Leishmania (L.) donovani complex possess abundant, developmentally regulated cathepsin L-like cysteine proteases. Previously, we have reported the isolation of cysteine protease gene, Ldccys2 from Leishmania (L.) chagasi. Here, we have further characterized this cysteine protease gene and demonstrated its role during infection and survival of Leishmania (L.) chagasi within the U937 macrophage cells.The parasitic protozoa belonging to L. (L.) chagasi and L. (L.) donovani have identical gene organization, as determined by southern blots. In vivo expression analyses by Northern blots showed that Ldccys2 is amastigote specific. Western blot using anti-Ldccys2 antibody confirmed the amastigote specific protein expression. Recombinant expression of Ldccys2, a 30 kDA protein, was functionally active in a gelatin assay. Results from Ldccys2 heterozygous knockout mutants showed its role during macrophage infection and in intra-macrophage survival of the parasites. Since attempts to generate null mutants failed, we used antisense RNA inhibition to regulate Ldcccys2 gene expression. Not surprisingly, the results from antisense studies further confirmed the results from heterozygous knockout mutants, reiterating the importance of amastigote specific cysteine proteases in Leishmania infection and pathogenesis.The amastigote specific Ldccys2 genes of Ldccys2 is a developmentally regulated gene and that Ldccys2 is expressed only in infectious amastigote stages of the parasite. The collective results from both the heterozygous knockout mutants and antisense mRNA inhibition studies shows that Ldccys2 helps in infection and survival of L. (L.) chagasi amastigotes within the macrophage cells. Finally, antisense RNA technique can be used as an alternate approach to gene knockout, for silencing gene expression in L. (L.) chagasi, especially in cases such as this, where a null mutant cannot be achieved by homologous recombination.The study shows that  Leishmania are the etiological agents of a variety of disease manifestations, collectively termed as leishmaniasis. Visceral leishmaniasis caused by Leishmania (L.) donovani and Leishmania (L.) chagasi is a serious health problem in many tropical and subtropical countries [Leishmania, it alternates between gut of sand fly vector as an extra cellular promastigote and in the acidic phagolysosome of macrophage as an intracellular amastigote. However, the most intriguing question is the capacity of Leishmania to withstand the hydrolytic conditions of the macrophages, the mechanism of which is still unclear. Thus, identifying the genes expressed specifically in the amastigote stage of the parasites and elucidating their biological function is very important as it would provide new insights into the role of these gene products in the intracellular life cycle of the parasites. Further, this will also help in designing specific drugs and identifying vaccine candidates.ountries -3. DurinPlasmodium falciparum [Entamoeba histolytica [Leishmania (L.) mexicana [Trypanosoma cruzi, within the macrophages, in vitro [L. (L.) mexicana have shown that cysteine proteases not only are virulence factors but also act as modulators of host immune responses [T. cruzi, P. falciparum and L. (L.) major [L. (L.) major cysteine proteases CPB and CPA, partially protected against leishmaniasis [Lesihamania causing the cutaneous forms of leishmaniasis. The members of L .(L.) donovani complex also possess multiple classes of cysteine proteases, which are developmentally regulated [Cysteine proteases play an important role in the infection, replication, development and metabolism of protozoan parasites ,5. They lciparum  and constolytica . Cysteinmexicana ,9 and rein vitro . Knockouesponses ,12. Thus.) major -15. Immumaniasis . So far,egulated ,18 and aEntamoeba and Leishmania, to elucidate the functions of cysteine protease [Ldccys1 and Ldccys2 from promastigote and amastigote specific cDNA libararies of L. (L.) chagasi [Ldccys1, a member of multi gene family was characterized both in L. (L.) chagasi (Ldccys1) and L. (L.) donovani (Lddcys1) parasites [Ldccys2) of L. (L.) chagasi. We have generated Ldccys2 heterozygous knockout mutants of L. (L.) chagasi by homologous recombination. As an alternative approach, antisense mRNA expression was also employed. Results obtained from both, gene disruption and antisense mRNA expression shows that Ldccys2 plays an important role in the survival of amastigotes within the U937 macrophages.Studies aimed at defining the function of protozoan parasite components have often used gene disruption approach by homologous recombination. Recently an alternative antisense RNA method was followed that may easily and rapidly answer the complex biological questions. Anti sense RNA approach has been used to study the functions of certain gene products in protease ,20, A2 pprotease  and gp63protease . Previou chagasi . Ldccys1arasites . In the Ldccys2 is a single copy gene in L. (L.) chagasi. In order to compare the genomic organization of Ldccys2 gene in L. (L.) donovani and L. (L.) chagasi, the members of L. (L.) donovani complex, Southern analysis of genomic DNA was performed. Genomic DNA was digested with different restriction enzymes as shown in Figure Ldccys2 coding region as a probe. Identical hybridizing bands were present in both L. (L.) chagasi and L. (L.) donovani  donovani genomic DNA showed identical nucleotide sequences, indicating that L. (L.) chagasi and L. (L.) donovani had identical Ldccys2 cysteine protease genes.From our earlier studies it is known that i Figure . PCR ampLdccys2 cysteine protease genes, Northern blot analysis was performed using L. (L.) chagasi total RNA from log and stationary phase promastigotes and amastigotes. The 3'UTR region of Ldccys2 used as a probe, hybridized to a 2.5 kb transcript in amastigote  donovani (data not shown). It is important to note that this result is in contrast to the earlier report (17), wherein Ldccys2 was identified as promastigote specific cysteine protease gene and Ldccys1 was thought to be amastigote specific. We have confirmed the expression pattern and rectified this error in the following publication (18).To study the expression of e Figure BI unlikeL. (L.) chagasi. The anti-Ldccys2 antibody detected a 30 kDa band in amastigotes alone  chagasi.However, a single 46 kDa band seen in log and stationary phase promastigotes  vector and transfected into High Five insect cells (invitrogen). The supernatant was harvested and analyzed for the presence of recombinant protein by Western blot analysis using JHE and Ldccys2 antibodies. Western blot analyses with α-JHE antibody  were selected for further analysis. The PstI restriction enzyme digested genomic DNA from wild type L. (L.) chagasi and Ldccys2 KO were subjected to Southern analysis and results are presented in figure Ldccys2 allele had an additional PstI site that is present within the hyg gene. As shown in the figure Ldccys2KO, probe-a hybridized to two bands (3.5 kb and 2.8 kb) and probe-b hybridized to 3.5 kb and 3.0 kb bands. As expected, one of the bands corresponds to the wild type allele whereas the other band corresponded to the disrupted allele. The hyg specific probe-c resulted in no hybridizing band with PstI digested wild type genomic DNA, while in Ldccys2KO, two bands (2.8 kb and 3.0 kb) corresponding to the disrupted allele were detected .In order to investigate the function d Figure , probe cLdccys2 gene in wild type and heterozygous knockout mutants (Ldccys2 KO), amastigote RNA was isolated from U937 cells infected with wild type as well as Ldccys2KO mutant L. (L.) chagasi promastigotes. Northern blot analysis with Ldccys2 coding region probe detected a single transcript of 2.5 kb from wild type as well as Ldccys2 KO amastigotes  chagasi. There was no change in the level of α-tubulin expression either in the Ldccys2KO or in the wild type amastigotes  chagasi amastigotes were analyzed using antiserum raised against Ldccys2 protein. Wild type L. (L.) chagasi amastigotes showed a major band of 30 kDa . Figure Ldccys2.The effect of a Figure , panel 1Ldccys2KO (data not shown). Next, we performed an infection assay with differentiated U937 macrophage cells using with wild type and Ldccys2KO L. (L.) chagasi promastigotes. Interestingly, twelve hours after infection, the number of amastigotes in Ldccys2KO infected macrophages were reduced by half as compared to that of wild type infected macrophage cells. This trend remained same at all the time intervals tested (24, 36 and 48 hours post infection), indicating that the intracellular amastigotes were not able to multiply efficiently  chagasi promastigotes. The presence of plasmids in these transfectants was confirmed by Southern analysis with Ldccys2 probes (data not shown). Northern blot analyses of amastigotes expressing different plasmid constructs were carried out using specific sense and antisense probes. As expected, Ldccys2 antisense mRNA was expressed only in amastigotes with antisense constructs  in the levels of Ldccys2, however, the expression of sense mRNA did not increase the levels of Ldccys2 compared to amastigotes containing the control plasmid  chagasi amastigotes within the macrophages was assessed in vitro, using U937 cells. Results from three individual experiments are pooled and the collective data is presented in figure Ldccys2 sense mRNA did not have any effect on their capacity to infect and survive inside the macrophages. The number of amastigotes per macrophage cell remained the same at initial stages of infection (data not shown). However, amastigotes expressing Ldccys2 antisense mRNA showed significant reduction  in terms of the initial infection and intra macrophage survival starting from 12 hours post infection and remained consistent up to 48 hours after infection  chagasi amastigotes.Survival of n Figure . The perL. (L.) chagasi amastigotes inside the macrophage cells using two different approaches, gene disruption and antisense mRNA expression. Leishmania (L.) chagasi cysteine protease, Ldccys2 is a single copy gene [L. (L.) donovani also possesses an identical gene. Expression studies performed demonstrates that Ldccys2 is expressed only in amastigotes of L. (L.) chagasi and L. (L.) donovani. A similar single copy gene in L. (L.) mexicana (Lmcpa) has a significant sequence identity (80%) to Ldccys2. Lmcpa gene of L. (L.) mexicana which is the closest homolog of Ldccys2 is differentially expressed in amastigotes [Ldccys2 gene of L. (L.) chagasi is expressed specifically in the amastigote stage. In addition, the major amastigote specific cysteine proteinase gene of L. (L.) mexicana, cpb2.8 is a multicopy gene, located within the lmcpb cluster [Ldccys2 and lmcpb2.8. Therefore, gene organization and expression of amastigote specific cysteine proteases in L. (L.) mexicana and L. (L.) donovani complex are not identical. Interestingly, Llacys1, an amastigote specific cysteine protease from L. (L.) amazonensis was identified recently, which has high homology (88%) with Ldccys2 and like Ldccys2, is only expressed in amastigote stages [Ldccys2 was expressed as JHE-fusion protein in an insect cell expression system. The recombinant protein hydrolysed gelatin in activity gels.In this study, we have shown that amastigote cysteine protease, Ldccys2 necessary for macrophage infection and for survival of opy gene  and is estigotes , whereas cluster  and there stages . In ordeLeishmania by following gene disruption approach [hyg gene as the selectable marker. Genomic Southern analyses of the hygromycin B resistant clones using specific probes confirmed the replacement of 500 bp region within the ORF of the Ldccys2 by hyg/dhfr-ts. Northern and Western blot analyses Ldccys2KO amastigotes showed a clear decrease in the endogenous levels of Ldccys2 compared to that of wild type L. (L.) chagasi amastigotes  chagasi. Since gene amplification and increase in ploidy level has been reported for Leishmania species [Ldccys2 is an important gene necessary for the initiation of macrophage infection and survival of amastigotes within the macrophages. Therefore we used the Ldccys2 heterozygous knockout parasites for further functional studies. The U937 macrophage cells infected with Ldccys2 heterozygous knockout mutants showed a significant decrease in the initial infection and survival within the macrophage cells, compared to wild type  mexicana exhibited reduction in macrophage infectivity and survival [Ldccys2 null mutant prevented us from a detailed study on the role of Ldccys2 in survival and pathogenesis, using animal models.Recently, functions of many genes have been established in approach ,27. To ss Figure , demonst species , we anale Figure . This trsurvival . HoweverLdccys2 null mutants prompted us to use an alternative approach of antisense mRNA inhibtion in order to confirm the results obtained from Ldccys2 heterozygous knockout studies. Using antisense RNA technique it has become possible to investigate the contribution of cysteine proteases to virulence mechanism of E. histolytica [L. (L.) donovani amastigotes in macrophages [L. (L.) amazonensis gp63using Leishmania- specific P6.5 vector provided evidence for a role both in binding of macrophages and the intracellular survival and replication [L. (L.) chagasi amastigotes expressing Ldccys2 antisense mRNA has clearly down regulated the endogenous Ldccys2 mRNA levels  chagasi amastigotes inside the macrophage cells. Based on the data obtained in this study, there is a clear justification for targeting the Ldccys2 gene in L (L.). chagasi and carrying out further studies in animal models. Currently, we are working towards generating null mutants of Ldccys2 in L. (L.) chagasi and to further understand the role of this protein in the pathogenesis of L. (L.) chagasi parasites.The out come of this study shows that L. (L.) chagasi strain MHOM/BR/74/PP75 promastigotes were grown in HOMEM, pH 7.4 (Gibco BRL) supplemented with 10% (v/v) heat inactivated calf serum, at 26°C. Promastigote cultures were seeded at 1 × 106parasites/ml and harvested in logarithmic or stationary growth phase, as defined by cell concentration. L. (L.) chagasi promastigotes were transformed to axenic amastigotes using the standardized protocol as described earlier [ earlier . All theL. (L.) chagasi. To isolate amastigote RNA, human macrophage cell line (U937, ATCC) was infected with promastigote parasites, which were subsequently converted into intracellular amastigotes [Ldccys2 cDNA clone was labeled with [α32P] dCTP. The sense and antisense probes were generated by 5'end-labeling the specific sense and antisense oligonuecleotide primers with γ-32P ATP, using T4 polynucleotide kinase according to the standard protocol.Total RNA was isolated using trizol (GibcoBRL) as recommended by the manufacturer. Promastigote RNA was obtained from logarithmic and stationary growth phase parasites of stigotes . RNA (10Ldccys2 cDNA clone using specific 5' (5'CAGACAGGATCCG CGGCCGCCATGGACGACTTCATTGCC 3') and 3' primers (5' GGCCGCGGATCCGCG GCCGCCTATGAGGTGTTGGAGTCGTC 3') and the PCR product was sub-cloned into BamHI site of pcDNA 3.1 (Invitrogen). After confirmation by sequencing the plasmid constructs were amplified in E. coli and DNA was isolated using endotoxin free maxi prep kit (Qiagen). 50 μg of plasmid DNA in 100 μl of saline was injected into the hind limb quadriceps muscles of BALB/c mice. All the mice were boosted after 14 days and tested for antibody response seven days after the boost. Mice with positive response were boosted one more time and after a week sera was collected and stored at -20°C for further use.Direct DNA immunization  was usedDenaturing gel electrophoresis was carried out according to the standard protocols. Following SDS-PAGE, proteins were transferred onto to Hybond P membrane (Amersham Pharmacia Biotech) using semi-dry transblot apparatus (BioRad). Membranes were blocked for 2 hours with 5% (w/v) skim milk solution and probed overnight at 4°C with 1:200 dilution of anti-Ldccys2 antiserum. Membranes were washed three times with phosphate buffered saline containing 0.1% (v/v) Tween and incubated with 1:5000 dilution of anti-mouse horseradish peroxidase linked secondary antibody (Molecular Probes). Enhanced chemiluminescent kit (Amersham Pharmacia Biotech) was used for detection.Ldccys2 cysteine protease gene, the coding region was amplified by PCR. The digested PCR product was cloned in-frame into unique NotI site of pIE1/153A.jhe (6hep) vector [5 cells/ml (2 ml/well) and transfected for 5 h with 0.5 ml of transfection solution containing 30 μg/ml lipofectin (Life Technologies) and 6 μg/ml total plasmid DNA in basal IPL-41 medium. Then the transfection solution was removed, the cells were rinsed with basal media and 2 ml IPL-41 + 10% FBS was added and incubated for sixty hours. The supernatant was then harvested and stored at -20°C for analysis. Enzyme activity was analyzed on gelatin SDS-PAGE containing 0.2% gelatin and 8% (w/v) acrylamide as described previously [In order to express ) vector  and conf) vector . Brieflyeviously . For inhpXLdccys2-HygKO was designed to replace 500 bp mature region of Ldccys2 coding region and constructed as outlined below  and the antisense primer Ldccys2 Xho I (5'GAAGGCCTCGAGCGAGCCGCACATTCCCTG-3') were used to amplify a 500 bp region from Ldccys2 cDNA and cloned between HindIII – XhoI sites in pX63-Hyg (kindly provided by Dr. S. M. Beverley) for the 5' end homologous recombination. A 450 bp 3' end homologous recombination fragment was amplified using the sense primer Ldccys2 SmaI (5'CCGGAGCCCGGGCCCACGGCGCTTGTGCAG-3') and antisense primer Ldccys2 BglII (5' ACTGTCAGATCTGCTGTGCGCCAGATCGCG-3') and cloned between SmaI and BglII sites of pX63-Hyg. The plasmid construct pXLdccys2-HygKO was confirmed by sequencing and digested with HindIII and BglII. The resulting 3.8 kb linear fragment containing hygromycin phospotransferase/dihydrofolate reductase-thymidylate synthase (hyg/dhfr-ts) gene sequence and the homologous recombination fragment was purified and used for transfection.Gene replacement vector, w Figure . The senLdccys2 cDNA clone was isolated from an amastigote specific cDNA library of L. (L.) chagasi. Ldccys2 was earlier reported as promastigote specific cDNA clone [Ldccys2 was inserted at BamHI site of Leishmania specific vector P6.5 [(nagt), which was used as the selective marker for tunicamycin resistance. Plasmid constructs were confirmed by sequencing. P6.5 plasmid and the sense and antisense plasmid constructs were amplified in E. coli and isolated with a DNA maxi prep kit (Qiagen) and the purified DNA samples were used for transfection.The NA clone . Howevertor P6.5 , a kind L. (L.) chagasi promastigotes. Briefly, 4 × 107 log phase promastigotes were pelleted, washed twice with phosphate buffered saline and resuspended in 0.4 ml of electroporation buffer in a 0.2 cm cuvette. Twenty-five microgram of P6.5 plasmid DNA constructs was used for episomal expression, whereas for homologous recombination 5 μg of linearized DNA fragment was transfected. DNA was electroporated using BioRad Gene Pulser Unit at 0.45 kV, capacitance of 25 μF and resistance of 250 ohms. Following electroporation, cells were resuspended in drug-free media for 24 hours. Parasites with P6.5 plasmid DNA constructs were selected for tunicamycin resistance at 5, 10 and 20 μg/ml tunicamycin (Sigma). Parasites transfected with DNA fragment for homologous recombination were selected for hygromycin B (GibcoBRL) resistance at 100 μg/ml.All the transfections were carried out using L. (L.) chagasi stationary promastigotes at a host to parasite ratio of 1:10 [6 cells/ml) in 10 ml RPMI 1640 medium supplemented with 10% (v/v) heat inactivated fetal bovine serum, 2 mM L-glutamine and 50 μg gentamycin (Gibco BRL) were allowed to grow at 37°C for 3 days in 10% (v/v) CO2. The cells were then induced to adhere with 7.5 ng/ml of phorbol myristate acetate (PMA) and allowed to recover for 72 hours. The macrophage cells were then infected with stationary phase promastigotes and allowed to infect for 6 hours. The unbound parasites were washed away with RPMI medium and the infected macrophage cells were fed with fresh RPMI medium and left for 4 days. At every 12 hours interval up to 72 hours, cells were scraped and Diff-Quick stained slides were prepared for microscopic counting,The U937 macrophage cells were infected with  of 1:10 . BrieflyHyg, hygromycin; JHE, juvenile hormone esterase; kb, kilobase; KO, knock-out; ORF, open reading frame; UTR, untranslated regionVM carried out all the plasmid constructs for gene disruption and antisense mRNA inhibiton studies and performed the Southern and Northern blot analyses shown in Figures


The astounding diversity of life—different body shapes and sizes, physiologies, and behaviors—stems from the accumulation of genetic changes through the process we call evolution. But catching a glimpse into the process of evolution at the gene level is difficult, mostly because significant changes to the plant and animal species of today happened a long time ago. Nevertheless, biologists are keen to understand exactly how evolution progresses. For example, how many genes must be altered before noticeable shifts in appearance can be seen? Is evolution the result of changes in many genes with small additive effects, or of just a few mutations that exert a strong influence?To tackle these questions, Pamela Colosimo and colleagues turned to threespine stickleback fish, a longtime favorite model system of evolutionary biologists because of its relatively youthful evolutionary history. At the end of the last ice age 10,000 years ago, when glaciers all over the Northern Hemisphere began to melt, small populations of these originally marine-dwelling fish became trapped in newly formed lakes. There, isolated stickleback colonies adapted to new ecological conditions—different predators, food availability, water chemistry, and temperature—and now look distinctly different from their marine ancestors. One of the most obvious changes in appearance is in their body armor—they come in three distinct types, or “morphs.” Marine sticklebacks are covered from head to tail with rows of tightly packed boney plates (a complete morph), while those found in freshwater lakes have fewer body plates  or almost none at all (a low morph). Colosimo and colleagues found that a single region of the genome is largely responsible for the dramatic changes in plate morph, and that this is true for two widely separated populations of independently evolving freshwater sticklebacks.To uncover the genomic regions that affect armor, Colosimo's team crossed fully armored marine sticklebacks from Japan with deep-water, or benthic, low morph fish from Paxton Lake in British Columbia, Canada. They then “mapped” the full genome of second generation offspring using 160 known genetic markers, or loci, as guideposts for distinct regions of the genome—loci that are inherited along with differences in the overall type of plating, and individual plate number and size.AA, were almost always fully plated. Those that inherited two copies of the allele from their benthic progenitors, aa, were mostly low morphs with very little plating. And Aa heterozygous fish  had mostly full or partial plates. Colosimo and colleagues also found three other regions in the genome that significantly affected the number and size of plates. These modifiers had an additive effect—the more benthic alleles inherited, the fewer and smaller the plates; more marine alleles caused a trend toward greater armor.The team found that one such locus explained 75% of the variation in plate morphs. Offspring that carried two alleles—versions of the gene—from their marine grandparents, genotype But is this genetic architecture the same for every independently evolving population of lake-bound sticklebacks in North America? Or did the geographically isolated freshwater groups loose their plates through mutations in different genes? Colosimo and colleagues mapped the genome of a population of sticklebacks from Friant, California, which is 800 miles away from Paxton Lake, and found that the same major locus seemed to be controlling plate morph there as well. Crossing a low morph from Friant with a low morph from Paxton yielded only offspring with very little armor. Further, some of the modifiers uncovered in the Paxton fish were also acting on the Friant sticklebacks. So, though these two populations of fish have been separated for 10,000 years, loss of armor in both groups probably stemmed from changes in the same genetic pathway.Without knowing the precise sequence of these genes, it is impossible to tell exactly how and when the alleles that reduce armor arose. Small numbers of individuals with genes causing less plating could have been present in ancestral populations of marine sticklebacks when they were originally locked in newly formed lakes. Alternatively, reduced armor could have arisen independently in different lakes following isolation if, for example, some genes that control armor are predisposed to mutation, or certain armor-related mutations are more advantageous than others. But however it happened, this study clearly shows that dramatic morphological evolution can result from a small number of genetic changes. Further study of this classic system should provide a detailed picture of the genes involved, and of the molecular events that underlie morphological changes in natural populations evolving in new environments.


FU is the human homologue of the Drosophila gene fused whose product fused is a positive regulator of the transcription factor Cubitus interruptus (Ci). Thus, FU may act as a regulator of the human counterparts of Ci, the GLI transcription factors. Since Ci and GLI are targets of Hedgehog signaling in development and morphogenesis, it is expected that FU plays an important role in Sonic, Desert and/or Indian Hedgehog induced cellular signaling.FU gene was identified on chromosome 2q35 at 217.56 Mb and its exon-intron organization determined. The human developmental disorder Syndactyly type 1 (SD1) maps to this region on chromosome 2 and the FU coding region was sequenced using genomic DNA from an affected individual in a linked family. While no FU mutations were found, three single nucleotide polymorphisms were identified. The expression pattern of FU was thoroughly investigated and all examined tissues express FU. It is also clear that different tissues express transcripts of different sizes and some tissues express more than one transcript. By means of nested PCR of specific regions in RT/PCR generated cDNA, it was possible to verify two alternative splicing events. This also suggests the existence of at least two additional protein isoforms besides the FU protein that has previously been described. This long FU and a much shorter isoform were compared for the ability to regulate GLI1 and GLI2. None of the FU isoforms showed any effects on GLI1 induced transcription but the long form can enhance GLI2 activity. Apparently FU did not have any effect on SUFU induced inhibition of GLI.The FU gene and its genomic structure was identified. FU is a candidate gene for SD1, but we have not identified a pathogenic mutation in the FU coding region in a family with SD1. The sequence information and expression analyses show that transcripts of different sizes are expressed and subjected to alternative splicing. Thus, mRNAs may contain different 5'UTRs and encode different protein isoforms. Furthermore, FU is able to enhance the activity of GLI2 but not of GLI1, implicating FU in some aspects of Hedgehog signaling.The  Drosophila, Hh signaling to the transcription factor Cubitus interruptus (Ci) is mediated by a protein complex consisting of Ci and three other cytosolic proteins. These are the costal 2 (cos2), suppressor of fused (su(fu)) and fused (fu), where fu is a kinase domain containing protein with positive regulatory activities in Hh induction of Ci mediated transcriptional activation. Hh binds to its receptor patched (ptc), a 12 membrane spanning protein, leading to the activation of another membrane protein smoothened (smo) [Drosophila genes and proteins have been identified during the last decade. To a large extent the signaling pathway has been conserved in vertebrates. However, the picture is more complicated since some of the Drosophila genes have two or more vertebrate homologues. There are three Ci homologues in vertebrates, GLI1, GLI2 and GLI3. GLI1 has activation properties whereas GLI2 and GLI3 have both activation and repression activities [reviewed in [The signaling molecule Hedgehog (Hh) and components of its intracellular signaling pathway have been the subject of intensive research in several species from fruit fly to man during recent years. Numerous developmental and morphogenic processes are controlled by the Hedgehog family of proteins. Much effort has been directed at identifying components of the signaling pathway and their respective roles and interactions [for an extensive review see ]. In Droed (smo) . Smo is iewed in ]. It is iewed in -7. In a iewed in . FU is aPTCH1, the human counterpart of ptc, underlie the Nevoid Basal Cell Carcinoma Syndrome (NBCCS) [SMO and SUFU mutations as well as overexpression of GLI1 or GLI2 can lead to BCC or medulloblastoma [Interestingly, it was discovered that mutations in  (NBCCS) ,10. Pati (NBCCS) ,12. Alsoblastoma -16. ThusFU gene, as well as for construction and subcloning of FU expression vectors. Using the available public databases the FU gene was found to be present in a sequenced BAC clone from chromosome 2. FU is located in the same region of chromosome 2q34-q36 to which the human limb malformation disorder Syndactyly type 1 (SD1) has recently been mapped in a large German pedigree [FU gene in an affected member from the German family [FU has been determined using an RNA array and Northern blots. FU is expressed in all 72 tested tissues. It is clear that not only a single transcript is expressed. Instead transcripts of different sizes are seen and some tissues apparently express more than one major transcript. From the genomic structure and the cDNA clones it was possible to predict several alternative splicing events and consequently the likely expression of different protein isoforms. Two of the isoforms were expressed in HEK293 cells and tested for their ability to regulate the activity of GLI1 and GLI2, showing positive effects on GLI2 but not on GLI1.Here three FU cDNA clones have been identified and used for sequence analysis, identification and structural description of the pedigree  and confpedigree . Its posn family . The tisFU gene in a 200 kb BAC clone (AC009974) from chromosome 2. The gene is localized to 2q35 at 217.56 Mb using the Ensembl [FU structure and named the gene STK36 (serine/threonine protein kinase 36). Chromosome 2q35 is the locus of several genetically based disorders. Both Syndactyly type 1 (SD1) and Brachydactyly type A1 (BDA1) have been mapped to this region [IHH (Indian Hedgehog) one of the vertebrate Hh homologues [IHH is located in the vicinity of FU on chromosome 2 (217.94 Mb) less than 400 kb away. In order to determine if alterations of FU are responsible for SD1, the FU coding region (exons 3–29) and the flanking intronic regions were sequenced using genomic DNA from an affected member of an SD1 family whose trait maps to the 2q34-q36 region [FU mutations were detected in this study, although three single nucleotide polymorphisms were identified. These included a T to C transition in intron 10, 17 bp 5' of exon 11 (IVS10-17T>C), causing gain of a BstNI site, and a G to A transition in exon 16, 17 bp 5' of the end of the exon (1748G>A), causing substitution of glutamine for arginine at amino acid 583 (R583Q) and loss of an AciI site. The altered restriction sites created by these sequence changes were tested in 44 CEPH unrelated individuals. The results showed that both changes are normal sequence variations as previously reported in the NCBI SNP database. The third change was a G to A transition in exon 27 (3008G>A), causing substitution of aspartic acid for glycine at amino acid 1003 (G1003D). By sequencing exon 27 in 8 affected and 6 unaffected members of the SD1 family [The sequence information derived from the FU cDNA clones 1HFU, 2HFU and Ngo3689 (see Methods) allowed the identification of the  Ensembl  annotatis region ,20. ReceFU exons, but they allow determination of the exon-intron organization of FU. Figure FU gene. The cDNA clones are outlined to account for the predicted structure. Only clone 1HFU and Ngo3689 contain exons from the 5'non-coding region. To the 5' side of the sequence encoded by exon 3 these clones are different, indicating that alternative 5' untranslated regions (UTR) from different exons can be used. Exons 3 to 9 encode the N-terminal kinase domain. None of the cDNA clones encodes the FU protein that has previously been described [None of the obtained cDNA clones contain sequence from all + RNA from 72 human tissues was hybridized with a labeled probe 3' of the kinase domain. It was clear from this array that all examined tissues express FU to some extent. The highest amount of FU transcripts were detected in testis and pituitary (not shown). This is in agreement with the previous results by Northern blotting, showing highest FU expression in testis [A tissue array with poly An testis . This ann testis . The Norn testis . Since on testis .To examine more specifically the expression of exons that are involved in alternative splicing events, nested PCR was performed on cDNA probes generated from whole tissue RNA by reverse transcription. Sequences containing segments including exons 8, 13 and 24 were amplified and analyzed on agarose gels .We investigated the possibility that FU expression have shown that transcripts are detected in all tissues examined. For the first time evidence is presented showing that more than one transcript can be expressed from this gene. The Northern blots clearly show that FU transcripts of different sizes indeed exist. Here the transcripts are estimated to be slightly bigger than previously reported and in some tissues more than one transcript is evident. It is clear from the available cDNAs and RT/PCR based transcript analyses that alternative splicing occurs. Additionally, it is also clear that different 5'UTRs are present in the transcripts. At least two protein isoforms, besides the previously described L-FU [Analyses of bed L-FU , may be bed L-FU . WhetherThe assessment of functionality revealed that S-FU was not able to regulate GLI1 or GLI2 when expressed in 293 cells. In contrast, both L-FU and a variant lacking a full kinase domain (2HFU) were able to enhance GLI2 induced transcription. These results are qualitatively similar to those previously reported in C3H/10T½ cells . L-FU anFU is localized on chromosome 2q35 very close to IHH. Though SD1 has been mapped to this region, we have not identified a causative role for FU in this disorder. FU consists of 29 exons of which 1 and 2 encode 5'UTRs and 3 to 9 encode a kinase domain. For the first time it is shown that transcripts of different sizes are expressed and alternative splicing takes place, probably leading to the generation of different protein isoforms. FU protein is likely to be involved in the Hedgehog signaling pathway since it can enhance the activity of GLI2. In contrast, it has no effect on GLI1 and an effect on SUFU cannot be observed in 293 cells.+. The human BAC clone AC009974 was obtained from Research Genetics (Huntsville, AL). The human GLI, human SUFU, 12GLI-RE-luciferase reporter and β-galactosidase vectors have been described previously [Two almost full-length human FU clones were identified in the Incyte database. Both 1HFU and 2HFU were cloned in the vector pINCY. A third clone was available from Kazusa DNA Research Institute (Chiba, Japan) and termed Ngo3689 (Gene name KIAA1278). This clone was in the vector pBluescript II SKeviously .R DNA polymerase (New England Biolabs, Beverly, MA). The cDNA for the long form of FU (L-FU) was subcloned into pCDNA3.1-HisB using the NotI and XbaI sites. 2HFU and the short FU (S-FU) cDNAs were subcloned into pCDNA3.1-HisC using the KpnI and XbaI sites.Expression constructs for different isoforms of FU was obtained by direct PCR or extension overlap PCR, using end-primers having specific restriction sites and the high fidelity VentAll PCR generated products were analyzed by DNA sequencing. The Big-Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA) was used according to instructions. Sequencing was performed at CyberGene AB (Huddinge, Sweden). Sequence alignments were done using the DIALIGN program  availablFU either as single exons with flanking intronic sequences or as products containing two exons with flanking intronic sequence and the complete intervening intron. The primer sequences can be obtained upon request. PCR was performed in a standard fashion and products were sequenced using either the Thermosequenase CyTM5.5 Dye Terminator or DYEnamic ET Dye Terminator Cycle Sequencing kits (Amersham Biosciences, Piscataway, NJ). Electrophoresis and analysis were performed on either an Automated Laser Fluorescence (ALF) DNA sequencer or MegaBACE DNA sequencer (Amersham Biosciences) after purification with Autoseq columns (Amersham Biosciences). For exon 27, the PCR product was purified using the enzymatic ExoI-SAP purification method, sequenced using the Terminator Cycle Sequencing kit (Amersham Pharmacia Biotech) and analysed on an ABI 3100 genetic analyzer (Applied Biosystems). PCR products containing exon 11 or exons 15/16 were digested with BstNI or AciI, respectively, and the bands resolved on 3–4% agarose gels to confirm sequence changes in the patient with SD1 and to determine their frequency in a panel of 44 CEPH individuals.After informed consent was obtained, blood was taken from affected and unaffected family members and DNA extracted from peripheral blood leukocytes according to standard methods. Intronic primers were designed to amplify exons 3–29 of 32P-ATP using the High Prime DNA labeling kit (Boehringer Mannheim, Mannheim, Germany) according to instructions. Hybridization of Northern blots was done with labeled DNA probes in ExpressHyp (Clontech) at 68°C according to instructions. The blots were then analyzed with a Fujix Bas 2000 phosphoimager (Fuji Photo Film, Tokyo, Japan).Commercially available Human MTN 12-lane Blot 2, Human Fetal MTN Blot II and Human Endocrine System MTN Blot Northern blots (Clontech, Paolo Alto, CA) were obtained and used with PCR generated hybridization probes. DNA probes were made by direct PCR, amplifying the sequences corresponding to exon 13 and 28. The generated fragments were then labeled with The expression of exon 8, 13 and 24 sequences in mRNA was assessed by nested PCR on RT/PCR generated cDNA samples from eight different tissues as provided in Human Multi Tissue cDNA Panel II (Clontech). Two sets of primers were made for each exon to be investigated. The outer pairs were used in a first PCR using 5 μl of the cDNA and Vent polymerase. In a second PCR 0.5 μl of the first PCR products was used together with the inner primer pairs. These pairs were also used for PCR of FU cDNA clones that served as controls. The primer pairs are listed in Table The cDNA clones were used in transfections of HEK293 cells in 24 well culture plates. Basically this was done as previously described . In shorTØ contributed to the experimental design; participated in sequencing, sequence analysis and subcloning; did the gene analysis, Northern blots, nested PCR, cell experiments; and made the manuscript draft. DBE and CES designed and carried out the patient analysis; and contributed to the manuscript. MM provided clones; contributed with subcloning; made the array analysis; and contributed to the manuscript. MMN and RCB provided the SD1 patient material; performed segregation analyses in the SD1 family; and edited the manuscript. PGZ contributed to the experimental design and subcloning; and edited the manuscript. RT contributed to the experimental design; did data base analysis; and edited the manuscript.


Previous smallpox ring vaccination models based on contact tracing over a network suggest that ring vaccination would be effective, but have not explicitly included response logistics and limited numbers of vaccinators.We developed a continuous-time stochastic simulation of smallpox transmission, including network structure, post-exposure vaccination, vaccination of contacts of contacts, limited response capacity, heterogeneity in symptoms and infectiousness, vaccination prior to the discontinuation of routine vaccination, more rapid diagnosis due to public awareness, surveillance of asymptomatic contacts, and isolation of cases.We found that even in cases of very rapidly spreading smallpox, ring vaccination (when coupled with surveillance) is sufficient in most cases to eliminate smallpox quickly, assuming that 95% of household contacts are traced, 80% of workplace or social contacts are traced, and no casual contacts are traced, and that in most cases the ability to trace 1–5 individuals per day per index case is sufficient. If smallpox is assumed to be transmitted very quickly to contacts, it may at times escape containment by ring vaccination, but could be controlled in these circumstances by mass vaccination.Small introductions of smallpox are likely to be easily contained by ring vaccination, provided contact tracing is feasible. Uncertainties in the nature of bioterrorist smallpox (infectiousness, vaccine efficacy) support continued planning for ring vaccination as well as mass vaccination. If initiated, ring vaccination should be conducted without delays in vaccination, should include contacts of contacts (whenever there is sufficient capacity) and should be accompanied by increased public awareness and surveillance. Concerns about intentional releases of smallpox have prompted extensive preparations to improve our ability to detect and respond to an outbreak of smallpox ,3,4,2. MThe Centers for Disease Control and Prevention (CDC) established ring vaccination , a straMathematical models can advance our understanding of how a smallpox outbreak might progress. Several mathematical and computer models address the question of smallpox transmission -13. The Because none of the available models includes both network structure (with explicit contact tracing) and response logistics limited by the number of available disease control investigators , we inclSmallpox was primarily a disease of close contact, especially household contacts . Such coSome evidence suggests that vaccination soon after exposure may lessen the severity of the resulting case of smallpox or possibly prevent disease entirely -20.Ring vaccination may involve not only vaccinating contacts of cases, but also contacts of contacts of cases ,22 – potLimited case-finding and vaccination capabilities lead to the possibility that it may be impossible to find newly exposed individuals and vaccinate them in time, resulting in a "race to trace" .Mild, ambulatory cases of smallpox may spread disease because such cases may be harder to recognize.Vaccination of individuals prior to the discontinuation of routine vaccination may provide some, possibly considerable, protection against infection ,23,24, aPublic awareness may lead to more rapid detection of cases.We use this model to determine what factors promote or hinder the success of ring vaccination during a smallpox outbreak, and whether ring vaccination is useful in the presence of a mass vaccination campaign. In particular, the goal of this paper is to examine the control of smallpox by contact tracing and ring vaccination using a network model which includes response logistics .focal rash, preceded in many cases by oropharyngeal lesions. In fatal cases of ordinary smallpox, death often occurred between the tenth and sixteenth day of symptoms; among survivors, most scabs had separated by day 22–27 of illness [We briefly review relevant features of the natural history and epidemiology of smallpox ,25,8,28. illness .The course of smallpox varied widely between individuals, and several different clinical classifications were developed -31,17,26Vaccination with vaccinia virus provided substantial protection against infection. Dixon assessed the risk of infection for an individual successfully vaccinated 3 years prior to exposure to be 0.1% the infection risk of an unvaccinated individual . HoweverSmallpox was largely a disease of close contacts ,26,33, sMathematically, we represent the course of smallpox according to Figure We simulate the transmission of smallpox on a "small-worlds" network (highly clustered, but with short characteristic path lengths) . SpecifiIn general, in a network-structured model, the number of secondary cases caused by an index case in a completely susceptible population is not a useful index of epidemic potential ,42  of household contacts are assumed to be traceable (see below). We assume that the fraction of workplace/social contacts that are traceable is less than the fraction of household contacts that are traceable; we assume that no casual contacts are traceable.High contact-finding rates may be plausible; we examined San Francisco Department of Public Health records of contact investigations for meningococcal disease . Records were available from December 2001 to April 2002; 13 such investigations during this period resulted in identification of 62 household contacts, all of which were contacted; out of 38 workplace/social contacts identified, 32 were contacted (84%).Kr per day for ring vaccination. We assumed one of two strategies for contact tracing: (1) tracing only of direct contacts of diagnosed cases, and (2) tracing of contacts of contacts of diagnosed cases as well as direct contacts.In our model, we assume that identified asymptomatic contacts are vaccinated, quarantined, and monitored for symptom development, while symptomatic patients are isolated and treated as necessary ; thus, tb and c are household contacts of individual a, so that if individual a were a smallpox case, an attempt would be made to find and vaccinate individuals b and c as household contacts of a case. If individuals a and b were both cases, then two attempts could be made to find individual c. We have modeled the effect of multiple contact-finding attempts conservatively in the sense that if the first attempt to find an individual as a household contact (of a case or of a contact) is determined to fail, no further attempts will be made. This maintains the failure rate of contact tracing  even in large households. Similar considerations apply to workplace/social groups.The contact structure of the network is illustrated in Figure We analyzed the model in three ways. First, we selected a Latin Hypercube sample -46 of paTo determine which of the input parameters were most important in determining the total number of smallpox cases, we selected a Latin Hypercube sample of size 1000 from the input parameter ranges indicated in Appendix 2 [see additional file 2] and simulated the mean number of cases within 250 days in a population of 10000. We then computed the partial rank correlation coefficient  , as shown in Figure Because we assumed nonzero diagnosis probabilities during the prodromal period for all individuals in Figure In Figure Finally, in Figure In Figure Finally, in Figure Because our baseline hazard for infection of individuals may be larger than would be expected for naturally occurring smallpox, we examined the effect of more realistic values of this hazard. In particular, we chose different levels of ring vaccination capacity (10, and 20), and of the relative hazard for workplace/social contacts, and then chose values of the baseline hazard for infection varying from 0.5 per day (for a mean time to infection of 2 days) to 2 per day , and introduced 10 index cases into a population of 10000. We then repeated this 100 times, and reported the fraction of scenarios in which the number of infections ultimately exceeded 500 (as before, chosen as a cutoff to indicate the ultimate "escape" of containment of the epidemic). These results, shown in Table Because of considerable uncertainty in the model parameters, we chose a collection of parameter values, and for each, estimated the containment probability . We estimated this containment probability by simulating the smallpox epidemic 100 times for the same parameter values, and computing the frequency out of these 100 realizations for which fewer than 500 index cases resulted within 250 days. .2), the monitored diagnosis rate φ , and the contact tracing/ring vaccination capacity per day (Kr). We then performed 100 realizations beginning with 10 index cases, and computed the containment fraction (fraction showing fewer than 500 cases in 250 days, beginning with 10 index cases). Thus, for each of the two choices each of υ2 and φ, and for each value of Kr we examined, we obtained 1000 values of the containment fraction. We use the resulting distributions in Figure One thousand scenarios chosen from a Latin Hypercube sample were analyzed, and as indicated before, we chose the hazard for close contact transmission and the hazard for random transmission to guarantee that between 2 and 5 secondary cases per case occur, and that no more than 5% of cases are attributable to random transmission . Having chosen this collection of 1000 parameter sets, we considered two levels of two different control parameters which were applied to each (so that each of the 1000 parameter sets were simulated under four different control conditions). The first of the two control parameters was the probability of workplace/social group contact finding; we chose values of 0.8 and 0.9 for this parameter . The second of the control parameters was the rate of diagnosis  from the community of cases developing among previously identified and traced contacts who were initially asymptomatic (we refer to this as the monitored diagnosis rate); we assumed first a low level corresponding to a mean diagnosis time of one day from the onset of symptoms, and a high level corresponding to a mean time of 3 hours from the onset of symptoms (high levels of the monitored diagnosis rate correspond effectively to isolation of contacts). Finally, we assumed a doubling of the diagnosis rate after the beginning of widespread community awareness of smallpox. We then computed the containment fraction at different levels of ring vaccination capacity (contact tracing capacity per day). Thus, for each of 1000 scenarios (parameter set choices), we assigned the workplace/social group contact tracing success probability (υ-1 and 8 day-1). For low levels of ring vaccination (traceable contacts per day), the epidemic is almost never contained, but for ring vaccination levels near 50–60 per day (5–6 per index case per day), the average containment fraction became close to 1. However, this average conceals the fact that for some scenarios , control remains difficult or impossible even at high levels of ring vaccination. Therefore, in Figure In Figure fast scenario, 238 infections occurred on average and the (estimated) containment probability was 99%; for the slow scenario, on average 3587 infections occurred and the (estimated) containment probability was only 1%.Rapid contact tracing in ring vaccination may play an important role in suppressing the epidemic, since the longer it takes to trace a contact, the less likely the vaccine is to be efficacious, and the more opportunities the infected individual may have to transmit disease before they are finally located, isolated, and vaccinated if appropriate. We illustrate this possibility in Figure While Figure Public awareness of smallpox, leading to more rapid isolation and identification, may play an important role in eliminating the epidemic, as illustrated by the scenarios in Figure In many cases, however, more rapid diagnosis was not required for ring vaccination to be effective. As before, we simulated smallpox epidemics for each of 1000 calibrated scenarios, performing 100 realizations each beginning with 10 index cases, and computed the fraction of scenarios for which the epidemic was always contained (as defined earlier), assuming no change in diagnosis rates. We assumed 80 ring vaccinators per day, contact finding probabilities of 0.95 for households and 0.8 for workplace/social contacts , most individuals may already be infected when identified through contact tracing from an infective. Using the scenario we examined in Figure a = 1 hour, so that cases arising in previously contacted persons almost never transmit the infection further). We chose different levels of household and workplace/social contact finding probabilities and different levels of ring vaccination capacity, and performed 100 replications of each of the 1000 different scenarios. In Table Finally, we used the "calibrated" scenarios (parameter set choices) to explore the levels of contact finding probability needed to contain the epidemic  Table . In thesa relative infectivity during entire period is one , b the relative infectivity just prior to the rash is the same as during the first week of the rash, but during the prodromal period is 4% ; in case b (high infectivity just prior to onset of rash), loss of containment occurs 36% of the time ). Scenario a  showed loss of control in every realization. Increasing the ring vaccination capacity from 40 per day to 80 per day (results not shown) led to containment in all of the realizations with high infectivity just prior to the rash and low infectivity during the prodromal period (case b), but made no difference if the infectivity was as high during the prodromal period as during the rash (case a). While intuitively adding additional infectiousness must increase the number of secondary cases and make control more difficult, these results do illustrate that even a small amount of increased infectiousness prior to the rash (when diagnosis is more difficult) may substantially increase the difficulty of smallpox control.Transmission prior to the rash makes epidemic control more difficult. In Figure a in the figure). Specifically, we assume that severe smallpox (hemorrhagic and flat) on average takes four times longer to diagnose and isolate than ordinary smallpox (case b), that no one in the population has prior vaccination protection (from before the discontinuation of routine vaccination, case c), that 10% more smallpox is too mild to diagnose (but still contagious, case d) compared to baseline, and finally that the vaccine is completely ineffective (case e). Each of these scenarios will be discussed further below.Finally, in Figure b was motivated by the possibility that individuals with severe forms of smallpox may be more difficult to diagnose, and thus remain infectious in the community longer (despite the much greater degree of illness of such patients), or that such patients may be more infectious. In this particular case, quadrupling the mean diagnosis time led to one additional replication out of 100 in which containment was not achieved (2/100, compared to the baseline of 1/100). However, we assumed that community awareness of smallpox leads to the same relative rate of increased diagnosis among severe cases as for ordinary cases, and that the most severe forms are relatively rare. In addition to the scenario shown in the figure, we also replicated the same 1000 "calibrated" simulations, assuming that in each case 40 contact tracings per day are possible and that the diagnosis time for severe cases was four times that of ordinary cases. Finally, we repeated each "calibrated" scenario 100 times assuming long diagnosis times for severe cases, and not making this assumption, and found that the difference in the decontainment fraction was not large (results not shown).Scenario c illustrates that vaccination prior to the discontinuation of routine vaccination does play a role in smallpox control by ring vaccination; there were more decontainment scenarios (5/100) when no prior protection exists in the population. The results suggest that prior vaccination aids in the control of smallpox, but that it is not strictly necessary for control (in this scenario, 95% of the replications exhibited containment). In Figure c of Figure Scenario d demonstrates that if 10% more smallpox infections . Here, with 40 contact tracings possible per day, 55% of the replications nevertheless exhibited containment even with a vaccine which offered no protection whatever. With 90 contact tracings possible per day, all replications exhibited containment even assuming no vaccine protection.Finally, scenario Although less efficient than ring vaccination in the sense that more vaccinations must be delivered to eliminate infection, comprehensive mass vaccination following the introduction of smallpox is sufficient to eliminate the infection. In Figure We constructed a simple network model of smallpox transmission, and addressed the question of what circumstances contribute to the success of a ring vaccination campaign designed to control smallpox. Our analysis focused on the use of contact tracing/ring vaccination to prevent a widespread epidemic following a deliberate release.We conducted a sensitivity analysis based on particular, but reasonable, ranges for the unknown parameters. Our results are consistent with prior vaccination models in identifying prior vaccination and ring vaccination capacity as significant factors in determining the spread of smallpox. Unsurprisingly, we also find that household size and ring vaccination speed are particularly important parameters; these results are intuitively plausible. The contact finding probability did not appear important in this analysis only because a narrow range of values was chosen.We illustrated smallpox control by presenting scenarios based on control of moderately severe smallpox epidemics. We find that swift, aggressive contact tracing and ring vaccination is is usually sufficient to bring the infection under control. Provided that there is sufficient capacity, vaccination of contacts of contacts is beneficial, and results in fewer infected individuals and more rapid elimination of infection; investigating contacts of contacts allows the chain of transmission to be outrun to some extent. When ring vaccination capacity is small, diversion of crucial resources away from contacts is harmful; contacts of contacts should only be traced and vaccinated provided that no resources are diverted away from contacts of cases. The increased surveillance (or isolation) of contacts, together with improved rates of diagnosis due to community awareness, play important roles in smallpox control; we note that in some cases, lowered diagnosis rates among severe cases contributed to a small extent to loss of epidemic control, and suggest that any public awareness campaign include information to help the public be more aware of the full spectrum of the clinical features of smallpox.One limitation of our analysis is that we chose not to explicitly incorporate the specific epidemiology of health care workers (or mortuary workers), who are likely to be exposed to infected individuals during any smallpox epidemic e.g. ,22), and, and22])While we analyzed the effect of contact tracing, case and contact isolation, and ring vaccination (together with mass vaccination), in a real smallpox epidemic, in practice, control efforts are unlikely to be limited strictly to vaccinating contacts  and isolating cases. Indeed, making vaccine available to individuals who believe they live near cases or to others on a voluntary basis occurred in smallpox control efforts in the past . VaccinaMore serious is the possibility that individuals who should be vaccinated or isolated would be missed; this could occur either because individuals or institutions did not cooperate with the disease control effort, or because the individuals simply could not be found. Our analysis suggests that ring vaccination need not be perfect to successfully contain the epidemic, and yet, under conditions where there is a high rate of infection among contacts, or a relatively high rate of casual transmission, high rates of contact finding (in excess of 90%), together with increased surveillance and contact isolation, are needed to contain the epidemic.Finally, the vaccination of individuals at low risk of contracting smallpox will cause harm due to adverse events of the vaccine; in our model, the assumed death rate due to vaccination was small compared to the probability of death from smallpox, and played essentially no role in the analysis. In practice, individuals suspected to be at high-risk for vaccine complications, but at relatively low risk for contracting smallpox, might simply be isolated or closely monitored even during an outbreak; while the presence of individuals in the population at higher risk for vaccine complications would increase the death rate during an outbreak, such individuals are unlikely to impair the containment of the epidemic .Our results support ring vaccination against epidemics of smallpox (even assuming high rates of transmission to close contacts), but do note that stochastically, for severe (rapidly transmissible) smallpox, scenarios of loss of control are seen, with resulting widespread epidemics. In scenarios in which the transmission potential of smallpox is smaller, such loss-of-control scenarios occur less frequently (results not shown). Mass vaccination campaigns, when conducted quickly and with very high coverage, do not result in loss of control in our model. Nevertheless, fewer deaths due to smallpox result when ring vaccination is conducted along with mass vaccination.Simulated smallpox epidemics with ring vaccination suggest that aggressive, fast ring vaccination can control epidemics of smallpox. To do so, however, smallpox must be identified quickly and contacts vaccinated promptly. We also identify public awareness of smallpox – leading to prompt identification of cases – as a major factor in smallpox control; in some simulations, it may play a role as significant as ring vaccination itself . Howeverwhich can facilitate more rapid identification and isolation of cases. We assumed that ring vaccination could be fast (little delay between identification of a case and vaccination of the contacts), effective , and available (there is sufficient capacity). To be effective, ring vaccination planning must yield a system capable of meeting these benchmarks; we should not only be able to assess the number of contact vaccinations that will be possible per day, but should have a plan in place to (1) identify contacts by working with individuals, employers, schools, community representatives, and authorities or businesses who may have access to information facilitating contact tracing, (2) rapidly investigate and vaccinate such individuals, perhaps using field teams managed by central dispatch. It is important to realize that for high-risk, transient, or unstably housed populations where reliable contact tracing is impossible, the conclusions of the model we present cannot be applied. It is important to note that while our model suggests that ring vaccination together with contact tracing and isolation is likely to be successful, we found that for some scenarios , epidemic containment required not only ring vaccination, but increased public awareness, the isolation of contacts, and tracing of contacts of contacts. For scenarios in which the smallpox was less transmissible, epidemic containment was possible at lower contact finding probabilities. Thus, while our simulations suggest that contact tracing/ring vaccination need not be perfect to succeed, because of uncertainties in our knowledge of the behavior of bioterrorist smallpox, it is impossible to know in advance how good it will have to be. Thus, that high contact finding rates, mass public awareness leading to early identification of cases, isolation of contacts, and investigation of contacts of contacts should all be conducted with maximum effectiveness to reduce the probability of a widespread epidemic.Because our model combines network structure with response logistics, our results support and complement the results of other investigators. Our results support the notion that prior vaccine protection may play an important role in slowing the epidemic , despiteWhile the possibility of smallpox uncontrollable by ring vaccination has made mass vaccination preparations wise, and while mass vaccination may be unavoidable in the event of a deliberate release of smallpox, we believe that ring vaccination is essential in any case. This is not only because individuals recently exposed to smallpox may be protected if they are vaccinated promptly, but because each contact identified potentially lies in the immediate future of the transmission chain. From the standpoint of epidemic control, it is far more valuable to vaccinate individuals next in the transmission chain than to vaccinate other persons. Our results support the idea that ring vaccination/case isolation may in many, if not most cases, eliminate smallpox even without mass vaccination, but also support planning for mass vaccination (so that the vastly more costly and difficult policy of mass vaccination will be available in the event of an explosive epidemic). When faced with the unknown, multiple redundant preparations are appropriate; case investigation/isolation may control smallpox even if the vaccine does not work at all, but mass vaccination is useful in the event of an explosive epidemic for which case tracking becomes impossible.None declared.TP, KH, SF, TA, RR, and DP performed the literature review , TP developed and implemented the model and simulation, TP performed the analysis of the simulation model and drafted the manuscript, DP performed analysis of contact tracing data, and KH conceived of the study. All authors contributed to, read and approved the final manuscript.The pre-publication history for this paper can be accessed here:For consistency, all references are included in the bibliography of the main text.Click here for fileFor consistency, all references are included in the bibliography of the main text.Click here for file


The physiological flux of oxygen is extreme in exercising skeletal muscle. Hypoxia is thus a critical parameter in muscle function, influencing production of ATP, utilization of energy-producing substrates, and manufacture of exhaustion-inducing metabolites. Glycolysis is the central source of anaerobic energy in animals, and this metabolic pathway is regulated under low-oxygen conditions by the transcription factor hypoxia-inducible factor 1α (HIF-1α). To determine the role of HIF-1α in regulating skeletal muscle function, we tissue-specifically deleted the gene encoding the factor in skeletal muscle. Significant exercise-induced changes in expression of genes are decreased or absent in the skeletal-muscle HIF-1α knockout mice (HIF-1α KOs); changes in activities of glycolytic enzymes are seen as well. There is an increase in activity of rate-limiting enzymes of the mitochondria in the muscles of HIF-1α KOs, indicating that the citric acid cycle and increased fatty acid oxidation may be compensating for decreased flow through the glycolytic pathway. This is corroborated by a finding of no significant decreases in muscle ATP, but significantly decreased amounts of lactate in the serum of exercising HIF-1α KOs. This metabolic shift away from glycolysis and toward oxidation has the consequence of increasing exercise times in the HIF-1α KOs. However, repeated exercise trials give rise to extensive muscle damage in HIF-1α KOs, ultimately resulting in greatly reduced exercise times relative to wild-type animals. The muscle damage seen is similar to that detected in humans in diseases caused by deficiencies in skeletal muscle glycogenolysis and glycolysis. Thus, these results demonstrate an important role for the HIF-1 pathway in the metabolic control of muscle function. Mice that can't express HIF-1α in skeletal muscle don't induce glycolytic genes and thus switch to aerobic metabolism allowing them to run uphill longer than the their wild-type littermates During exercise in normoxia, the partial pressure of oxygen in muscle tissue has been shown to dip to as low as 3.1 mm Hg, whereas in the capillary, it remains at 38 mm Hg . In ordeHIF-1α via the use of a conditionally targeted allele of the gene , the frequency of excision was evaluated through real-time PCR techniques. We saw deletion frequencies consistent with those described previously for this cre recombinase transgene . LDH activity was also increased in the WT mice, although the level did not reach statistical significance. Activities of both PK and LDH were not significantly changed in HIF-1α KO muscles following exercise. Increased activities of PK, and subsequently LDH, could be expected to lead to increased levels of lactate in the WT mice relative to HIF-1α KOs.In p = 0.10) toward an increased amount of hexose monophosphates (HMPs), which are pre-PFK glycolytic metabolites, following stimulation of the HIF-1α KO muscle. This increase was not due to differences in glucose uptake, since animals of both genotypes were able to significantly increase intramuscular glucose to a similar degree  and McArdle's disease . We analty acids . Increasty acids , althougty acids . This dep < 0.05).As mentioned above, patients with muscle glycolytic deficiencies demonstrate both increased exercise-induced muscle damage and a “second wind”; the latter phenomenon allows them to exercise for extended periods of time at submaximal levels. This is thought to be due to an increase in rates of oxidative ATP production, and a decreased utilization of and need for muscle glycogen . To assep < 0.01, Further testing was done to determine the parameters of this increased endurance. HIF-1α KOs were run on an enclosed treadmill, with a 5° incline and an initial velocity of 10 m/min, with an increase in velocity every 5 min. In their first runs, HIF-1α KOs again had significantly greater endurance, as shown by their consistently longer run times compared to WT controls (p < 0.05, p < 0.01); HIF-1α KOs did the reverse, and ran for significantly shorter times downhill than uphill (p < 0.05). Substrate utilization confirms the shift toward glycolytic fibers in downhill running; both genotypes had higher average respiratory exchange ratio (RER) values when running downhill compared with running uphill (As it has been shown that muscle groups and fibers respond differently to eccentric exercise (i.e., downhill running) than to concentric exercise (i.e., uphill running) , mice frg uphill D and 6E.p < 0.01, p < 0.05). Histological examination of gastrocnemius tissue following 1 d of recovery revealed significantly greater amounts of muscle damage in HIF-1α KO tissue than WT tissue (PFKD and McArdle's disease demonstrate significant myopathic effects in muscle, including soreness and cramping induced by bouts of exercise. After 1 d of recovery from endurance testing, HIF-1α KOs had increased levels of the MM isoform of creatine kinase in their serum (unpublished data), indicative of skeletal muscle damage. To further investigate this finding, mice were run on a treadmill daily for 4 d. By the second day, the trend for increased endurance in the HIF-1α KOs was absent, and by the final day, HIF-1α KOs were running for significantly shorter times than they had on the first day  and the ABI Prism 7700 Sequence Detector (Applied Biosystems). Conditions for the PCR were one 10-min incubation at 95 °C (polymerase activation), followed by 40 cycles of 15 s at 95 °C (denaturation) and 1 min at 60 °C . The degree of excision was calculated by comparing HIF-1α DNA levels to c-Jun DNA levels. HIF-1α real-time PCR primers and probe were as follows: forward primer, HIFLOX501/F 5′-CTATGGAGGCCAGAAGAGGGTAT-3′; reverse primer, HIFLOX574/R 5′-CCCACATCAGGTGGCTCATAA-3′; probe, HIFLOX/P 5′-(6FAM)AGATCCCTTGAAGCTAG(MGBNFQ)-3′.Mice from the above crosses were genotyped using DNA extracted from tail sections. DNA was then extracted from the gastrocnemius, heart, liver, and uterus of eight 4-mo–old, Paraffined gastrocnemius sections were deparaffinized and stained with Gill II hematoxylin. Sections were then washed successively in water, a bluing agent, water again, and 95% ethanol, and restained with eosin. Hematoxylin and eosin staining was performed by the University of California at San Diego (UCSD) Cancer Center Histology Resource . Imaging was performed on sections mounted on slides using Cytoseal 60 (VWR, West Chester, Pennsylvania, United States). Electron microscopy was performed by standard methods on gastrocnemius muscle. Briefly, fixation was by 25.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Postfix was in 1% osmium tetroxide. The section was stained in 2% uranyl acetate in sodium maleate buffer (pH 5.2), then placed in Epon resin (VWR, West Chester, Pennsylvania, United States), and cured overnight at 60 °C. Fiber typing was performed using the metachromatic dye ATPase method . PAS stan = 10; KO, n = 14) were run either on an Omnipacer treadmill (Columbus Instruments, Columbus, Ohio, United States) or on an enclosed-chamber modular treadmill (Columbus Instruments) with a 5° incline at an initial velocity of 10 m/min. Velocity was increased by 2 m/min every 5 min during the assessment. Exhaustion was determined to be the point at which the animal would not resume running when provoked through a low-voltage power grid. Gas flow (O2 and CO2) into and out of the enclosed chamber treadmill was monitored using the Paramax O2 sensor and a CO2 sensor (Columbus Instruments) and analyzed using Oxymax software (Columbus Instruments) to determine metabolic parameters. The downhill running assessment  was carried out in the enclosed-chamber modular treadmill at a 10° decline using the same protocol as above.Untrained, age-matched WT mice and HIF-1α KOs (WT, n = 8 for each class) was placed in a 30 °C water bath with mild turbulence. Exhaustion was determined to be the point at which the animal experienced three successive periods below the surface of more than 3 s.In the swimming exhaustion assessment, a second group of WT and HIF-1α KOs  and attached to a force transducer to record contractile force. Muscles were electrically stimulated through excitation of the sciatic nerve. Stimulation was in the form of 8–10-V direct titanic contractions using 200-ms trains at 70 Hz with 0.2 ms duration. Initial frequency of tetanic contraction was one every 8 s and was increased every 2 minutes to one every 4 s and one every 3 s, up to the end point of 6 min. Isolated muscles were then immediately harvested and snap-frozen for ATP, lactate, phosphocreatine, and glycogen analyses. Samples were freeze-dried and analyzed by enzymatic assay as has been previously described . Reverse transcription was performed using the Superscript First Stand Synthesis System for RT-PCR . Amplification was performed using the ABIPrism 7700 as described above. Reverse transcription real-time PCR primers and probes were as follows. For PGK-1: reverse primer, PGK/R 5′-CAGGACCATTCCAAACAATCTG-3′; forward primer, PGK/F 5′-CTGTGGTACTGAGAGCAGCAAGA-3′; probe, PGK/P 5′-(6∼FAM)TAGCTCGACCCA-CAGCCTCGGCATAT(TAMRA)-(phosphate)-3′. For VEGF-A: reverse primer, VEGF/R 5′-ATCCGCATGATCTGCATGG-3′; forward primer, VEGF/F 5′-AGTCCCATGAAGTGATCAAGTTCA-3; probe, VEGF/P (6∼FAM)TGCCCACGTCAGAGAGCAACATCAC(BHQ∼6∼FAM). For GLUT4: reverse primer, GLUT-4/R 5′-CCCATGCCGACAATGAAGTT-3′; forward primer, GLUT-4/F 5′-TGTGGCCTTCTTTGAGATTGG-3′; probe, GLUT-4/P 5′(6-FAM)TGGCCCCATTCCCTGGTTCATT(BHQ1-Q)-3′. For PFK-M: reverse primer, PFK-M/R 5′-AAGTCGTGCAGATGGTGTTCAG-3′; forward primer, PFK-M/F 5′-GCCACGGTTTCCAATAACGT-3′; probe, PFK-M/P 5′-(6-FAM)CCTGGGTCAGACTTCAGCATCGGG(BHQ1-Q)-3′. For LDH-A: reverse primer, LDH-A/R 5′-ATGCACCCGCCTAAGGTTCTT-3′; forward primer, LDH-A/F 5′-TGCCTACGAGGTGATCAAGCT-3′; probe, LDH-A/P 5′-(6- FAM)TGGCAGACTTGGCTGAGAGCAT(BHQ1-Q)-3′.n = 5; KO, n = 6) were run on a treadmill at 25 m/min for 30 min. Following the run, mice were euthanized and RNA was isolated and analyzed as described above.For changes in gene expression due to exercise, age-matched male mice  were run on a treadmill using the same protocol as for the gene expression analysis. Tissue was harvested after the run and from resting mice , and enzymes were extracted and analyzed spectrophotometrically as has been described  by the UCSD Animal Care Program Diagnostic Laboratory.Creatine kinase levels were analyzed from serum from WT mice and HIF-1α KOs 24 h after running-induced exhaustion using a kit from Sigma (St. Louis, Missouri, United States). Creatine kinase isoforms were analyzed enzymatically and then fractionated by gel electrophoresis. Serum lactate levels were analyzed by the UCSD Comparative Neuromuscular Laboratory from blood obtained by cardiac puncture from six WT mice and six HIF-1α KOs following 25 min running time on the treadmill ramp at 25 m/min. Hematocrit and hemoglobin levels were measured from resting mice  or glucose tolerance group . Experimental animals were injected with 0.3 g/ml glucose in PBS to achieve a dosage of 2 g/kg. Sham animals were injected with an equivalent amount of PBS. Blood was drawn from the tail at time intervals of 0, 15, 30, 60, and 120 min. Samples were then centrifuged to isolate plasma. Plasma blood glucose was quantified using the Infinity Glucose Kit (Sigma).Animals were assigned into either a sham  were mechanically dissected from the flexor brevis muscle and loaded with fura-2. Fibers were then stimulated while force generation and Ca2+ release were monitored.Intact individual muscle fibers  on the Omnipacer Treadmill or the enclosed-chamber modular treadmill using the same exhaustion protocol described above. Mice ran according to this protocol every day for 4 d with a minimum of 22 h of rest between trials. Following the fourth trial, mice were given 24 h of rest and then euthanized. Tissue was harvested and stained using hematoxylin-eosin (as described above) and α-PCNA  combined with a DAB Kit .Endurance was tested by running 24 animals  were carried out using StatView software (SAS Institute, Cary, North Carolina, United States) or Prism software .