figures were generated and analysed with the GraphPad Prism package (version 3.0). Statistical analyses (Student's DM initiated the study, carried out the zymography experiments, prepared tissue extracts and drafted the manuscript. KS prepared tissue extracts and carried out the fluorometric assays. AWM and NCB participated in the design and coordination of the study. All authors have read and approved the final manuscript.


Since small RNA molecules were discovered just over ten years ago, it's become clear that these once overlooked bits of genetic material play a decidedly large role in controlling gene expression. Though typically just 21 to 24 nucleotides long, small RNAs regulate a diverse array of cellular processes, from developmental patterning and genome rearrangement to antiviral defense. They typically accomplish these tasks by targeting specific nucleotide sequences to shut down gene expression.Found in both plants and animals, small RNAs come mainly in two classes—microRNA (miRNA) and short interfering RNA (siRNA). miRNAs arise from nonprotein-coding transcripts that adopt extended “fold-back” structures, which are then cleaved by enzymes called Dicer or Dicer-like (DCL). siRNAs, on the other hand, arise from perfectly base-paired double-stranded RNA, which are also cleaved by Dicer. Some siRNAs require enzymes called RNA-dependent RNA polymerases (RdRp). miRNAs and many types of siRNAs function post-transcriptionally—that is, they affect genes that have been expressed, or transcribed, into RNA—to guide cleavage or prevent translation into protein. In plants and some animals, this post-transcriptional RNA interference (RNAi) acts as an adaptive antiviral response, among other things. siRNAs can also “silence” gene expression by altering chromatin—the DNA-protein complex into which chromosomes assemble—and preventing transcription. It is thought that chromatin silencing acts as a genome defense mechanism, guarding against potential damage from mobile genetic elements or invasive DNA (say, from a virus) by keeping genes in the tightly coiled, and thus inaccessible, “heterochromatic” state.While much remains to be learned about the mechanisms and pathways that govern small RNAs, it's becoming clear that they add an important layer of regulation and flexibility to gene expression. Now a team led by James Carrington at Oregon State University and Steve Jacobsen at the University of California at Los Angeles demonstrates that plants have evolved multiple systems to produce distinct classes of small RNAs with specialized regulatory and defensive functions. The first generates miRNAs; the second produces siRNAs that regulate chromatin structure; and the third generates siRNAs in response to viral infections. Each system requires a unique spectrum of functions of three different DCL proteins; the siRNA systems each function in coordination with one of several RdRp proteins. The researchers propose that the expansion and subsequent diversification of these proteins, which occurred in plants but not in many animals, has contributed to the diversification of specialized small RNA-directed pathways.Arabidopsis thaliana, a favorite model organism for plant biologists, Zhixin Xie et al. analyzed a series of mutants with nonfunctional dcl and rdr genes, as well as a few other mutants of interest, to determine how the small RNAs responded to loss of these proteins. Two mutations (one in a dcl gene and one in another gene) affected the miRNAs, either impairing their function or reducing their populations. None of the RdRp proteins had any effect on miRNAs. The researchers performed the same type of genetic analyses on siRNAs and found that a different DCL mutant caused a reduction in one class of siRNAs and that an RdRp mutant nearly eliminated these populations of siRNAs.Working in Arabidopsis genome reveals an important role in genome maintenance, expression, and defense, the authors conclude. Given that large numbers of siRNAs arise from highly repeated sequences—such as those introduced by viruses or mobile genetic elements—it may be that the cell senses such “invasive” sequence duplication events and enlists siRNAs to run interference by silencing these potentially damaging sequences. In this way, chromatin-associated siRNAs may offer an additional line of defense against invasive sequences, on top of that offered by post-transcriptional RNAi—a dual adaptive advantage since a fast-spreading virus or over-proliferating transposon  could wreak havoc on a plant population.The diversity of siRNAs produced by the Whatever other roles small RNAs may play in genome regulation—they have also been implicated in regulating growth and development—their primary responsibility appears to be blocking gene expression. Whether they accomplish that by controlling chromosome activity to prevent gene transcription or by inhibiting or degrading RNA transcripts to block translation into protein, small RNAs appear to make wide-ranging contributions to the overall gene expression program of the cell.


To study the brain from molecules to behaviour, neuroscientists face the challenge of communicating an emerging wealth of information in coherent accessible forms The completion of the human genome project has ushered in a new era in which biology has become an information science. In this new era, sharing of information is quickly becoming a critical aspect of scientific discovery. As directors of National Institutes of Health (NIH) institutes dedicated to neuroscience, we recognize several areas of research where sharing of primary data will be necessary for us to reach our scientific goals, including brain-mapping, genetics, and clinical trials. Progress in each of these areas will require not only new tools for sharing information but a change in our scientific culture. Here we describe some of the recent progress in efforts to map the brain as an example of the potential and the challenge of sharing data in an era when neurobiology, like genomics, is becoming an information science.9 bases and the human brain has roughly 100 × 109 neurons; both the genome and the brain have embedded modules of functional units (genes versus circuits) that can be mapped in space; and localization of both genes and circuits requires computational power that can be distributed across laboratories. But the analogy breaks down quickly. Whereas fundamental genome data can be addressed as unidimensional text of four letters in varying order, a comprehensive map of the brain includes molecular, cellular, system, and behavioral data—all of which are dynamic, interacting, and interdependent. For example, brain circuitry is organized in three-dimensional space constantly changing in time, with each neuron having 103–104 synapses and with many of those synapses capable of plasticity that may, in turn, have significant functional consequences.In parallel to the worldwide effort to map the human genome, investigators in neuroscience have used a range of techniques to map the brain. The efforts share some superficial similarities: the genome has 3 × 10As we emerge from the “decade of the brain,” we are entering a decade for which data-sharing will be the currency for progress in neuroscience.As a testament to the complexity of brain data, a century after the classic age of neurohistology, there are continuing arguments about the taxonomy of neurons, depending on location, morphology, neurochemistry, or RNA profile. For instance, a population of neurons in a small region of the brain, the dorsal raphe, is the main source of the neurochemical serotonin that has been implicated in stress responsiveness and mood disorders. These serotonergic neurons can be subdivided according to rostralcaudal location, axon thickness, or projections . HoweverIndeed, advances in human brain-mapping, like discoveries in astronomy, have until recently largely depended on the tools available. The postmortem studies of the early 20th century provided delineation of cortical areas through light microscopic histology and gross connectional information. Neurochemical techniques in the last three decades yielded maps with cellular and subcellular resolution, identifying populations of cells usually by one or two neurochemical phenotypes. During the same period, electrophysiological approaches revealed the exquisite distribution of function across the brain, within particular brain subdivisions, and within neurons themselves. In the past two decades, direct study of the intact, functioning human brain in healthy and disordered states has been made possible by a variety of neuroimaging modalities. These studies have provided both structural and functional topography at increasing resolution, as well as neurochemical data and, most recently, information regarding neural connectivity . The advcollaboration of scientists who add value to the enterprise by working in multidisciplinary teams; coordination of efforts to attain a goal; and computation through the use of informatics, models, and simulations. The keystone in this new paradigm is, of course, meaningful data-sharing.The challenge now is to integrate this information into a coherent, accessible form that permits hierarchical analysis from RNA to protein to morphology to connectivity to function in a universal language while preserving fidelity. While earlier comprehensive maps in simpler nervous systems, such as the classic lineage maps of invertebrates , could bwww.gensat.org) will soon provide developmental and whole-brain maps of several hundred genes in the mouse nervous system using a bacterial artificial chromosome (BAC) transgenic strategy with fluorescent reporters to provide subcellular resolution. A digital atlas of the mouse brain and associated informatics tools have been developed to organize, visualize, and analyze such gene expression  data generated by researchers (http://www.loni.ucla.edu/MAP/index.html). We now have the capability to map the transcriptional expression of virtually the entire mouse genome in the adult and the developing mouse brain, registering these data to a common, digital atlas. Like the galaxy maps generated by the Hubble telescope, this transcriptional atlas will provide important temporal as well as spatial information, revealing genes that may be expressed only at critical stages of brain development. Similarly, the Human Brain Project (http://www.nimh.nih.gov/neuroinformatics/index.cfm) is an informatics effort funded through several federal agencies to develop databases, analytical and computational simulations, and other resources to assist human brain-mapping as well as other large-scale coordinated neuroscience programs.Several initiatives serving the brain and behavioral research communities are advancing cooperative research. The Gensat Project (http://www.becon.nih.gov/symposium2003.htm).While there are several initiatives at NIH aimed at overcoming the informatics barriers to sharing data and facilitating collaboration, coordination, and computation, we recognize that not all of the impediments to data-sharing are technical. The advent of neurobiology as an information science also demonstrates that the academic culture in which our science develops and the publication culture in which our science is communicated will need to change. Promotion decisions at major universities largely depend on the quality and quantity of first-authored or senior-authored papers. Multidisciplinary studies require teams of investigators in which hierarchical schemes for authorship may fail to reflect accurately the magnitude of each individual's contributions. Similarly, contributions to a research database may represent important scientific and scholarly achievements, but generally are underrecognized by promotions committees counting peer-reviewed publications. Indeed, the nature of publication itself needs to change in an era when some of the most important contributions will emerge from comprehensive descriptions of new landscapes  rather than tests of specific hypotheses. These cultural issues are not peculiar to brain and behavioral science, of course, and have recently been considered broadly at the NIH (Scientific publication, as we have known it in print, is slow and expensive, with access limited to those with either the funds to purchase an individual subscription or the proximity to a library with an institutional subscription. Data-sharing also means open-access publishing so that data, whether from mapping efforts or from hypothesis-driven experiments, become available quickly and freely to the scientific community. As we emerge from the “decade of the brain,” we are entering a decade for which data-sharing will be the currency for progress in neuroscience. Efforts driven by collaboration, coordination, and computation should yield the data, tools, and resources that neuroscientists will need in the coming decades. We hope that new electronic publications with open access will accelerate this change and provide the vehicle for disseminating the most exciting discoveries in neuroscience in a rapid, respected, and ready format.