In these regions, substitution rates are suppressed, which results in an overall substitution process that is not Poissonian. However, the simulation method of Bastolla et al. is representative only for cases in which the product of mutation rate μ and population size Ne is small. How the substitution process behaves when μNe is large is not known.A frequent observation in molecular evolution is that amino-acid substitution rates show an index of dispersion  substantially larger than one. This observation has been termed the overdispersed molecular clock. On the basis of in silico protein evolution as a function of mutation rate and population size. I find that the index of dispersion decays with increasing μNe, and approaches 1 for large μNe . This observation can be explained with the selective pressure for mutational robustness, which is effective when μNe is large. This pressure keeps the population out of low-neutrality traps, and thus steadies the ticking of the molecular clock.Here, I study the behavior of the molecular clock in μNe, and a mostly Poissonian one for large μNe. The former is relevant for the majority of organisms in the plant and animal kingdom, and the latter may be relevant for RNA viruses.The molecular clock in neutral protein evolution can fall into two distinct regimes, a strongly overdispersed one for small  Kimura has argued that the majority of nucleotide substitutions that accumulate in genes over time are selectively neutral, and go to fixation purely by chance . One majin silico, and found significant overdispersion in all cases they considered.An explanation for fluctuations in neutral mutation rate was recently proposed by Bastolla et al. -16. Diffμ and population size Ne is small . Since population size and mutation rate can be substantial in some species (most notably in RNA viruses), it is justified to ask how general this result is for arbitrary values of μNe. It is known that large populations and populations evolving under high mutation pressure experience a strong selective pressure to avoid regions of low neutrality, an effect that has been termed "evolution of mutational robustness" / [1 - exp(2sNe)], where s is the selective disadvantage of the deleterious mutation [μNe, as long as Ne is large in comparison to s.Throughout this paper, I have considered only neutral or lethal mutations. It is a reasonable question to ask if and how deleterious mutations would change my results. The answer is that they probably have only a minor impact, and the less so the larger mutation . TherefoThe present study supports the following conclusions:• Neutral drift of proteins can lead to an overdispersed substitution process for non-synonymous mutations, but not for synonymous mutations.μNe ≈ 10, and extends to values well above 100.• The amount of overdispersion in the non-synonymous substitution process depends strongly on the product of mutation rate and population size. As this product increases, the substitution process becomes more and more Poissonian. The transition region starts at Drosophila, the product of mutation rate and population size is close to one, which is well below the parameter region in which the substitution process turns Poissonian.• It is not clear whether there are any species that have a sufficiently large population size and mutation rate to prevent overdispersion through neutral drift. In by Goldstein and coworkers [n = 25 residues that fold into a maximally compact structure on a two-dimensional grid of 5 × 5 lattice points. There are 1081 distinct possible conformations in this model, and the partition function can be evaluated exactly by summing over the contact energies of all distinct conformations.I implemented a version of the 5 × 5 lattice protein model put forward oworkers -23,32. Ii isThe contact energy of a conformation  is the contact energy between amino acids  at location j and  at location k in the sequence, and  is 1 if the two amino acids are in contact in conformation k, and 0 otherwise. The partition function iswhere f if the contact energy for that conformation is lower than the contact energies of all other formations, Ef <Ei for all i ≠ f, and if the free energy of folding, which is defined aswhere the sum runs over all 1081 conformations. A sequence folds into conformation Gcut. Throughout this study, I used kT = 0.6 and ΔGcut = 0. The contact energies  where taken from Table VI in Ref. [is smaller than some cutoff Δ in Ref. .Ne. The fitness of a sequence was 1 if the DNA sequence translated into a peptide sequence that could fold into a chosen target structure, and had a free energy of folding smaller than Gcut. Otherwise, the fitness of the sequence was 0. All sequences had length L = 75. In each successive generation, sequences with fitness 1 were randomly chosen to reproduce, until the new generation had Ne members. At reproduction, the sequences were mutated, with an average of μ base pair substitutions per sequence. I let each population evolve for several thousand generations, and kept track of the full genealogic information of all sequences in the population. In order to measure the molecular clock of fixed mutations only, I studied the pattern of base substitutions in a window of τ generations along the line of descent backwards in time, starting from the most recent common ancestor of the final population.I simulated the evolution of populations of DNA sequences in discrete, non-overlapping generations. Population size is denoted by Ne , μ , and τ . For each set of parameters, I carried out 500 replicates , to obtain a distribution for the number of synonymous and non-synonymous substitutions Sd and Nd. Since there was some variation in the number of synonymous and non-synonymous sites across different target structures (on the order of approximately ± 5% variation from the mean), I then applied a correction factor to Sd and Nd to bring them into comparable units: I calculated the corrected number of synonymous substitutions  as  Here, S is the mean number of synonymous sites for the given replicate, and (S) is the average of S over all 500 replicates. Likewise, I calculated  . Similar correction factors have been used in sequence analysis [I varied the parameters To obtain an estimate for mean and standard error of the index of dispersion, I subdivided the 500 results into 10 blocks of 50 each, and calculated mean and variance of the number of substitutions for each block. The ratio of variance to mean for a given set of substitutions (synonymous or non-synonymous) in a block is the index of dispersion for this data set. I then calculated mean and standard error for the index of dispersion from the individual results of the 10 blocks.The total CPU time needed to carry out all simulations was several months on a small cluster of Pentium II 500 MHz machines.S and N and the number of synonymous and non-synonymous substitutions Sd and Nd according to the method proposed by Nei and Gojobori [si of a codon i is the fraction of possible substitutions to that codon that leave the residue unchanged. The number of non-synonymous sites ni for the same codon is ni = 3 - si. For the complete sequence, S and N are calculated as  and  where i runs over all codons in the sequence. The number of synonymous or non-synonymous substitutions si d,or ni d,between two codons is the average number of such substitutions, where the average is taken over all paths that lead from one codon to the other. The total number of synonymous or non-synonymous substitutions between two sequences is the sum over all individual constributions,  and  .I calculated the number of synonymous and non-synonymous sites Gojobori . In shorTo calculate the number of synonymous or non-synonymous substitutions along the line of descent, I simply summed up all synonymous or non-synonymous substitutions that occurred from generation to generation. Because the full evolutionary history was known, a correction for multiple mutations such as the Jukes-Cantor correction  was not COW carried out all aspects of this study.


Viruses are obligate intracellular parasites that rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies.5 independently disrupted genes, of which 111 contained mutations in previously characterized genes and functionally anonymous transcription units. Collectively, the genes associated with reovirus resistance differed from genes targeted by random gene entrapment in that known mutational hot spots were under represented, and a number of mutations appeared to cluster around specific cellular processes, including: IGF-II expression/signalling, vesicular transport/cytoskeletal trafficking and apoptosis. Notably, several of the genes have been directly implicated in the replication of reovirus and other viruses at different steps in the viral lifecycle.Candidate genes required for lytic reovirus infection were identified by tagged sequence mutagenesis, a process that permits rapid identification of genes disrupted by gene entrapment. One hundred fifty-one reovirus resistant clones were selected from cell libraries containing 2 × 10Tagged sequence mutagenesis provides a rapid, genome-wide strategy to identify candidate cellular genes required for virus infection. The candidate genes provide a starting point for mechanistic studies of cellular processes that participate in the virus lifecycle and may provide targets for novel anti-viral therapies. Cellular genes are likely to participate in all phases of viral life cycles including attachment to cellular receptors, internalization, disassembly, translation of mRNA, assembly and egress from the cells . The susGene traps are efficient mutagens as assessed by studies in mice of mutations induced originally in embryonic stem cells. In somatic cells, the approach assumes that loss-of-function mutations induced by gene entrapment may confer reovirus resistance as a result of gene dosage effects (e.g. haploinsufficiency), pre-existing heterozygosity or loss of heterozygosity. Following infection with the U3NeoSV1 retrovirus gene trap shuttle vector, libraries of mutagenized rat intestinal epithelial (RIE)-1 cell clones were isolated in which each clone contained a single gene disrupted by provirus integration . The entCtcf mutation, a transcriptional repressor of insulin growth factor II (IGF-II), was one of 4 mutations associated with reovirus resistance that affected IGF-II expression and/or signalling. Subsequent experiments demonstrated that enforced IGF-II expression is sufficient to confer high levels of reovirus resistance  are predicted to affect IGF-II expression and/or signalling. Cells containing the Ctcf mutation were subsequently found to express elevated levels IGF-II, while enforced IGF-II expression was sufficient to confer high levels of reovirus resistance. The resistance was caused, at least in part, by a block in virus disassembly [The genes associated with reovirus resistance can be grouped according to their presumed role in virus entry, disassembly, translation, and maturation. Reovirus enters the host via an endocytic pathway that requires acidification and proteolysis to remove the viral outer capsid. The presumptive roles of several candidate genes would be anticipated to affect virus replication by interfering with virus disassembly. For example the mannose-6-phosphate receptor/insulin-growth factor-2 receptor (lysosome  and acidAtp6v0c) . NH4Cl i A virus ,14. Fourassembly . By infeS100a6/S100a1) – proteins that may bind to each other [Cald1, Kif13b, Mapt, Mkln1, Stmn1, Tm9sf4, and Tpm1). Annexin-II associates with cytomegalovirus virions and anti-annexin-II antibodies have been found to prevent cytomegalovirus plaque formation [Additionally, several of the inserted mutations encode proteins that are thought to participate in trafficking of cargo in cells, and may participate in various stages of virus infection. These include mutations in genes encoding three annexins  and calcyclin (ch other -18 – andormation . Annexinormation ) [17,18,ormation .Jam)-1 [One of the clones has a disruption of a novel cell receptor, OL-16, which is a member of the immunoglobulin superfamily ,22. A prJam)-1 , whereasJam)-1 . OL-16 iJam)-1 . Forced Cstf 64 KaD subunit, which affects polyadenylation of mRNA, can be cross-linked to the mRNA of herpes simplex mRNA in infected HeLa cell extracts [Pts), a major regulator of interferon activity [Several of the candidate genes have products that interact with either reovirus or with other viruses (Supplementary Table see ). Cellulextracts . Other cextracts -30. For activity  associatactivity ,33.Ppm1a [Madh7 [Ube1c [Map3k7ip1 [Mapkbp1 and the proteosome [N-linked protein glycosylation, a process that may affect compartmentalization of proteins or ligand interactions.Many of the targeted gene products have roles involving the Golgi or endosomal compartments Figure , and addPpm1a , Madh7 [a [Madh7 -37, Ube17 [Ube1c ,39, and ap3k7ip1 -43 (Tablap3k7ip1 ). In addoteosome . We haveoteosome ), and thlacZ reporter as an immediate-early gene [Eif3s10, AnxaI, Mgat1, and Igf2r genes, were resistant to HSV-I infection  gene was readily apparent 4 h following infection, whereas lacZ expression was barely detected in Eif3s10 mutant cells 16h following infection. In all cases, levels of lacZ expression correlated with susceptibility to HSV-1 infection, suggesting that resistance involved early steps in the viral lifecycle.As several of the genes listed in the Supplementary Table see  have beerly gene . Data res Figure , c and eCtcf, a transcriptional repressor of IGF-II, was directly responsible for reovirus-resistance. In particular, cells containing the Ctcf mutation express elevated levels IGF-II, while parental RIE1 cells forced to express IGF-II acquired high levels of reovirus resistance. The mutation in Ctcf was chosen for further analysis because it was one of 3 mutations affecting IGF-II expression and/or signalling [Candidate genes required for lytic reovirus infections were identified by tagged sequence mutagenesis, a process that permits rapid identification of genes disrupted by gene entrapment. Since virus-resistant mutants may arise by a variety of mechanisms, additional experiments are needed to demonstrate that individual genes disrupted by gene entrapment actually contribute to the reovirus-resistant phenotype. Even so, several lines of evidence suggest that the genes collectively identified by tagged sequence mutagenesis include cellular activities that participate in the virus lifecycle. First, reovirus-resistant clones were selected at higher frequencies from entrapment libraries than from non-mutagenized cells, suggesting that reovirus-resistant phenotypes were induced by gene trap mutagenesis. Second, the genes associated with reovirus resistance differed from genes targeted in an unselected manner in mouse ES cells. Known mutational hot spots of the U3NeoSV1 were under-represented, and a number of mutations associated with virus resistance appeared to cluster within specific cellular processes and/or affected different components of multi-protein complexes that are likely to play roles in the virus lifecycle. These include IGF-II expression/signalling (3 genes), cytoskeletal/vesicular/trafficking (20 genes), signalling pathways (11 genes), and apoptosis 4 genes). Finally, we recently demonstrated that the disruption of  genes. FIgf2r. Alternatively, mutations induced by gene entrapment may confer reovirus resistance as a result of gene dosage effects (e.g. haploinsufficiency). For example, recent data suggests that the most common genetic disease in Caucasians, cystic fibrosis, involves mutations in the ABC-cassette transporter protein, CFTR, that confer resistance to infection by Salmonella typhi [Cryptosporidium parum [Pgy1 (Abcb1), encodes P glycoprotein  to disrupt all expressed genes, it seems unlikely that all the genes that are required for virus infection, and that can be targeted by tagged sequence mutagenesis, were identified in the present study.The circumstances that allow gene entrapment to disrupt the function of diploid genes illustrate that events secondary to provirus integration may be required for expression of some reovirus resistant phenotypes. Consequently, while the entrapment libraries were theoretically large enough  and Anxa2 genes was accompanied by a dramatic increase in immediate-early gene expression. This temporal enhancement of HSV-1 replication may reflect activities of calcyclin and annexin 2 proteins that suppress HSV-1 entry [Genetic alterations giving rise to reovirus resistant clones have variable effects on HSV-1 replication, with some reovirus resistant clones showing enhanced HSV1 replication. The reasons for this are unknown, although each of these two viruses enter cells by different mechanisms. The early steps in HSV replication require entry into cells, release of the capsid with migration to the nucleus for which virus and cellular proteins play roles ,54, wher-1 entry -57.Anxa1, clones with mutations in Eif3s10, Mgat1, and Igf2r also show decreases in transcription and translation of virus mRNA and cell death. Of these, mutations in the Igf2r are known to affect HSV replication [Eif3s10, Anxa1, and Mgat1 are novel. These data suggest that some of the candidate genes discovered in clones surviving reovirus infection may affect common cellular processes that are used by other viruses.In addition to the clone with a mutation in lication ,54,58; wStudies in a variety of systems indicate that resistance to infection may be found in nature or achieved in cultured cells. Results presented here support the hypothesis that pathways involved in reovirus infection can be identified through a functional genomics approach based upon insertional mutagenesis. Systematic selection of virus-resistant mutant cells in which the mutant gene can be easily identified may also identify targets for the development of anti-viral therapies. Drugs that disrupt cellular processes may circumvent the problem of virus resistance, generally observed with drugs against virus-encoded proteins. Moreover, since mutations associated with virus resistance are, by necessity, not lethal to the cell, drugs that target the same processes are not expected to have overtly toxic side effects. The fact that the resulting candidate genes may play roles in the replication of other viruses, suggests that different viruses may use similar host proteins for common steps required for virus entry, disassembly, transcription, translation, and reassembly. This view is supported by our studies with HSV-1 and by published reports implicating several of the genes disrupted in reovirus resistant cells (Supplementary Table see ) in the 3 mammalian genes, the definition of function will become the major task facing scientists interested in the relationship between host genes and viral disease over the next decade.The dramatic increase in the pace of the genome project has led to an explosion of information concerning the sequence of the genome of several species of animals and pathogenic organisms. However, most of the gene sequences have not been functionally ascribed with regard to host-parasite interactions. As there are approximately 30 to 50 × 10Candidate host genes that participate in lytic virus infections were identified utilizing insertional mutagenesis. Mutant cell clones were recovered that lost their capacity to support virus replication, but were able to proliferate. There was enrichment for genes that were involved in particular metabolic or signalling pathways, with many of the genes being selected more than once from independently derived libraries of RIE-1 cells. Several of the gene products are known to bind to each other. These genes or their products, which are identified by this process of selection, may provide targets for therapeutic intervention.Reovirus type 1, strain Lang, was initially obtained from Bernard N. Fields. Virus was passaged in L-cells and a third passaged stock was purified over a CsCl gradient as previously described and was used for these experiments . To deve4 gene entrapment events, were expanded until approximately 103 sibling cells represented each mutant clone. These cells were plated at a sub-confluent density and incubated in serum-free media for 3 days until they became quiescent, and infected with reovirus serotype 1, MOI of 35 plaque forming units (pfu) per cell. Eighteen hours following infection, the cells were detached with trypsin, and plated in DMEM medium containing 10% fetal bovine serum (FBS) . After 6 hrs, the medium was removed and cells were maintained in serum-free medium until only a few cells remained attached to the flask. On average, one to ten clones were recovered from a library consisting of 107 mutant cells, an enrichment for selected cells of six orders of magnitude. Cells that survived the selection were transferred to cell culture plates in media containing 10% FBS and cells were divided for extraction of DNA and cryopreservation.Following infection of RIE-1 cells with the U3neoSV1 vector, MOI of 0.1, mutagenized cells were selected for neomycin resistance in medium containing 1 mg/ml G418 sulfate  . Twenty lacZ, was determined by standard northern blot techniques and β-galactosidase assay, respectively.The transcription and translation of the HSV-1 immediate early gene reporter gene, Libraries of mutagenized cells were infected with reovirus serotype-1, strain Lang, to select for clones resistant to lytic infection. Selection of virus-resistant clones was performed in serum-free medium to suppress the emergence of persistently infected (PI) cells . This is-5 and the sequence was non-repetitive. In most cases, the matching gene was in the same transcriptional orientation as the provirus. Moreover, matches involving cDNA sequences were co-linear across exons present in the flanking genomic DNA and diverged at splice sites. As indicated, virtually all of the genes identified had matches to murine, rat, or human gene sequences with p < 10-10.Genomic DNA immediately adjoining the 5' end of the proviral insert in each of 130 cell lines was cloned by plasmid rescue . ApproxiELO, and JS conducted most of the laboratory work. HER provided the vectors and advice on their use. DHR discovered that persistently infected cells require serum to survive, allowing the selection of genetically resistant cell clones, and did the genetic analysis. Drs. Ruley and Rubin provided funding and supervision for the research, and prepared the manuscript. All authors have read and approved the final manuscript.Genes associated with resistance to lytic reovirus infection identified by tagged sequence mutagenesis A list of previously named genes disrupted by the insertional mutagen, U3neoSV1, and recovered in cell clones resistant to lytic infection is provided. The rat mRNA and vector insertion site accession number, rat chromosome location, human homologue chromosome location, link to the NCBI Entrez Gene and NCBI Nucleotide databases, and known virus interactions are listed.Click here for file


The complexity of parasitic infections requires novel approaches to vaccine design. The versatility of DNA vaccination provides new perspectives. This review discusses the use of prime-boost immunizations, genetic adjuvants, multivalent vaccines and codon optimization for optimal DNA vaccine design against parasites. DNA vaccination was introduced in 1990 by a study that demonstrated the induction of protein expression upon direct intramuscular injection of plasmid DNA in myocytes -4. DNA vSuccessful vaccines should be able to induce strong immune responses which are long-lasting and in most cases providing protection against different strains of the same pathogen. Progress has been made towards development of DNA vaccines against viral and bacterial pathogens showing protection and lasting immunity . ApplicaCurrent sub-unit vaccines predominantly induce strong antibody responses and weak cellular immunity. DNA vaccines in animal models can induce both strong humoral and cellular mediated responses, but although safe in humans, DNA vaccines do not produce the same magnitude of cellular immunity . In casePlasmodium yoelii DNA vaccine, mice were primed intramuscularly with DNA vaccine and granulocyte/macrophage colony stimulating factor (GM-CSF) plasmid and boosted with rVv encoding the same circumsporozoite protein (CSP) [P. falciparum [P. berghei challenge characterized by strong CD8+ T cell responses was observed in mice after intradermal adenovirus-prime-MVA-boost encoding CSP [P. falciparum pre-erythrocytic TRAP antigen were administered via gene-gun to healthy volunteers with no adverse effects [P. falciparum. Another group demonstrated that priming with DNA vaccine for P. falciparum CSP and boosting with a recombinant protein vaccine in adjuvant  induced the production of significant antibody and T cell responses in healthy volunteers [P. falciparum TRAP antigen is safe, well tolerated and induces responses greater than those observed in naive volunteers [To further improve the efficacy of a in (CSP) . This colciparum . Completding CSP . These s effects . The pol effects . Furtherlunteers . Phase Ilunteers . BoostinAdjuvants are used to strengthen the immune response to a vaccine and have been critical in modern vaccine development. Genetic adjuvants are expression vectors encoding biologically active molecules such as cytokines, chemokines and co-stimulatory molecules. These adjuvants can be encoded on the same vector as the antigen or expressed on a separate vector and co-injected with the vaccine. This method provides adjuvant activity at the site of antigen production, with lasting effect from transfected cells. Cytokines are chosen as genetic adjuvants because they regulate cells involved in host defense and can be used to modulate immune responses. Co-delivery of cytokines in DNA vaccine formulation has been used extensively for a wide range of infectious and parasitic diseases  and either mouse GM-CSF or IL-4 to determine their adjuvant effect in mice. GM-CSF may work as adjuvant through its activating effect on dendritic cells and macrophages. Intramuscular vaccination with Sm-p80 alone provided 39% protection and this protection was significantly increased to 44% with GM-CSF co-administration and 42% with IL-4. The addition of GM-CSF led to an increase in total IgG and IgG1 while Th1 type IgG2a antibody titers remained high in protected animals [Siddiqui et al.  generate animals . GreaterAnother advantage of DNA vaccines is the possibility to integrate several antigens into the plasmid or to administer a mixture of plasmid vectors. The development of multivalent vaccines consisting of several antigens is a novel approach to create broad range protection against different parasite strains and parasite life cycle stages (see Table Leishmania major-activated C kinase (LACK), thio-specific antioxidant (TSA), and L. major stress-inducible protein (LmST11) was able to induce complete and long lasting protection after parasite challenge in mice compared to killed Leishmania parasites and rIL-12 [L. major cysteine proteinase genes (Cpa/Cpb) was protective and characterized by IFN-γ production by spleen cells, while separate injections were not protective [Vaccination studies against leishmaniasis in mice have identified various parasite antigens with varying degrees of protection as protein vaccines. When combined into multivalent DNA vaccines these antigens have the ability to confer complete or enhanced protection. In fact, a DNA vaccine including a mixture of plasmids encoding three antigens, d rIL-12 . This prd rIL-12 . It was otective . The cysotective  as the nin vitro and antigen specific responses in vaccinated animals. In our laboratory, we have developed a codon-optimized DNA vaccine encoding a portion of the Entamoeba histolytica Gal-lectin [E. histolytica genes are rich in A:T codons, whereas mammalian codons are more G:C rich. Protein expression of the E. histolytica Gal-lectin protein using the wild type sequence was difficult and stable clones were difficult to obtain in mammalian cells. Codon optimization was performed to ultimately increase protein expression in gerbils, a model for experimental amoebiasis; therefore gerbil codon usage was used to re-write the Gal-lectin Hg1l gene. Transfection of Cos-7 cells with the optimized vaccine construct produced a protein which was immunoreactive with a Gal-lectin specific monoclonal antibody (3F4), demonstrating successful expression of this amoebic protein. Upon vaccination with this codon optimized DNA plasmid, mice developed antigen specific antibodies of the Th1 isotype and Gal-lectin specific cellular immune responses.Interspecies differences in codon usage are a major obstacle in DNA vaccine development. This is due to the fact that DNA vaccines use host cells for transcription and translation of proteins. Every species has a codon bias for which most genes are encoded and this use of selected codons is related to gene expression efficiency. Closely related species use similar codons. However, in cases where there is a great difference in codon usage between the pathogen and mammals, codon optimization may be required. This strategy involves the modification of codon usage for the genes encoded in a DNA vaccine to a suitable codon bias for increased expression in mammals. This method has proved effective in many systems ,49,50, il-lectin . E. histet al. [P. falciparum DNA vaccines when administered as a mixture of plasmids compared to single plasmid injections. Another study, however, demonstrated that a mixture of three plasmids encoding P. falciparum blood-stage antigens had no reduction in immunogenecity when co-injected [In this review, strategies for increased DNA vaccine efficacy against parasitic diseases to date, i.e. prime-boost immunizations, genetic adjuvants, multivalent vaccines and codon optimization, have been discussed. DNA vaccine technology provides the versatility required to separate protective components of immunity from counter-protective responses. As seen with genetic adjuvants, DNA vaccines can focus on the protective cytokines involved and include antigens that stimulate the production of specific cytokines. This allows designing vaccination strategies that are tailored to a particular infection or even a specific stage of infection. Parasitic diseases are complex, involving changes in immunological responses during the course of infection and changes in immunity to stage specific antigens. The advent of optimization strategies with DNA vaccines presents researchers with the tools to design effective vaccines with specific purposes. It is possible to enhance DNA vaccine efficacy, thus increasing immune responses and protection, through the use of these methodologies. However, it is important to note that these strategies need to be adjusted to the parasite system in order to provide the greatest benefit upon vaccination. For example, Sedegah et al.  reportedinjected . Therefoinjected ,24,32,54® XR1 is a needle-free powder injection system that delivers fine gold particles coated with the vaccine vectors directly into epidermal cells, specifically dendritic cells. This vaccination method is advantageous since it eliminates the cold chain requirement and reduces the chances of needle-borne contamination. Moreover, the gene-gun method is safe and seems as immunogenic as intramuscular injections in these trials [The method of vaccine delivery is an important variable in vaccination design. DNA vaccination has been successful through a variety of injection routes, including intradermal, intramuscular, and intranasal. Although intramuscular injections are most common and give consistent responses, alternative routes of delivery may be desired depending on the disease model. Mucosal DNA vaccine immunizations, against intestinal parasites for example, are effective to generate mucosal immune responses at the site of infection. For leishmaniasis, where the disease manifests itself as cutaneous lesions, an intradermal injection targeting Langerhans' cells may be optimal . The gene trials .Leishmania parasites in mice have improved cellular immune responses when plasmids expressing IL-12 and IL-18 are co-injected [P. cynomolgi antigens (CSP/TRAP) resulted in lower peak parasitemia and higher antibody and cellular responses than controls [The greatest challenge in designing DNA vaccines against parasites is making the vaccine suitable for humans while providing strong, long lasting immune responses. Many studies in laboratory animals are successful but the results cannot be replicated in humans. The prime-boost strategy has shown the most success as a delivery technique in larger animals or humans. Field trials with prime-boost malaria vaccines are ongoing and will provide experts with insight with regards to the safety and the immune responses required for protection in humans. Meanwhile, other groups are reporting improved responses in mice or larger mammals with other vaccines, suggesting that this vaccination strategy may be applicable to many other parasitic diseases ,29. A vainjected . A DNA pcontrols . Taken tThe author(s) declare that they have no competing interests.CI and KC produced the manuscript together. All authors read and approved the final manuscript.


Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line.Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c) were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members.One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20) with three members  in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate The FAM20 family represents a new family of secreted proteins with potential functions in regulating differentiation and function of hematopoietic and other tissues. The Fam20a mRNA was only expressed during early stages of hematopoietic development and may play a role in lineage commitment or proliferation. The expansion in gene number in different species suggests that the family has evolved as a result of several gene duplication events that have occurred in both vertebrates and invertebrates. Hematopoietic differentiation is a complex process whereby multiple functionally and morphologically distinct cell types arise from a population of pluripotent hematopoietic stem cells (PHSCs) . The accWe have used a pair of factor-dependent murine cell lines to identify novel genes expressed within distinct hematopoietic lineages as an approach to the identification of novel candidate genes for development of diagnostic and therapeutic approaches to leukemia. The EML and MPRO cell lines were both established by infecting murine bone marrow cells with a retrovirus expressing a dominant negative retinoic acid receptor α (RARα) protein ,5. The iWe have employed the representation difference analysis technique  to identTotal RNA was prepared from EML cells grown in the presence of SCF alone (0 hour) or in medium supplemented with IL-3 and atRA for 72 hours. The RNA was converted to cDNA and subjected to three rounds of RDA as previously described . Six difThe initial cDNA isolated by RDA was a 273 bp fragment that appeared to contain the coding sequence of the C-terminus of a protein that was not present at that time in public databases. To identify the full open reading frame of this cDNA, we first performed rapid amplification of cDNA ends (RACE) in both the 5' and 3' directions. Extension in the 3' direction revealed the presence of a consensus polyadenylation signal located 154 nucleotides downstream of the putative translation stop codon. Extension in the 5' direction yielded an additional 443 bp of sequence containing a contiguous ORF. Comparison of this extended sequence to public databases identified a cDNA (NM_017565) that was identical to clone 1623 in the region of overlap. This cDNA was isolated from a mouse mammary tumor but no functional analysis had been performed. We designed PCR primers based on the published sequence and confirmed that the cDNA isolated from EML cells was identical to the published sequence. The full length ORF was 1623 bp in length and encoded a protein of 541 amino acids Figure . The pronamed four-jointed [The full length cDNA and the encoded protein were compared to sequences in public databases. We reported previously a weak similarity to a protein named Fjx1, which is the mouse orthologue of a Drosophila protein -jointed . HoweverCiona intestinalis is a model for a basal chordate organism and has emerged as a powerful model for evolutionary and developmental studies [C. intestinalis and thus the identification of an orthologue in this organism can provide useful information for evaluating the evolutionary origin of the members of a gene family. Consistent with this concept, we identified a single cDNA and the corresponding genomic locus in C. intestinalis that displayed significant sequence similarity to the mammalian FAM20 genes and proteins (Table Drosophila melanogaster and Anopheles gambiae with one family member in Caenorhabditis elegans (Table Fugu rubripes) and zebrafish (Danio rerio) revealed the presence of six and five family representatives, respectively  was compared and revealed obvious inter and intra orthologue similarities figure . Each FAC. intestinalis clustered with FAM20B proteins, suggesting that this represents the ancestral branch of the FAM20 family. A single protein from Fugu and zebrafish clustered with the FAM20A and FAM20B family members while two Fugu and two zebrafish proteins clustered with FAM20C members. However, two Fugu proteins and one zebrafish protein clustered on a separate branch between FAM20A and FAM20B. In order to determine the subfamily to which these proteins belonged, we made use of the high degree of conservation of exon size and number noted in the mammalian genes  and either retained there or processed and transported into the Golgi apparatus and secreted from the cell. Many proteins are glycosylated during their transit through the ER and Golgi apparatus and the mouse Fam20a protein contains four potential sites for N-glycosylation (indicated in red type in figure 14–Leu15 to Asp-Glu) were predicted to disrupt the signal sequence without grossly altering the protein structure. Again the mutant protein displayed nuclear staining and was absent from the ER . Therefore, we examined the expression patterns of the three genes in a panel of cDNAs derived from various human tissues. FAM20A displayed the most restricted expression pattern with high levels in lung and liver and intermediate levels in thymus and ovary Figure . Low levSeveral classes of secreted proteins, including the colony stimulating factors or hematopoietins, are important regulators of hematopoietic differentiation and function . These mAt the present time, the specific function(s) of the FAM20 proteins is unknown. Analysis of the sequences of the proteins from various species failed to reveal obvious similarities to known functional domains except for an N-terminal signal sequence. Expression studies clearly demonstrated that the mouse Fam20a protein is a secreted protein and that disruption of the signal sequence prevented the detection of the glycosylated form of the protein in cell media. Surprisingly, disruption of the signal sequence resulted in redistribution of intracellular Fam20a from a cytoplasmic compartment that is likely to be the ER to the nucleus. It is unclear whether this redistribution is functionally significant and is presumably due to the presence of a cryptic nuclear localization signal (NLS) in the protein. NLSs are generally comprised of stretches of basic residues  and seveOverall, the FAM20 proteins vary in length between 400–670 amino acids with the FAM20B family members being the shortest and the FAM20C members generally being the longest. The variation in length is due to differences in the length of the less conserved N-terminal region. The FAM20 proteins are further characterized by a highly conserved C-terminal region of approximately 350 amino acids that we named the conserved C-terminal domain (CCD). We noted the presence of three regions that are more highly conserved amongst all family members, with the most invariant extended sequence being the DRHHYE heptapeptide within conserved region 2. The function of the peptide is as yet unknown but three possible functions can be proposed. First, the histidines within this sequence could be involved in the coordination of metal ions that may be required for FAM20 protein function. Second, FAM20 proteins may be enzymes and this sequence may be a component of the active site of the enzyme. Interestingly, a weak match was detected between this region of certain FAM20 family members and a conserved domain within the phosphatidylinositol 3- and 4-kinases ; howeverD. melanogaster and A. gambiae), a simple chordate , three mammals  and two fish (F. rubripes and D. rerio). In each case, the identification of these genes as bona fide transcription units is supported either by direct experimental evidence for the human and mouse genes, or by the existence of multiple EST sequences in public databases. A single FAM20 gene was identified in C. intestinalis, which is considered to be a representative of a basal chordate related to the common ancestor of humans and other higher chordates [C. intestinalis genome encodes approximately 16,000 genes and generally contains single representatives of superfamilies in higher vertebrates, thereby permitting the elucidation of likely evolutionary origins of genes within these families [Tremendous progress has been made over the past decade in the sequencing of genomes from species at different positions on the evolutionary tree -25. Thishordates . The C. families . The C. families . This pafamilies . In the A further round of gene duplication has been proposed to have occurred in fish  and the Expression analysis of the three FAM20 family members in mammalian tissues and hematopoietic cells showed that FAM20A is expressed in a much more restricted pattern that the other two members. Importantly, Fam20a was also the only member to display obvious differential expression in hematopoietic cells undergoing myeloid differentiation. Fam20a mRNA levels were highest during intermediate stages of differentiation of EML cells, at a time when many cells are becoming committed to the myeloid lineages and undergoing extensive proliferation ,8,29. EM-5 M, Sigma) for 72 hours. Terminal granulocytic differentiation was induced by removing IL-3 and SCF and adding 10% BHK-HM5 CM as a source of granulocyte/macrophage-colony stimulating factor (GM-CSF). MPRO cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum  and 10% BHK-HM5 CM. Terminal differentiation was induced by adding 1 – 10-5 M atRA to the culture medium. For collection of RNA samples at different timepoints, 10 cm dishes were seeded with 150,000 cells and cultured under identical conditions. Cells were harvested from individual wells at 24 hour intervals for RNA preparation.EML and MPRO cells were cultured as described previously ,30. Brie+ RNA prepared from EML cells at zero hours (stem cell stage) and 72 hours (promyelocyte stage) was converted to double stranded cDNA and three rounds of RDA was performed as described previously [Total RNA was prepared from isolated cells using a modified guanidium isothiocyanite/phenol extraction procedure as described previously . Poly A+eviously . cDNA cleviously ,8 and cl4 per well in six well plates and transfected using the Effectene Transfection Reagent  under conditions recommended by the manufacturer.Cos-1 cells were maintained in Dulbecco's modified Eagle's medium  containing 10% FBS and Penicillin/Streptomycin (BioWhittaker) under standard cell culture conditions. For transfections, cells were plated at a density of 2.5 × 10Analysis of the sequence of the original 1623 cDNA isolated from the RDA experiments revealed that it contained the coding sequence from the C-terminus of an uncharacterized protein. 5' and 3' rapid amplification of cDNA ends (RACE) was performed using Marathon-Ready cDNA from mouse spleen using the Advantage cDNA PCR kit (both from Clontech) as described previously . The RAC. Genome specific searches were performed either through NCBI or Ensembl  or through genome specific websites . Searches were performed either as nucleotide-nucleotide (blastn) searches or as protein-translated nucleotide (tblastn) searches. In general, these searches were sufficient to identify gene regions encoding the most highly conserved regions of the proteins, particularly in the fish and Ciona genomes. To assemble complete genes, genomic regions containing the identified regions of similarity were downloaded into VectorNTI and searches were performed for individual exons, initially derived from mammalian genes but subsequently, from potential orthologues in fish or lower vertebrates, depending on the sequence being examined. Putative exons were examined for the presence of consensus splice donor and acceptor sites and complete sets of exons were assembled into cDNAs for translation and further comparisons. These results were compared against genes assembled by two gene prediction programs, FGENESH:  and GENSCAN: , however, these programs often made incorrect assignments that required manual assessment of the results. The protein sequences derived from each of the examined species were primarily used in the comparisons to define subfamily assignments and alignments were performed in the AlignX program in VectorNTI using the blosum62 scoring matrix. Protein domain searches were performed using the Simple Modular Architecture Research Tool (SMART)  and/or Profilescan . Signal sequence searches were performed using the SignalP analysis program [All basic sequence manipulations were performed using the VectorNTI suite of sequence analysis programs . Identification of FAM20 members from different species was initially achieved using standard BLAST searches at NCBI  program . SequencXhoI and HindIII restriction sites were engineered into the PCR primers . Similar strategy was used to clone Fam20a and Fam20aΔ23 in the pcDNA3.1 vector . The Fam20a(SSmut) was generated using a PCR-based site-directed mutagenesis kit . All of the sequences were verified by sequencing using an ABI automated sequencer.The Fam20a and Fam20aΔ23 were constructed in the pEGFP-N1 vector by PCR cloning. Whole cell extracts were prepared from transfected COS-1 cells by lysing directly in 2X Laemmli Sample Buffer as described previously . Purifie2PO4-H2O (pH 8.0), 150 mM NaCl, 10 mM Imidazole). His-tagged proteins were purified using Ni-NTA Magnetic Agarose Beads  according to manufacturer's recommendations. Following the purification step they were either used directly in western blot analysis or for deglycosylation experiments. Purified proteins were subjected to enzymatic deglycosylation using the GlycoPro deglycosylation kit  following the manufacturer's protocol.COS-1 cells were transfected as described above and media were collected after 48 hours and mixed with freshly made 10X binding buffer (500 mM NaH2HPO4 (pH 7.3)) containing 1% Triton X-100 for 10 minutes to permeabilize the cell membranes. They were then transferred to PBST (PBS containing 0.1% Tween-20) and incubated for 30 minutes. Mouse monoclonal anti-Myc antiserum (Santa Cruz) was used at 1:2,000 dilution in PBST containing 1% bovine serum albumin (1 hour at room temperature). Coverslips were then washed with PBST three times and incubated with the secondary antibody  for 1 hour. Coverslips were washed three times in PBST and once in water for 15 minutes each, and were mounted on glass slides. The cells were observed by epifluorescence microscopy using an Axiovert 135 TV microscope . Images were captured with a Kodak DC290 zoom camera and analyzed with MetaMorph 6.0 .COS-1 cells were plated on glass coverslips and transfected with the indicated constructs the following day. After 48 hours of incubation, the cells were fixed in 1% formaldehyde for 30 minutes at room temperature. Coverslips were incubated in PBS , 0.125 mM dNTPs, 10 mM DTT, and 1 μM oligo (dT)15 in a total volume of 50 μl for 1 hour at 37°C. 2 ul of the RT reaction was then mixed with 1 ul of 10X PCR Buffer (500 mM Tris-HCl (pH8.3), 2.5 mg/ml crystalline BSA and MgCl2 at 10, 20 or 30 mM , 0.2 mM each dNTP, 0.05 μM of each primer and 1.25 Units Taq DNA polymerase  in a total volume of 10 μl. Reactions were loaded into a capillary tube and PCR cycles were carried out using the Rapidcycler Thermal cycler (Idaho Technology). Annealing temperatures and Mg2+ concentrations were initially optimized for each primer. A commercial set of tissue cDNAs, (Human Multiple Tissue cDNA (MTC) Panels, Clontech, Palo Alto, CA) was used for the RT-PCR analysis of human FAM20 family members.Total RNA was purified as described above from EML and MPRO cells at 24 hour timepoints during the differentiation process of each cell line The primers were designed using the Vector NTI sequence analysis suite of programs  and were follows: MmFam20a forward 5'-catagaggcccacggcgagcg-3' and reverse 5'-atggaatggggcaacag gggc-3'; Mmfam20b forward 5'-tggacaggattctgggtttc-3' and reverse 5'-ccagggatgtcgatgtttct-3'; MmFam20c forward 5'-agcagacgagagagcaggag-3' and reverse 5'-cggatctccttggtcatgtt-3'; HsFAM20a forward 5'-ctggcaggaaaagagtg-3' and 5'-cccgaacttggtgaacatct-3', HsFAM20b forward 5'-ccctgaagagacaccagaagagc-3' and reverse 5'-gaaacccagaatcctgtcca-3', HsFAM20c forward 5'-ggctcacgttccacattggt-3' and reverse 5'-aaagtcagggggtgtctcct-3'.; mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward 5'-aatggtgaaggtcggtg tgaac-3' and reverse 5'-gaagatggtgatgggcttcc-3'; human (GAPDH) forward 5'-tgaaggtcggagtcaacggatttggt-3' and reverse 5'-catgtgggccatgaggtccaccac-3'. GAPDH was used as control for cDNA integrity. Single stranded cDNA was reverse transcribed from 2 μg of total RNA using 400 Units of Superscript RNase HFAM20: family with sequence similarity 20; EML: Erythroid, myeloid, lymphoid cell line; MPRO: mouse promyelocyte cell line; GM-CSF: granulocyte/macrophage-colony stimulating factor; PHSC: pluripotent hematopoietic stem cell; HGF: hematopoietic growth factor; RAR: retinoic acid receptor; atRA: all trans retinoic acid; SCF: stem cell factor; IL-3: interleukin-3; RDA: representational difference analysis; RACE: rapid amplification of cDNA ends; CCD: conserved C-terminal domain; GFP: green fluorescent protein; CM: conditioned medium.DN isolated full length Fam20a cDNA, performed transfection experiments and drafted the manuscript. HY performed RT-PCR assays. IN assisted in construction of expression vectors. SS performed genomics analyses. EC and EGB provided reagents and assistance in performing RT-PCR assays. YD performed the original representational difference analysis and SCW directed the project and performed genomics analyses. SCW also wrote the final draft of the manuscript and all authors read and approved this version.


Since the mid-1980's there has been a worldwide resurgence of severe disease from group A streptococcus (GAS), with clonal clusters implicated in Europe and the United States. However GAS associated sepsis and rheumatic fever have always remained at high levels in many less developed countries. In this context we aimed to study GAS necrotising fasciitis (NF) in a region where there are high background rates of GAS carriage and disease.We describe the epidemiology, clinical and laboratory features of 14 consecutive cases of GAS NF treated over a seven year period from tropical northern Australia.emm type 1 and two emm type 3 strains.Incidence rates of GAS NF in the Aboriginal population were up to five times those previously published from other countries. Clinical features were similar to those described elsewhere, with 7/14 (50%) bacteremic and 9/14 (64%) having associated streptococcal toxic shock syndrome. 11/14 (79%) had underlying chronic illnesses, including all four fatalities . Important laboratory differences from other series were that leukocytosis was absent in 9/14 (64%) but all had substantial lymphopenia. Sequence typing of the 14 NF-associated GAS isolates showed no clonality, with only one While NF clusters can occur from a single emergent GAS clone, this was not evident in our tropical region, where high rates of NF parallel high overall rates of GAS infection from a wide diversity of strains. The specific virulence factors of GAS strains which do cause NF and the basis of the inadequate host response in those patients who develop NF on infection with these GAS require further elucidation. Streptococcus pyogenes  alone, or in association with Staphylococcus aureus or Staphylococcus epidermidis [Necrotising fasciitis (NF) is an aggressive and rapidly destructive soft tissue infection resulting in high mortality and significant long term morbidity. Two groups of infectious agents have been described – Type I which are polymicrobial, involving anaerobic bacteria and streptococci other than serogroup A, and Type II from dermidis . The latdermidis  and has Since the mid-1980's a worldwide increase in infections due to GAS has been noted. In affluent populations where GAS disease is uncommon aside from pharyngitis in childhood, increasing numbers of necrotising fasciitis and streptococcal toxic shock syndrome (STSS) have been seen, as well as an upsurge of acute rheumatic fever apparently restricted to parts of the United States -6. This Recent case series of GAS NF have been published from Norway and OntaThe Royal Darwin Hospital services 135,000 inhabitants of the NT, with around 100,000 located in the city of Darwin. Indigenous Aboriginal Australians account for 24% of the population[Fourteen cases fulfilling the case definition were identified between May 1994 and April 2001. A chart review was conducted to identify epidemiological characteristics, clinical features, laboratory results, treatment and outcomes. Age, gender, ethnicity, urban or rural residence and any concurrent medical conditions were documented. The dates of presentation, presumptive diagnosis of NF and of surgery were documented. Symptoms, signs, hemodynamic status and temperature were documented for these dates, as was the presence of STSS as defined by the Working Group on Severe Streptococcal Infections. AntibioIncidence rates of GAS NF for this region of the NT were calculated using regional population numbers from census data. There was possibly an increase in case ascertainment after 1998 and incidence rates were also calculated by year.emm sequence typing and analysis of emm-specific PCR products were carried out as previously described[emm sequences deposited in GenBank and in the Centers for Disease Control (CDC) Streptococcus pyogenes emm sequence database[Procedures for described. DNA seq database.Approval for the study was obtained from the Health Research Ethics Committee of Royal Darwin Hospital and the Menzies School of Health Research.Table From 1994 to 2001, 14 cases of confirmed GAS NF were treated at Royal Darwin Hospital. The number of cases per year ranged from zero in 1995 and 1998 to five in 2001. This gives a maximum yearly incidence of 3.8 cases per 100,000. Half of the cases occurred in Aboriginal patients, with an incidence of 5.8 per 100,000 for Aboriginals in the 2001 cluster of cases. Eight cases occurred in women (57%), five of whom were Aboriginal. Ages ranged from 27–61 years in males (mean 41.5 yrs) and 27–59 years in females (mean 41.5 yrs). Eleven cases were community acquired and three nosocomial. All seven Aboriginal patients had multiple concurrent medical conditions, whereas three of the seven non-Aboriginals had no additional pathology.Patients presented most consistently with pain in the affected area. The other common complaints were swelling, and systemic symptoms of fever and rigors. Signs on presentation were typically induration and tense swelling of the area. Only four of the fourteen cases (29%) were noted to have extensive skin erythema, but nine (64%) had local pre-existing wounds or ulceration. Ten patients had only limb involvement, two had chest wall involvement, one had extension from right leg to the abdominal wall and one had extension from the left buttock to perineum/labia. STSS occurred in nine patients. Ten patients required inotropic support, six had a documented coagulopathy and eleven had renal impairment. Thirteen patients (93%) required ICU stay of 1–24 days. Eleven patients were ventilated, and six received renal replacement therapy.9/L) in only 5/14 (36%) patients, although lymphocyte count was globally reduced, being <1.0 × 109/L in all patients, and <0.5#215;109/l in nine cases (64%). Renal function was frequently impaired, with 11/14 (79%) patients having raised urea  and 12/14 (86%) having raised creatinine . All patients were hypoalbuminemic with 11 having albumin <30 g/L  Plasma creatine kinase (CK) ranged from 25 U/L to 2526 U/L (NR <220 U/L), being elevated in 5/9 (56%) patients measured. C-Reactive Protein (CRP) was substantially raised in all 11 where measured, being between 108–522 mg/L (NR<10). Blood cultures were positive for GAS in seven cases (50%). GAS was isolated from all operative tissue specimens, with four also positive for S. aureus.Total white cell count (WCC) was found to be raised , one being emm sequence type 3.2, and the other three isolates in this cluster were all different emm types.One GAS isolate could not be sequenced. GAS from the other 13 patients showed a large diversity of All patients underwent surgery 0–7 days after presentation. Debridement alone was conducted in nine patients, and combined with fasciotomy in one case. Fasciotomy alone was done in one case. Three patients underwent limb amputation. At surgery it was established that three patients (21%) had concurrent myonecrosis. Antibiotic therapy was instituted with a beta lactam in all patients . Clindamycin was also given in eleven patients. Adjuvant hyperbaric oxygen therapy was given in four cases, and intravenous immunoglobulin (IVIG) in one.Four patients (29%) died, all being Aboriginal females with co-morbidities. Two of the four patients with truncal infection died in comparison to only two of the ten with only peripheral involvement. The mean time to surgery from presentation was 4.25 days in patients who died and 3.7 days in survivors (not significant). Blood cultures were positive in 3/4 fatalities with STSS in 3/4. All four were in ICU for 1–5 days until death, with all requiring inotropes and mechanical ventilation, two given renal replacement therapy and one IVIG. Two received clindamycin in addition to beta-lactam antibiotic therapy. Surgery in the fatal cases consisted of debridement and/or fasciotomy, with no amputation.We describe a series of 14 cases of GAS necrotising fasciitis in a region with a high background incidence of GAS disease. Our incThe mean age of our patients 41.5 years) is lower than other reports showing an average age of 56–58 years, with notable increases in incidence with increasing age1.5 years,15. 79% 9/L, 64% of those being <0.5 × 109/L. Low albumin was also universal.Two studies have looked at the value of early blood test results in predicting NF as opposed to non-NF soft tissue infection. One found that CK >600 U/L achieved PPV of 58% for NF. HoweverSTSS occurred in 64% of our cases in comparison to 40–46% in other series, and reiCase-fatality rates for NF previously reached 50%, with mortality of 80–100% in GAS myositis. Recent Immediate, extensive surgical debridement of all necrotic tissue is considered essential for optimal treatment of NF, with one early observational study showing a mortality of 0% with aggressive early surgery in comparison to 50% in historical controls,20. SurgCombination therapy with benzylpenicillin and clindamycin is now recommended treatment for proven GAS NF and STSS-23. ExpeHyperbaric oxygen (HBO) therapy has been advocated as adjuvant therapy for both microbiological types of NF. Although there are no randomised trials, NF is an approved Undersea and Hyperbaric Medical Society indication for HBO therapy. It is sThe clinical picture of STSS has been attributed to superantigens produced by GAS, including pyrogenic exotoxins, streptococcal superantigen SSA and a mitogenic exotoxin. This isIn summary, there is in vitro and in vivo experimental data to suggest a benefit for clindamycin, HBO therapy and IVIG in GAS NF and myositis. This is supported by limited case series but not by randomized controlled trials. The primary role of early, aggressive surgery in GAS NF remains probably the most critical factor in minimizing mortality. Clindamycin is recommended for GAS NF but whether clindamycin alone is as effective as penicillin plus clindamycin remains unclear. Benefit from HBO remains unproven, while there is emerging evidence for the use of IVIG, especially if STSS is present.emm type 1. This reflects previous findings of enormous diversity amongst organisms causing GAS bacteremia in our region, with no evidence of a dominant clone[Following a reported resurgence in invasive GAS infections in the 1980's, epidemiological studies proposed that dissemination of a new, more virulent strain of M1 serotype GAS was the cause for a large proportion of these infections. In repoant clone.Emm sequencing typing of GAS isolates showed none to be the same and only 1/13 to be emm1. GAS NF in our region is not clonal but occurs from a wide diversity of GAS strains that reflect the very high background rate of both uncomplicated GAS infection and invasive disease. While NF case clusters can occur from a single emergent GAS clone, this is not evident in our tropical region. The specific virulence factors of the GAS which do cause NF and the basis of the inadequate host response in those patients who develop NF on infection with these GAS require further elucidation.In conclusion we have described a case series of necrotising fasciitis due to GAS for a unique mixed population of patients living in both affluent and disadvantaged society. Our overall incidence rates of GAS NF are higher than previously reported, with rates in the Aboriginal population up to five times those previously published. The majority of those with NF had underlying chronic illnesses and mortality was also higher in this group. Important laboratory findings were that leukocytosis was absent in more than half of cases but all had substantial lymphopenia. The authors declare they have no competing interests.MH collated and analysed the case data and was principal writer of the manuscript, PF carried out the molecular studies, PC participated in the study design and patient management, BC conceived the study and participated in the study design and coordination and patient management. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Studies on the effects of ethylene on CO2 metabolism have shown conflicting results. Increase or inhibition of photosynthesis by ethylene has been reported. To understand the physiological processes responsible for ethylene-mediated changes in photosynthesis, stomatal and mesophyll effects on photosynthesis and ethylene biosynthesis in response to ethephon treatment in mustard (Brassica juncea) cultivars differing in photosynthetic capacity were studied.The stimulatory effect of CON), stomatal conductance (gS), carbonic anhydrase (CA) activity, 1-aminocyclopropane carboxylic acid synthase (ACS) activity and ethylene evolution were similar in both the cultivars. Increasing ethephon concentration up to 1.5 mM increased PN, gS and CA maximally, whereas 3.0 mM ethephon proved inhibitory. ACS activity and ethylene evolution increased with increasing concentrations of ethephon. The corresponding changes in gs and CA activity suggest that the changes in photosynthesis in response to ethephon were triggered by altered stomatal and mesophyll processes. Stomatal conductance changed in parallel with changes in mesophyll photosynthetic properties. In both the cultivars ACS activity and ethylene increased up to 3.0 mM ethephon, but 1.5 mM ethephon caused maximum effects on photosynthetic parameters.The effects of ethephon on photosynthetic rate (PS were a response maintaining stable intercellular CO2 concentration (Ci) under the given treatment in both the cultivars. Also, the high photosynthetic capacity cultivar, Varuna responded less to ethephon than the low photosynthetic capacity cultivar, RH30. The photosynthetic capacity of RH30 increased with the increase in ethylene evolution due to 1.5 mM ethephon application.These results suggest that ethephon affects foliar gas exchange responses. The changes in photosynthesis in response to ethephon were due to stomatal and mesophyll effects. The changes in g N) have been reported. It has been shown to increase PN [N with ethylene-releasing compounds was due to the increase in chlorophyll per unit leaf area [N in response to ethephon treatment. For that, PN, stomatal conductance (gS) and carbonic anhydrase (CA) activity were recorded. To find a possible relationship of ethylene-mediated changes in foliar gas exchange parameters, activity of ACS and ethylene evolution were also determined. The work was carried out in two cultivars of mustard previously shown to have different photosynthetic capacity [Photosynthesis is controlled by several intrinsic and extrinsic factors. Of these, plant hormones have received considerable attention in the past in photosynthetic responses of plants. Ethylene is a phytohormone that influences every aspect of plant growth and development . It is srease PN -7 or decrease PN ,9, but neaf area  or by greaf area . In my eeaf area . The goacapacity .N, gS and CA were found significant in both the cultivars  decreased the characteristics in both the cultivars. The ratio of intercellular to ambient CO2 concentration (Ci/Ca) was constant.The effects of ethephon on P Figures , 2. EtheN, gS and CA. Ethylene evolution with 3.0 mM ethephon proved inhibitory for photosynthetic parameters.Ethephon application significantly affected ACS activity and ethylene evolution, and were greatest with 3.0 mM ethephon Table . Low phoN due to ethephon has been reported [s and CA values in both the cultivars showed stomatal and mesophyll effects on photosynthesis. Mesophyll effects are characterized as a product of CO2 binding capacity and the electron transport capacity. The carboxylation capacity determines the mesophyll effects [2 fixation exhibited the enhanced carboxylation reaction [2 concentration (Ci) under the given treatment. Thus, stomatal and mesophyll processes contributed to the increase in PN in response to ethephon. The ethephon-induced effects on photosynthetic parameters were mediated by ethylene evolved due to ethephon treatment. Taylor and Gunderson [S and ethylene-enhanced PN. Higher concentration of ethephon (3.0 mM) decreased the PN and gS. Such condition of inhibition of PN by ethylene-releasing compound has been observed by Kays and Pallas [N due to its effect on gS. Mattoo and White [2 assimilation and the plant responded depending on the tissue concentration. On the similar lines, Dhawan et al. [et al. [2 regulated PN and was related to ethylene evolution. In the present study, low photosynthetic cultivar, RH30 responded more to ethephon than the high photosynthetic cultivar, Varuna. In control plants, lesser ethylene evolution in RH30 than Varuna was responsible for lesser PN. As the ethylene evolution increased with ethephon application, the capacity of RH30 for PN also increased resulting in higher per cent increase in PN than the Varuna. An increase of 75% ethylene in RH30 due to 1.5 mM ethephon increased PN by 41.8%, whereas 52.6% increase in ethylene in Varuna due to the same treatment increased PN by 31.8%. Earlier strong positive correlation between ACS activity and PN has been shown [Maximum rates of photosynthesis were found with 1.5 mM ethephon. The increase in Preported -6. Incre effects ,14. Incrreaction -17. The underson  showed ad Pallas  and Rajad Pallas . In all nd White  reportedn et al. , Kao andn et al.  and Grod [et al.  reasoneden shown .It therefore, appears possible that the threshold value for ethylene with 1.5 mM ethephon was comparable to that which elicits the ethylene-mediated hormonal responses, which differ with the cultivars inherent capacity of physiological processes. It is that there is some requirement of ethylene for optimum response. Low and high concentration represent the two ends of an optimum curve, promoting at low concentration and inhibiting at high.N in both high and low photosynthetic capacity cultivars, Varuna and RH30. In both the cultivars, changes in PN were due to stomatal and mesophyll effects. Ethephon-induced PN was attributed to ethylene evolution. The high photosynthetic capacity cultivar, Varuna responded less to ethephon than the low photosynthetic capacity cultivar, RH30. The low PN of RH30 was due to low level of ethylene. The low photosynthetic capacity of RH30 could be enhanced to give higher PN through increase in ethylene evolution. However, for both the cultivars there is a range of physiologically active concentration of ethylene beyond which it exerts inhibitory effects.This study shows that ethephon affects PBrassica juncea L. Czern & Coss.), namely Varuna (high photosynthetic capacity) and RH30 (low photosynthetic capacity) were grown from seeds in 10 m2 field plots in complete randomized design with five replications. At seedling establishment a plant population of 12 plants m-2 was maintained and recommended plant cultivation procedures were adopted. A uniform recommended soil application of 18 g N, 3 g P and 3 g K m-2 was given at the time of sowing so as the nutrients were non-limiting.Two cultivars of mustard  was sprayed with a hand sprayer. Ethephon is a direct ethylene source when applied to plants and elicits response identical to those induced by ethylene gas ,24. SincN, gS, Ci, CA activity, ACS activity and ethylene evolution were determined.At 45 d after sowing (15 d after ethephon treatment) PN, gS and Ci were measured using infrared gas analyzer  on fully expanded upper most leaves at saturating light intensity on four plants from each replicate. The atmospheric conditions during the experiment between 1100–1200 h were: photosynthetic active radiation about 1050 μmol m-2 s-1, relative humidity 64% and temperature 23°C, atmospheric CO2 concentration 360 μmol mol-1.P2 during the catalytic action of enzyme on sodium bicarbonate was estimated by titrating the reaction mixture against 0.05 N hydrochloric acid.The leaves used for photosynthesis measurement were selected for CA activity determination. CA was measured by the method of Dwivedi and Randhava . Leaves et al. [et al. [2 followed by 0.1 mL of 1:1 mixture of saturated NaOH/NaCl and incubated on ice for 10 min, and ethylene evolution was measured on a gas chromatograph. For control set AdoMet was not added. For ethylene evolution 5 mL of gas phase was removed with a syringe and ethylene was measured on a gas chromatograph  equipped with 1.8 m Porapack N (80/100 mesh) column, a flame ionization detector and an integrator. Nitrogen was used as carrier gas. The flow rates of nitrogen, hydrogen and oxygen were 0.5, 0.5 and 5 mL s-1, respectively. The oven temperature was 100°C and detector was at 150°C. Ethylene identification was based on the retention time and quantified comparing with the peaks from standard ethylene concentrations.Activity of ACS was measured adopting the methods of Avni et al.  and Woes [et al. . Leaf tiData were analyzed statistically and standard error of the mean value was calculated. Analysis of variance was performed to identify the significant differences among treatments at P < 0.05 .i – intercellular CO2 concentration; gS – stomatal conductance; PN – net photosynthetic rateAdomet – S-adenosyl methionine; ACS – 1-aminocyclopropane carboxylic acid synthase; CA – carbonic anhydrase; C


For two centuries, vaccination has been the dominating approach to develop prophylaxis against viral infections through immunological prevention. However, vaccines are not always possible to make, are ineffective for many viral infections, and also carry certain risk for a small, yet significant portion of the population. In the recent years, FDA's approval and subsequent market acceptance of Synagis, a monoclonal antibody indicated for prevention and treatment of respiratory syncytial virus (RSV) has heralded a new era for viral infection prevention and treatment. This emerging paradigm, herein designated "Biochemical Prevention and Treatment", currently involves two aspects: (1) preventing viral entry via passive transfer of specific protein-based anti-viral molecules or host cell receptor blockers; (2) inhibiting viral amplification by targeting the viral mRNA with anti-sense DNA, ribozyme, or RNA interference (RNAi). This article summarizes the current status of this field. A landmark in the battle against viral infectious diseases was made in 1798 when Jenner first inoculated humans against smallpox with the less virulent cowpox. For about two centuries since then, humans relied almost exclusively on vaccines for protection against viruses. Only in the recent years, new strategies for controlling viral infectious diseases have emerged, which have so far led to a couple of viral prophylaxis/therapeutics on the market. These strategies are fundamentally different from vaccines in that they attempt to directly interrupt viral infectious life cycle at molecular level by using proteins or oligonucleotides. To differentiate them from the conventional vaccines that prevent viral infection by boosting immune system, we refer the new antiviral approaches as "Biochemical Prevention and Treatment" . Soluble receptor drug candidates have gradually lost favor over the past several years due to issues relating to low potency and cost. Peptide-based drug candidates are limited by insufficient efficacy and unfavorable pharmacokinetics. MAbs have increasingly gained favor in large part because of the development of chimeric, humanized, and human antibodies have reduced the immunogenicity of antibody therapies. The MAbs that are currently in clinical trials for viral infection prophylaxis and treatment are listed in Table The respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in infants and young children producing bronchiolitis and pneumonia worldwide. RSV infection leads to more than 90,000 hospitalizations and a 2% mortality rate among infants nationwide . ApproxiThe most successful approach to date has been Biochemical Prevention and Treatment with anti-viral antibodies. In 1996, RespiGam™  became available for use in children less than two years of age with high-risk factors -10. The in vitro studies have reported effective viral inhibition by receptor-blocking MAbs. However, these works have not yielded yet any approved drug on the market.Human rhinovirus (HRV) causes over 80% of the common cold in the fall . Developin vitro [In HRV infection, about 90% of HRV serotypes utilize a single cell surface receptor exclusively, which is the intercellular adhesion molecule-1 (ICAM-1), for viral attachment and subsequent viral entry ,18. As sin vitro -21. Admiin vitro -24. For in vitro .in vitro assays, CFY196 consistently and significantly outpaced the best commercial anti-ICAM-1 MAbs in preventing HRV infection as measured by reduction of cytopathic effects and HRV viral titers [High avidity is achieved by multivalency. To improve avidity of HRV receptor blocking antibody, a novel tetravalent recombinant antibody, CFY196, has been generated against ICAM-1 . CFY196 l titers . The prel titers . Further®. A number of clinical trials of drug candidates from these technologies are currently ongoing.Targeting viral mRNA is one of the most active areas of research and development. Several strategies have emerged over the years and are being tested pre-clinically and clinically. They include: antisense-oligonucleotides (AS-ONs), ribozymes, and recently, RNA interference (RNAi). All these strategies share the features of conceptual simplicity, straightforward drug design and quick route to identify drug leads. However, the challenges have been to improve potency, pharmacokinetics and, most importantly, intracellular delivery of the drug candidates. As the oldest strategy, AS-ON technology has produced to date one drug in the market place, VitraveneAntisense-oligonucleotides (AS-ONs) are short synthetic oligonucleotides that form complementary pair with specific viral mRNA targets. AS-ONs inhibit viral protein production by both blocking viral mRNA translation and triggering its degradation. Since the discovery of viral inhibition effect of AS-ONs by Zamecnik and Stephenson in 1978 , antisenVitravene is the first AS-ON based drug approved by FDA. Vitravene, or fomivirsen sodium, is a 21-base phosphorothioate oligodeoxynucleotide complementary to the messenger RNA of the major immediate-early region proteins of human cytomegalovirus, and is a potent and selective antiviral agent for cytomegalovirus retinitis, a herpes-like eye disease that afflicts the immune-suppressed ,30. A nufirst generation' DNA analogs. The 'second generation' ONs contain nucleotides with alkyl modifications at the 2' position of the ribose. They are less toxic than PS-DNAs and have a slightly enhanced affinity. DNA and RNA analogs with modified phosphate linkages, or different sugar residues substituting the furanose ring have been referred as 'third generation' [Phosphorothioate (PS) oligodeoxynucleotides are the 'eration' . For insRibozymes (Rz) are catalytically active ONs that both bind and cleave target RNAs. They were discovered after the AS-ON technology. Initial findings on ribozymes raised the hope that they may offer a more potent alternative to AS-ONs. Many cell based and animal tests have performed on anti-viral effects of ribozymes, including HIV, hepatitis B, hepatitis C, influenza, etc. Results from these tests have shown that ribozymes are promising viral inhibitors -38. Howein vitro synthesized small interfering RNAs (siRNAs) of 21 to 23 nucleotides in length can effectively silence targeted genes in mammalian cells without triggering interferon production [RNA interference, or RNAi, is the inhibition of expression of specific genes by double-stranded RNAs (dsRNAs). It is becoming the method of choice to knockdown gene expression rapidly and robustly in mammalian cells. Comparing to the traditional antisense method, RNAi technology has the advantage of significantly enhanced potency; therefore, only lower concentrations may be needed to achieve same level of gene knockdown. RNAi gained rapid acceptance by researchers after Tuschl and coworkers discovered that oduction ,41. In moduction .Several initial studies, which test the potential application of synthetic siRNAs as antiviral agents, have shown very promising results. To date, RNAi has been used effectively to inhibit the replication of several different pathogenic viruses in culture, including: RSV  , influenCurrently, our understanding of the biological mechanisms underlying RNAi lags behind the movement to apply this technology to human diseases such as viral infections. Some major technical hurdles need to be overcome before siRNA-based anti-viral prophylaxis and treatments move into the clinics. Especially, intracellular delivery of siRNA needs to be greatly improved. The next few years of research will indicate whether RNAi technology will realize its potential as the next wave of Biochemical Prevention and Treatment.Dr. Hervé Le Calvez declares that he has no competing interest. Dr. Mang Yu and Dr. Fang Fang are the co-founders and current share holders of Perlan Therapeutics who has developed CFY196.


Molecular Cancer offers Open Access to all of its content, thereby providing a platform to present information to specialists and the public in order to further promote free exchange of ideas, concepts and findings in all fields of cancer-related biomedical science. All the published articles in the journal are determined by the peer review process.Open Access is one of the attempts to maximize the exchange of information, and therefore benefits the scholar communication . MoleculOpen Access has following broad benefits for science and the general public:• All articles become freely and universally accessible online; so an author's work can be read by anyone at no cost.• The authors hold copyright for their work and grant anyone the right to reproduce and disseminate the article, provided that it is correctly cited.• A copy of the full text of each Open Access article is permanently archived in an online repository separate from the journal, such as PubMed Central, the US National Library of Medicine's full-text repository of life science literature, the repositories at the University of Potsdam in Germany, at INIST in France and in e-Depot, the National Library of the Netherlands' digital archive of all electronic publications.• Authors are assured that their work is disseminated to the widest possible audience. This is accentuated by the authors being free to reproduce and distribute their work, for example by placing it on their institution's website. It has been suggested that free online articles are more highly cited because of their easier availability .• The information available to researchers will not be limited by their library's budget, and the widespread availability of articles will enhance literature searching.• The results of publicly funded research will be accessible to all interested readers and not just those with access to a library with a subscription. As such, Open Access could help to increase public interest in, and support of, research. Please note that this public accessibility may become a legal requirement in the USA if the proposed Public Access to Science Act is made law .• A country's economy will not influence its scientists' ability to access articles because resource-poor countries (and institutions) will be able to read the same material as wealthier ones, although creating access to the internet is another matter.Molecular Cancer published a number of interesting papers, and the list of the top ten most accessed articles is available at . All papers accepted by Molecular Cancer appear as 'accepted manuscript' on the web pages and are subsequently included in PubMed. A fully formatted portable document file is available approximately two to three weeks after acceptance along with a web-version of the article.The on-line publication, to the exclusion of print, has many advantages: Coloured pictures can be presented along with large sets of supporting data  without additional charges. In addition, the on-line submission process allows a fast and effective handling of papers and allows authors to check the status of their submitted manuscript(s). There is no limitation in space, but concise papers are more likely to be read.Molecular Cancer, all members of the editorial board and the reviewers for their ongoing support for Open Access publishing and for aiming higher standards for Molecular Cancer.The peer review policy, described in , ensuresMolecular Cancer did not change significantly, compared to the last report [Molecular Cancer after revisions. In addition to indexing in PubMed, PubMed Central and other search engines, Molecular Cancer is working closely with the Institute for Scientific Information to ensure that citation analysis of our articles will be available.The acceptance rate of t report . One outPJC is Editor-in-Chief and CS is Deputy Editor of this journal. Both do not receive any remuneration for their efforts but they are exempted from the article processing fee for this journal.


Propagation of repolarization is a phenomenon that occurs in cardiac muscle. We wanted to test whether this phenomenon would also occur in our model of simulated action potentials (APs) of cardiac muscle (CM) and smooth muscle (SM) generated with the PSpice program.gj) across the four cell junctions.A linear chain of 5 cells was used, with intracellular stimulation of cell #1 for the antegrade propagation and of cell #5 for the retrograde propagation. The hyperpolarizing stimulus parameters applied for termination of the AP in cell #5 were varied over a wide range in order to generate strength / duration (S/D) curves. Because it was not possible to insert a second "black box" (voltage-controlled current source) into the basic units representing segments of excitable membrane that would allow the cells to respond to small hyperpolarizing voltages, gap-junction (g.j.) channels had to be inserted between the cells, represented by inserting a resistor (Rm) to within the range of the sigmoidal curve of the Na+ conductance (CM) or Ca++ conductance (SM) terminated the AP in cell #5 in an all-or-none fashion. If there were no g.j. channels (Rgj = ∞), then only cell #5 repolarized to its stable resting potential . The positive junctional cleft potential (VJC) produced only a small hyperpolarization of cell #4. However, if many g.j. channels were inserted, more hyperpolarizing current was required (for a constant duration) to repolarize cell #5, but repolarization then propagated into cells 4, 3, 2, and 1. When duration of the pulses was varied, a typical S/D curve, characteristic of excitable membranes, was produced. The chronaxie measured from the S/D curve was about 1.0 ms, similar to that obtained for muscle membranes.Application of sufficient hyperpolarizing current to cell #5 to bring its membrane potential (VJC) developed in the narrow junctional clefts. Because it was not possible to insert a second black-box into the basic units that would allow the cells to respond to small hyperpolarizing voltages, only cell #5 (the cell injected with hyperpolarizing pulses) repolarized in an all-or-none manner. But addition of many g.j. channels allowed repolarization to propagate in a retrograde direction over all 5 cells.These experiments demonstrate that normal antegrade propagation of excitation can occur in the complete absence of g.j. channels, and therefore no low-resistance pathways between cells, by the electric field (negative V In c muscle ,14,15, aJC) was large and sufficient to allow transfer of excitation to the contiguous cell, without the requirement of gap-junction (g.j.) channels. Propagation of excitation can occur by the EF mechanism alone, even when the excitability of the cells was made low. In connexin-43 (heterozygous) and Cx40 knockout mice, propagation in the heart still occurs, but it is slowed [We recently modeled propagation of APs of cardiac muscle and smooth muscle using the PSpice program for circuit design and analysis -18. Likes slowed -22 as prThe present experiments were carried out to study propagated repolarization in this model of simulated action potentials (APs). Propagation of repolarization is a phenomenon that occurs in cardiac muscle . It has JC) was placed in the junctions between adjoining cells. The basic units were connected internally by the intracellular longitudinal resistance (ri). The basic units were connected externally with the extracellular resistance (RO), broken down into a longitudinal component (Rol) and a transverse  component (Ror). RO was connected to ground as depicted in Figure The methods used and PSpice program  have been described in detail previously, including the circuit ,18. In b+ compared to Na+ (cardiac muscle) or Ca++ (smooth muscle) were set to give resting potentials (RPs) of -80 mV for cardiac muscle and -55 mV for smooth muscle. During excitation, the action potentials (APs) overshot to +32 mV and +11 mV, respectively.The myocardial cell was assumed to be a cylinder 150 μm long and 16 μm in diameter, and the smooth muscle cell a cylinder 200 μm long and 5 μm diameter. Since in vascular smooth muscle (VSM), the muscle fibers run in a circular direction, if transverse velocity is calculated, the fiber diameter should be used. The values of the capacitive and the resistive elements in each basic unit were set to reflect the input resistance (ca 20 MΩ) and input capacitance (ca 100 pF) of the individual cells, and the junctional units were prorated, with respect to the surface units, based on relative areas represented. At rest, the resistance of KS1) were always applied internally to the first cell of the chain (cell A1). Rectangular depolarizing current pulses of 0.25 nA amplitude and 0.50 ms duration were applied. The delay time before the IS1 pulse was applied was usually set to 1.0 ms in SM. A second stimulus (IS2) that was hyperpolarizing was applied to the inside of the last cell (A5) of the chain when the APs of all 5 cells were in their plateau phase. The intensity and duration of the IS2 pulses were varied over a wide range in order to generate strength / duration (S/D) curves.Electrical stimulations (I+ or Ca++ conductance during excitation), this function had to be done with a V-controlled current source (our "black-box"). The sigmoidal relationship between conductance and membrane potential (VM), over a relatively narrow VM range, was mimicked by the black-box. The Na+ or Ca++ current required for excitation had to be calculated for several VM values and inserted into the GTABLE function.Because the PSpice program does not have a voltage-dependent resistance (to generate the increase in Nagj) across each cell-to-cell junction. This resistor connected the inner surface of the prejunctional membrane with the inner surface of the postjunctional membrane. This Rgj shunt resistance was varied between 10,000 MΩ (1 tunnel), 1000 MΩ , 100 MΩ (100 tunnels), 10 MΩ , and 1.0 MΩ . Each tunnel was assumed to have a conductance of 100 pS.Experiments were done with a single chain of 5 cells or 2 cells. There were no gap junctions between the cells of the chain under initial conditions. The presence of gap junction connexons (tunnels) was represented by adding a variable shunt resistance (RS1) produced propagation of APs down the chain. At the plateau (peak) of the APs, a repolarizing pulse applied intracelluarly to cell A5, if of sufficient intensity (duration constant), produced a sudden repolarization of only cell A5  repolarization of the stimulated cell (A5) of the 5-cell chain in both cardiac muscle Fig.  and smoocal case -25.+ leg of the basic circuit, because the PSpice program became erratic. Therefore, in order to achieve propagation of the repolarization of cell A5 in the retrograde direction, it was necessary to insert gap-junction channels between the cells of the chain . This corresponded to adding resistive shunts between the cells across the junctions (Rgj) of 10000, 1000, 100, 10, and 1.0 MΩ (assuming each channel has a conductance of 100 pS).As indicated in the Methods section, it was not possible to insert a second black-box in the KThe results of doing such an experiment are shown in Fig S2) applied to cell A5 were varied over a wide range in order to generate strength / duration curves. This was done when Rgj was infinite  and when Rgj was 10 MΩ (1000 channels) for strong coupling. The pulse duration was initially constant at 1.0 ms  and then lowered to 0.5 ms and to 0.25 ms. The current intensity was varied until the sharp endpoint occurred, namely the stable repolarization of all cells in the chain. These results are plotted in Figure Panel A is the strength / duration (S / D) curve for when Rgj was infinite (0 channels), and Panel B is the S/D curve for when Rgj was 10 MΩ(1000 channels). Note that the IS2 intensity was about 8–10-fold greater when the cells were well-coupled, because the applied hyperpolarizing current had to spread to all 5 cells of the chain. Regardless, the chronaxie values were about the same (ca. 1.0 ms).The intensity (strength) and duration of the rectangular hyperpolarizing current pulses  injected with repolarizing current (IS2) was able to repolarize in an all-or-none manner. The neighboring cell (A4) exhibited only a slight repolarization of <1 mV when cell A5 had repolarized completely back to the RP (-80 mV for CM and -55 mV for SM). This fact emphasized that there were no low-resistance connections between the cells under our initial conditions.In principle, the addition of a second black-box into the Kgj values of 1.0, 10, and 100 MΩ) did allow propagation of repolarization to occur. The borderline value was 10 g.j. channels (1000 MΩ Rgj), e.g., repolarization propagated part-way down the chain in SM and almost succeeded in CM. Of course, inserting the g.j. channels required that the IS2 repolarizing current applied be much greater. This is because the IS2 current had to spread down the entire chain, with the threshold current required to cause all cells to repolarize being determined by sufficient current entering distal cell A1 to repolarize it to the GTABLE sigmoidal region. Thus the proximal cells, like A5 and A4, became hyperpolarized beyond the level required for their repolarization.However, addition of 10,000, 1,000, or 100 g.j channels  and when all 5 cells repolarized . Plots of current intensity (ordinate) versus current duration (abscissa) gave the typical hyperbolic strength/ duration curve for excitable membranes. The chronaxie values were about 1.0 ms, for which a time constant τm of about 1.44 ms could be calculated. The S/D curves for the two conditions (Rgj = ∞ and Rgj = 10 MΩ) show that the current intensity required was about 8–10-fold greater when there were many gj-channels, in both CM and SM.The repolarizing Ir) varied with the number of gj-channels  and about 50 cm/s in the less coupled case (100 channels) (Table r) was much lower than the velocity for antegrade propagation (θa.). In the 2-cell chain, the calculated velocities of propagated repolarization were similar to those for the 5-cell chain . Third, it shows for the first time that, in the PSpice model, the membranes exhibit the characteristic strength/duration curves. Fourth, it shows that the PSpice program has some serious limitations.The present study provides some new and important information about the PSpice simulations. First, it verifies that propagation (orthodromic) can occur in the complete absence of gap-junction channels, as previously reported ,4,16-18.S2 current was able to repolarize. Since the potential change in the neighboring cell was only about 1 mV or less, this emphasizes that there were no low-resistance connections between the simulated cells under initial conditions. Propagation in the orthodromic direction occurs by the electric field (EF) discussed in previous papers . The repolarizing IS2 current gave S/D plots that were typical rectangular hyperbolic curves for excitable membranes, with chronaxie values of about 1.0 ms, both for CM and SM. The calculated velocity for propagated repolarization was greater when the number of gj-channels was increased. The antidromic (retrograde) propagation velocity was usually considerably slower than the orthodromic (antegrade) propagation velocity for depolarization. The present findings do not necessarily imply that, in biological tissue, gap junctions are required for propagated repolarization to occur.In summary, because of technical difficulties with the PSpice program, it was necessary to insert gj-channels in order to produce propagation of repolarization. Otherwise, only the modeled cell injected (A5) with the repolarizing I