Several studies have been conducted where minimum inhibitory concentration  of rifampin has been measured and partial DNA sequences have been determined for rpoB in different isolates of M. tuberculosis. However, no model has been constructed to predict rifampin resistance based on sequence information alone. Such a model might provide the basis for quantifying rifampin resistance status based exclusively on DNA sequence data and thus eliminate the requirements for time consuming culturing and antibiotic testing of clinical isolates.Mutations in rpoB and associated MIC of rifampin for different isolates of M. tuberculosis were taken from studies examining rifampin resistance in clinical samples from New York City and throughout Japan. We used tree-based statistical methods and random forests to generate models of the relationships between rpoB amino acid sequence and rifampin resistance. The proportion of variance explained by a relatively simple tree-based cross-validated regression model involving two amino acid positions (526 and 531) is 0.679. The first partition in the data, based on position 531, results in groups that differ one hundredfold in mean MIC (1.596 μg/ml and 159.676 μg/ml). The subsequent partition based on position 526, the most variable in this region, results in a > 354-fold difference in MIC. When considered as a classification problem (susceptible or resistant), a cross-validated tree-based model correctly classified most (0.884) of the observations and was very similar to the regression model. Random forest analysis of the MIC data as a continuous variable, a regression problem, produced a model that explained 0.861 of the variance. The random forest analysis of the MIC data as discrete classes produced a model that correctly classified 0.942 of the observations with sensitivity of 0.958 and specificity of 0.885.Sequence data for amino acid positions 511–533 of Highly accurate regression and classification models of rifampin resistance can be made based on this short sequence region. Models may be better with improved (and consistent) measurements of MIC and more sequence data. Mycobacterium tuberculosis resistant to rifampin occur at low to moderate frequencies in many regions of the world [rpoB, the gene encoding the β subunit of DNA-dependent RNA polymerase, are associated with rifampin resistance. In the laboratory, drug resistance is quantified in terms of minimum inhibitory concentration (MIC), which is defined as the minimum concentration of the antibiotic in a given culture medium below which bacterial growth is not inhibited.Rifampin, one of the principal drugs used in tuberculosis treatment, is a semi-synthetic antibiotic that inhibits transcription by preventing RNA synthesis. Isolates of he world . MutatiorpoB in different isolates of M. tuberculosis [M. tuberculosis.Several studies have been conducted where MIC of rifampin has been measured and partial DNA sequences have been determined for rculosis -6. Howevrculosis . Both trrculosis  and cytirculosis . The sucβ subunit of RNA polymerase of M. tuberculosis? What particular positions, and what distribution of amino acids at those positions, are associated with most of the variance in MIC? Alternatively, the response variable could be cast in discrete terms: resistant or susceptible. This is possible by assuming a threshold value for MIC above which an isolate is considered resistant to rifampin. Among the specific questions we can answer with such a model are the following: What particular positions, and what distribution of amino acids at those positions, allow for distinguishing rifampin-susceptible and rifampin-resistant isolates of M. tuberculosis? What is the misclassification error rate associated with susceptibility prediction for these data? We address these questions and evaluate the ability to predict MIC from protein sequence data (inferred from DNA sequence data) using tree-based regression and classification methods. We find that these methods generate highly accurate models of rifampin resistance.The response variable is a set of continuously distributed values for MIC, which makes the problem one of regression. These data are used to answer the following questions: What proportion of the variance in MIC is attributable to sequence differences in positions 511–533 of the μg/ml to > 512 μg/ml. The 173 sequences are distributed among 24 genotypes, 11 of which occur uniquely in the data set. The plurality genotype is represented by 69 samples; 98 samples differ from the plurality by one amino acid; and the remaining 6 samples differ from the plurality by two amino acids. Some genotypes defined by the partial sequence of rpoB are associated with several different phenotypes . Also, some genotypic states are associated with large effects, while some have little or no effect on MIC phenotype. Finally, some changes in MIC are not associated with changes in the sequence region examined. These genotype data are short (69 bp) partial sequences of a single gene, and thus they may not contain all phenotypically relevant genetic information. Indeed, there is evidence that amino acid changes outside of the examined region are associated with changes in MIC for rifampin [The data set used in the study consists of 173 observations with 60 distinct genotype-phenotype combinations  is the mean of all isolates at that node. The first split of the topmost node (root node) consists of the entire sample and is based on the amino acid at position 531, with those sequences having serine (S) going to the left child node, and those having leucine (L) or tryptophan (W) going to the right child node. The best split for each node is that which gives the largest decrease in the error. Here error is measured as the deviance, which for a continuous variable is a constant multiple of the residual sums-of-squares. Reported values were determined using 10-fold cross-validation. Moving down the tree the error decreases, as the sum of the deviance for each pair of child nodes is less than the deviance of the parent node. Given the hierarchical nature of trees and the criterion used to choose splits, the first split, that based on position 531, explains the highest proportion of the overall phenotypic variance. This bisection of the data results in groups that differ one hundredfold in mean MIC (1.596 μg/ml and 159.676 μg/ml). The subsequent partition based on position 526, the most variable in this region, results in a > 354-fold difference in MIC. The proportion of the variance in MIC explained across all splits involving the two amino acid positions (526 and 531) is 0.679. All proportions of variances explained by the model as reported here are those estimated through cross-validation and are not based on re-substitution, and thus represent appropriately conservative estimates.The regression tree for the relationship of des Fig. . At eachM. tuberculosis is determined to be rifampin resistant then rifampin is replaced with another antibiotic. Although blood serum concentration of rifampin reaches levels of 6 – 7 μg/ml about 1.5 – 2 hours after ingestion [μg/ml as susceptible and values > 1 μg/ml as resistant, a definition consistent with conventional standards [From a clinical perspective it may be most relevant to consider the level of drug resistance as a two-state categorical variable (susceptible or resistant) rather than as a continuously distributed variable. In clinical practice, if an isolate of ngestion , a clinitandards . With thP < 0.0001) based on permutation tests.The predictor variables are again the unordered categorical designations of amino acids at polymorphic positions. The classification tree for these data Fig.  has two The random forest analysis, which aggregates results over many tree models, each constructed on subsamples of the data, produced markedly better models as compared to the single tree-based models. The random forest analysis of the MIC data as a continuous variable, a regression problem, produced a model that explained 0.861 of the variance. The random forest analysis of the MIC data as discrete classes (susceptible and resistant), a classification problem, produced a model that correctly classified 0.942 of the observations with corresponding sensitivity of 0.958 and specificity of 0.885.Although both the regression and classification random forest results are markedly better than the single tree-based models, they do lack the ease of interpretation of a tree model. However, variable importance can be assessed in random forests by measuring the increase in group purity based individual models containing the variable. As might be expected, the results for both regression and classification are similar and identify the same amino acid positions as being most important in determining response to rifampin as did the single tree models: primarily 531 and 526, and much less so for 513 and 516  such as microsatellites, restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs), and similar data. Tree-based and random forest analyses can also be applied directly to DNA sequence data including single nucleotide polymorphisms (SNPs). In general, tree-based statistical and random forest models are applicable to all cases where the goal is to examine the relationship between genotype and phenotype.M. tuberculosis. These models demonstrated that only a few variable positions in the β subunit of DNA-dependent RNA polymerase were responsible for most of the variation in rifampin resistance. Such models might provide the basis for quantifying rifampin resistance status based exclusively on DNA sequence data and thus eliminate the requirements for time consuming culturing and antibiotic testing of clinical isolates. More generally, the results of this study demonstrate the usefulness of tree-based statistical models and random forests in genetic analysis.Relatively simple models provided accurate predictions of rifampin resistance in rpoB and associated MIC of rifampin for different isolates of M. tuberculosis were taken from studies examining rifampin resistance in clinical samples from New York City and throughout Japan [Sequence data for amino acid positions 511–533 of ut Japan -4. Minimlog2 transforms. Values given in the original sources as <x were set to log2(x - 0.5), and those values given as >x were set to log2(x + 0.5). The MIC values are converted back to μg/ml in figures to be consistent with Table The predictor variables are unordered categorical designations of amino acid at polymorphic positions, and the response variable are continuous values for MIC represented by their rpoB amino acid sequence and rifampin susceptibility was done through the use of tree-based statistical models [recursive partitioning) [xi ∈ A? Here A is a region of the variable space. Thus answering the question for all observations produces two groups of observations; those for which the answer is yes (those in region A) and those for which the answer is no . The specific criteria for choosing among the possible partitions (questions) is based on the change in deviance, which for regression problems is equivalent to least squares. Subsequent binary partitioning continues until stopping criteria (variously defined) are met. The result is a classification or regression tree: a hierarchical series of data bifurcations, which depicts the partition definitions and describes the resulting data subsets defined by each partition.Analysis of the relationships between l models , also knl models . Analysetioning)  using thtioning)  for the tioning) . Tree-ban-1 -1 possible partitions, where n is the number of different amino acids observed. For example, in the case of amino acid position 526 of rpoB analyzed here there are 9 observed amino acids resulting in 255 possible partitions to be evaluated. The preferred way to construct an appropriately sized tree is to first build a large tree and subsequently prune it [cost-complexity [For unordered categorical covariates, such as amino acid designation, the search through possible splits is exhaustive. For each variable amino acid position there are 2prune it ,13. PrunPerformance of tree-based models can be assessed in a number of ways depending on the goals of the analysis. One way is to evaluate the fit of the data used to generate the model, which is known as the re-substitution error. The use of re-substitution error may be justified when the principal goal of the analysis is to explain the observations in hand. However, the re-substitution error provides an underestimate of the error if the goal is to produce a model for future prediction. Another scheme to assess performance is to partition observations into a subset for model building, the training set, and a subset to evaluate the model, the test set. To remove biases this general scheme can be expanded in the form of cross-validation. Typically 10-fold cross-validation is used, where the data are randomly divided into 10 equal or near equal portions. Nine of these portions are used to generate the model and the remaining portion, the test set, is used to evaluate the model. This step is repeated until all test sets have been used in model evaluation.4 permutations is the estimate of the probability associated with the observed result.We assessed the significance of our tree-based statistical models through permutation where the predictor variables are randomized with respect to the response variable . The freIn a series of recent papers -21, BreiRandom forests seek to effect such correlation reduction by a further injection of randomness. Instead of determining the optimal split of a given node of a (constituent) tree by evaluating all allowable splits on all covariates, as is done with single tree methods or bagging, a subset of the covariates drawn at random is employed. Breiman ,21 argue4 individual trees constructed by sub-sampling eight predictor variables (regression) or two predictor variables (classification) at each node. Variable importance was assessed by measuring the increase in group purity when partitioning data based on a variable. We used the R package randomForest [Here, our random forests comprised 1 × 10omForest .MPC conceived of the study, and participated in its coordination. Both authors participated in study design, carried out the statistical analyses, wrote and approved the final manuscript.


To study the apoptosis and its mechanism at the fetal-maternal interface of early gestation, localization of apoptotic cells in the implantation sites of the rhesus monkey on day 17, 19, 28 and 34 of pregnancy were first examine by using the TUNEL technique. The expression of Ki67, a molecular marker of proliferating cells, and two apoptotic proteins, B cell lymphoma/leukaemia-2 (Bcl-2) and P53, were then studied by immunohistochemistry. Apoptotic nuclei were observed mainly in the syncytiotrophoblast. Ki67 was confined almost exclusively to cytotrophoblasts. The localization of Bcl-2 protein follows that of the apoptotic nuclei and its expression level increased as the development of the placenta progressed on. P53 was detected to some extent in cytotrophoblasts and syncytiotrophoblast covering the basal feet of the anchoring villi during the late stage of placentation. Based on these observations, it might be suggested that Bcl-2 could be possible to play an interesting role in limiting degree of nuclear degradation and sustaining cell suvival in the multi-nucleated syncytiotrophoblast cells during early pregnancy, and P53 could also be essential in regulating the trophoblastic homeostasis by controlling its proliferation or apoptosis. Apoptosis plays important roles in placentation and embryonic development . The celThe expression of Bcl-2 and P53 human placenta has been studied ,13. HoweMacaca mulatta) were purchased from the monkey colony of the Primate Research Center (PRC), Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences (CAS). All experimental procedures were approved by the Animal Ethics Committees of both the Institute of Zoology and PRC. The animals were caged individually and were evaluated daily by visual examination of the perineum for menses, with the onset of menses defined as Day 1 of the menstrual cycle. Adult female monkeys with regular menstrual cycles of approximately 28 days were chosen for this study. Female monkeys on Day 11 of their menstrual cycle were caged with a male monkey of proven fertility from previous mating for 3 days. Vaginal smears were examined the next morning for the presence of sperm. The day when the smear was detected as positive for sperms was designated as Day 1 of pregnancy (D1). The presence of a conceptus was confirmed by ultrasound examination. The monkeys were anesthetized by pentobarbital sodium , and the uteri were removed surgically from early villous to villous placenta stages: on D17, D19, D28 and D34 of pregnancy respectively and cut into pieces, the specimens were quickly washed in cold phosphate-buffered saline (PBS) to remove adherent blood, then placed in cold 4% paraformaldehyde fixative for 16 h at 4°C and further processed through graded dehydration, clearing and embedding in paraffin for immunohistochemistry and TUNEL assay. Part of the specimen was cryopreserved at -70°C for Western blot analysis.Healthy adult male and female rhesus monkeys .Primary antibodies including rabbit anti-human P53 (SC-6243) and mouse anti-human Bcl-2 (SC-7382) were obtained from Santa Cruz Biotechnology . Their quality and specificity were confirmed by the result of the Western blot analysis of D34 placental proteins Figure . Rabbit ® West Pico substrate, then exposed on x-ray film. For negative controls, primary antibodies were replaced with normal IgG of the same concentration and origin.Western blot was done as previously described  with sliApoptotic cells were identified by using the TUNEL technique ,16. The Serial 4 μm sections of tissue were deparaffinized and rehydrated through degraded ethanol. Antigen retrieval was performed by incubating the sections in 0.01 M citrate buffer (pH 6.0) at 98°C for 20 min followed by cooling at room temperature for 20 min. Non-specific binding was blocked with 5% (v/v) normal goat serum in PBS for 1 h. The sections were incubated with primary antibodies specific for P53 (1 μg/ml), Bcl-2 (2 μg/ml) or Ki67 (2 μg/ml) respectively in 2% goat serum overnight at 4°C. Sections were then washed three times with PBS (10 min each) and incubated with biotinylated secondary antibody (2 μg/ml) at RT for 30 min. 3 × 10 min successive washes were followed by incubation with avidin-AP complex . Sections were developed with standard substrates (337.5 μg/ml NBT and 175 μg/ml BCIP) or Vector Red AP substrates according to the manufacturer's protocol after another three washes. Endogenous AP activity was inhibited by supplement of 1 mM levamisole into substrate. The sections stained with Vector Red substrates were counter-stained using haematoxylin. Sections incubated with normal IgG instead of primary antibody served as negative controls.2O2 in methanol for 10 min at room temperature to quench endogenous peroxidase after antigen retrieval treatment as described above. To detect the cytokeratin signal, the sections were washed (3 × 10 min in PBS), blocked for nonspecific signals, incubated sequentially with primary anti-human cytokeratin anbibody , secondary biotinylated goat anti-rabbit IgG , and avidin-peroxidase complex , and developed with DAB substrate solution in a similar way as described above. To detect actin signal, the procedure was repeated one more time with anti-human actin antibody  as primary antibody, AP conjugated horse anti-mouse IgG  as secondary antibody, and Vector Red developing AP substrate. As a result, the trophoblast cells were labeled brown and the blood vessel wall red.A double immunostaining technique using the antibodies to cytokeratin and actin was performed to localize the extravillous endovascular trophoblast cells. De-paraffinized sections were incubated with 3% HPlacental samples from three individual monkeys for each group were analyzed. Experiments were repeated at least three times, and one representative from at least three similar results was presented. The mounted sections were examined using a Nikon microscope. For Ki67, the percentages of immunoreactive cells were assessed on at least 2000 cells in each tissue section; For TUNEL, the percentages of positive nuclei were assessed out of at least 2000 nuclei in each tissue section; For assessment of Bcl-2 staining intensity in cells of different compartments, semi-quantitative subjective scoring was performed by three blinded investigators using a 4-scale system with "-"= nil; "+/-"= weak; "+" = moderate; and "++" = strong as described by Yue et al. .The TUNEL technique was used to identify cell types that underwent apoptosis in the implantation site of rhesus monkey on D17, D19, D28 and D34 of pregnancy. On D17 and D19, apoptotic nuclei were observed in the syncytiotrophoblast layer covering the basal feet of the anchoring villi Figure , arrow, Ki67 is a protein expressed in cycling cells from G1 to M phases and is widely used as a roliferative marker . As shown in Figure In order to study the mechanisms of the apoptosis observed at the fetal-maternal interface, the expression of Bcl-2 was investigated by using immunohistochemistry. At the early stages of placentation D17, D19), Bcl-2 was only detected in the syncytiotrophoblast covering the cell columns Figure  and 4B a7, D19, BThe expression profile of P53 was also acquired by using the immunohistochemistry. On D17 and D19, the expression of P53 was only confined to a small number of nuclei in the syncytiotrophoblast Figure . On D28 For the first time in present study, we investigated the expression of Bcl-2 and P53 in relation to apoptosis at the fetal-maternal interface of rhesus monkey at the very early stages D17-D34) of gestation. Villous trophoblasts consist of cytotrophoblasts and syncytiotrophoblast. While cytotrophoblasts possess a brisk mitotic activity during the first trimester of gestation in human, the syncytiotrophoblast is incapable of cell division despite of a metabolic activity 7-D34 of ,22. Our In our previous study, Bax expression was found at the Fetal-Maternal Interface of Rhesus Monkey . Bax is P53 was partly identified in some nuclei of the syncytiotrophoblast with the same position of apoptotic nuclei, in the basal feet of the anchoring villi in particular, but it is not clear whether the P53 was co-localized with the apoptotic signals. Activation of P53 in some cell types leads to either the cessation of cell growth or apoptosis . TherefoIt is known that early pregnancy is unique in its methods of cell proliferation control, the existing data suggest that some growth factors and transcription factors from the embryo and endometrium, such as CSF-1, VEGF, and transcription factors of the helix-loop-helix family, provide at least part of this control . In addi


Down's syndrome (DS) is characterized by several immunological defects, especially regarding T cell compartment. DS is considered the best example of accelerated ageing in humans. Direct observations of the thymus have shown that in DS this organ undergoes severe histological and morphological changes. However, no data on its capacity to generate T cells are present in the literature. Here, using a new technology based upon real time PCR, we have investigated the capacity of the thymus to produce and release newly generated T lymphocytes  in children with DS.i.e. peripheral blood lymphocytes that express the T cell receptor rearrangement excision circles (TREC).We studied 8 children affected by DS, aged 2–7 years, compared with 8 age- and sex-matched healthy controls. Flow cytometry was used to determine different lymphocytes subsets. Real time PCR with the Taqman system was used to quantify the amount of RTE, In comparison with control children, those with DS had a significant lower number of TREC+ peripheral blood cells. Moreover, in DS children but not in controls, a strong negative correlation between age and the levels of TREC+ cells was found.The direct measure of thymic output indicates that the impairment of the organ results in a reduced production of newly generated T cells. This observation could suggest that cytokines able to modulate thymic function, such as interleukins, could be useful to improve the functionality of the organ and to treat the immunodeficiency present in DS subjects. Down's Syndrome (DS) is the most common chromosomal abnormality in humans, that occurs in 1 out of every 800–1,000 births. It is an autosomal disorder resulting by triplication of chromosome 21. Many characteristics are commonly seen in DS, including some degree of intellectual impairment, that varies widely from individual to individual, heart defects, hypotonia, hyperuricemia, and the development of Alzheimer disease-type neuropathology beginning at about 40 years of age [DS subjects also present alterations of the immune system which are similar to those of aged people, including increased susceptibility to bacterial and viral infection and to the onset of different types of haematological malignancies, along with a high frequency of autoantibodies. Alterations of B lymphocytes, T cell subsets, and natural killer cells, defective phagocytosis and chemotaxis of polymorphonuclear leukocytes and interleukin-2 production by activated T cells were also reported . QuantitT cell receptor Rearrangement Excision Circles), generated during the intrathymic rearrangement of the α-chain locus of the T cell receptor (TCR).Several studies have focused their attention on the role of the thymus, and have described a variety of structural and anatomic alterations present in DS . Few datGenes for the δ-chain of the TCR are distributed within the genomic region that codifies for the α-chain, and are removed in two steps during the recombination of Vα with Jα. Thus, a thymocyte that starts to rearrange the α-chain produces the first TREC, called signal-joint (sj)-TREC, then proliferates three or four times, and finally completes the rearrangement of Vα with Jα, so producing the second TREC, called coding-joint (cj)-TREC. The removal of genes for the δ-chain from the α region does not imply their elimination, as such DNA remains into the nucleus as a circle that is not able to replicate. As a consequence, when a cell undergoes a division, TREC are passed only to one of the two daughter cells. During the following cell cycles, TREC are then diluted into the population that origins from the first cell. Several data indicated that the percentage of TREC+ cells is a marker of thymic activity, that TREC+ cells are almost present within the subset of virgin T lymphocytes, and that their number consistently declines with age .We could analyze blood samples from 8 children with DS , 7 males and 1 females. As control group, we studied 8 children with a mean age of 4.75 years , 7 males and 1 female. All subjects were in good conditions, and had no acute or chronic disease affecting the immune system. Written informed consent was obtained by their parents, according to the Italian laws.Peripheral blood mononuclear cells (PBMC) were separated from a minimum of 8 mL freshly collected blood according to standard procedures.Blood samples were stained with different monoclonal antibodies for the cytometric analysis, as described . The qua6 PBMC using the QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer's protocols and stored in sterile water at -20°C until use.DNA was extracted from 5 × 10The percentage of PBMC containing sjTREC was measured by an original method that has been recently developed (patent pending) using a Real Time PCR approach. This assay was performed by two parallel polymerase chain reactions (PCR), that quantify sjTREC or nuclear DNA (nDNA) in a given sample, carried out in two different reaction tubes, but in the same plate, in order to have similar reaction conditions.In the first reaction, we quantified sjTREC using a mix that consisted of: Supermix Biorad 1X, primers for sjTREC 500 pmol and we added 3 μl of the sample in each tube. The sjTREC primers we used were: sjTREC Dir (5'-CAC ATC CCT TTC AAC CAT GCT-3') and sjTREC Rev (5'-GCC AGC TGC AGG GTT TAG G-3'). TaqMan probe for sjTREC (5'-FAM-ACA CCT CTG GTT TTT GTA AAG GTG CCC ACT – BLACK HOLE-3') was included in the reaction mixture at the concentration of 200 nM, as a real time detector for the amplified product. One cycle of denaturation (95°C for 6 min) was performed, followed by 50 cycles of amplification . The same approach was used to quantify nDNA, which was required to obtain the number of cells present in the sample. In this case, primers GenDir (5'-GGC TCT GTG AGG GAT ATA AAG ACA-3') and GenRev (5'-CCA ACC ACC CGA GCA ACT AAT CT-3'), designed on FasL gene sequence, present in two copies in the human genome (one in each chromosome 1), were used at the concentration of 600 and 400 nM respectively, and the TaqMan probe GenProbe (5' TexasRed – CTG TTC CGT TTC CTG CCG GTG C – BlackHole Quencher2 3') was included in the reaction mixture at the concentration of 300 nM. One cycle of denaturation (95°C for 3 min 30 sec) was performed, followed by 45 cycles of amplification .. The length of amplified fragments is 104 pb for nDNA and 101 pb for sjTREC.All of the aforementioned primers and probes have been designed using the program "primer3", available on the internet address: PCR was performed by using an iCycler Thermal cycler , that monitors changes in the fluorescence spectrum of each reaction tube during the annealing phase. The fluorescence signal was processed using the "Real-time detection system iCycler iQ" software, that calculates the threshold and the threshold cycles of each sample. All reactions were carried out in triplicate.i.e., those of sjTREC and nDNA) were purified and cloned tail to tail in a vector , to have a ratio of 1:1 of the molecules used as reference.To optimize the precision of the assay, whose results derive from the comparison between the threshold cycles of two different real time PCR reactions, we have developed a new approach. Indeed, the regions used as template for the two amplifications (The nDNA region used has been excised as a SacI-ApaI fragment and cloned in the pGEMT easy vector (Promega), obtaining the pGEM11Z-nDNA vector. The sjTREC region used has been excised as a PstI-SacI fragment and cloned in the same vector, obtaining the pSJ vector we used. The plasmid has been purified, quantified with the spetrofotometer and linearized using SacI. Then a series of reactions has been performed for nDNA and sjTREC with serial dilution of the standard. All the times the threshold cycles of nDNA and sjTREC were the same, proving the ratio 1:1 of the two fragments.Then, serial known dilutions of this vector, amplified in triplicate, were included in each PCR run to generate a standard curve from which the relative copy number of either sjTREC or nDNA present in the unknown samples was determined. The measured values for sjTREC and nDNA were always within the range of the standard curve, whose correlation coefficient was always >0.990. The values of sjTREC and nDNA present in each sample were calculated using the mean of the threshold cycles of the three replicates.versus the same vector), multiplied by 2 (as two copies of the nuclear gene are present in a cell).Then, the percentage of PBMC containing sjTREC in each sample was simply obtained from the ratio between the relative values of sjTREC  and nDNA obtained  complex, elevated numbers expressing TCRγδ and a decreased proportion of CD4+, CD45RA+ naïve T cells, along with a high number of cells with NK phenotype, suggesting that the DS thymus is inefficient in the release of functionally mature T cells . An age-i.e. the dysfunction of thymus. Indeed, defects in the capacity to produce thymic hormones has been described several years ago [All of the above mentioned alterations have a common substrate, ears ago ,18. Furtears ago . In addiears ago . DS thymears ago . Taken tIn conclusion, in this paper we show that the aforementioned alterations of the thymus result in the reduced production and output of newly generated lymphocytes, that can be directly measured by the assay we have developed and used. The quantitative analysis of TREC+ cells in the periphery is a relatively new and sensitive marker of thymic functionality, which is able to provide information on the status of the organ in different pathological conditions, and on its capability to generate new T cells -25. A ma


Helicoverpa zea moths is manifested as insects that are either sterile "agonadal" individuals with malformed reproductive tissues or fertile asymptomatic carriers which are capable of transmitting virus on to their progeny. Virus infected progeny arising from eggs laid by asymptomatic carrier females may themselves be either sterile agonadals or asymptomatic carriers.Hz-2V infection of female By injecting virus into female moths, a correlation was established between virus doses administered to the females and the levels of resulting asymptomatic and sterile progeny.The results of these experiments indicate that high virus doses produced a higher level of agonadal progeny and lower doses produced higher levels of asymptomatic carriers. Helicoverpa zea originating at the USDA-ARS in Stoneville, MS [in vitro and in genome structure and size [The insect virus, Hz-2V originally named gonad-specific virus (GSV)  was firsille, MS ,2. Insecand size -5.et al. [et al. [While examining progeny from eggs laid by infected female moths, Hamm et al.  identifi [et al. , were abet al. [et al. [Hamm et al.  presente [et al.  were unaet al. [et al. [Raina et al.  showed t [et al. .In this study we have used the approach of injecting virus into healthy female moths to examine the relationship between virus dose and the level of infected, agonadal and asymptomatic carrier progeny insects hatching from eggs laid on successive ovipostion days. The results presented here demonstrate that virus dose affects both the level of infected progeny and the kind of infection found in insects hatching from eggs laid by virus infected females, indicating a direct correlation between virus dose received by females and the level of infected progeny they produce. Also demonstrated here is the fact that for each virus dose, as the level of agonadal insects hatching from eggs laid on successive oviposition days increase, the level of asymptomatic carrier progeny decreases.5, 2 × 106, 2 × 107, or 2 × 108 TCID50 units of Hz-2V were dissected and the reproductive tissues examined for signs of virus pathology. The PCR products of DNA samples from reproductive tissues of all apparently healthy progeny moths were examined for the presence of Hz-2V DNA via slot blot hybridization , and uninfected progeny moths hatching from eggs laid on each oviposition day was recorded.5 to 2 × 108 TCID50 units (figure 7 and 2 × 108), whereas the lowest percentage (about 60%) was produced by females infected with the lowest doses of virus (2 × 105 and 2 × 106 TCID50).The analysis of these results showed that the percentage of total infected progeny  at all doses tested increased with each successive oviposition day, and the level of infected progeny increased as virus dose increased from 2 × 10s figure . For indVirus infected progeny moths arising from eggs laid on each oviposition day by females infected with different virus doses were divided into agonadal and asymptomatic carriers and these results are presented in figure For all F1 insects hatching from eggs laid on day two, figure  the percIn order to better illustrate the relationship between the two types of infections and to emphasize the effects of virus dose upon the proportions of asymptomatic and agonadal infections, percentages of asymptomatic carriers and agonadal progeny for only the highest and lowest dose are presented in figure et al. [Injecting Hz-2V into female moths results in experimentally infected insects that resemble asymptomatic females and females that have become infected with the virus during copulation, not unlike the females infected during mass-matings by infected males in transmission experiments conducted by Hamm The results presented in figure The percentage of agonadal progeny also increases with each successive oviposition day, approaching 100% agonadal progeny by day three at all virus doses tested, and all progeny moths arising from eggs laid on oviposition day four in all groups were agonadal. Based on the correlation between virus titer and percent agonadal progeny observed in these experiments, the increase in agonadal progeny per oviposition day is likely due to an increase in the titer of virus transmitted to the eggs, suggesting that the titer of virus increases in the parent female moths with each successive oviposition day.in vitro revealed a rapid increase in virus titer by 24 hours post infection in Tn-368 and Ld-652Y cells [in vivo in the epithelial cells of agonadal female oviduct tissue has been described previously by Rallis and Burand [in vitro also occurs in vivo, resulting in a significant daily increase in the titer of Hz-2V in these experimentally infected female moths. Although the precise site of virus replication in these experimentally infected females is not known, the increase in virus titer in these individuals almost certainly results in an increase in virus being transmitted to the progeny with each successive oviposition day and ultimately in the patterns of infection reported here.Studies of Hz-2V replication If, as we have proposed, low virus doses result in asymptomatic carrier moths, and high virus doses produce agonadal progeny, then asymptomatic carrier progeny would likely arise from eggs produced on the earliest oviposition days and decrease with each day, as the virus titer in the egg-laying female moth increases. In fact, the percentage of asymptomatic carrier progeny in these experiments does decrease with each successive oviposition day to 0% by day four. The percent asymptomatic carriers is highest in progeny that receive the lowest virus dose, specifically progeny from oviposition day one and progeny arising from the parent female moths that were experimentally infected with the lowest dose of virus. This is directly opposite of what is observed for agonadal progeny, which is at its highest level at the highest virus dose, specifically on the later oviposition days (days three or greater) and in progeny arising from parent female moths that were infected with the highest virus dose. Interestingly, the lowest percentage of asymptomatic carrier progeny arose from eggs laid by the group of female moths that received the highest virus dose of Hz-2V figure. . These dThe results presented here clearly show that there is a direct correlation between virus dose and the relative percentage of agonadal and asymptomatic progeny. That is, increasing the virus dose causes an increase in the percentage of agonadal progeny, but a decrease in the percentage of asymptomatic progeny. At the present time, it is unknown how the development of an infected individual into an agonadal adult or an asymptomatic carrier is regulated. It is likely that a minimum titer of Hz-2V is needed at a key point in larval development to produce a viral factor(s) within the larval tissues at a threshold level required to reprogram the development and differentiation of the reproductive tissues into the agonadal structures. If this threshold is equaled or exceeded at this point in development, the progeny will exhibit the agonadal condition. However, if this threshold level is not attained, then the reproductive tissues are not reprogrammed and the infected insect becomes an apparently healthy, fertile, asymptomatic carrier of Hz-2V.H. zea has resulted in the ability of the virus to produce two different types of infections in the insects that enable the virus to replicate to high titers in the reprogrammed reproductive tissues in sterile agonadal moths, while maintaining itself in a population in asymptomatic carrier moths. This replication strategy appears to be essential for the continued existence of Hz-2V, since the development of the sterile, agonadal condition in all infected moths would lead to the extinction of the insect host, and the possibly the virus as well. The production of asymptomatic carrier moths ensures that some fertile, infected moths exist that can mate and produce infected progeny, enabling an Hz-2V-infected population to sustain itself, as in the case of the Stoneville colony.The evolution of Hz-2V infection in H. zea were obtained from the USDA-ARS in Stoneville, MS. Insects were reared on artificial diet and maintained as outlined previously [Corn earworm larvae used to start a laboratory colony of healthy eviously .Hz-2V for infecting female moths was prepared as described previously and purified via sucrose gradient centrifugation .5, 2 × 106, 2 × 107, and 2 × 108 TCID50 units.Newly emerged adult female moths were prepared and injected with Hz-2V as outlined by Rallis and Burand . The fem4 Tn368 cells were seeded into each well of a 96-well plate. Between 6 and 13 serial dilutions were made from each virus sample assayed and 10 or 20 wells were plated with 10 ul for each dilution. Plates were incubated at 27°C for 3 to 4 days and examined for the appearance of cytopathic effect (CPE). The numbers of wells with CPE were counted and the TCID50 calculated [Tn368 cells were cultured as per Burand & Lu  and 100 DNA was extracted from the reproductive tissues of adult moths by first homogenizing dissected tissues in 200 ul of TE buffer  followed by a 2-minute incubation in a boiling water bath. The homogenate was then chilled on ice, after which Ribonuclease A (10 ug/ul) was added to each sample, which was then incubated at room temperature for 15 min. The samples were then clarified by centrifugation at 15,600 × g for 2 min.Viral DNA used as template for PCR reactions was extracted from purified virus using 1% SDS in TE containing 1 mg/ml Protease K as outlined by Burand and Lu .® PCR reagent premix kit with 1 unit of Taq DNA polymerase. Each reaction was carried out in10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl2 and 40 mM KCl, containing 250 uM of each of the four dNTP's, with 100 pM of P4-1 forward and P4-2 reverse primers, and 10 ng of purified viral DNA as template. These primer set and reaction conditions were also used to amplify viral DNA sequences in approximately 100 ng of DNA from reproductive tissues of moths thought to be asymptomatic carriers of Hz-2V.Two sets of primers were used to amplify Hz-2V genomic DNA to prepare a probe for use in slot blot analysis of insect reproductive tissues. The first set  was designed to amplify a 434 bp sequence of the Hz-2V genome . PCR rea2), 100 pM of both P4-3 and P4-4 primers, a hexanucleotide mixture containing DIG-labeled dUTP , and 100 pM of Hz-2V genomic DNA. The DIG-labeled PCR product was purified on a 0.8% agarose gel using the Qiagen gel electrophoresis purification kit.The second set of primers  was designed to amplify a 350 bp region directly interior to that of the P4-1 and P4-2 amplified sequence. These primers were used to generate a DIG-labeled probe for Hz-2V to be used in slot blot hybridization assays. PCR reactions for production of the DIG-labeled probe were carried out in a final volume of 50 ul using the Boehringer Manheim DIG High Prime DNA Labeling and Detection Kit, with 1X concentrations of Taq Polymerase buffer , and 50% Formamide). Slot blots were hybridized with 150 ng DIG-labeled Hz-2V probe at 37°C for 12–14 hrs. Following washing, chemiluminescent detection was carried out as recommended by the DIG High Prime Labeling and Detection Kit Manual for DNA Hybridization (Boehringer Mannheim).To prepare the DNA for slot blot analysis, 15 ul of the P4-1 and P4-2 PCR amplified DNA from insect samples was denatured by incubating with NaOH (0.4 M)/ EDTA  at 100°C for 10 min., then applied to a Hybon-N+ membrane prewashed with 500 ul 5X SSC buffer  in a Manifold II slot blotter (Schleicher & Schuell). After applying the DNA, the membrane was baked at 88°C for 2 hrs under vacuum and prehybridized for 6 hrs. at 42°C in 50% formamide prehybridization buffer  N-laurylsarcosine, 1% (w/v) NaIn order to confirm that the PCR products that hybridized to the viral DNA probe contained an amplified DNA fragment of the appropriate size (434 bp), representative samples were analyzed by electrophoresis on 0.8% agarose gels with 0.5X TBE buffer at 100 volts for approximately 1 hr, then stained with EtBr to visualize DNA bands under ultraviolet light.The author(s) declare that they have no competing interests.CPR participated in the design of the study, carried out the work with the insects, coordinated the project and assisted in the molecular analysis and drafting of the manuscript. JPB conceived the study, designed and supervised the experimental work and drafted the manuscript. All authors read and approved the final manuscript.


Climate is a major driving force behind malaria transmission and climate data are often used to account for the spatial, seasonal and interannual variation in malaria transmission.This paper describes a mathematical-biological model of the parasite dynamics, comprising both the weather-dependent within-vector stages and the weather-independent within-host stages.Numerical evaluations of the model in both time and space show that it qualitatively reconstructs the prevalence of infection.A process-based modelling structure has been developed that may be suitable for the simulation of malaria forecasts based on seasonal weather forecasts. The importance of climate as a driving force of malaria transmission has been known since the earliest days of research on this devastating parasitic disease. However, it is only with the advent of effective weather forecasting techniques that this knowledge may be implemented numerically. Seasonal climate forecasting (with up to six months lead time) has developed rapidly in recent years with a number of atmospheric climate modelling groups showing evidence of skill and reliability in their systems. Because of the chaotic nature of the atmosphere, seasonal forecasts are necessarily probabilistic. These probabilistic predictions are derived from multiple integrations of deterministic climate models. These models successfully predicted the onset and demise of the 1997/1998 El Nino event and its impact on weather in Africa  and, con was aimed to advance the concept of seasonal climate forecasts based on multi-model ensembles. The DEMETER coupled models and the DEMETER retrospective forecast (hindcast) integrations are described elsewhere [ is being used to test the accuracy ("skill") of the hindcasts. Central to the DEMETER project is an evaluation of the potential of seasonal climate forecasts for end-user communities, such as those concerned with agricultural output and malaria epidemic control[The DEMETER project lsewhere . The Eurc control. ERA is The aim of the MALSAT group at the School of Tropical Medicine along with the Department of Geography of the University of Liverpool was the assessment of the methodological issues raised by driving a dynamic malaria model with seasonal climate forecasts. In this paper, the first phase is presented, namely the formulation of the model and the development of a dynamic mathematical model of malaria transmission, which can be driven by daily meteorological variables . Additional research assessed the future risk of malaria epidemics in probabilistic terms[Mathematical models of malaria span nearly a century and are well established. Macdonald reformulet al. [Anopheles maculipennis to transmit Plasmodium vivax malaria. Martens [et al. [Climate (as distinct from weather) models of malaria transmission have been developed in recent years to improve our understanding of the likely impact of climate change on malaria transmission. For example, Craig et al.  developeet al.  used a s Martens  used a r Martens  used a s [et al.  analysed [et al.  used a s [et al. .In order to be able to predict within-season and between-year variation in weather-related malaria risk, the model must be driven by varying weather. This paper describes in detail the development of a weather-driven dynamic mathematical malaria model, the final output of which is new infections in the human host. Preliminary results of its numerical evaluation in time and space are presented. The choice of a causal mathematical model rather than a statistical model is based on the knowledge that the former is better suited to extrapolations to novel situations (e.g. when interventions are introduced), and for investigating the non-linear impacts of short-lived changes in driving meteorological determinants. The present malaria model is designed to be used for two distinct but related functions (a) to determine the impact of weather variables on model output  for given interventions and (b) to determine the impact of specific control interventions on model outputs by modifying model parameters.For the former function, the model can be driven with meteorological variables, from ground-based observations, satellite or modelled weather data, seasonal climate forecasts  climate change scenarios. In the second function, the value of a malaria early warning system (MEWS) in terms of triggering earlier or scaling up intervention efforts  in epidemic years may be assessed. In both cases, the biological processes are included as a series of interlinked sub-models and thus represented as coupled delay differential equations. Each new item of knowledge  may be immediately used in the model. New developments such as, for example, the ominous spread of drug resistance may also be immediately incorporated into the model, as, in this example, a reduction in the parasite clearance rate. The implications of all such changes may then be assessed as a quantitative amendment to the prediction. This is done independently for each location and, therefore, the model can be fitted to local conditions, where relevant data are available, or to regional parameters when such data are lacking. Sensitivity analysis can also be used to establish the relative importance of obtaining more accurate data on each parameteret al. [Thus, central to the analysis is the development of a new weather-dependent mathematical dynamic model which no longer attempts to calculate a single constant epidemiological per-case multiplication rate, but follows the temporal progress of the prevalence of infection within a population through seasons and years, To simulate the stochastic elements of the model, delay differential equations based on probabilistic transition between groups rather than on Monte Carlo modelling, as has been undertaken by Gu et al. , have be. These three- to six-hourly data are stored on the ECMWF site and extracted by local software at the site by the user. Weather variables were extracted from the database as gridded data at one degree (~111 km.) and twelve-hour resolution for the African region. Ideally, daily averaged temperature, accumulated rainfall and humidity would have been the variables. However, reanalysis-based daily average temperature correlated poorly with station data , and daily minimum temperature proved even worse, and humidity too was poorly modelled. Thus, the analysis used surface (2 m above ground level) daily maximum temperature, offset by -5°C (to roughly represent mean temperature) and total rainfall estimates as input variables. As accumulated rainfall (puddles etc.) is more important than daily rainfall, Rd , the sum of the previous 10 days of rainfall., were used. There are four malaria parasites (Plasmodium spp.), which cause disease in humans. The focus here is on Plasmodium falciparum, as it is the principal life-threatening parasite species and is most common in Africa. The temporal resolution of the model was based on the nocturnal activity of the vector and the fact that empirical vector observations are usually made at no better than daily time-resolution. Therefore, the simulation time-step is a single day (24 hours).The ERA-40 weather reanalysis data set was chosen because it is the reference data for DEMETER and can provide daily estimates of a range of potentially significant weather variables for the whole globe. This data set was prepared by ECMWF and consists of weather reanalysis data for the whole globe for 40 years (1960–2000) and builds on previous reanalysis data ERA-15 which had been used in an earlier analysis of multi-model ensembles seasonal forecasts It was assumed that the dynamics at the grid-points used do not interact significantly and can be treated as independent, as the distance between grid-points is far greater than the normal mosquito flight distance of roughly 1 to 2 km -23. To sHuman malaria disease is caused in the individual by an infective mosquito biting a non or semi-immune human. After some two weeks the first gametocytes are produced, independent of ambient temperature. A second mosquito biting the infected human thereafter may ingest gametocytes, which after fertilization pass through the gut wall, develop and ultimately produce sporozoites which become infective when they migrate to the mosquito salivary glands. This process is ambient temperature-dependent. As transmission is less dependent on the number of parasites than on the infective status of the carriers, human and arthropod, only the infection and infectiousness status of the carrier populations are simulated (see Table Anopheles gambiae (s.l.) [Plasmodium hosts), and the mosquito's anthropophilic tendency is an important factor in establishing the intensity of transmission. While the anthropophily varies between regions, at this stage it is assumed constant. The male does not bite and, therefore, does not transmit the disease, and as there are always sufficient males to impregnate the mature females, there is no need to simulate the males' dynamics.The most common vector of falciparum malaria in Africa is e (s.l.) ,25. As tThe female life is divided into two major parts: the immature stages , and the mature stage, where onset of maturity is defined as the time of the first flight, which is shortly followed by the first bite. The importance of this division is twofold. First, the immature mosquitoes do not participate in the infection cycle and are, thus, basically in a waiting period, which limits rapid vector population growth. Second, the survivorship (defined as the probability to survive 24 hours) and development rate (part of stage completed in 24 hours) have a different dependence on weather conditions for mature and immature mosquitoes. A schematic representation of the mosquito life cycle is presented in Fig. Hitherto, the immature population dynamics have not been involved explicitly or clearly in malaria modelling. The immature forms are water-bound and are thus totally dependent on the existence of water bodies. High temperatures in breeding sites and evaporation  are generally lethal (see below). Unfortunately, reports giving quantified relationships of temperature and rainfall/humidity dependence of the mosquito dynamics are in short supply, although some data are available . Their fd filling the pool. Thus a mosquito's probability to oviposit, and for the larvae to survive, is proportional to the amount of water it finds. The inclusion of hydrology and soil typemay improve the understanding of the connection between rainfall and breeding-sites. This dependence on rainfall is valid for areas in which the surface water is dependent only on rainfall. However, in constant mosquito habitats such as stable pools or rice-fields, lack of rain may not limit growth of larvae, but natural predators may curb the growth of larval populations. As the movement between the human location  and the ovipostion site is dangerous and energy consuming, this distance is a major factor in determining the probability of oviposition. Future numeric data for this would be of great value.Eggs are posited by mosquitoes in pools. As a mosquito must find water to reproduce, the oviposition rate is roughly assumed to be proportionate to both the ovipositing mosquito number and to the dekadal (ten-daily) rainfall Ret al. [e, larvae L1 and pupae Lp) of An. gambiae s.l. in 11 different breeding habitats , in which the daytime water temperature was measured. Maturation rate, m, is defined as the fraction of the total immature stage covered in a single day, and is the inverse of the sum of the duration of the immature stages[There is a shortage of detailed information on the varied survivorship and stage-dependent developmental progress in natural habitats as functions of weather conditions. There are, however, certain limited sources for derivation of information,28. Jepsre stages,30e+L1 + Lp)     (1).m = 1/ which would allow the introduction of this element.Thus the maturation rate is a function of temperature for these stages. Even though it is known that high temperatures are detrimental to larvae survival, there are no published numerical data . New eggs (box number 1) are laid as a fraction of the number of ovipositing adult females . Thus, at each time step, t, the immature population at stage s, I, has the dynamics:The immature population is, thus, simulated as a set of ν virtual boxes with populations I(n) . At each simulated day the whole population of each box is multiplied by the I = σ I     (2).Maturing pupae (reaching age n and above) are removed and enter the mature mosquito dynamics according to:i.e. all immature mosquitoes within one day of maturation (at stages above ν(1-m)) will become mature mosquitoes the next day, if they survive(s).i.e. blood meals. Although sometimes more than one initial blood meal is required before egg maturation can occur, and fed mosquitoes that are unable to develop mature eggs are best described as pre-gravid[An. maculipennis  and hence also of the time for biting, which may be expressed asMature mosquitoes begin their adult life with their first, nuptial flight, during which fertilization occurs. In the model, a small constant trickle (trickle2) of young uninfected mosquitoes (representing a new imported population) is added to the population of maturing mosquitoes. Afterwards, the development of the fertilized eggs requires the intake of protein, re-gravid, this frre-gravid detectedC = 1+Dd/(T-Tc)     (4);Gd is the number of degree-days required, Tc the threshold beneath which development halts and T is the daily average temperature. Both Dd and Tc are dependent on humidity. In highly humid conditions, Dd = 37 from Detinova's data. In the tropics GC is typically about three days depending on temperature. The temperature dependence of An. gambiae is assumed similar until further data become available.where DR = 1/GC is calculated. This assumes that the temperature dependence of the rate is constant throughout the gonotrophic cycle, which is implicit in the degree-day concept.As temperature is not assumed constant , a daily progress rate (part of gonotrophic cycle covered in one day): PPR values reaches 1. 37 "boxes" (corresponding to the 37 degree days) are constructed, between which the mosquitoes progress in steps of PR reduced by multiplication by the survival rate . At the end of a gonotrophic cycle , each mosquito oviposits and then begins a new cycle. The success of oviposition is dependent on the existence of water-bodies, and hence on dekadal rainfall, and we assume that each ovipositing female lays γRd viable eggs, where γ is a constant. The following day these eggs begin the immature mosquito cycle (as above). It seems that the survivorship of mosquitoes is only weakly dependent on their age[per diem survival is thus calculated by P = αc1/G. As GC is weather-dependent, so is the daily survival. It was assumed that survivorship is independent of the infective state[The completion of a cycle may be established when the sum of daily their age,9,37-39,their age. The statheir age. Estimattheir age-43. The ive state,44, evenm) is the difference between the new mosquitoes maturing (and not dying in the period) and the fraction of the mature mosquitoes dying (the daily cycle completion rate 1/GC multiplied by the death rate 1-α):Combining these, it may be possible to write for δφ, the daily change in the total number of mosquitoes (Ni) rather than non-infectious (Hn) bitten is the human infectious ratioAs mentioned, the parasite within-vector dynamics is superimposed on the mosquito dynamics. Mosquitoes are assumed to bite human hosts randomly (independent of their infective status) and thus the proportion of infectious humans (Hi/(Hi+Hn)     (6).r = HNon-random biting by mosquito vectors is well described and thisAn. gambiae s.s. (even though the tendency varies between strains and regions) and generally much lower for zoophilic Anopheles arabiensis[The preference for human biting over cattle is described by the human blood index , which is high (0.6+) for anthropophilic rabiensis. Of courIP = χ B r     (7)MC)  for P, falciparum lasts 111 degree-days above 18°C[R = 111/SC. The infectivity of a specific mosquito over its lifetime is dependent on the number of bites it makes after the completion of a sporogonic cycle following the first bite of an infective host.It is generally assumed that infected mosquitoes stay so for life. The sporogonic cycle , at stage s of the gonotrophic cycle and at stage Ss of the sporogonic cycle (in sporogonic-cycle degree days) at time t  progresses each day by gonotrophic rate PR and by the sporogonic rate SR:To combine the gonotrophic and sporogonic processes each of the 37 box-stages of the gonotrophic cycle are sub-divided into 112 sub-sections, numbered 0 to 111, representing progress in degree-days. The 0 subsection reflects an uninfected mosquito. The mosquito population is governed by the following dynamics. An infected mosquito sub-population, M = pM     (8).M(s+PA finite fraction (1-p) of the mosquito population which dies and thus does not make the transition. Upon completion of the gonotrophic cycle, the process restarts. Upon the completion of the sporogonic cycle the mosquito remains at the infectious stage. If the mosquito is not infected at biting, it remains uninfected throughout the gonotrophic cycle:R,0,t+1) = pM (cycle without infection);     (9a)M = pMMIP (new infection)     (9b)M = pM(l-MIP).     (9c)M by two processes, either just after maturation or else by completing an uninfected gonotrophic cycle:New eggs are laid by mosquitoes completing a gonotrophic cycle:R from the end of the gonotrophic cycle will oviposit. Their number must be summed over all infection states 0...111. As discussed above, the average brood size is dependent on rainfall by a multiplicative constant γ. In the present report all the initial mosquitoes were non-infected.This means that all mosquitoes located less than PObviously, the focus is on the infected host population dynamics, which reflects the diseased population. The simulation of this population is based on the following assumptions:All hosts and mature mosquitoes are equivalent except for their infection status. Acquired immunity is not accounted for. Thus, the model reflected the prevalence of malaria infection in the population unless the population is largely non-immune in which case it reflects the prevalence of malaria disease. Immune individuals are assumed to be potential carriers, even though not at personal risk. This issue is contended within the modelling community, and future models will allow for both possibilities.The crude human death rate is taken as low enough to be unimportant over the time scale. Specifically, the malaria-induced death rate does not influence transmission patterns.Newly infected patients are not infectious for two weeks, during the intra-hepatic phase of the disease and the early erythrocytic stage, before gametocytaemia rises sufficiently for significant transmission:H = δH (13>h>l)     (12)trickle = 0.01 into a population of 100 every four days). This has the technical benefit of not changing the total population considerably over the simulation period, but limiting the influence of initial conditions found when a large infected population is assumed, or the dying out of the disease after a short initial dry period. This reflects the constant pressure of low-scale transmission by migration (infected migrant workers or troops for instance). This would be quite similar to the case of static communities with low-level external contact [IR, of a human being bitten by an infectious mosquito each day. This rate depends on the abundance of infectious mosquitoes and of human hosts [Malaria clearance is a slow process. Patients may become uninfected at a constant rate (first order process). The rate selected  enables contact . A humanan hosts .There are three components of the human infectious population at time t+1, H: (i) Individuals remaining so from time t, (ii) those who complete the hepatic latent period and (iii) new imports:H = +H)(δ) + trickle     (13a). IR), or else (2) being an infected host )) and clearing his/her infection with probability (1-δ):A host may be uninfected at time t+1, by either (1) remaining uninfected with probability (1-HIR)H+(1-δ)S)     (13b)H = .     (13c)H = H) using Victoria Falls weather station (WMO ID 678430). Weather values for days with missing data  were filled by averaging data from adjacent days.The described modelling process was used to establish the fit of the model to a time series of clinical data from Hwange District, Matabeleland, Zimbabwe . Due to Using the steps described above, the model was run using the ERA-40 weather reanalysis for every grid point covering the African continent  over the time period 1987–2000. To allow a spin-up period for the model weather data for 1987 was run twice, while not storing the daily output for the first run, but allowing the first run's output of host, parasite and mosquito situation at the end of the year to be the initial conditions for the "real" run. This allows the simulation of more climatologically realistic starting conditions. Variations in the length of the spin-up period gave similar results. The average prevalence and incidence for the period is then established. Next, the variation of incidence during the period from interannual means was calculated, and hence the standard deviation of annual incidence. This value serves as an estimate of the extent of anomalous malaria, thus reflecting epidemics, beyond holoendemicity and seasonal variation.et al. [Fig. et al. . The x-am = 0.011 (± 0.001) T-0.2 (± 0.26) (1/day)     (14)Note that the report is for water temperature in shallow pools, which may be significantly higher than the ambient temperature. A few points suggest themselves. To begin with, the intercept with the X-axis is around 18°C. Even though the variation for this value is large, it suggests a lower limit for larval development. Beyond this point, it seems that the assumption in the theoretical methodology of linearity of the development rate with temperature is justified. New data being collected may allow a more thorough validation.m), the per-cycle rain-dependent survivorship is simply Sm1/. The survivorship for the immature stage, by temperature for different values of S, is depicted in Fig. Using the proposed rain-dependent daily survivorship (S) and the length of cycle  for the range of climatic variables predicted by the hindcasts in the DEMETER multi-model system. In addition, this reanalysis-based dataset is used to derive the initial conditions of the hindcasts and verification dataset for DEMETER hindcasts. Thus an assessment of the value of the biological malaria model driven by ERA weather data represents the potential value of a "perfect" forecast of climatic conditions to the prediction of malaria epidemics. The assessment of this ability of the weather-driven model to describe interannual variability in malaria infection rates given a perfect forecast, is the basis for a realistic assessment of the benefit associated with the use of operational  seasonal climate forecasts in the context of a malaria early warning system.i.e. chaos. Chaotic behaviour may, however, be limited by the application of saturation limits to certain model parameters (such as a self-limiting proliferation rate). In addition, the knowledge of the equations themselves is often limited, being typically a linearization of behaviour of empirical data. Thus, there is considerable uncertainty in the result. The dynamic, deterministic method may, however, be used numerically, experimentally, by using small steps so as to validate the stability of the equations. For this reason, the improvement of the understanding of both the ability of weather forecasts to predict weather and of biological models to predict disease, presents a path to the understanding of probabilistic solutions to non-linear epidemic prediction problems.The dynamic approach claims that total knowledge of the initial state and of the equations, as well as of the external driving forces, allows total knowledge of all future states. However, in real situations, this precise knowledge is often lacking. In many cases, bifurcations of the motion in phase-space due to minute perturbations of initial conditions, may result in large differences in the result, The capability of seasonal climate forecasts to predict anomalous seasonal climate conditions has improved considerably over the last decade. In particular, the El Niño/La Niña cycle has been correlated with extreme weather conditions throughout the globe. This cyAn. gambiae s.l. In some regions, the existence of permanent waterbodies, such as slow-flowing rivers, lakes or swamps may provide suitable breeding sites, and thus make up for the shortage in rainfall, as far as the larvae are concerned. Even though the mature mosquito survival will be considerably reduced by the low humidity[This stage is, however, limited to reconstructions. The good fit in Fig.  humidity, malaria humidity. These efalciparum malaria, unless the temperature remains in the high 20s (°C) for considerable periods. For this reason falciparum malaria is associated heavily with tropical regions, while in pre-eradication malarious Europe P.. vivax was the dominant malaria parasite species.The requirement for high temperatures for malarial transmission is further illustrated by the plots in Figs. Further improvements should be added to the model. These may be achieved as new relevant numerical information becomes available on the biological processes, which were here handled somewhat heuristically. For example, the relationship between rainfall and larvae survivorship was simplified due to the lack of data. Laboratory and field, meteorological and entomological data may establish its true form.The relative importance of the various parameters, assessed by sensitivity analysis by variation of individual parameters is of great interest. This will allow field scientists to focus their efforts in establishing the values of the most critical parameters. In a parallel paper this has been presented. However, a full multivariate evaluation is still underway, using novel parallel computing methods.The model currently ignores both antiparasitic immunity (immunity to infection) and antitoxic immunity (immunity to disease) – the sharp distinction between which may not exist in reality,55. NeitHowever, where the model may be used to predict the impact of an intervention amongst a semi-immune or immune population then both forms of immunity may be significant factors in determining the transmission dynamics of malaria infection. Thus, iNumerical evaluations of the model in both time and space show that it has a good first order approximation to the prevalence of infection across the continent. It captures well both the seasonality and interannual variability of infection at the test site in Zimbabwe. Note that there is a large level of under-reporting of clinical cases  and also over-diagnosis of malaria, which often confound correlations  ,58.The model, when run with the following inputs Table , is ableStronger variation, both between seasons and interannual, may be expected. Thus malaria transmission will continue further north than suggested, but will also be less stable.2) means that the model can only represent the most marked changes in transmission potential over large geographic regions – appropriate to seasonal climate forecasts. This is expressed in the poor spatial resolution as regards Madagascar, in which the spatial variation of prevalence is not represented.There are other points to consider. The most obvious discrepancy between our results and those of previous workers is that of high prevalence rates being predicted for Somalia – an area where malaria transmission in normally low. The results are created from a relatively short time series and may be disproportionately influenced by the anomalously high rainfall recorded in 1997/1998 in this area, and in East Africa in general, which corresponded with unusually high rates of transmission. The malDespite the limitations, which may be overcome in specific regions by using daily weather data at a finer spatial resolution formed by downscaling , the model is capable of describing in general terms the spatial, seasonal and inter-annual variability of malaria transmission in Africa.max-5 as a surrogate for daily temperature was an ad hoc attempt to create a single value for a wide spectrum varying in time and place. The diurnal variation is, for example, usually far greater in dry regions than in humid conditions. Our attempt was to use a single value which bears relation to something measurable, namelyTmax. In fact, the two-metre model temperature value, was has been used may not have been the ideal, as larval development is closer to ground, but that would have required detailed soil data, which was beyond the scope of this research. The usage of reanalysis data is obviously inferior to high quality station data. Convective precipitation is highly local and is not well correlated to the averaging required by meteorological reanalysis. For example, the heavy rains in north-western Zimbabwe (end 1995) were under-expressed in ERA-40, partly due to the scale of the reanalysis data, and thus the local malaria incidence using that data is too low. The integration of the data in a reanalysis model in itself makes numerous physical assumptions. In some cases, the result is poor quality of even some large-scale processes, such as the El-Niño of 1997 in East Africa.The usage of T will allow development of coupled malaria and meteorological data sets, which will be more effective for future analysis. Development of seasonal climate forecasting tools over large geographic areas, however, will remain for some time partly dependent on verification by reanalysis. Thus, malaria epidemic seasonal early warning will also be linked to this imperfect data source, though perhaps improved by novel downscaling methods.High quality station data at the pan-African level is, however, not available. The stations are irregularly dispersed, and not all produce complete data sets, having many missing days. As the model requires daily values, interpolation over large areas with varying orographic characteristics is required with the inherent uncertainty this brings. In addition, the relation between mosquito habitat microclimate and station data too is unknown. High temporal and spatial resolution of weather data will improve the modelling attempts. Among other issues, the continuous measurement of weather data at malaria epidemiological and entomological research and surveillance sites now introduced This paper presents a first step in the preparation of a weather-driven dynamical model of malaria transmission, for use with both observed weather data and seasonal climate forecasts. The model incorporates the stages of the malaria vector and their dependence on temperature and rainfall, and part of the within-host parasite population dynamics. Some of these elements lack concrete theoretical and empirical development, requiring further input. Further work, under work at present, will enable the employment of such a model in the prediction of outbreaks based on skilful weather forecasts.MBH formulated the mathematical model, prepared the code and ran the program. APM lead the applications model work package in DEMETER.. Both authors read and approved the final manuscript.


Data regarding the impact of cigarette smoking on trace elements are scarce and inconsistent. In this study, we evaluated the effect of smoking on serum concentrations of trace elements among adult males with heart disease.This cross-sectional study included 100 adults hospitalized with heart disease in Bangladesh. The major variables of interest included mean serum concentrations of trace elements and proportion of subjects with bacterial growth on throat swab culture.Streptococcus β-hemolyticus in smokers than controls.Smokers had significantly lower serum concentrations of retinol, alpha-tocopherol, selenium, and zinc and increased concentrations of copper. Throat swab cultures were more often positive for Smoking decreases serum concentrations of trace elements. Smoking control programs are needed in Bangladesh to improve health and nutrition of the people who are already nutritionally deficient. Smoking is a widely accepted practice in Bangladeshi men and is associated with socialising, sharing, and male identity . Accordin = 20), non-smokers; Grade I (n = 20), 10 – 15 sticks/day; Grade II (n = 30), 16 – 20 sticks/day; Grade III (n = 30), 21 and more sticks/day.A cross-sectional study was conducted among 100 male patients admitted to the National Institute of Cardiovascular Disease (NICVD), Dhaka, Bangladesh from January through December 1998, after obtaining informed consent from the participants. The study protocol was reviewed and approved by the Human Subjects Ethical Committee of the NICVD. The patients who had a history of smoking 10 or more cigarettes per day were considered smokers, and those who never smoked were controls. All the patients, including controls, were admitted with heart disease. The study did not include females in this study because smoking is not a norm among females in that society. All the smokers and the first 20 of the non-smokers who met the selection criteria were eligible for the study. This study included only heavy smokers who smoked at least 10 sticks per day, and excluded mild or casual smokers to leave a buffer zone of comparison between smokers and non-smokers. Patients were stratified according to their smoking status as follows: Control  was measured to the nearest 0.1 kg, with participants wearing light clothing and no shoes and using a beam balance with non-detachable weights. Height (cm) was measured with a stadiometer to the nearest 0.5 cm. Body mass index (BMI) was calculated using the following formula: weight (kg) / height (m)18 column using methanol-water  as the mobile phase. Coefficient of variation (CV) values of ten replicates from a pooled serum sample for retinol and α-tocopherol were 2.3 and 3.3% respectively.Serum retinol (vitamin A) and serum α-tocopherol (vitamin E) concentrations were determined by high performance liquid chromatography (HPLC) according to Bieri et al. . In brieSerum zinc was measured by flame atomic absorption spectrophotometry  using a modification of the method described by Kirgbright [Data were analyzed using SPSS for windows, version 11.0 . Descriptive statistics of the major variables of interest were calculated to determine the distribution of the data. All the variables except serum iron concentrations were normally distributed. Variables between smokers and non-smokers were compared by Student t-test for continuous data and by Chi-square test for categorical data. A probability level of 5% was considered statistically significant.Of the 110 patients enrolled, 10 dropped out; eight had incomplete data, one withdrew early, and one left the hospital without notice. The mean ± SD age of the study patients was 43.67 ± 6.79 y . The groups did not differ significantly in terms of age and anthropometric measurements  among all smokers and the majority (60%) of the controls. Table Streptococcus β-hemolyticus , catalase, and glutathione peroxidase (GSH-Px) were measured in tobacco smokers and compared with those of nonsmokers . Plasma Several studies documented that smoking may increase oxidative stress and impair oxidant defense system . Serum sIn a later study, Kim et al. (2004) further evaluated the influence of short– and long-term cigarette smoking on blood antioxidant status among Korean teenage girls (aged 14 to 18 y) and adult males (aged 36 to 51 y) . ExtraceIncreasing evidence suggests that smoking is a causal factor for coronary heart disease and stroke. In a prospective study in Japan , 19,782 Streptococcus β-hemolyticus among our study subjects, because it is a cross-sectional study. However, epidemiologic evidence has suggested a modifying role for antioxidant micronutrients, including tocopherols and carotenoids, in atherosclerosis and heart disease. In an experimental study, Handelman et al. (1996) exposed freshly obtained human plasma to the gas phase of cigarette smoke to assess its effects on tocopherols, carotenoids, and retinol. Exposure to cigarette smoke led to the depletion of most of the lipophilic antioxidants in human plasma [One of the limitations of our study is that it is difficult to establish any causal association of heart disease and deficiency of trace elements or increased isolation of n plasma .In addition to the impact on health, tobacco smoking represents a major economic burden for impoverished Bangladeshis. Average male cigarette smokers spend more than twice as much on cigarettes as per capita expenditure on clothing, housing, health and education combined. The typical poor smoker could easily add over 500 calories to the diet of one or two children with the daily tobacco expenditure . It may This study demonstrated that increasing amount of cigarette smoking negatively impact serum concentrations of retinol, alpha-tocopherol, selenium, and zinc. Cigarette smoking may act as an important adjunct to the deficiency of those trace elements in a population who are already nutritionally compromised.The authors declare that they have no competing interests.SKB participated in the design of the study and collected the samples. AKM performed the statistical analysis and drafted the manuscript.


Plasmodium falciparum, represents a major public health problem in the developing world. The P. falciparum genome has been sequenced, which provides new opportunities for the identification of novel drug targets. Eukaryotic protein kinases (ePKs) form a large family of enzymes with crucial roles in most cellular processes; hence malarial ePKS represent potential drug targets. We report an exhaustive analysis of the P. falciparum genomic database (PlasmoDB) aimed at identifying and classifying all ePKs in this organism.Malaria, caused by the parasitic protist P. falciparum and just one member in some other Apicomplexan species.Using a variety of bioinformatics tools, we identified 65 malarial ePK sequences and constructed a phylogenetic tree to position these sequences relative to the seven established ePK groups. Predominant features of the tree were: (i) that several malarial sequences did not cluster within any of the known ePK groups; (ii) that the CMGC group, whose members are usually involved in the control of cell proliferation, had the highest number of malarial ePKs; and (iii) that no malarial ePK clustered with the tyrosine kinase (TyrK) or STE groups, pointing to the absence of three-component MAPK modules in the parasite. A novel family of 20 ePK-related sequences was identified and called FIKK, on the basis of a conserved amino acid motif. The FIKK family seems restricted to Apicomplexa, with 20 members in The considerable phylogenetic distance between Apicomplexa and other Eukaryotes is reflected by profound divergences between the kinome of malaria parasites and that of yeast or mammalian cells. Modulation of protein phosphorylation through the antagonistic effects of protein kinases and protein phosphatases is a major regulatory mechanism of most cellular processes. Dysregulation of protein phosphorylation in human cells plays a major role in many diseases such as cancers and neurodegenerative disorders . This haPlasmodium falciparum, the species responsible for the lethal form of human malaria, begins with the bite of an infected Anopheles mosquito, which delivers sporozoites into the bloodstream. These cells establish an infection inside hepatocytes, where they undergo an intense multiplication generating several thousand merozoites, a process called exo-erythrocytic schizogony. The merozoites invade erythrocytes, where they also undergo schizogony, the process that is responsible for malaria pathogenesis. Some merozoites, however, arrest the cell cycle and differentiate into male or female gametocytes, which are infective to the mosquito. Once ingested by the insect, the gametocytes develop into gametes  and fuse into a zygote. Further development in the mosquito involves a process of sporogony, producing sporozoites that accumulate in the salivary glands and are now ready to infect a new human host .Malaria parasites have a complex life cycle. Infection of human beings by Plasmodium falciparum  , clearlyP. falciparum genome (see below). Two additional sequences [PFC0385c and PF11_0464] form a separate cluster attached to the base of the AGC branch. There appears to be no clear member of the PKC subfamily.Five malarial kinases cluster within this group, three of which have been characterized: the cAMP-dependent PfPKA [PFI1685w] , the cGMThe main branch of the tree that contains the human CamKs also contains 13 PfePKs, which underlines the importance of calcium signalling in the parasite . A tightPlasmodium kinome. Interestingly, in other eukaryotic systems a majority of CMGC kinases are involved in the control of cell proliferation and development, and their relative abundance in the P. falciparum kinome may reflect the variety of successive proliferative and non-proliferative stages which constitute the life cycle of malaria parasites. Six enzymes are related to the cyclin-dependent kinase family, 5 of which were identified previously , the laiewed in -33 and Piewed in , clusteriewed in  and Pfcriewed in ,37, formiewed in . The comFive malarial enzymes appear in the vicinity of the TyrK-like group, including two [MAL6P1.191 and PFB0520w] that display maximal homology to MAPKKK-related or MLK (mixed-lineage kinases) enzymes upon BLASTP analysis. PFB0520w clusters with the TGFβ receptor (TGFβ1). The malarial sequence is much more similar to TGFβ receptors than to mammalian Raf, and furthermore, in common with TGFβ receptors, the malarial enzyme has a predicted transmembrane sequence N-terminal to the kinase domain. Mammalian TGFβ receptors assemble as heterodimers, and it remains to be seen whether the malarial enzyme forms a homodimer or has the capacity to coassemble with a mammalian subunit.in vitro and in silico attempts at identifying MAPKK malarial homologues , is able to specifically phosphorylate Pfmap-2  in vitro, and to act in synergy with Pfmap-2 towards the phosphorylation of exogenous substrates , an enzyme previously described as related to the SNF1 family , with twL7P1.91) , which fSo far, four PfePKs have been described as appearing as "composite" enzymes displaying features from more than one established ePK family. As mentioned above, PfPK6 [PF130206] and Pfcrk-4 [PFC0755c] both display relatedness to CDKs and MAPKs, and this is confirmed by their position on the tree. The MAPKK-like activation site of Pfnek-1 [PFL1370w] has been discussed above. The fourth example is that of PfPK7 [PFB0605w], an enzyme whose C-terminal region carries a sequence which is conserved in MAPKKs but whose N-terminal region is more closely related to that of PKAs . This seP. falciparum genome, we carried out a more thorough analysis in which the 18 malarial kinases belonging to this group were compared with comprehensive sets of related kinases from the yeast and human genomes  previously reported as belonging to the ERK family clustered, as expected, with the MAP kinases. However, in contrast to previous suggestions brought forward before the full complement of mammalian ERKs had been characterised  appears orthologous to yeast cdc28 and to human CDK1-3. PfPK5 displays similar levels (60% identity) of overall homology to both mammalian CDK1 and CDK5; however, in our analysis this enzyme clearly clusters with the former, lending support to the idea that this enzyme is a functional homologue of the major cell cycle control kinases, as previously suggested   and PfcrSchizosaccharomyces pombe, but not Saccharomyces cerevisiae) [P. falciparum kinases (PFC0105w and PFl4_0408) cluster with SPRK. Both these kinases have an insertion between domains VIb and VII that is a distinctive feature of SRPKs. Previously described PfLAMMER [PF14_0431]   associat14_0431]  and the 14_0431] .P. falciparum kinome. As detected on the general tree  is half the size of the P. falciparum genome (24.8 megabases), encodes approximately twice as many enzymes of this family.That only 65 typical ePKs were identified in this search is somewhat surprising, as P. falciparum genome showed that they share most of the residues that are conserved in the ePK catalytic domain. Indeed, with the exception of the Glycine triad in subdomain I, all residues which are crucial for phosphotransfer or structural stability of protein kinases, and therefore well conserved throughout the family, are present in all members of this family   that exMalaria parasites possess two organelles with extra-chromosomal DNA: the apicoplast and the mitochondrion. The apicoplast is a four-membrane organelle carrying a circular 35 kb DNA whose structure is very similar to that of plastid genomes. It is specific to the Apicomplexa (hence its name), and thought to originate from secondary endosymbiosis . As is tProteins devoid of kinase activity but which are known to associate with, and regulate the activity of, ePKs have been identified in PlasmoDB. These include four previously characterised cyclins  which have been demonstrated to associate with histone H1 kinase activities in parasite extracts ,55, a PKHomo sapiens as queries. GeneDB was also used to look for relevant Pfam domains . Two members of the RIO kinase family were found: PFL1490w (RIOK1-like) and PFD0975w (RIOK2-like). Enzymes of this family are involved in rRNA processing in S. cerevisiae , in agreement with experimental studies  and the P. falciparum transcriptome during development are available on the PlasmoDB database. We compiled these data to obtain a general picture of PfePK gene expression during erythrocytic development . A full alignment of the kinase domains is shown in P. falciparum sequences. Only gap-free conserved regions of the alignment were used for the construction of the tree (164 amino acid positions). Bootstrap values supporting the branches of the tree are rather low; this is to be expected given the diversity of the protein kinase family.The set of predicted peptides of the nome 3D7  was downnome 3D7 . A Hiddenome 3D7  of the pnome 3D7 . In addinome 3D7  of 'prot Artemis . Eight pin silico and in vitro analyses of the FIKK family. JP generated the HMM-derived alignments, constructed the ePK phylogenetic trees and contributed significantly to their description in the text. CD coordinated the study and wrote the larger part of the manuscript. All authors read and approved the manuscript.PW performed most of the database searches for ePK-related sequences and constructed the FIKK phylogenetic tree; LE contributed to the searches for aPKs, compiled expression data and performed the P. falciparum ePKs used for constructing the tree in Fig. 2. Please see the Methods section for details on how the alignment was generated.Alignment of the 65 "typical" Click here for filepartial sequence of the cDNA for the gene PF14_0733/PF14_0734.Click here for fileAlignement of the 20 FIKK sequences used to construct the tree in Fig. 6.Click here for file