Pseudomonas lung infection on alveolar function.While the functional consequences of acute pulmonary infections are widely documented, few studies focused on chronic pneumonia. We evaluated the consequences of chronic P. aeruginosa, included in agar beads, was instilled intratracheally in Sprague Dawley rats. Analysis was performed from day 2 to 21, a control group received only sterile agar beads. Alveolar-capillary barrier permeability, lung liquid clearance (LLC) and distal alveolar fluid clearance (DAFC) were measured using a vascular (131I-Albumin) and an alveolar tracer (125I-Albumin).th day. Stimulation with terbutaline failed to increase DAFC. Eradication of the pathogen with ceftazidime did not restore DAFC response.The increase in permeability and LLC peaked on the second day, to return to baseline on the fifth. DAFC increased independently of TNF-α or endogenous catecholamine production. Despite the persistence of the pathogen within the alveoli, DAFC returned to baseline on the 5th day and remains unresponsive to stimulation. This impairment of DAFC may partly explain the higher susceptibility of chronically infected patients to subsequent lung injury.From these results, we observe an adequate initial alveolar response to increased permeability with an increase of DAFC. However, DAFC increase does not persist after the 5 Pseudomonas aeruginosa is a Gram negative bacteria producing a wide array of virulence factors frequently responsible for chronic airway infections in cystic fibrosis (CF) or chronic obstructive pneumonia disease (COPD) patients, as well as acute nosocomial airway infections in intensive care units [re units -3.P. aeruginosa pneumonia, the functional consequences, and particularly lung fluid movements, have been studied extensively. Lung fluid balance is the result of fluid movements following active ion transport by functional alveolar cells, and permeability of the alveolar capillary barrier. In P. aeruginosa-induced acute lung injury (ALI), distal airspace fluid clearance (DAFC) is typically increased at 24 hours through a TNF-α pathway [In acute  pathway ,6. Lung  pathway . We prev pathway  reflectiP. aeruginosa embedded in agar beads [P. aeruginosa chronic lung infection. In order to elucidate these functional aspects we studied lung fluid transport in an experimental model of chronic P. aeruginosa lung infection in the rat. After the validation of the experimental model, we studied alveolar function: alveolar-capillary barrier permeability, lung liquid clearance, distal airspace fluid clearance and its pharmacologic stimulation.In chronic infection, these functional consequences on lung fluid balance are less clear. In the 70's, Cash developed an experimental model of chronic pneumonia by intra tracheal injection of ar beads . Most ofar beads -12. To tad lib. All experiments were performed with approval of the Lille Institutional Animal Care and Use Committee.Specific pathogen-free Sprague Dawley rats (n = 280) (230–270 g), (Depre, St Doulchard, France) were housed in the Lille University Animal Care Facility and allowed food and water P. aeruginosa (PAO1 strain) was incubated in 125 ml of tryptic soy broth at 37°C in a rotating shaking water bath for 8 hours. The culture was then washed twice, and resuspended in phosphate-buffered saline. The resulting bacterial suspension was 1 × 109 CFU/ml. A sample of 1 mL of this suspension was mixed in agarose and mineral oil (Sigma Diagnoses, St Louis, USA) at 56°C. The resulting oil-agar emulsion was cooled to obtain agar beads. Dilutions of the final suspension were cultured to determine the size of the final inoculum.The methodology was adapted from Cash et al . BrieflyUnder a short general anesthesia with ether , with sterile surgical conditions, a small midline incision was made on the neck ventral surface after swabbing it with ethanol. The trachea was exposed by blunt dissection. Using a 28-gauge needle, 0.1 mL of agar beads followed by 0.5 mL of air were inoculated intra-tracheally.After exsanguination of the animal, the lungs were isolated and homogenized in 2 mL of sterile isotonic saline. Bacterial culture after serial dilutions was performed and bacterial colonies counted after 12 h at 37°C.In a subgroup of animals, ceftazidime (GlaxoSmithKline, Marly-le-Roi, France), 100 mg/kg, was administered in the peritoneal cavity every 8 hours during 72 hours. Lungs were harvested, homogenized and cultures were performed to confirm bacterial eradication. Serum ceftazidime levels were measured in HPLC.Broncho-alveolar lavage (BAL) was performed by cannulating the trachea. Lungs from each experimental group were lavaged with a total of 20 ml in 5-ml aliquots of PBS with EDTA (3 mM). BAL fluid samples were filtered and immediately frozen at -80°C. A cell count was performed directly. Cellular monolayers were prepared with a cytocentrifuge and stained with Wright-Giemsa stain. Cellular morphotype differential was obtained by counting 200 cells/sample and expressing each type of cell as a percentage of the total number counted. Protein concentration in the BAL was measured with an automated analyzer (Hitachi 917, Japan).After a vascular flushing with sterile isotonic saline through the pulmonary artery, the lungs were removed. Samples were fixed by intratracheal instillation of paraformaldehyde 10 %. Samples were included in paraffin and sections of 5 μm were realized. Analysis was performed after coloration with Hematoxyline-Eosine-Safran (Zeiss, LEO 906).Levels of tumor necrosis factor α (TNF-α), in the serum, and the BAL fluid, were determined by use of commercial immunoassay kits (ELISA) specific for rat cytokines (Quantikine Murine rat TNFα, R&D Systems, Abingdon OX, UK). The reading was performed with a microplate reader Digiscan .Blood and broncho-alveolar lavage fluid were collected on heparin/Na-metabisulfite coated tubes. The samples were centrifuged (2500 g, 4°C), supernatants were frozen (-80°C).Catecholamines are specifically fixed on alumina (pH = 8.7), the eluent is analyzed with an inversed phase H.P.L.C (Coulochem II ESA). The results are expressed in μg/L.2 fraction of 1.0, a peak airway pressure of 8–12 cmH2O, and a positive end expiratory pressure of 2 cmH2O. The animals were placed in left decubitus position until the end of the protocol. The body temperature was maintained at 37°C.Sprague-Dawley male rats were anesthetized with pentobarbital (Sanofi, Libourne, France). A catheter (PE-50) was inserted into the left carotid artery in order to monitor systemic arterial pressure (Acqknowledge Software v 3.7.1, Biopac systems, Santa Barbara, CA, USA) and obtain blood samples. An endotracheal tube (PE-220) was inserted through a tracheostomy. The rats were ventilated with a constant volume pump (Harvard Apparatus, South Natick, MA) with an inspired O-4 M)  was added to the instillate or injected intra-peritoneally to the animals.The test solution, used for alveolar instillation, was prepared as follows : briefly, a 5% bovine albumin solution was prepared using Ringer lactate and was adjusted with NaCl to be isoosmolar with the rat circulating plasma ,14. A sa131I-labeled human albumin, was injected into the bloodstream [131I-HSA was prepared in our institution according to a standardized technique. Administration of the instillate (3 ml/kg) was performed into the left lung over a 2-min period, using a 1-ml syringe and polypropylene tube (PE 50, Intramedic, Becton Dickinson, Sparks, MD, USA)[For all ventilated rats experiments, the following general protocol was used. After the surgical preparation, heart rate and blood pressure were allowed to stabilize for 1 hour. To calculate the flux of plasma protein into the lung interstitium, a vascular tracer, 1 μCi of odstream ,15. 131IOne hour after the beginning of the alveolar instillation, the rat was exanguinated. The lungs were removed, and fluid from the distal airspaces was obtained (aspirate). The total protein concentration and the radioactivity of the liquid sampled were measured. Right and left lungs were homogenized separately for water to dry weight ratio measurements and radioactivity counts.• Hemodynamics, pulmonary gas exchange, and protein concentration2 was used to quantify the oxygenation deficit [Systemic arterial pressure and airway pressures were measured continuously. Arterial blood gases were measured at one hour intervals. The arterial PO deficit ,14. Samp• Albumin flux across endothelial and epithelial barriers131I-albumin, in the alveolar and extravascular spaces of the lungs. Endothelial permeability was assessed by measuring the ratio of 131-iodine radioactivity in the aspirate to the radioactivity obtained in the plasma (Asp/plasma), it reflects the leak of the vascular tracer in the alveolar compartment. We estimated the quantity of plasma that entered the instilled lungs by measuring the transfer of the vascular protein tracer, 131I-albumin, into the extravascular spaces of the instilled lung using the equation of plasma equivalents previously described [The flux of albumin across the lung endothelial and epithelial barriers was used to evaluate the permeability. This method requires measurement of the vascular protein tracer, escribed ,13,14.• Extravascular lung water (EVLW) and lung liquid clearance (LLC)The EVLW was estimated by gravimetry: 300 μL of the lung homogenate were weighed, to determine the wet weight, and dessicated at 45°C during 7 days, to obtain the dry weight. The blood fraction was calculated from the homogenate hemoglobin supernatant content. The wet to dry weight ratio (W/D) was estimated using the values of the right lung which was not instilled ,16. Lung• Distal Airspace Fluid Clearance (DAFC):A change of native bovine albumin concentration over the study period (1 h) was used to measure alveolar fluid movement. DAFC was calculated from the ratio of the final unlabeled alveolar protein concentration, compared to the initial instilled alveolar protein concentration.15 experimental groups were constituted for the study:- A control group (Ctr), which received an intratracheal instillation of sterile saline at the beginning of the protocol- 7 Sterile groups (St) received an intratracheal instillation of sterile beads and were studied at different days after inoculation: St 1, St 2, St 5, St 8, St 15, St 21 and St 28.Pseudomonas containing beads and were studied at different days after inoculation: Pn 1, Pn 2, Pn 5, Pn 8, Pn 15, Pn 21, Pn 28.- 7 Pneumonic groups (Pn) received an intratracheal instillation of t-test. Comparisons between more than two groups were made using a one way analysis of variance with post hoc test for multiple comparisons. A value of p < 0.05 was considered as significant. The data are expressed as means ± SD.Comparisons between two groups were made using an unpaired, two tailed Student's P. aeruginosa beads infected animals compared to the sterile beads groups  and the 3rd week (1.67 ± 1.63 105 CFU/mL).Prior to the instillation, the size of the inoculum was 7.9 10nd day mostly polymorphonuclear's neutrophils (PMN). From the 8th day, the relative number of PMN progressively decreased as alveolar macrophages increased. All the results are summarized in Table Total broncho-alveolar lavage (BAL) cells slightly increased in the sterile beads group, the difference was however not statistically significant compared to the control group, the analysis showed that the number of cells peaked on the second day and was constituted, at that time, of 25% polymorphonuclear cells and 75% macrophages. The results were not statistically different over time and therefore pooled in Table nd day, large numbers of PMNs were observed, mostly centered on the alveoli .No variation in permeability or clearance was observed between St groups, so all the results were included in a single group (St) for the analysis . Alveolar-capillary barrier permeability, evaluated by the leakage of the vascular marker into the alveoli (Asp/plasma ratio), was increased in infected animals on the second day compared to the control group (0.59 ± 0.08 vs 0.11 ± 0.02). This ratio came back to control values from the fifth to the 28• Extra-vascular lung water and Lung liquid clearance (LLC)th day. A moderate but not statistically significant increase was observed in the Pn15 group (p = 0.13).As shown in Table • Distal alveolar fluid clearanceth day and remained comparable to both the St and the Ctr groups.Distal alveolar fluid clearance increased in the Pn2 group Figure . This rand and 21st days in the serum or the alveolar compartment. Similarly, neither epinephrine nor nor-epinephrine could be detected in the alveolar compartment at 48 hours. The levels recovered in the plasma were comparable between control and pneumonic animals on the 2nd and the 5th days . This result is consistent with the literature, Nacucchio et al showed that agar beads alone could not reproduce the same level of injury than P. aeruginosa in agar beads [Since, in this model, ar beads .P. aeruginosa is adequate, based on clinical, bacteriological, cytological and histological data.From this first part of our work, we concluded that the model of chronic infection with P. aeruginosa [nd day post infection. We observed an increase of alveolar-capillary barrier permeability, DAFC and overall lung liquid clearance. A normal lung wet to dry weight ratio was a consequence of this adequate alveolar response. This contrasts sharply with the data we obtained in an acute lung injury model where LLC dramatically decreased and W/D weight ratio increased at 4 and 24 hours after Pseudomonas instillation [Although a clinical study has reported increased lung permeability in COPD patients infected by ruginosa , few stuillation .th day.In our chronic model, following the increase in both permeability and lung liquid clearance, we observed an improvement in permeability with a return to baseline of these 2 parameters on the 5The St group presented a moderate increase in permeability (Asp/plasma ratio: 0.31 ± 0.04), it has previously been reported that agar beads could alone be responsible for a moderate increase in permeability . HoweverP. aeruginosa pneumonia, increased DAFC can be related to either Pseudomonas exoproducts [nd or the 5th day. TNF-α is produced during the early phase of pneumonia, and its short half life probably explains the absence of detectable levels at 48 hours. A dynamic evaluation of TNF-α production with serial samples or antibody neutralization experiments would be helpful to precisely study the role of TNF-α. We therefore did not rule out that TNF-α may have triggered an inflammatory response which could be responsible for the increased DAFC. Other potential mechanisms such as Transforming Growth Factor β remain to be investigated [Our results showed an increase of the DAFC at 48 hours post infection. In acute lung injury, the initial alveolar response is usually towards an increase of DAFC which many authors have documented in septic shock , or afteproducts  or to a stigated .th day post infection in response to known pharmacological stimuli [th day, terbutaline intratrachéal administration did not change DAFC. However the lack of effect may be due to airway inflammation and an inability to adequately deliver the drug, we therefore decided to use intraperitoneal administration with the same agent. Our results also show the absence of DAFC increase. We then hypothesized that the absence of response to the stimulation might be related to the persistence of the pathogen in the alveoli. To test this hypothesis, we injected the animals with ceftazidime to sterilize the lungs on the 5th day. Sterilization was achieved but failed to restore DAFC stimulation with terbutaline. To explain this impairment of DAFC, different hypotheses still remain to be investigated concerning these agonist's receptors and their regulation. Other authors have shown in different situations that either an internalization or a decrease of affinity of the receptors [Surprisingly, on the fifth day, DAFC returned to baseline along with the improvement in permeability. Although it is logical to see an improvement in permeability, consistent with a decrease of the bacterial burden and an adequate host response, DAFC was expected to remain increased. The persisting presence of the pathogen in the alveoli and many factors only related to its presence would normally lead to a persistent increase of DAFC . We ther stimuli ,25. In t stimuli . We obseeceptors  could beeceptors .If this unresponsiveness exists in patients, the absence of an adapted DAFC response in chronic lung infection could lead to major damage in the presence of any new lung injury. Although chronic lung infection has not been isolated, per se, as an aggravating factor associated to mortality in COPD patients admitted in an intensive care unit, a pre-existing underlying pathology is associated with a worsening of the prognosis in community and nosocomial pneumonia ,30. DAFCP. aeruginosa pneumonia is characterized initially at 48 hours by an increased alveolar-capillary barrier permeability and an adapted host response with an increased DAFC and LLC preserving a normal lung wet to dry weight ratio. On the 5th day, DAFC remains non responsive to pharmacological stimulation even after bacterial elimination. This impairment of DAFC could represent one of the factors responsible for the increased susceptibility of chronically infected patients to other respiratory insults.In conclusion, chronic 131). EK was involved in the revision of the manuscript and the English editing. XL performed all the histological analysis. BG was involved in the acquisition of the data, the design and the conception of the study as well as the drafting of the article. All the authors read and approved the final manuscript.SB and FA were responsible for the acquisition of the data. KF and MOH made substantial contributions to the drafting of the manuscript and the analysis of the data. TP performed the radioactive labelling of the albumin (I


For disease outbreak detection, the public-health community has historically relied on the watchful eyes of doctors and other health-care workers, who have reported individual cases or clusters of cases of particular diseases to health-care and other authorities. The increased availability of electronic health-care data, however, raises the possibility of more automated and earlier outbreak detection and subsequent intervention. Besides diagnoses of known diseases, pre-diagnostic syndromic indicators—such as the primary complaints of patients coming to the emergency room or calling a nurse hotline—are being collected in electronic formats and could be analyzed if suitable methods existed. Martin Kulldorff and colleagues have been developing such methods, and now report a new and very flexible approach for prospective infectious disease outbreak surveillance.Their method, which they call the “space–time permutation scan statistic,” is an extension of a method called scan statistic. All previously developed scan statistics require either (i) a uniform population at risk (with the same number of expected disease cases in every square kilometer), (ii) a control group (such as emergency visits not due to the disease of interest), or (iii) other data that provide information about the geographical and temporal distribution of the underlying population at risk, such as census numbers. The new method, because of a different probability model, can be used for the early detection of disease outbreaks when only the number of cases is available. It also corrects for missing data and makes minimal assumptions about the spatiotemporal characteristics of an outbreak. To make it widely accessible, the method has been implemented as a feature of the freely available SaTScan software.In their article, Kulldorff and colleagues illustrate the utility of the new method by applying it to data collected from hospital emergency departments in New York City. The researchers analyzed diarrhea records from 2002, and did both a “residential analysis” (based on the home address of the patients) and a “hospital analysis” . The former has more detailed geographical information, the latter maybe be better able to detect outbreaks not primarily related to place of residence but, for example, school or workplace. They found four highly unusual clusters of diarrhea cases, three of which heralded citywide gastrointestinal outbreaks due to rotavirus and norovirus.Since November 2003, the space–time permutation scan statistic has been used daily to analyze emergency department data in New York City in parallel with other methods, and it seems to perform well. As the authors discuss, as any other surveillance method, theirs has limitations. Because it adjusts for purely temporal clusters, the method can only detect outbreaks if they start locally (not simultaneously across the entire surveillance area). The less geographically compact an outbreak is, the less power there is to detect it. And some outbreaks, for example, those caused by exposure to an infectious agent in the subway, will be hard to cluster by place of residence or choice of emergency department.In the present study, Kulldorff and colleagues have applied their method to infectious disease surveillance in a metropolitan area in the United States. As they state, however, “the ability to perform disease surveillance without population-at-risk data is especially important in developing countries, where these data may be hard to obtain.”


A comprehensive study of the transcriptional response of mononuclear phagocytes to cytokines reveals distinct classes of cytokines that elicit either the classical or alternative pathway of monocyte activation. Mononuclear phagocytes (MPs) stand at the crossroads between the induction of acute inflammation to recruit and activate immune effector cells and the downmodulation of the inflammatory process to contain collateral damage. This decision is extensively modulated by the cytokine microenvironment, which includes a broad array of cytokines whose direct effect on MPs remains largely unexplored. Therefore, we tested whether polarized responses of MPs to pathogens are related to the influence of selected cytokines or represent a mandatory molecular switch through which most cytokines operate.+ MPs were exposed to bacterial lipopolysaccharide (LPS) followed by exposure to an array of cytokines, chemokines and soluble factors involved in the immune response. Gene expression was studied by global transcript analysis. Two main classes of cytokines were identified that induced a classical or an alternative pathway of MP activation. Expression of genes affected by NFκB activation was most predictive of the two main classes, suggesting that this pathway is a fundamental target of cytokine regulation. As LPS itself induces a classical type of activation, the most dramatic modulation was observed toward the alternative pathway, suggesting that a broad array of cytokines may counteract the pro-inflammatory effects of bacterial components.Circulating CD14This analysis is directly informative of the primary effect of individual cytokines on the early stages of LPS stimulation and, therefore, may be most informative of the way MP maturation may be polarized at the early stages of the immune response. This heterogeneity spans a continuous spectrum that can be polarized into two extremes recently described by Mantovani et al. . Pathogeet al. ,3. Treatet al. , polarizet al. .It has been suggested that the extreme dichotomy between a classical M1 and an alternative M2 polarization of macrophage function may not take into account intermediate regulation by cytokines such as IL-10, transforming growth factors (TGF-α and TGF-β), macrophage colony-stimulating factor (M-CSF), IFN-α and IFN-β and tumor necrosis factor (TNF) . Most imin vitro for 7 days with IL-4 and GM-CSF (immature dendritic cell, (DC)) to pathogen components [A recent study analyzed the transcriptional profile induced by the exposure of circulating MP conditioned mponents . Bacteri+ MPs would react similarly to DCs on engagement with infectious agents. Thus, a preliminary aim of this study was to evaluate the kinetics of the response of non-conditioned circulating CD14+ MP to LPS. The results suggested that these cells respond to LPS similarly to immature CD14- DCs, with a surge in transcriptional activity that peaks around 3 hours after stimulation and in which the activation of genes associated with a classical activation of innate immune mechanisms predominates [The response of circulating MP-derived DCs is short-lived as these cells can exhaust their production of effector molecules (cytokines and chemokines) within a few hours of LPS stimulation . The traominates .in vitro by studying a few illustrative cytokines or whether they represent a mandatory molecular switch through which most cytokines operate. Thus, we stimulated non-conditioned CD14+ MPs with LPS for 1 hour. The MPs were then exposed to an array of different cytokines that may be expressed in distinct pathologic conditions by different immune-cell subsets. The 1-hour interval was empirically selected to induce a biphasic model in which the presumed modulation by cytokines occurred during an ongoing reaction to LPS. This allowed mapping of cytokines into conditional subclasses based on their effects on the global transcriptional changes responsible for MP activation and differentiation.The stringent dichotomy describing a classical activation of MPs into mature antigen-presenting cells caused by IFN-γ and an alternative induction into macrophages induced by IL-4 and IL-13 may not apply to physiological conditions in which the microenvironment responds to pathogen exposure with a broad array of cytokine secretion. We therefore investigated whether polarized responses of MPs to pathogens are extreme behaviors that can be observed Two main classes of cytokines were identified that induced a classical or alternative pathway of MP activation, respectively. An intermediate class (including IL-10) was also identified, while TNF-α, TNF-β and GM-CSF displayed a quite distinct behavior from the other cytokines. Expression of genes affected by NFκB activation was most predictive of the two main classes, suggesting that in most cases the NFκB pathway is a central target of cytokine regulation that modulates the cascade of events following LPS stimulation. Overall, it seems that MP maturation/differentiation goes through a molecular switch that is partly independent of the fine differences in the stimulatory properties of the various cytokines and, with few exceptions, is pre-programmed towards a classical or an alternative route. As LPS itself induces a classical type of activation, the most dramatic modulation in this model was observed toward the alternative pathway, suggesting that a broad array of cytokines may counteract the pro-inflammatory effects of bacterial components.+) were exposed to LPS. Total RNA extracts were obtained 4 and 9 hours after. These time points were selected to catch salient stages of the biphasic response of MP to LPS described by others [-, immature DCs to pathogen components [+ MPs (data not shown). This finding suggests that the differential expression of the LPS co-receptor CD14 between the two cell populations has a relatively minor impact on the transcriptional regulation of the innate immune response [Enriched MP preparations . These three samples were prepared at times 0, 4 and 9 hours to parallel the culture conditions used for stimulation. This finding suggested that the transcriptional profile of CD14+ MPs 9 hours after LPS stimulation and 8 hours after treatment with most cytokines converges toward a less reactive metabolic state closer to that of unstimulated MPs. Several cytokines, however, maintained a more active metabolic profile and after 9 hours retained a transcriptional footprint relatively close to that of samples treated for 4 hours (9"). This group included the genes for most IFN-α isoforms, IFN-β, vascular endothelial growth factor (VEGF), FLT-3 ligand, TGF-α, the chemokine RANTES (CCL5), IL-2, IL-4, IL-15 and the chemokines MIP1α (CCL3) and MIP1β (CCL4), suggesting that these cytokines may have relatively prolonged kinetics of MP activation.Aliquots of CD14ustering  was applThe average number of genes whose expression was increased compared to unstimulated MPs was higher (420 genes) after 4 hours than after 9 hours 265 genes). About half of the genes upregulated after 4 hours remained upregulated at 9 hours (223 genes). This is in accordance with Huang's observation [5 genes. Although LPS alone strongly affected the transcriptional program of MPs, it was possible to discern the contribution of individual cytokines. This analysis was limited to samples treated for 4 hours, when the most dramatic effects on gene modulation were noted. Genes differentially expressed in samples treated with cytokines compared to those treated with LPS alone were identified. Stringent criteria were applied to select genes expressed in at least 80% of samples with a threefold or greater increase or reduction in expression over LPS in at least one of the cytokine-treated samples. This gave 2,057 genes that were deemed most relevant to the analysis. Using these genes, all cytokine-treated samples were subjected to unsupervised clustering to evaluate their relatedness Figure .+ MPs [Two main classes of cytokines were identified. One class  to the global, unfiltered 17K gene dataset Figure . This an2, which is the one most commonly used in clinical trials as a pro-inflammatory cytokine, clustered with the classical cytokines adjacent to IFN-γ.Particular mention should be made of the erratic behavior of various IFN-α subtypes, which clustered indiscriminately between the two main cytokine classes. Interestingly, however, alignment of the IFN-α protein sequences through the EMBL-EBI Clustal W database identified, with the exception of IFN-αG, a close relationship among the IFN-α subtypes that clustered with the alternative type of cytokines (data not shown). This subclassification was also supported by the phylogenetic relationship among interferons described by Henco . This inSignatures associated with several pathways of immune-cell activation were constructed by selecting genes from the global pool of 17K clones according to literature information without pre-existing information about the association of their expression to either class of cytokines. Signature genes were then subjected to supervised clustering according to the cytokine classification shown in Figure The same 121 genes were used for unsupervised class prediction by reclustering cytokine-treated samples Figure . This inAmong the additional pathways tested, those mediated through STATs, Janus kinases (JAKs), and interferon regulatory factor (IRF) did not appear consequential to the experimental conditions tested in this study (data not shown).Matrix metalloproteinases (MMP) are tightly connected to MP activation. MMP released by MPs contribute to normal and pathological tissue remodeling and MP migration. In addition MMP function as regulatory proteins by promoting the activation or degradation of cytokines. Finally, MMP are susceptible to cytokine stimulation. Thirty-six MMP and MMP-related genes (filtered from a larger group of 184 MMPs, disintegrins, α-defensins, TGF-β, TNF-α, insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), fibroblast growth factor (FGF), IL-1 and monocyte chemotactic protein-3 (CCL7/MCP-3) genes) ,17 were t-test, p2-value). Genes associated with immune function were proportionally over-represented. A selection of genes relevant to MP function is shown in Figure Comparison of gene-expression patterns induced by the two cytokine classes  identified 2,007 genes that were differentially expressed at a less than 0.001 significance level  cells, DCs, helper T lymphocytes (Th2) and B lymphocytes. Several genes known to be associated with alternative MP activation ,23 were This early transcriptional profile underlines the primary role of MPs during the acute phases of the response to pathogen as effector cells that can kill pathogens, take up antigen and migrate to local regional lymph nodes to recruit adaptive immune responses . In contrast, alternative cytokines may be produced in the microenvironment to maintain a resident MP phenotype rich in chemokine production, which can attract Th2-type immune responses while continuing pathogen clearance through retention of phagocyte properties and promoting tissue remodeling.Surprisingly, genes of the IL-1 family and its receptors, TNF and IL-6 were consistently upregulated by the alternative class of cytokines, suggesting that LPS alters MP polarization with regard to these cytokines ,10. PartTo define the validity and accuracy of our global microarray analysis, quantitative TaqMan real-time PCR was performed on amplified RNA material isolated after stimulation of monocytes obtained from five additional normal donors with representative cytokines/soluble factors selected from the alternative and from the classical groups. Comparison of monocyte stimulation with a candidate cytokine from the alternative/M2 (IL-4) and the classical/M1 (IFN-γ) group, respectively, is shown in Figure Reproducibility of the estimates of mannose receptor, NFKb-p105/50 and TRAF gene expression in MPs obtained from the same five donors was evaluated after stimulation with four cytokines/soluble factors selected from the alternative (IL-13, IL-1α, IL-4, TGF-β) and four from the classical groups (FLT-3 ligand, IFN-γ, IL-3, CD40L) Figure . In all It has been suggested that MP activation and maturation progresses through a polarized mechanism whereby two extreme products result that promote inflammation on one side and tissue repair on the other ,5. The fIn this study we evaluated whether MP commitment is dependent on an early bipolar switch through which most cytokines operate. This was done by testing in parallel a library of 42 stimulatory molecules possibly present in the tissue microenvironment following a pathogenic insult. This modulation was tested on MPs triggered by a pathogenic stimulus, exemplified in this case by LPS. This was done on the assumption that in most circumstances resident or migratory MPs reaching an infected area are exposed concomitantly to a pathogen and to the cytokine milieu resulting from the infection. The transcriptional profile identified by this study cannot distinguish between the direct effect of each soluble factor analyzed and the downstream activation of transcriptional pathways by secondary paracrine or autocrine secretion of biological modifiers by MPs. However, the main goal of this study was to identify the overall effect on MPs of the exposure to individual cytokines. Future analyses, focused on specific cytokine patterns described here, should possibly include the addition of blocking antibodies to segregate secondary from primary MP responses.yin and yang of the MP transcriptional program. In fact, most cytokines preferentially induced one or other pattern of transcriptional activation. We, therefore, mapped most of the cytokines within a classical or an alternative classification according to their effects on CD14+, LPS-induced MPs. In particular, it appeared that transcriptional programs down-stream of NFκB activation [2b and GM-CSF to the classical group and the alternative type II cytokines with the alternative group, suggesting that NFκB may be central to MP polarization.The results suggest that MP activation is in most cases a bipolar process regulated by an internal switch through which cytokines modulate the tivation  were mostivation ,11,28,29This information cannot of course be generalized to other conditions as the model tested is strongly biased by NFκB induction by LPS. This is underlined by the unexpected alternative upregulation of genes associated with IL-1, TNF and IL-6 . This obet al. [Cytokine and chemokine effects on MP function are tightly intertwined with the enzymatic activities of MMP. Membrane-bound cytokine receptors and adhesion molecules can be released from the cell surface by MMPs acting as 'sheddases' or 'convertases'. This, in turn, can downregulate cell-surface signaling by removal of receptors, or induce paracrine activity by release of soluble proteins. Not surprisingly, IL-13 overexpression results in production of several MMPs . For inset al. , who notEnhanced transcription of gap junction/connexin 26 by the alternative cytokines is also of particular interest in view of the hypothesized junctional communications among MPs or between MPs and endothelial cells ,37. The in vitro or, most importantly, in vivo conditions. Secondary, autocrine and paracrine modulation through the cytokine network following a primary stimulation may introduce novel on and off switches that could override the original signal. Nevertheless, this analysis is directly informative on the primary effect of individual cytokines on the early stages of LPS stimulation and, therefore, may be most informative on the way MP maturation may be polarized at the early stages of the immune response. The clustering of most cytokines into two main groups suggests that their control of central switches (NFκB), or regulatory molecules (cytokines, MMPs, gap junctions, cytotoxic molecules, migratory markers) is essentially bimodal. This polarization program turns MPs to a 'cytotoxic' or a 'symbiotic' phenotype [The early-phase transcriptional profile presented in this study may not comprehensively parallel the plethora of biological effects that a given cytokine can induce under henotype . In phys2 tissue-culture flasks (Costar) in complete medium (CM) consisting of Iscove's medium (Biofluids) supplemented with 10% heat-inactivated human AB-serum (Gemini Bioproducts), 10 mM HEPES buffer , 0.03% l-glutamine (Biofluids), 100 U/ml penicillin/streptomycin (Biofluids), 10 μg/ml ciprofloxacin (Bayer), and 0.5 mg/ml amphotericin B (Biofluids). Adherent and non-adherent cells were gently removed from the flask and centrifugated. MPs were separated by negative selection using the MP isolation kit and an autoMACS system (Miltenyi). Before and after separation cells were stained with anti-CD14-FITC (Becton Dickinson), and analyzed using a FACScalibur flow cytometer and CellQuest software (Becton Dickinson).Peripheral blood mononuclear cells (PBMCs) from an HLA-A*0201-positive healthy caucasian male donor age 35 were collected at the Department of Transfusion Medicine, NIH. PBMCs were isolated by Ficoll gradient separation and frozen until analysis. After thawing, PBMCs were kept overnight in 175-cm+ cells were washed twice with serum-free OPTI-MEM (OM) medium (Gibco-BRL) prepared similarly to CM. CD14+ cells were then seeded at a concentration of 1 × 106/ml in 10 ml OM in 25 cm2 flasks  and stimulated with 5 μg/ml LPS (Sigma) for 1 h. LPS was used at 5 μg/ml to simulate maximal pathogen exposure as in [2 flask, according to the manufacturer's protocol.Negatively selected CD14Total RNA was isolated using RNeasy minikits (Qiagen). Amplified antisense RNA (aRNA) was prepared from total RNA (0.5-3 μg) according the protocol previously described by us ,39. Test2 ≥ 1.2: 2.3 ratio in at least one of the samples) selected 10,370 genes for further analysis. Clustering of experimental samples according to Eisen et al. [Hybridized arrays were scanned at 10-μm resolution on a GenePix 4000 scanner (Axon Instruments) at variable PMT voltage to obtain maximal signal intensities with less than 1% probe saturation. Resulting jpeg and data files were analyzed via mAdb Gateway Analysis tool  and Partn et al.  was basen et al. .MPs separated from peripheral blood by adherence were stimulated for 1 h with LPS and for an additional 30 min with cytokines selected from the alternative group (IL-4, IL-13, IL-1α) or the classical group (IFN-γ, IL-6, IL-3). In addition, TNF-α was tested. After 90 min stimulation, cytoplasmic cell extracts were isolated using a cytoplasmic and nuclear extract kit (Active Motif), and the TransAM NFκB transcription factor kit (Active Motif) was used to detect activation of NFκB subunits p50, p52, p65, c-Rel and RelB, according to the manufacturer's protocol.max = 518 nm) and at the 3' end with the quencher dye molecule TAMRA . The following are the sequences for forward (f) and reverse (r) primer and probe (p) pairs:MPs obtained from PBMC of five normal caucasian donors  were stimulated with four cytokines/soluble factors selected from the alternative group (IL-13, IL-1α, IL-4, TGF-β) and four from the classical groups (FLT-3L, IFN-γ, IL-3, CD40L). TaqMan real-time PCR was performed on amplified RNA material isolated after stimulation for 4 h in conditions identical to those applied for the cDNA array study to validate the expression of the following 10 genes: TRAF binding protein, NFκB-p105/50, MMP9, MMP19, MCP-1, mannose receptor, IL-24, IL-1R, IL-1A and FADD-MORT. An ABI Prism 7900 HT sequence detection system with 384-well capability (Applied Biosystems) was used for detection. Primers and TaqMan probes (Biosource) were designed to span exon-intron junctions and to generate amplicons of less than 150 bp. TaqMan probes were labeled at the 5' end with the reporter dye molecule FAM  and reverse transcribed using random dN6 primers (Boehringer Mannheim). Amplified cDNA standards were quantified by spectrometry and the number of copies was calculated using the Oligo Calculator software [Standards for the selected genes were amplified by reverse transcriptase primer-specific amplification of 6 μg antisense RNA obtained from PBMCs stimulated software . Six mic5 [Linear regression analyses of all standard curves were 0.98 or greater. Standard curve extrapolation of copy number and quantity means were performed using the ABI Prism SDS 2.1 software (Applied Biosystems). Normalization of samples was performed by dividing the quantity mean of the gene of interest run in duplicate by the quantity mean of reference actin filament associated protein (AFAP) gene × 105 .The following additional data are available with the online version of this article. Additional data file A spread sheet containing microarray raw data (intensity ratios of test versus reference samples) subsequently subjected to cluster analysisClick here for additional data file


Dr. Octopus, the villain of the movie "Spiderman 2", is a fusion of man and machine. Neuroscientist Jose Carmena examines the facts behind this fictional account of a brain- machine interface Spider-Man comic, is the ultimate characterization of a brain–machine interface (BMI) on the big screen. In Spider-Man 2, the brain is that of nuclear physicist Dr. Otto Octavius, who dreams of harnessing nuclear fusion. The machine is a harness of four mechanical arms designed with tentacle-like flexibility, gripping and vision capabilities, and an artificial intelligence module that gives them some autonomy. The interface between the machine and the brain is at the spinal cord level, with an “inhibitor chip” to prevent the artificial intelligence module in the mechanical arms from taking over Octavius's brain. Controlling this mechanical device with his own thoughts, Octavius is able to manipulate hazardous materials during his fusion experiments. However, things go terribly wrong during the exhibition of one of these experiments: the mechanical arms fuse to Octavius's body while the inhibitor chip is disabled, resulting in the machine gaining partial control of his brain. Unable to subvert the machine to his will and conscience, Octavius, together with the BMI, becomes the villainous Dr. Octopus. At the end of the movie, in a flicker of sanity and heroism, Octavius dramatically sacrifices his life as the only way to terminate the evil machine and save the world.“Dr. Octopus,” the villain that terrorizes the city in the most recent film of the popular Star Trek, Firefox, and many other science-fiction scenarios, the scientific community has targeted clinical applications, such as neuroprostheses for restoring motor function after traumatic lesion of the central nervous system. The current BMI approach is based on the idea that a human user could enact voluntary motor intentions through a direct interface between his brain and an artificial actuator in virtually the same way that we see, walk, or grab an object with our own natural limbs. Proficient brain control of an external device or actuator should be achievable through training using any combination of visual, tactile, or auditory feedback. As a result of long-term use of the BMI, the brain should be able to “incorporate” (or adapt to) the artificial actuator as an extension of its own body. With these goals in mind, the last five years have witnessed a dramatic increase in BMI-related studies in academic institutions around the world. Subjects have learned to utilize their brain activity for different purposes, ranging from electroencephalogram- and electrocorticographic-based systems  of current BMIs. Even if significant improvements are made in the algorithms used to decode neural activity by, for example, incorporating knowledge from neurophysiological experiments of how motor signals that underlie movements are encoded in the brain, current BMI bandwidth still may not be sufficient to reach the performance level an injured patient would desire.Spider-Man 2 is innovative in the sense that it is a hybrid system that incorporates both neuronal and artificial control signals. It makes perfect sense to take advantage of the fields of engineering (control theory) and artificial intelligence to build better BMIs—part brain and part robot. In principle, these hybrid BMIs would allow a patient to accomplish a task more efficiently than those relying on neuronal signals alone. For example, in a common task such as reaching for and grasping a glass of water, a hybrid BMI would be fed with both brain and machine control signals; the intention of movement would be decoded directly from neuronal signals, leaving obstacle avoidance and grasping stabilization to the artificial control module of the system. Such a module would get inputs from sensors embedded in the robot, and would produce a control signal that would fuse with the neuronal control signal to augment the final output command.What does this mean for second-generation BMIs? We may find some inspiration in Dr. Octopus. The fictional BMI in What ratio of neuronal versus artificial signal would be needed for optimal control of a BMI? In the movie, Octavius's crisis is a severe unbalance in favor of machine control. Science fiction aside, we see the more realistic potential problems of having a physical device gaining autonomous control. Technically, this could be analyzed as too much gain in the artificial control signal, which, in a realistic scenario, would likely result in oscillating behavior, jerky grasping, etc. Hence, safeguarding measures (characterized in the movie as the inhibitor chip in Octavius's brain stem) would be needed to avoid dangerous situations when a chronic neuroprosthesis freely interacts with the real world. For both science and science fiction, the question is the same. Brain and machine: which one gets the power?


To investigate the species' evolutionary history and to establish objective methods for subspecies recognition, voucher specimens of blood, skin, hair, and/or skin biopsies from 134 tigers with verified geographic origins or heritage across the whole distribution range were examined for three molecular markers: (1) 4.0 kb of mitochondrial DNA (mtDNA) sequence; (2) allele variation in the nuclear major histocompatibility complex class II DRB gene; and (3) composite nuclear microsatellite genotypes based on 30 loci. Relatively low genetic variation with mtDNA, DRB, and microsatellite loci was found, but significant population subdivision was nonetheless apparent among five living subspecies. In addition, a distinct partition of the Indochinese subspecies P. t. corbetti into northern Indochinese and Malayan Peninsula populations was discovered. Population genetic structure would suggest recognition of six taxonomic units or subspecies: (1) Amur tiger P. t. altaica; (2) northern Indochinese tiger P. t. corbetti; (3) South China tiger P. t. amoyensis; (4) Malayan tiger P. t. jacksoni, named for the tiger conservationist Peter Jackson; (5) Sumatran tiger P. t. sumatrae; and (6) Bengal tiger P. t. tigris. The proposed South China tiger lineage is tentative due to limited sampling. The age of the most recent common ancestor for tiger mtDNA was estimated to be 72,000–108,000 y, relatively younger than some other Panthera species. A combination of population expansions, reduced gene flow, and genetic drift following the last genetic diminution, and the recent anthropogenic range contraction, have led to the distinct genetic partitions. These results provide an explicit basis for subspecies recognition and will lead to the improved management and conservation of these recently isolated but distinct geographic populations of tigers.Eight traditional subspecies of tiger  Genetic analysis provides the basis for subspecies recognition among tigers, and will lead to improved conservation strategies for these endangered animals Historically tigers inhabited much of Asia, including the regions between the Caspian and Aral Seas, southeastern Russia, and the Sunda islands , Caspian (P. t. virgata), and Javan (P. t. sondaica) tiger subspecies were eradicated by the 1940s, 1970s, and 1980s respectively ((P. t. tigris) exist in Bangladesh, Bhutan, western China, India, western Myanmar, and Nepal  survive in eastern Russia, northeastern China, and Korea ((P. t. amoyensis) now exist in captivity only ((P. t. sumatrae) occur in Sumatra ((P. t. corbetti) live in Cambodia, China, Laos, Malaysia, east Myanmar, Thailand, and Vietnam . In addition, the presence of Numt, a nuclear pseudogene insertion of cytoplasmic mitochondrial DNA (mtDNA) in tiger autosomes , has madDRB gene; and (3) allele size variation of 30 hypervariable short tandem repeat loci or microsatellites. Observed patterns of population genetic variation replicated with different gene families form the basis of interpretation of the tiger's evolutionary history and recommendations for its management.To establish proper biological reference specimens, samples from 134 tigers of known geographic origin were collected. Three genetic markers were examined: (1) 4 kb of mtDNA sequence derived from primer pairs that excluded Numt amplification, (2) allele variation in the major histocompatibility complex (MHC) n = 13), south China (n = 4), northern Indochina (n = 30), Malayan Peninsula (n = 22), Sumatra (n = 16), and the Indian subcontinent (n = 15). The mtDNA sequences specified 54 variable sites defining 25 haplotypes -specific primer pairs . The fraplotypes . Thirty plotypes , and 29 (P. t. sumatrae) formed a monophyletic group (80% MP, 70% ME, and 66% ML bootstrap support). A second monophyletic cluster of six haplotypes (TIG1 to TIG6) from 15 Bengal tigers (P. t. tigris) also received high bootstrap support (93% MP, 82% ME, and 90% ML). The rest of the mainland Asian haplotypes grouped together and partitioned into three distinct geographic groups: (1) a genetically invariant Amur tiger lineage  represented by a single haplotype in 13 individuals, (2) a northern Indochinese lineage (P. t. corbetti I) of individuals originating from south China to the Indochinese countries north of the Isthmus of Kra, and (3) a paraphyletic assembly of haplotypes from tigers from Malayan Peninsula (P. t. corbetti II). Support for subdividing the conventional Indochinese subspecies of tigers P. t. corbetti into two clusters was high . The COR1/AMO3 haplotype, represented by 22 individuals from Vietnam (n = 2), Cambodia (n = 14), northeast Thailand (n = 5), and south China (n = 1), was the only haplotype found in two classical subspecies lineages (P. t. amoyensis and P. t. corbetti) , minimum evolution (ME), and maximum likelihood (ML) approaches produced congruent topologies that defined major geographic partitions A. Eight orbetti) .P. t. amoyensis, fell into two very distinct phylogenetic origins. Two tigers from the Suzhou Zoo  in all phylogenetic analyses (P. t. corbetti I phylogenetic lineages. In contrast, two P. t. amoyensis tigers (Pti-219 and Pti-220) from the Chongqing Zoo collection had a haplotype (AMO1) that formed a separate lineage that was ten nucleotide substitutions from its nearest sequence   B. Haplot(P. t. sumatrae) formed a monophyletic clade with 97% bootstrap support, and Amur tigers  grouped with 76% bootstrap support. The remaining tiger genotypes partitioned into two weakly supported monophyletic lineages  and a paraphyletic assemblage of northern Indochinese P. t. corbetti I. For example, three individuals from Thailand (Pti-296, Pti-297, and Pti-301) clustered with samples from the India subcontinent, blurring the distinction between P. t. corbetti I and P. t. tigris. The three South China tigers from the Suzhou Zoo, China, that had clustered with P. t. corbetti I by mtDNA (Pti-217, Pti-218, and Pti-222) also associated more closely with P. t. corbetti I from northern Indochina by microsatellite analysis  from the Chongqing Zoo, China, likewise formed a distinct lineage in the microsatellite analysis  were excluded in this subdivision analysis due to our small sample size. In our first hypothesis, two groups were considered: the P. t. sumatrae island population and all contemporary mainland populations . This recently proposed model ((P. t. sumatrae), the Amur tigers , which presently are isolated from other tiger populations by more than their maximum known dispersal distance , (2) P. t. corbetti, including Indochina and part of south China, (3) Bengal tigers (P. t. tigris), and (4) Sumatran tigers (P. t. sumatrae). The fourth scenario, based on the results of the mtDNA phylogenetic analyses  with both mtDNA haplotypes and microsatellite genotypes . P. t. aed model  presumesdistance , and a gyses see  and the yses see , furtherst  and Rst  values. Under this model, 31% of the microsatellite variation discriminated between the five groups, while the balance, 69%, occurred within each group. For mtDNA the Fst was very high (0.838), indicating that 84% of the variation was partitioned among the different phylogeographic subspecies. Each of the five subspecies showed highly significant population genetic differentiation (p < 0.0001) by pairwise Fst and Rst with 10,000 permutations  four tigers from China and Vietnam; (2) nine tigers from Cambodia; and (3) 20 tigers from Cambodia and northern Thailand (K = 7, Pr[K] = 0.993). In this scenario, most individuals were assigned to a cluster with high probability (q > 0.90), indicating very low level of gene flow between the groups. However, because this additional substructure within P. t. corbetti I had little geographic or ecological basis, and because AMOVA analysis based on this population subdivision resulted in lower Fst and Rst values than that in the five-group scenario (unpublished data), the distinction was not considered to be a consistent basis for subspecies classification and may reflect additional population differentiation within a subspecies.An alternative analysis of the combined microsatellite and mitochondrial haplotype data using a Bayesian approach  as imple(P. pardus) ((Oncifelis geoffroyi), Pampas cat (O. colocolo), or tigrina (Leopardus tigrinus) ((Puma concolor) (P. t. tigris, P. t. sumatrae, P. t. corbetti I, and P. t. corbetti II) showed moderate nucleotide diversity (π), ranging from 0.0001 to 0.0070  , Geoffroigrinus) , but comoncolor)  in perceo 0.0070 . The P. n = 17) (P. t. corbetti I (p < 0.00167), possibly reflecting further population subdivision in this region. Expected heterozygosity in tigers ranged from 0.456 in P. t. altaica to 0.670 in P. t. corbetti I (P. t. tigris (4.94) and P. t. corbetti I (3.58) and lowest in P. t. altaica (1.93).Parameters of microsatellite variation have been shown to provide sensitive measures of historic demographic perturbations in felid and other species . Estimatn = 17) . After Brbetti I . AverageP. t. amoyensis to 14.6% in P. t. corbetti I . There were a total of seven electrophoretic allele variants (A–G). This is a relatively low MHC-DR diversity compared to human and domestic cat, which possess 126 and 63 DRB alleles, respectively, for the same gene segment in samplings of 251 humans and 37 cats, respectively (DRB variation among tigers, there was detectable population differentiation. Three mainland subspecies P. t. tigris (n = 1), P. t. altaica (n = 5), and P. t. corbetti I (n = 2) were genetically identical for DRB-A allele sequence. Three additional DRB alleles (B, C, and D) were found only in P. t. corbetti II (n = 2), while three others (E, F, and G) were unique to P. t. sumatrae (n = 11)   and 8 (W(n = 11) .–8 substitutions per site per y, or one substitution every 14,000 y in the segment examined. According to this rate, the estimated coalescence time of mtDNA variation for extant tiger lineages was 72,000 y (95% CI = 39,000–104,000 y). An older fossil record calibration of 3 MY for the separation of leopards and tigers produced a rate of 1.53 × 10–8 substitutions per site per y, or one substitution every 20,000 y. According to this substitution rate, mtDNA diversity of modern tigers originated about 108,000 y (95% CI = 59,000–157,000 y) ago. Based on either calibration, the Amur tigers probably experienced a genetic reduction or founder event more recently (less than 20,000 y), as no variation was detected within the population.The mtDNA sequence divergences in a combined data set of 3,217 bp, of which homologous sequences from the tiger and leopard were both determined see , were usn > 15), ranged from 9,900 y in Amur tigers P. t. altaica to 18,437 y in northern Indochinese tigers P. t. corbetti I.The estimate of microsatellite variance in average allele repeat-size can also be used as a surrogate for evolutionary time based on the rate of allele range reconstitution subsequent to a severe founder effect . Using aNeμT, and Ne is the long-term effective female population size, μ the substitution rate per site per year, and T the generation time. From a coalescent-based simulation of the mitochondrial sequences, the average estimate of θ was 0.00255 per nucleotide site, with a 95% CI from 0.00147 to 0.00417. With the substitution rate calibrated from this study (1.91 × 10–8 bp–1 y–1) and an average generation time of 5 y for tigers   . MHC clastic cat . By contstic cat  (Culver stic cat .(Panthera onca) lineages diverged approximately 280,000–510,000 y ago (The oldest tiger fossils, around two million y (MY) old, are from northern China and Java . By the 00 y ago . Our coa00 y ago , which h00 y ago  and to a00 y ago .; northern Indochinese tigers (P. t. corbetti I); southern Indochinese tigers (P. t. corbetti II), which are confined to the Malayan Peninsula; Sumatran tigers (P. t. sumatrae); Bengal tigers (P. t. tigris); and, if its uniqueness is affirmed by more extensive sampling, South China tiger (P. t. amoyensis). These conclusions are based on significant genetic structure among tigers from these different geographic regions with the MHC, mtDNA, and microsatellite data, and extremely limited gene flow as shown by disjunct distributions of genetic variation  and the high mtDNA Fst and microsatellite Rst values. In addition, each subspecies has an allopatric geographical distribution , human-induced population fragmentation and random lineage loss from a single panmictic population is not supported by the strong geographical partitioning of the mitochondrial lineages or by differences in measures of nucleotide diversity within each subspecies. Mismatch analysis  of pairwP. t. corbetti I has a large number of mtDNA diagnostic sites (three), the largest number of unique microsatellite alleles (19 out of 130), and the highest overall microsatellite diversity , evidence of a fairly stable demographic history, and alleles found in the other subspecies were almost always a subset of those found in P. t. corbetti I.The differences in molecular genetic patterns among the six hypothesized subspecies are dramatic . Furtheriversity  and 8. IP. t. corbetti I region  was indistinguishable from northern Indochinese tigers  in the gion see , as therrbetti I . The ultgers see  and 4, pgers see , of histP. t. corbetti I Indochinese tigers  supportThe isolation of Sumatran tigers from mainland populations is supported by multiple unique characters, including two diagnostic mtDNA nucleotide sites, eight mtDNA haplotypes, and 11 (of 108) unique microsatellite alleles . CracrafThe Bengal tigers are defined by three distinct mitochondrial nucleotide sites and 12 unique microsatellite alleles. The pattern of genetic variation in the Bengal tiger corresponds to the premise that tigers arrived in India approximately 12,000 y ago . This reP. t. corbetti is located in northern Vietnam (P. t. jacksoni, to honor the contributions of Peter Jackson, the former Chair of the the World Conservation Union (IUCN) Cat Specialist Group, who tirelessly labored for more than 40 y on behalf of tiger conservation. We designate the type specimen of the Malayan tigers to Pti-163 from the Zoo Melaka, Malaysia, and the taxonomic diagnosis will be described elsewhere. The present status of tigers from northern Indochina and from Malayan Peninsula is uncertain, urging more extensive study and conservation.Our results have several implications for tiger conservation. Management strategies for the tiger, both in situ and ex situ, have been historically influenced by perceptions of its geographical variation and subspecific taxonomy , and sev Vietnam , and no Our results also show that, although modern tigers have a relatively young history, ecological, demographic, and biogeographic factors have led to recognizable subdivisions among otherwise closely related populations. We therefore might expect that more extensive geographic sampling would reveal additional phylogenetic divisions among populations, especially in the Indian Subcontinent and the Indochina bioregions, or alternatively, would blur the apparent phylogenetic subdivisions and reveal a clinal distribution of genetic variation across different subspecies. Further sampling of modern and historic specimens will also help clarify whether the patterns we have observed are attributable to the recent substantial population decline throughout the range in tigers, or whether the observed differentiations among tigers occurred earlier.A total of 134 tiger individuals were sampled throughout the distribution range see . Of thesPanthera species is complicated by the presence of a large 12.8 kb nuclear mtDNA fragment that transposed to chromosome F2 in an ancestral Panthera species approximately 3 MYA (ND1, ND2, ND5, and ND6), cytochrome B(CytB), control region(CR), 12S rRNA(12S), cytochrome C oxidase subunits I and II(COI and COII), and ATPase8 (ATP8)  see . The priTP8) see .2, 1.0 mM dNTPs, 0.25 units of AmpliTaq Gold DNA polymerase , and 1× PCR buffer II; the amplification protocol was: denaturation 10 min at 95 °C, a touch-down cycle of 95 °C for 30 s, 52 °C for 30 s decreased by 1 °C in the next cycle for 10 cycles, 72 °C for 45 s, then 35 amplification cycles of 95 °C for 30 s, 52 °C for 30 s, and 72 °C for 45 s, followed by an extension of 10 min at 72 °C. PCR products were purified using Microcon PCR filters (Millipore, Billerica, Massachusetts, United States) and were directly sequenced in both directions using BigDye Terminator kits (Applied Biosystems) and run on an ABI 377 sequencing apparatus. Sequences were inspected using SEQUENCHER (Gene Codes, Ann Arbor, Michigan, United States), unambiguously aligned using Clustal-X  using AMOVA as implemented in ARLEQUIN 2.0. Statistical significance was tested using 10,000 permutations.Phylogenetic relationships among mtDNA haplotypes were assessed using three approaches implemented in PAUP . An MP aH = 2μT, where H was the branch height in the linearized tree correlated to the average pairwise distance among haplotypes, μ the substitution rate, and T the divergence time. Since there was no comparable sequence from other Panthera species available, we generated from PCR and GenBank a chimera-homologous sequence of 3.2 kb  from three leopard individuals. The divergence time between leopard and tiger was used as a calibration point, and two fossil dates were chosen. Two million y was a commonly used lower bound for the Panthera lineage radiation and the date for the earliest reported tiger in the fossil record ((Felis catus) was used as an outgroup. Coalescent-based simulation of the population parameter θ estimation and its 95% CI was conducted in DnaSP 4.0  (Thirty polymorphic microsatellite loci (FCA005, FCA008, FCA032, FCA043, FCA044, FCA069, FCA077, FCA090, FCA091, FCA094, FCA096, FCA105, FCA123, FCA126, FCA129, FCA139, FCA161, FCA176, FCA201, FCA211, FCA212, FCA220, FCA229, FCA242, FCA290, FCA293, FCA304, FCA310, FCA391, and FCA441) originally designed in the domestic cat . catus)  were amp. catus) . Two loc. catus) . These mhttp://wbiomed.curtin.edu.au/genepop/) (st, sum of square size differences) were derived from ARLEQUIN 2.0. The statistical significance of Rst values, sum of squared size differences, was tested with 10,000 permutations as implemented in ARLEQUIN. A Bayesian clustering method implemented in the program STRUCTURE ((K) without prior information of the origin of individuals. A series of tests was conducted using different numbers of population clusters to guide an empirical estimate of the number of identifiable populations, assuming an admixture model with correlated allele frequencies and with burn-in and replication values set at 50,000 and 106, respectively. Each test yielded a log likelihood value of the data (Ln probability), the highest of which would indicate which test was closest to the actual number of genetically distinct populations. These tests also provided an alpha value, the measure of admixed individuals in the data set.Tests for genotypic linkage disequilibrium and deviations from Hardy-Weinberg equilibrium for each locus in each population were performed using GENEPOP web version of 3.1c (enepop/) . Measureenepop/) . Pairwisenepop/) . AssessmTRUCTURE  was usedDRB gene was assessed in five Amur, two northern Indochinese, two Malayan, 11 Sumatran, and one Bengal tiger. Conserved PCR primers designed from the human DRB sequence were used to amplify homologous DRB sequences (of around 238 bp) in 21 tiger voucher DNAs with primers 61a (5′-CCGCTGCACTGTGAAGCT-3′) and 219a (5′-CCACACAGCACGTTTCTT-3′). Products were screened for polymorphisms using SSCP, a method that detects single-basepair substitutions in 100–300-bp DNA fragments. For SSCP, PCR products were mixed with a solution of 120 μl of formamide, 20 μl of TAMRA (Applied Biosystems) lane standard, and 20 μl of a blue dextran loading dye (from a stock solution of 50 mg/ml with 25 mM EDTA), then denatured at 95 °C for 3 min. The electrophoresis ran at 2,000 volts, 400 amps, and 25 watts in 1× TBE buffer through a 6% denaturing polyacrylamide gel (19.5:1 acrylamide:bis). The SSCP fragments were visualized by autoradiography, and alleles were scored by eye  with the (1 – kf) option in MICROSAT . Numbers(108 KB DOC).Click here for additional data file.Figure S26 replicates. Each individual is represented by a thin vertical bar, which is partitioned into K colored segments that represent the individual affiliation to each of K clusters. Here shows the population structure when K = 7, which produced the highest probability among other choices of K. Three STRUCTURE runs produced almost identical individual affiliation.Data obtained from microsatellite genotype and mitochondrial haplotype data were analyzed using STRUCTURE . Simulat(62 KB DOC).Click here for additional data file.Table S1(59 KB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/) accession numbers of the mtDNA fragments discussed in this paper are AY736559–AY736808.The GenBank (


Cellular functions are coordinately carried out by groups of genes forming functional modules. Identifying such modules in the transcriptional regulatory network (TRN) of organisms is important for understanding the structure and function of these fundamental cellular networks and essential for the emerging modular biology. So far, the global connectivity structure of TRN has not been well studied and consequently not applied for the identification of functional modules. Moreover, network motifs such as feed forward loop are recently proposed to be basic building blocks of TRN. However, their relationship to functional modules is not clear.E. coli. By studying the global connectivity structure of the regulatory network, we first revealed a five-layer hierarchical structure in which all the regulatory relationships are downward. Based on this regulatory hierarchy, we developed a new method to decompose the regulatory network into functional modules and to identify global regulators governing multiple modules. As a result, 10 global regulators and 39 modules were identified and shown to have well defined functions. We then investigated the distribution and composition of the two basic network motifs (feed forward loop and bi-fan motif) in the hierarchical structure of TRN. We found that most of these network motifs include global regulators, indicating that these motifs are not basic building blocks of modules since modules should not contain global regulators.In this work we proposed a top-down approach to identify modules in the TRN of E. coli possesses a multi-layer hierarchical modular structure without feedback regulation at transcription level. This hierarchical structure builds the basis for a new and simple decomposition method which is suitable for the identification of functional modules and global regulators in the transcriptional regulatory network of E. coli. Analysis of the distribution of feed forward loops and bi-fan motifs in the hierarchical structure suggests that these network motifs are not elementary building blocks of functional modules in the transcriptional regulatory network of E. coli.The transcriptional regulatory network of  Escherichia coli and Saccharomyces cerevisiae [E. coli TRN aggregated into homologous motif clusters that largely overlapped with known biological functions and further formed a giant motif supercluster which comprised about half of the nodes in the giant component of the whole network. This study provided interesting information for understanding the organization principle of regulatory networks. A different approach for studying network organization is the so called "top-down view"[Genome sequencing and high-throughput technologies of functional genomics generate a huge amount of information about cellular components and their functions in an unprecedented pace. These advances make it possible to reconstruct large scale biological networks  at a whole cell level -4. One orevisiae ,7,8. Recrevisiae . Networkrevisiae ,17. Feedrevisiae  reportedown view". It starown view"-25. The own view",26-30. Aown view" the globE. coli as an example. For this purpose we first showed an uncovered global hierarchical structure. Global regulators and modules with clearly defined functions were then identified by a new network decomposition method based on the hierarchical structure. We further investigated the distribution of the two basic network motifs, feed forward loop and bi-fan motif, in the network hierarchical structure and examined their relationship to functional modules.In this work we demonstrated the applicability of a top-down approach for the identification of functional modules in TRN with the well established transcriptional regulatory network of E. coli considered in this work is based on RegulonDB [E. coli. This means that there are no regulatory cycles  in the TRN of E. coli. This result implies an acyclic structure of the E. coli TRN in which the nodes can be placed in different layers according to their depth. To identify such a hierachical structure we rearranged the operons in the following way: (1) operons which do not code for transcription factors (TFs) or code for a TF which only regulates its own expression (auto-regulatory loop) were assigned to layer 1 (the lowest layer); (2) then we removed all the operons in layer 1 and from the remaining network identified TFs which do not regulate other operons and assigned the corresponding operons in layer 2; (3) we repeated step 2 to remove nodes which have been assigned to a layer and identified a new layer until all the operons were assigned to different layers. As a result, a five layer hierarchical structure was uncovered as shown in Fig. The transcriptional regulatory network of egulonDB  and compegulonDB . It consegulonDB . A subseegulonDB . HoweverE. coli TRN implies that no feed back regulation exists at transcription level. We noticed that Shen-Orr et al have also reported that there was no feed back loop in the E. coli regulatory network [lac operon may be used as an example to further illustrate this point. Lac operon is not expressed unless lactose is available for the cell because it is repressed by the lac repressor. Lac repressor (the protein but not the gene) is the control element of the system. Its existence (expression) is necessary for the cell to properly response to environmental changes (i.e. the presence and absence of lactose). Therefore, for cells to quickly and properly response to changes of environmental conditions it is of advantage to keep a set of proteins expressed in all conditions and through them to regulate the expression of other genes in a hierarchical way. Feedback control of gene expression may be mainly through other interactions (e.g. metabolite and protein interaction) rather than through transcriptional interactions between proteins and genes. In fact, many transcription factors can bind small molecules to gain or loss their ability to bind DNA.The multi-layer hierarchical structure of the  network . We furt network  and founIHF → OmpR → FlhDC → FliA and further to seven operons  and these operons.The five-layer hierarchy shown in Fig. E. coli. We find that the giant component preserves the five layer hierarchical structure of the whole network. It also includes the single large motif super cluster found by Dorbin et al. [Based on the uncovered hierarchical organization structure we propose a new method to identify functional modules in TRN. As discussed above, there is a giant weakly connected component in the whole TRN of n et al.  and thusgcvTHP which codes enzymes for glycine cleavage. We checked the function of the operons in the two separated parts by using EcoGene database [First we removed all the operons in the top three layers and the operons which are regulated only by them, resulting in a network with 221 nodes and 186 links as shown in Fig. database . The lefaceBAK is also repressed by FruR, the regulator for fructose uptake. This makes the cell not to use acetate as a substrate in the presence of fructose. Therefore, the two different pathways are actually functionally related from a regulation viewpoint. The other three modules (module 4, 23 and 32) which include operons with different functions are actually linked by certain global regulators (fis, arcA and rpoN respectively). They are not connected by any local regulators with specific functions. Thus, it is not strange that they are not closely functionally related. One reason for this problem is probably the information incompleteness of the network. The regulatory network considered in this work contains only about twenty percent of the genes in the E. coli genome. With more and more information available we can include more interactions and genes in the network to obtain more reasonable modules by structural analysis. Identifying these functional modules can help us to gain a general view of the function (or ability) of organisms. Furthermore, we can compare these structure based modules with modules from hierarchical classification results of microarray experiments to find unknown regulatory relationships.To investigate if the modules identified from anaylisis of the network structure are really functionally related, we checked the functions of the genes in the individual modules by using database EcoGene . Most geCRP, IHF, FNR, HNS, ArcA) have been identified in all the three studies. The other five regulators have also been recognized as global in either one or two of the three studies. Our definition of global regulators is directly linked to the identification of functional modules. Modules are sets of genes with closely related function. An important criterion for a regulator to be regarded as global is that it regulates genes with diverse but concerted functions. Therefore, determination of global regulators by the number of regulated modules is more reasonable than that solely by the number of genes or operons. From Fig. We compared the ten global regulators with those found in three previous studies by considering the number of directly or indirectly regulated genes (operons) and their structure and function diversity ,10,11. FE. coli. In agreement with the results of Dobrin et al [FNR and CRP respectively) which regulates genes with various functions. We further investigated the distribution of the 42 FF loops in the hierarchical structure and find that 32 of them contain one of the ten global regulators. Because modules are defined as subsets of genes with closely related functions, while global regulators tend to regulate functionally far related genes, clusters formed from network motifs which contain global regulators are not proper candidates for modules. For the four consistent motif clusters, three of them are formed from the ten FF loops that do not contain global regulators. Cluster four (osmoregulated porin gene) contains the global regulators IHF and OmpR. As shown in Fig. To investigate if network motifs, which are considered to be the elementary building blocks of the whole network , are basin et al , the 42 in et al  compriseE. coli transcriptional regulatory network presently known possesses a multi-layer hierarchical structure with no feedback regulation at transcription level. Regulators in the top layers of the hierarchical structure can be considered as global regulators that often act together with local regulators to regulate genes in the bottom layer. Based on the hierarchical structure a new decomposition method is proposed which can be used to identify functional modules in the network. Analysis of operon composition of the two well-known network motifs (feed forward loop and bi-fan motif) and their distribution in the hierarchical structure suggests that they are not elementary building blocks of functional modules in the transcriptional regulatory network of E. coli.The E. coli was obtained from the website of Alon's research group[gatR_1, rcsA and rotA) because they are either the same with another operon or inside another operon. GatR_1 has been merged with gatR_2 in the updated annotation of E. coli genome in the database EcoGene [RcsA is part of the rcsAB operon, while rotA is the same with ppiA. Another operon, nycA, was not found in any E. coli genome database. We searched the original literature [nycA operon from the network. There are also six operons (emrRAB, gatYZABCDR, hipBA, idnDOTR, moaABCDE and mtlADR) in the network that are only autoregulated and hence do not connected with other operons. Therefore, we ignored these operons as well when analyzing the network connectivity structure. The resulting network consists of 413 nodes (operons) and 576 directed links (regulatory relationships). The 54 autoregulatory relationships in the network are represented as loops in the graph.The original transcriptional regulatory database of rch group. This darch group and comprch group. We remo EcoGene . RcsA isterature  for thisterature  and coulCalculations for the network structure analysis were carried out by using the software Pajek . The numFrom the hierarchical structure, feed forward loops are easily found by searching for all the fully connected triads which are located in different regulatory layers (not necessary to be three nearby layers). Bi-fan motifs are searched by using the subgraph searching algorithm in Pajek .HWM performed the analyses and drafted the manuscript. JB contributed to the concept and promoted the work. APZ is the project leader. He contributed to the concept, supervised the study and was involved in writing the manuscript. All authors have read and approved the final manuscript.E. coli transcriptional regulatory network. Supplenmentary fig E. coli transcriptional regulatory network.Supplenmentary table Click here for file


The number of g-j channels per junction was varied from zero to 100, 1,000 and 10,000 (Rgj of ∞, 100 MΩ, 10 MΩ, 1.0 MΩ, respectively). The longitudinal resistance of the interstitial fluid (ISF) space between the parallel chains (Rol2) was varied between 200 KΩ  and 1.0, 5.0, and 10 MΩ. The higher the Rol2 value, the tighter the packing of the chains. It was found that adding many g-j channels inhibited transverse propagation by blocking activation of all 5 chains, unless Rol2 was greatly increased above the standard value of 200 KΩ. This was true for either method of stimulation. This was explained by, when there is strong longitudinal coupling between all 5 cells of a chain awaiting excitation, there must be more transfer energy (i.e., more current) to simultaneously excite all 5 cells of a chain.The effect of adding many gap-junctions (g-j) channels between contiguous cells in a linear chain on transverse propagation between parallel chains was examined in a 5 × 5 model  for cardiac muscle. The action potential upstrokes were simulated using the PSpice program for circuit analysis. Either a single cell was stimulated (cell A1) or the entire chain was stimulated simultaneously (A-chain). Transverse velocity was calculated from the total propagation time (TPT) from when the first AP crossed a V The parameters that affect the magnitude of Vjc include the size of Rjc, the transverse resistance of the junctional cleft. This results in excitation of the postjunctional cell, after a brief junctional delay. The total propagation time consists primarily of the summed junctional delays. This results in a staircase-shaped propagation, the surface sarcolemma of each cell firing almost simultaneously [We have developed an electric field hypothesis for the mechanism of transmission of excitation from one cell to the next that does not require gap-junction channels -4. In thaneously .+ channels are localized in the junctional membranes of the intercalated discs of cardiac muscle [There are no low-resistance connections between the cells in several different cardiac muscle and smooth muscle preparations (reviewed in ,4. Propac muscle -15 Sperec muscle ,4,1,2,13c muscle , as predc muscle . It was c muscle .inhibited transverse propagation. This fact was briefly mentioned as an unpublished observation in that paper. The purpose of the present study was to do a thorough investigation of that strange phenomenon. The results showed that, in cardiac muscle, transverse propagation was inhibited when many g-j channels were added between cells of each chain. This was true when either a single cell in the first chain was stimulated (cell A1) or the entire chain (A-chain) was stimulated simultaneously.Subsequently, we published a series of papers on the longitudinal and transverse propagation of action potentials in cardiac muscle and smooth muscle using PSpice analysis ,20,24. IDetails of the methods used and modeling of the PSpice analysis were given in our previous papers, including the limitations -24. The The circuit used for each unit was kept as simple as possible, using only those ion channels that set the resting potential (RP) and predominate during the rising phase of the AP. We wanted to only inscribe the rising phase of the APs to study propagation in the 2-dimensional sheet of myocardial cells. It was not possible to insert a second or third "black-box" into the basic excitable units, because the system became erratic, therefore the dynamic behavior of the cardiac cell membrane was an approximation. The RP was -80 mV, and the overshoot potential was +30 mV (AP amplitude of 110 mV). Propagation velocity was calculated from the measured total propagation time (TPT) (measured as the difference between when the APs of the first cell and last cell crossed -20 mV) and cell length.+ ions in myocardial cells during depolarization and excitation, this function had to be simulated by a V-controlled current source (our "black-box") in each of the basic circuit units  of this fluid was divided into two components: a radial (or transverse) resistance (Ror) and a longitudinal resistance (Rol). The longitudinal resistance values (Rol2) between the upper chain and interior chains were increased over a wide range to reflect packing of parallel chains into a bundle of fibers, with different degrees of tightness of the packing  × L (cm) /Ax (cm2), where ρ is the resistivity of the ISF, L is the length, and Ax is the cross-sectional area of the ISF space. The transverse resistance of the interstitial fluid (ISF) space (Ror2) also reflects the closeness between the chains; the lower the Ror2 value, the closer the chains are packed. In the present 5 × 5 model, there were five parallel chains (chains A, B, C, D, and E) of five cells each (cells 1–5). Electrical stimulation (rectangular current pulses of 0.25 nA and 0.50 ms duration) was applied to the inside of either the first cell of chain A (cell A1) or all 5 cells of the A-chain. Under initial conditions, the cells in each chain were not interconnected by low-resistance pathways (gap junction channels), so that transmission of excitation from one cell to the next had to be by the EF developed in the narrow junctional cleft. Then gj-channels were added (100, 1,000 and 10,000 channels per junction) to determine what effect they would have on transverse propagation. Since the number of functional gj-channels per junction have been estimated to be about a 1000 [The upper chain of cells was assumed to be bathed in a large volume of Ringer solution connected to ground. The external resistance  was recorded across Rjc, the radial (or transverse) resistance of the narrow and tortuous junctional cleft. Under standard conditions, Rol2 was 200 KΩ, Ror2 was 100 Ω, and Rjc and was 25 MΩ (50 MΩ ÷ 2).As shown in Figure gj) of infinite) to 100 (Rgj = 100 MΩ), 1000 (Rgj = 10 MΩ), and 10,000 (Rgj = 1.0 MΩ). Experiments were done with electrical stimulation (0.5 nA, 0.5 ms) of only the first cell of the first chain  and with simultaneous stimulation of all 5 cells of the A-Chain (each cell receiving 0.5 nA current). This second method of stimulation was done to obtain a more accurate assessment of strictly transverse propagation.The 5 × 5 model  of cardiac muscle was used to examine whether addition of gap-junction (gj) channels between the cells in each chain would affect transverse propagation of simulated (PSpice) action potentials. The number of gj-channels was increased from zero  and Rol2 of 200 KΩ . As shown, all 5 chains (25 cells) responded. The total propagation time (TPT) was 4.2 ms (measured as the elapsed time between when the first and last APs crossed -20 mV). When 10,000 g-j channels were inserted into the cells of each chain (Rgj = 1.0 MΩ), then the last 2 chains (D and E) failed to respond (panel B). The 5 cells of each chain (A, B, and C) that responded now fired simultaneously because of the high degree of cell coupling, thereby giving only three AP traces, as shown. However, raising Rol2 to 10 MΩ allowed all 5 chains to respond (Panel C), and propagation velocity was increased. These data are summarized in Table part A. Hence greatly elevating Rol2 could overcome the impaired transverse propagation caused by the gj-channels.Figure Panel A shows the standard conditions: Rgj = ∞ and Rol2 = 200 KΩ. As shown, all 5 chains responded (TPT was 3.6 ms). Note that all 5 cells of the B – E chains did not respond simultaneously. When 10,000 gj-channels were added to the contiguous cells of each chain (Rgj = 1.0 MΩ), then the last 3 chains (C, D, and E) failed to respond (Panel B). The 5 cells of each chain that responded (A, B) now fired simultaneously because of the strong coupling. However, elevating Rol2 to 10 MΩ allowed all 5 chains to respond (Panel C). These data are summarized in Table Figure Panel A) and stimulation of the entire A-chain (Panel B). The data include Rgj values and Rol2 values not illustrated in Figures ol2 reverses this inhibition.Figure ol2 , reflecting tighter packing of the chains.The results demonstrated that, in a cardiac muscle model, the insertion of many gj- channels, between abutting cells in each longitudinal chain of cells, actually inhibited transverse propagation of excitation between the parallel chains. This was true both for stimulation of only a single cell of the first chain  and for stimulation simultaneously of the entire 5 cells of the A-chain. The inhibition produced by the gj-channels could be overcome by greatly increasing the value of Rpanel A, in which Rgj was 1.0 MΩ (10,000 gj-channels) uniformly in all 5 chains, chains D and E failed to fire. When Rgj of just chains D and E were changed to infinity (0 channels), then all 5 chains fired (panel B). Therefore, the inhibition of transverse propagation can be reversed by uncoupling the cells of the chains that failed.This finding surprised us at first. We were expecting either that there would be no effect on transverse transmission of excitation or that transverse transfer of excitation would be enhanced. But on further refection, the inhibition produced might be predicted. This is based on the fact that, with strong longitudinal coupling between cells, the entire chain of 5 cells must be simultaneously stimulated to threshold. Therefore, if the transverse transfer energy available is limited and is near threshold for a give chain, it is likely that some chains will fail to fire. Thus, the problem of strong coupling is not in the chains that are already excited, but rather in the quiescent adjacent chain which is in process of trying to become excited  and Rgj of 100 MΩ (100 channels); adding more channels (Rgj of 10 MΩ (1000 channels) or 1.0 MΩ (10,000 channels)) did not further decrease TPT. Similar results were found when the entire A-chain was simulated . Therefore, the dynamic behavior of the cardiac cell membrane was only a close approximation.We previously found (unpublished observation) that the transverse velocity was greater when size of the model was increased (eg, 3 × 4, 5 × 5, 7 × 7) up to a presumed maximum, indicating that the boundary conditions affected behavior of the model. Therefore, the present experiments should be repeated on larger-sized models. But we believe that the qualitative findings would be much the same. In addition, 2-dimensional activation maps should be made in the future to better elucidate how the wavefront spreads.These findings might have important clinical implications, especially for the genesis of arrhythmias in pathophysiological situations. Any pathology that altered the number of functioning gj-channels, not only would affect longitudinal velocity of propagation, but also the transverse propagation ability and velocity. Therefore, the genesis of some arrhythmias, e.g., the reentrant type, could be promoted under such conditions.


The hypothesis that human cancers express antigens that can be specifically targeted by cell mediated immunity has become a scientifically justifiable rationale for the design and clinical testing of novel tumor-associated antigens (TAA). Although a number of TAA have been recognized and it has been suggested that they could be useful in the immunological treatment of cancer, the complexity of human beings leads us to reflect on the need to establish new criteria for validating their real applicability. Herein, we show a system level-based approach that includes morphological and molecular techniques, which is specifically required to improve the capacity to produce desired results and to allow cancer immunotherapy to re-emerge from the mist in which it is currently shrouded. Although considerable advances have been made in terms of our molecular and cellular knowledge, for most human disease states a fundamental understanding of causal disease onset, disease mechanism and progression, and optimal treatment is still significantly limited.In part, this advancement has been hampered by our inability to fully and rapidly delineate complex cellular metabolic processes and molecular pathways.Organisms are complex self-organizing entities made up of such parts: organs, tissues, cells, organelles and ultimately molecules and atoms. One question that arises, concerns the relationship between the whole and its component parts. The issue at stake is sometimes called "the question of reduction" or "the problem of reductionism" .cause and effect.The inefficacy of contemporary science to describe biological systems, consisting of non-identical parts that have different and non-local interactions has tended to limit progress in the human healthcare. Many biological systems remain incomprehensible because their multifarious nature has been combined with a reductionist approach based on the linear conception of holistic multidimensional system level-based approach may provide new insights into the understanding of disease processes and mechanisms of action of therapeutical agents [The use, however, of a more l agents .Herein we aim to introduce a system level-based approach that includes morphological and molecular techniques for validating the appropriateness of using novel tumor-associated antigens (TAA) for clinical purposes. This approach might be easily implemented for identifying prognostic, diagnostic and alternative biomarkers.cost-effective) design of clinical trials [Finally, this type of analysis of appropriately designed cohorts might also provide a key to understanding the differences in patients who do or do not respond to any particular therapy. This information may be helpful for a more effective  that interrelate differently with each other to form networks of growing complexity. The concept of anatomical entities as hierarchy of graduated forms, and the increasing number of known structural variables, have highlighted new properties of organized biological matter and raised a series of intriguing questions. In order to understand biology at the system level, we need to examine the structure and dynamics of the functions of organisms rather than the characteristics of their constitutive isolated parts [However, although a number of TAA have been discovered and it has been suggested that they could be useful in the immunological treatment of cancer, the o define . Human bed parts -13.information provided by these approaches is limited by the fact that the phenomena observed at each level of anatomical organization have properties that do not exist at a lower or higher level: RT-PCR and qrt-PCR may offer a satisfactory qualitative/quantitative description of small-scale structures, but this is likely to be irrelevant when it comes to large-scale features. The above considerations, in conjunction with the complexity of tumor-host interactions within the tumor microenvironment caused by temporal changes in tumor phenotypes and an array of immune mediators expressed in the tumor microenvironment [The expression of TAA in biological materials has mainly been studied at the level of gene expression and gene level measurement by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis and the Quantitative real-time PCR (qrt-PCR) technology -17. Howeironment  might clsystem level-based approach  by ultra-structural morphological visualizations.• Mapping candidate antigen expression in all of the organs making up the apparatuses.• Mapping candidate antigen expression in all of the apparatuses making up the living organism.• Estimating the percentage of normal cells and their neoplastic counterparts expressing the candidate antigen.• Evaluating the dynamics of candidate antigen expression at the level of the cell cycle, the physiological status of the organism  and the process of aging.• in vitro laboratory results  and their in vivo application , and it is necessary to adopt a more complete experimental approach that forcefully includes both morphological and molecular techniques [In order to advance our knowledge in a currently widely debated field of investigation, a clearer distinction must be made between chniques .emergence in other words the appearance of unexpected structures and/or the occurrence of surprising behaviors in large systems composed from microscopic parts, whether physical or biological. By unexpected and surprising we mean structures and behaviors which are not intuitive and are not simply predictable.Translational science which is aimed to test, in humans, novel therapeutic strategies developed through experimentation  should bSince our understanding of complex human disease such as cancer, is still limited and pre-clinical models have shown a discouraging propensity ,6 to faihuman cancer, which takes into account the complexity of the human being as a system.Although the model presented here is based on a multidisciplinary system-level approach probably within the reach of only very large and multi-talented laboratories, it is aimed to introduce a different way of investigating The use of a holistic approach, which enables a more accurate selection of immunotherapeutic target antigens in the first phase of the experimental research, will reduce the notable fragmentation of the biological information in the post-genomic era, and will facilitate a more accurate transfer of the acquired knowledge to the bedside.energy, time, or resources for immunotherapeutic treatments and to allow cancer immunotherapy to re-emerge from the mist in which it is currently shrouded.Further, this new multidisciplinary approach is specifically required to improve the capacity to produce desired results with a minimum expenditure of