Mycobacterium tuberculosis have evolved strategies for coping with the pressures encountered inside host cells. The ability to coordinate global gene expression in response to environmental and internal cues is one key to their success. Prolonged survival and replication within macrophages, a key virulence trait of M. tuberculosis, requires dynamic adaptation to diverse and changing conditions within its phagosomal niche. However, the physiological adaptations during the different phases of this infection process remain poorly understood. To address this knowledge gap, we have developed a multi-tiered approach to define the temporal patterns of gene expression in M. tuberculosis in a macrophage infection model that extends from infection, through intracellular adaptation, to the establishment of a productive infection. Using a clock plasmid to measure intracellular replication and death rates over a 14-day infection and electron microscopy to define bacterial integrity, we observed an initial period of rapid replication coupled with a high death rate. This was followed by period of slowed growth and enhanced intracellular survival, leading finally to an extended period of net growth. The transcriptional profiles of M. tuberculosis reflect these physiological transitions as the bacterium adapts to conditions within its host cell. Finally, analysis with a Transcriptional Regulatory Network model revealed linked genetic networks whereby M. tuberculosis coordinates global gene expression during intracellular survival. The integration of molecular and cellular biology together with transcriptional profiling and systems analysis offers unique insights into the host-driven responses of intracellular pathogens such as M. tuberculosis.Intracellular pathogens such as  Mycobacterium tuberculosis on global health is undeniable, with ∼2 million deaths and ∼9 million new cases of tuberculosis each year. A key to the success of M. tuberculosis as a persistent, intracellular pathogen is its ability to survive for extended periods within professional phagocytes. Sustained growth within macrophage phagosomes requires avoiding or resisting antimicrobial mechanisms and adapting to replicate in a stressful, nutrient-restricted environment. Our understanding of the survival strategies, metabolism, and physiology of M. tuberculosis during intracellular growth remains incomplete. We employed multi-disciplinary approaches to gain new insights into adaptive responses that M. tuberculosis mobilizes to secure a productive infection. We simultaneously quantified replication and death rates, used electron microscopy to evaluate bacterial integrity, and determined the temporal changes in bacterial gene expression during a 14-day infection. By overlaying this temporal transcriptome dataset onto an extended Transcriptional Regulatory Network model, we identified regulatory pathways, stress responses, and metabolic adaptations activated during key physiological transitions over the 14 days of infection.The impact of  Mycobacterium tuberculosis (Mtb) has evolved successful strategies to survive in the human host and thwart the attempts of the immune system to eradicate it. The astounding morbidity and mortality caused by Mtb – with roughly 9 million new cases and 2 million deaths annually – is a testament to the success of this enduring pathogen. Its remarkable resistance to killing by both innate and acquired immune effectors and successful adaptation to constantly changing environments allows Mtb to maintain infections for decades. The ability of Mtb to survive and replicate within macrophages (Mφ) is central to the pathogenesis of tuberculosis Mtb survives its interaction with the Mφ Mtb survives better through escape into the cytosol Mtb must adapt to succeed.A considerable body of literature has been devoted to understanding how Mtb's survival is its ability to sense its environment and modulate genetic pathways controlling resistance mechanisms, metabolism, and other processes in a timely manner. The importance of gene regulation during intracellular survival and in vivo infection is illustrated by the attenuated phenotypes of numerous mutants in which key transcriptional regulators have been inactivated Mtb immediately following Mφ invasion Mtb within resting Mφ over the course of a 14-day infection. During this extended timeframe the bacteria appear to experience diverse and changing environments and adopt differing physiological states. Shortly after infection, the bacterial numbers decline indicating a period of acute stress as the macrophage attempts to eradicate the infection. This is followed by a second period during which the bacterial numbers remain relatively constant, presumably while the bacterium adapts to, and tries to modulate its environment. Finally, approximately 5–6 days post-infection (p.i.) the bacterial population increases, indicating that conditions to support survival and replication have been established. This model affords us unique access to unraveling Mtb's adaptation strategies to the changing stresses and environmental cues experienced during the establishment of a productive infection. We have exploited a range of technical approaches to define the growth states of the bacterium through the course of this 14-day infection, and to correlate these physiological states with transcriptional profiles of the surviving bacteria to identify genetic networks consistent with survival and the establishment of a productive infection.A characteristic essential for Mtb. To appreciate the adaptations required by Mtb to enter this replicative state one has to employ a temporal approach that accurately correlates bacterial numbers, including the assessment of death versus growth, with changing transcriptional profiles. We employed three different methods to probe the growth states of Mtb during a 14-day intracellular infection model.The establishment of a productive infection in the macrophage is not dependent merely on the short-term response following phagocytosis but the subsequent stages of adaptation that lead to a net increase in intracellular Mtb at a low multiplicity of infection (MOI) of 1∶1. At two-day intervals, infected monolayers were lysed and dilutions plated to determine viable colony forming units (CFU). Supernatants were also diluted and plated (prior to lysis of Mφ) to monitor extracellular bacilli that could contribute to reinfection. An initial 0.5 log decrease in cell-associated viable CFU at day 2 was followed by a period of minimal change (days 2–6) and then a more steady increase in CFU (days 6–14)  suggesting that re-infection is minimal.First, resting primary Mφ isolated from C57BL/6 mice were infected with ys 6–14) . This prMtb was also monitored by parallel transmission electron microscopy (TEM) analysis of fixed infected Mφ samples  to 10.3 Mtb/cell by day 4 (∼1.7-fold increase/day). From day 4 to day 14, the average bacterial burden increased at a notably slower rate (∼0.3-fold increase/day) (Mtb replication following Mφ invasion coincided with the marked decrease in viable CFU (The intramacrophage growth of  samples . Enumeraase/day) . Interesable CFU . This inMtb growth and death rates during long-term infection of resting Mφ based on the loss of an unstable replication clock plasmid Mtb retaining the clock plasmid pBP10 was determined by plating bacteria on media with or without 25 µg/ml kanamycin. The profile of pBP10 plasmid loss  and colocalize with colloidal gold as early as 2 hr p.i. .Consistent with previous observations ell wall . While idal gold , ∼25% of hr p.i. . At 2 daysosomes , consistmid data , and themid data  suggestsmid data  with up Mtb appeared to reside during long-term infections of Mφ, including “replicating” Mtb in typical tight niches, small fused P-L, and putative double membrane-bound autophagosomes that colocalized with gold . Fluorescent amplified RNA (aRNA) targets from each time-point were prepared from linearly amplified total Mtb RNA and hybridized against a 2 hr “no Mφ” control as previously described aprAB, hspX, bfrB, icl, groEL2, katG, whiB7) was conducted to confirm temporal array profiles  methodology , stress response regulation (sigB), anaerobic respiration , Fe-S cluster assembly , regulation of acetate and glyoxylate cycle metabolism (Rv0465c) , prpD (Rv1130)), and the starvation response (lat). This temporal profile indicates that cues driving these responses are likely most acute as the bacteria try to maintain rapid early intracellular growth amidst an array of host-derived pressures.The transcriptome of bacilli sampled every 2 days largely reflected this transition, with gene expression changes of maximal magnitude and complexity occurring at day 2 . We propet al. previously reported the intra Mφ gene expression of Mtb strain 1254 over a short-term infection out to 2 days p.i. Mtb to the phagosomal environment, the majority of which remain elevated for the duration of the 14-day infection. These included genes known to be involved in general stress responses , carbon metabolism , oxidative stress , iron storage (bfrB), hypoxia and nitrosative stress , and secreted antigens . This consistency with previously reported datasets from short-term infection models argues the general validity of the approach and the significance of the data from later time points in our extended infection model.Schnappinger Mtb CDC1551 genome whose intracellular expression has not been reported previously (aprABC), which showed rapid induction upon acid stress or Mφ invasion and is known to regulate carbon flux into cell wall lipids to support intracellular survival We also detected 137 additional genes upregulated at day 2 that were specific to this study including 17 members of the PhoPR regulon eviously . The difwhiB7 and adjacent ORF MT3290.2 at Day 2 followed by a return to baseline levels  will indicate the metabolic pathways and survival mechanisms required for establishment of an environment supportive to bacterial growth. For example, pckA, icl, ald, Rv0448c-0449c), KstR-dependent cholesterol metabolism (igr), secreted antigens (mpt83-Rv2874-mpt70), and regulators (Rv2359). In a previous study, we characterized the functional impact of genetic diversity on Mtb-host interactions and gene expression by analyzing the intracellular transcriptome profiles of a globally diverse panel of M. tuberculosis complex (MTC) clinical isolates The temporal changes in rplF, rplI, rplJ, rplV)  with a profile of early, sustained repression including numerous ribosomal genes  . This coMtb may encounter novel host-derived pressures at later time points or that prolonged growth within the phagosomal environment requires additional bacterial adaptation. This genelist included several transcriptional regulators  and fatty acid metabolism genes . In contrast to early responses likely triggered by immediate stress, Mtb may also encounter more insidious pressures linked to evolving metabolic requirements.Although transcript levels for the vast majority of genes were altered by day 2, we did identify a set of genes that exhibited a delayed “bell-curve” pattern of expression . This prIn addition to the temporal alterations in the transcriptional profile, we also examined transcription profiles of genesets linked to some of the more well-characterized environmental responses to probe how they matched the physiological transitions we have proposed occur during the 14-day infection. We have focused on the DosR regulon, regulation of carbon flux, and the response to pH, as some of the better characterized physiological themes.in vitro remained unchanged in resting Mφ phagosomes, perhaps reflecting distinct threshold levels of sensitivity to inducing signals. It is conceivable that all three known cues of DosRS/T signaling are acting in concert within the resting phagosome. Greiss assay analysis of supernatants from 14-day infected Mφ showed a spike in nitrate levels at day 2 p.i. that rapidly declined to baseline levels by day 4 (data not shown), implicating nitric oxide as a signal of early DosR regulon induction. Decreased intraphagosomal oxygen concentrations in macrophages dosR, dosS, and dosT  driving the response. Alternatively, this profile may reflect the staged adaptation of Mtb to phagosomal stresses such as hypoxia, NO, and/or CO. Balazsi et al. reported similar induction of the DosRS regulon followed by a downward shift at ∼day 8 in an in vitro hypoxia model et al.  and cholesterol is a key component of Mtb intracellular adaptation. We noted early, sustained induction of pckA (phosphoenolpyruvate carboxykinase) and concomitant repression of pca (pyruvate carboxylase)  (icl (discussed below). Previous studies have demonstrated the ability of Mtb to utilize cholesterol as a source of energy and carbon mce4igrMtb likely has ready access to cholesterol within resting Mφ  . pckA isynthase) , suggeststing Mφ . KstR isMtb is equipped with three pathways to detoxify and utilize propionyl-CoA as either a carbon source or building block for methyl-branched fatty acid components of the cell wall: methylcitrate cycle (MCC), methylmalonyl pathway (MM), and synthesis of methyl-branched fatty acids. To identify propionyl-CoA detoxification mechanisms activated during long-term intracellular survival, we overlayed temporal expression profiles of relevant metabolic genes (shown as heat maps) onto the pathway map , the bifunctional icl1 is also a 2-methylisocitrate lyase (MCL) that interacts with prpC (encoding methylcitrate synthase) and prpD (encoding methylcitrate dehydratase) in the MCC icl1, prpC, and/or prpD were unable to grow on propionate as a sole carbon source. In contrast to the normal growth of ΔprpCD Mtb in vivo, a Δicl1 strain was markedly attenuated in mice prpCD-generated toxic propionyl-CoA metabolites in the absence of MCL activity rather than the inability to utilize fatty acids due to interruption of the glyoxylate shunt. In a recent study, Griffin et al. confirmed the important role for the MCC enzymes prpCD regulated via Rv1129c for cholesterol utilization during intracellular survival A potentially deleterious side-effect of cholesterol catabolism and β-oxidation of odd-chain length fatty acids is the generation of toxic metabolites including propionyl-CoA and downstream intermediates p et al.. The conmutAB  transcript levels, we did not detect coordinated upregulation of the MM pathway during Mtb infection of resting Mφ. This may be due to inactivation of the MM pathway in our infection model due to the absence of vitamin B12, a cofactor required for MutAB activity Mtb (pks2-papA1 (SL) and pks3-pks3-papA3-mmpL10  upon internalization of Mtb by Mφ. We have previously linked the rapid activation of these loci within minutes of phagocytosis to acidification of the vacuole Other than a small increase in vity Mtb , we noteMtb for early intracellular gene induction pks2-papA1 (sulfolipid (SL) biosynthesis) and pks3-pks4-papA3  biosynthesis)  , suggestnthesis)  further whiB3 to phagosomal acidification aprABC (MT2466-MT2467-Rv2396), which exhibits early sustained induction, mediates intracellular survival via unknown mechanisms by influencing gene expression and lipid metabolism Mtb may rely on phagosomal acidification as a cue of Mφ invasion to coordinate the timely expression of intracellular virulence mechanisms. For example, acid induction of espABC (Rv3614c–Rv3616c), a locus required for ESX-1 mediated secretion of virulence factors espABC  underlying pathogen survival during the 14-day infection. This network-based approach incorporates extensive a priori information on Mtb gene regulation and network topology, combined with expression data, to assess network responses in surviving bacteria elicited by the Mφ.In addition to analyzing the transcriptional profiles according to temporal dynamics and known functional themes we also conducted a systems-level analysis to characterize the behavior of the Mtb TRN containing gene regulatory interactions extracted from both experimental and computational datasets i) literature mining; (ii) MtbRegList database iii) inference from orthology with Escherichia coliiv) We added orthology-based interactions inferred from the closely related Corynebacterium glutamicum available in the MycoRegNet database, which considerably expanded the TRN by adding 425 new interactions  Further, we expanded the TRN by adding 114 protein-DNA interactions discovered by a new bacterial one-hybrid reporter system termed TB1Hybrid vi) Finally, we performed operon-based network expansion, propagating a TF's regulatory effect to all members of the operon containing a given TG. The final expanded Mtb TRN contained 1133 genes (28% of the genome) and 1801 regulatory links, more than a half of which were experimentally determined  to quantify the expression change within the module (subnetwork) that we define as the total genes directly regulated by a given TF. A significant Cov-score indicates subnetwork response, when the expression levels of the subnetwork's gene members are either down- or upregulated during consecutive time points . Alternatively, simultaneous change of a TF's direct target genes may also be a surrogate of the TF's activity. Under this assumption, NetReSFun may recognize TFs that are “turned on” through posttranslational modifications such as phosphorylation and metabolite binding, but may or may not show increased expression levels themselves.We improved the earlier method called NetReSFun (Network Response to Step Functions), which identifies responsive TF-regulated subnetworks from time course microarray data The temporal map of network responses  depicts Bacillus subtilisStrikingly, the map reveals that the dynamic utilization of the TRN occurs in a defined pattern that can be mapped back to distinctive phases of intracellular growth. In the first 2 days p.i. - which corresponds to the stress phase in the growth curve of sigH caused reduced immunopathology and lethality in mice In contrast, after ∼6 days inside Mφ, we observed a reciprocal scenario where the TRN reflects a significant downmodulation of target genes, many of which had been induced immediately after invasion. This pattern is especially evident for a number of stress-responsive subnetworks that shift into downregulation during the slow growth phase, including RegX3, HspR and DosR. This “repressive” transcription phase indicates that the surviving bacteria have either adapted to stress, or they reside in a less hostile niche. For example, the SigH-controlled subnetwork displays a strong negative response only late in the time course (∼8 days and onwards). As a global regulator, SigH modulates the transcription of SigE and SigB, as well as its own promoter. Although SigH is not required for growth in Mφ, mutants lacking hspR strains exhibited attenuated growth in the chronic infection Importantly, we observed responsive subnetworks throughout the entire time-course, which indicates their importance for establishing productive infection. The presence of sustained responders such as HspR and DosR can have two possible implications. First, the opposite trends in the early phase of the infection (primarily upregulation) and later phases of infection (primarily downregulation) may indicate that the stress to which these modules respond initially is ameliorated at later time points. Alternatively, sustained responders may be necessary both to counteract initial phagosomal stress during the early phase of infection as well as for driving the persistor phenotype encountered in later phases of infection. This is the case of DosR, which is crucial for maintaining redox balance and energy levels during transitions into and out of dormancy-like conditions that perturb aerobic respiration, electron transport, or menaquinone pools Mtb adaptation within the intracellular compartment. For example, the Rv2034-controlled subnetwork (inferred from C. glutamicum orthology) contains multiple fadE homologs implicated in β-oxidation of fatty acids and redox homeostasis. Notably, Rv2034 was recently characterized as an activator of the phoP virulence regulator in mycobacteria Finally, TRN analyses revealed novel sustained responders that might be critical for Mtb gene expression during productive infection of macrophages.By overlaying the genome-scale temporal expression data onto the TRN, we revealed the activation of additional regulons during macrophage survival not readily apparent in our supervised analyses. Thus, by leveraging the behavior of multiple TG as a readout of TF activity, TRN analysis of time-course microarray data further enhances the ability to detect adaptive changes in Mtb transitions on its way to the establishment of a productive infection in its host macrophage. This analysis revealed several unexpected findings. Firstly, the initial phase of infection is marked by rapid bacterial replication coupled with effective bacterial killing by the macrophage. This is a period of marked stress for Mtb, which is illustrated by the greatest transcriptional response with respect to both up-regulated and down-regulated genes. Subsequently, the rate of replication slows and the bacterial number appears constant or at equilibrium, during which period the expression of many genes returns closer to control levels, whilst the divergent level of expression of others is sustained. Finally, the bacterial numbers start to increase indicating that the rate of replication exceeds that of death. At this time there is a marked down-regulation of many of the genes linked to general stress supporting the contention that Mtb has entered into a productive phase of infection characterized by enhanced intracellular survival. This is an important functional framework on which to hang the transcriptional profiles to determine which responses and/or metabolic themes impact which phase of infection.The process of infection is extremely dynamic as both host and pathogen seek to respond to the stimuli that they sense at their interface. In the current study we applied multiple analytical tools to establish a link between the transcriptional responses and physiological states through which Mtb TRN also highlights stress responses and survival mechanisms deployed during distinct phases of host interaction. Among other things, our results indicate adaptive changes in lipid and energy metabolism akin to those observed in various dormancy models. Based on this, it is tempting to speculate that early infection of resting macrophages may serve to prepare Mtb for conditions encountered within granulomas after the onset of the adaptive immune response. Detailed understanding of the sensory and regulatory pathways required for Mtb virulence remains rudimentary at best. This point is illustrated by the large number of “genes of unknown function” which are actively regulated during intracellular survival.Our examination of dynamic alternations of gene expression across the Mtb cells sensing and responding in concert to phagosomal cues in the intracellular environment. Clearly, the microarray profiles in this study capture an average behavior over time of a population of intracellular bacilli that, as we have shown, exist in distinct vacuolar niches and presumably metabolic states. It could be argued that the changes in gene expression represent the minority of Mtb that fail to block P-L fusion mounting dramatic stress responses. However, our data appear to be inconsistent with this view. For example, we know that Mtb in P-L encounter Fe-deplete conditions whereas the average behavior of Fe-responsive signature genes reflects an Fe-replete environment (data not shown). Alternatively, the apparent up- and down-regulation may be due to the selective enrichment of pre-existing phenotypic variant cells with randomly upregulated stress response subnetworks, similar to the cell-to-cell heterogeneity reported by Aldridge et al.Mtb are altering gene expression in response to host-derived cues.In the Mtb to survive across diverse host environments experienced by each individual bacterium. To address this, we have begun to exploit fluorescent reporter strains responsive to specific environmental cues to gain a high-resolution view of Mtb intracellular adaptation and cell biology. Interestingly, the expression of an acid-inducible locus required for normal intracellular survival, aprABC, is expressed at distinct levels by individual bacilli within the same host cell Mtb-host interactions and pathogenesis has yet to be determined. Previous work has shown that stochastic variation  can aid survival in stressful environmental conditions Thus, our data suggest that the dominant transcriptional profiles highlighted here represent the adaptive responses of the majority population. Analysis at the single-cell level will be required to explore the strategies employed by in vivo survival mechanisms, however, there are some limitations to this methodology that can be addressed by transcriptional profiling. Array-based mutant screens such as Transposon Site Hybridization (TraSH) pckAicl  with Mtb CDC1551. After 2 hr (t = 0) and at two day intervals up to 14 days, intracellular Mtb released from monolayers by lysis with ddH2O+0.05% Tween-80 were serially diluted, and plated on 7H10+cycloheximide agar. CFU were enumerated after ∼3 weeks incubation at 37°C. The overall integrity of Mφ monolayer was verified by microscopy.To monitor the survival/growth of Mtb replication and death during Mφ infection, a replication clock plasmid, pBP10, was used (generous gift of Dr. David R. Sherman). As previously described Mtb CDC1551 harboring pBP10 (introduced by electroporation) were conducted as described above except that aliquots were plated on 7H10+cycloheximide agar both with and without 25 µg/ml kanamycin. Based on the loss of kanamycin resistance over time, rates of replication and death were quantified using the mathematical model of Gill et al.In order to measure the dynamics of Mtb infections for survival assays and microarray analysis, confluent monolayers of Mφ were infected with Mtb CDC1551 at a low MOI (1∶1) as described above. At select times p.i. , samples were fixed in buffered glutaraldehyde solution , rinsed with 0.1M sodium cacodylate buffer (see above), post-fixed with 1% osmium tetroxide , rinsed with buffer, soaked in 1% aqueous uranyl acetate, and then rinsed with water. Samples were then dehydrated in a graded ethanol and propylene oxide series, followed by gradual infiltration of Spurr's resin. Blocks were then polymerized and ultrathin sections (∼70 nm) were cut and contrasted with both lead citrate and uranyl acetate. In independent experiments to determine phagosome-lysosome fusion by the colocalization of Mtb with colloidal gold, at various time-points after infection  infected monolayers were pulsed with 15 nm colloidal gold (Aurion) for 2 hr, washed, and then chased for 45 min in infection media before fixation as above. In assessment of the morphology of intracellular Mtb, bacilli were considered intact if they maintained a rod shape  or circular shape (cross sections), if ultrastructural organization and electron opacity of the cytoplasm was preserved, and if no breaks in the cytoplasmic membrane or cell wall were detected. Otherwise, bacilli were counted as damaged.In parallel with Mφ-Mtb including RNA isolation, linear amplification, and hybridization has been described previously M. tuberculosis CDC1551 from 2 hr up to 14 days . In controls, aliquots of the same bacterial samples were incubated in flasks without a Mφ monolayer for 2 hr. Addition of guanidine thiocyanate-based lysis buffer selectively lysed Mφ and stabilized bacterial RNA while leaving mycobacteria intact. Pelleted bacilli were lysed in 65°C Trizol using a BeadBeater and 0.1 mm silicon beads. Total RNA was isolated from Trizol lysates by chloroform extraction and Qiagen RNeasy column purification. To generate array targets, 250 ng of total RNA was amplified using the MessageAmp-II Bacteria RNA Amplification system (Ambion). Amino-allyl UTP was incorporated into aRNA during transcription to allow labeling with Alexa dyes.The methodology for transcriptional profiling of intramacrophage 2O and isopropanol. Labeled targets were denatured at 95°C for 5 min, cooled to 60°C, and hybridized to microarrays at 45°C for 16–18 hr. Following hybridization, arrays were washed and processed for scanning as described previously Amino-allyl modified aRNA were labeled with Alexa Fluor 555 and Alexa Fluor 647 (Invitrogen) and purified using a MegaClear kit (Ambion). 10 µg of Alexa-labeled aRNA from paired samples was dried and resuspended in 75 µl of hybridization buffer . Slides were prehybridized for 1 hr in 25% formamide, 5× SSC, 0.1% SDS, 1% BSA and washed with HExtraction and analysis of Differential Gene Expression) methodology of Storey et al. to identify time-dependent transcriptional changes Microarrays were scanned with a GenePix 4000B instrument  with preliminary image analysis, spot intensity determination, background measurements, spot quality assessment and flagging conducted using Imagene software . Poor quality spots with signal intensities less than three standard deviations above background were excluded from further analysis. Genes that were not flagged as Present in at least 14 of 16 slides were omitted from further analyses. Subsequent normalization, statistical analysis, and visualization of array data were performed with Genespring 7.3 (Agilent). We utilized the EDGE . The density estimation of the c.d.f is done in a standard fashion by assigning probability mass 1/1000 for each observed random score; linear interpolation is applied to transform the discrete I to all other subnetworks, is given byWe modified the tool NetReSFun (89) in order to identify TF-regulated subnetworks sequentially responsive during TB lifespan in the macrophage environment. Consider a set of oral map , we firsFigure S1Quantitative electron microscopy analysis of long-term Mtb-macrophage interactions. At each timepoint, all visible intracellular Mtb in 100 macrophages were enumerated and categorized based on morphological criteria as described in Mtb. The proportion of intact, morphologically normal bacilli to damaged Mtb remained relatively constant over the 14-day infection. (B) Change in distribution of Mtb burden over time. The number of Mtb per macrophage increases steadily over time. Note, the large set of cells in which no Mtb were detected at early time points likely reflects the absence of detectable bacilli within the plane of section rather than uninfected cells.(TIF)Click here for additional data file.Figure S2Mtb occupies heterogeneous intracellular niches during macrophage infection. (A) Electron microscopy image shows dividing bacilli in “tight” vacuole, day 10 p.i. (B) Morphologically intact Mtb in spacious lysosome-like compartments surrounded by granular debris at day 6 p.i. This suggests Mtb can survive at least for a time in vacuoles that have fused with lysosomes. (C) Mtb surrounded by double-membrane (arrow) vacuole containing colloidal gold, consistent with an autophagosome (day 14 p.i.).(TIF)Click here for additional data file.Figure S3Temporal expression profiling reveals novel genes differentially regulated within macrophage phagosomes. (A) Gene tree (Euclidean distance measure) of 137 genes significantly upregulated at 48 hr p.i. novel to this study (compared to expression profiles at same time point reported in (TIF)Click here for additional data file.Figure S4Long-term intracellular expression of the core transcriptome. Gene tree showing the distinct expression profiles of 215 genes comprising a core intracellular transcriptome previously defined based on their conserved presence and induction across a diverse panel of Mtb clinical isolates at 24 hr p.i. of resting macrophages (TIF)Click here for additional data file.Figure S5Genes of the Enduring Hypoxic Response (EHR) exhibit distinct expression patterns during long-term adaptation within macrophages. Gene tree showing the transcriptional patterns of genes comprising the EHR, as identified by Rustad et al.(TIF)Click here for additional data file.Figure S6Selective up-regulation of members of the KstR-dependent cholesterol regulon during intracellular growth. (A) Gene tree showing the relative transcript levels of genes proposed to be directly controlled by KstR Click here for additional data file.Figure S7Regulation of cell wall synthesis and composition in response to phagosomal cues. Gene trees showing the sustained downregulation of genes involved in mycolic acid synthesis (A) and pthiocerol dimycocerosate (PDIM) (B).(TIF)Click here for additional data file.Figure S8Regulation of ESX secretion systems during intracellular growth.  Early induction of the ESX-1 secretion system including the main ESX-1 locus (A) as well as the EspR regulator and accessory factors Rv3614c-3616c encoded outside the RD-1 locus (B). These gene products function coordinately to facilitate secretion of ESAT-6/CFP-10 complex. (TIF)Click here for additional data file.Figure S9The enlarged Mtb Transcriptional Regulatory Network (TRN). (A) Number of regulatory links from each data source and corresponding overlaps. Links with experimental evidence originate from literature, as well as from the MtbRegList database and the TB1H assay. (B) Distribution of interactions based on their inference method. (C) Overview of the TRN, depicting protein-DNA interactions as edges linking TFs (blue triangles) to TGs (yellow circles).(TIF)Click here for additional data file.Table S1Genes with peak induction at Day 2 followed by gradual decline toward control levels.(XLSX)Click here for additional data file.Table S2Genes significantly upregulated (>1.5X) at 48 hr novel to this study .(XLSX)Click here for additional data file.Table S3Temporal expression profile of “MT genes”, predicted ORFs annotated in strain CDC1551 genome not originally annotated in the H37Rv genome. Note, the gene order does not match the accompanying gene tree in (XLSX)Click here for additional data file.Table S4enes with Early, transient induced temporal profile. (XLSX)Click here for additional data file.Table S5(XLSX)Click here for additional data file.Table S6Subsets of previously identified core intracellular transcriptome display distinct temporal expression profiles during 14-day macrophage infection.(XLSX)Click here for additional data file.Table S7 (black) - Genes with Early Repressed, Sustained temporal profile.(XLSX)Click here for additional data file.Table S8enes with Steady, sustained induced temporal profile.(XLSX)Click here for additional data file.Table S9enes with Steady, sustained repression temporal profile.(XLSX)Click here for additional data file.Table S10enes with Delayed induced temporal profile.(XLSX)Click here for additional data file.Table S11enes with hspX-like temporal profiles.(XLSX)Click here for additional data file.Table S12M. tuberculosis Expanded Transcription Regulatory Network (TRN).(XLSX)Click here for additional data file.


Sc) particles and their fragmentation, a process that provides new replication centers. The current study introduced an experimental approach that employs Protein Misfolding Cyclic Amplification with beads (PMCAb) and relies on a series of kinetic experiments for assessing elongation rates of PrPSc particles. Four prion strains including two strains with short incubation times to disease (263K and Hyper) and two strains with very long incubation times (SSLOW and LOTSS) were tested. The elongation rate of brain-derived PrPSc was found to be strain-specific. Strains with short incubation times had higher rates than strains with long incubation times. Surprisingly, the strain-specific elongation rates increased substantially for all four strains after they were subjected to six rounds of serial PMCAb. In parallel to an increase in elongation rates, the percentages of diglycosylated PrP glycoforms increased in PMCAb-derived PrPSc comparing to those of brain-derived PrPSc. These results suggest that PMCAb selects the same molecular features regardless of strain initial characteristics and that convergent evolution of PrPSc properties occurred during in vitro amplification. These results are consistent with the hypothesis that each prion strain is comprised of a variety of conformers or ‘quasi-species’ and that change in the prion replication environment gives selective advantage to those conformers that replicate most effectively under specific environment.Prion replication is believed to consist of two components, a growth or elongation of infectious isoform of the prion protein (PrP Sc), which is capable of propagating itself in an autocatalytic manner by recruiting and converting the normal, cellular form of the prion protein (PrPC) in vitro have been inflaming debate about the biochemical nature of the prion infectious agent. In 2001, Soto and colleagues introduced the first experimental approach, referred to as Protein Misfolding Cyclic Amplification (PMCA), that permits amplification of mammalian prions in vitroin vitro were produced using PMCA Prion diseases are a group of fatal age-dependent neurodegenerative maladies that can either arise spontaneously or via transmission of a prion infectious agent Sc particles into smaller fragments, whereas the incubation intervals between sonication cycles are believed to be required for the growth or elongation of PrPSc particles. Co-factors including RNA and polyanions were found to stimulate prion conversion in PMCA Despite significant advances brought to the field with the development of PMCA, our understanding of the mechanism underlying prion replication remains limited. PMCA consists of repetitive cycles of sonication and incubation Sc properties in crude brain homogenate, it is difficult to gain mechanistic insight into prion replication. What are the rate limiting steps in PMCA/PMCAb? Is a PrPSc population transformed during PMCA/PMCAb? Does PMCA/ PMCAb selectively amplify PrPSc particles with certain physical features? While previous studies focused on characterization of strain-specific conformational stability and aggregation states of PrPScSc and, specifically, its elongation rate. Four prion strains, two of which cause disease within very short incubation times (263K and Hyper (HY)), while the other two induce it within very long incubation times (SSLOW and LOTSS) were tested. SSLOW and LOTSS are two synthetic strains that were generated in Syrian hamsters by inoculating amyloid fibrils produced from hamster full-length recombinant prion protein Sc growth or elongation rate was strain-specific. Furthermore, the elongation rates of PrPSc populations were found to increase after serial PMCAb for all four strains. These results suggest that PMCAb selects the same physical features regardless of the strain initial characteristics and that convergent evolution of PrPSc properties occurred during in vitro amplification.Due to technical limitations in assessing PrPSc in PMCA or PMCAb is believed to involve two alternating steps: (i) sonication-induced fragmentation of PrPSc particles and (ii) their elongation or growth through recruiting and converting PrPC. In standard PMCAb format, the sonication cycles are separated by 30 minute incubation intervals, during which PrPSc particles grew in size. To test whether the PrPSc elongation rate is strain-specific, we designed a series of kinetic experiments, in which the sonication conditions were kept constant, whereas the length of incubation intervals between the sonication cycles was reduced from 30 minutes to 10 or 5 minutes  digestion followed by Western blot  but not at high PrPSc intensities.For this study, we selected four strains that represent the two extremes of prion disease phenotypes, but are known to replicate in PMCAb with reasonably high efficiency. 263K and HY cause disease within very short incubation times, whereas SSLOW and LOTSS are characterized by very long incubation times of more than 300 days ern blot . Notablymembrane . The splTwo different formats were employed for presenting the same sets of data: strain-specific amplification yield was plotted as a function of cycle number  or as a Sc.For HY, only a slight dissociation between the three kinetic curves collected at 30, 10 or 5 min incubation intervals was observed . In factSc than those for HY, an indication that 263K PrPSc elongates with a slower rate than HY PrPS. For SSLOW, the amplification efficiency at both 10 and 5 minute intervals were significantly lower than that at 30 minute intervals  reactions. In general, PrPSc subjected to several PMCAb rounds showed more robust and efficient amplification than brain-derived PrPSc. To elucidate these changes in more detail, brain-derived PrPSc from animals inoculated with HY, 263K, SSLOW or LOTSS were subjected to six standard sPMCAb rounds, then resulting PMCAb-derived PrPSc was used in a series of kinetic experiments with 30, 10 or 5 minute incubation intervals as described above  were found to display higher elongation rates than the strains with long incubation times (SSLOW and LOTSS). However, the potential relationship between elongation rates and incubation time should be considered with a great caution, because an increase in elongation rates for PMCAb-adapted PrPSc does not lead to shorter incubation times.In previous studies, significant effort has been spent in an attempt to establish a relationship between incubation time to disease and PrPThe current studies illustrated that a high yield in prion amplification could be achieved by considerably shortening the length of incubation intervals in PMCAb, at least for some strains. For optimal results, the length of incubation intervals should be optimized individually for each strain. Despite of such inconvenience, the approach introduced by the current study is worthy of effort in developing rapid prion detection assays.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the University of Maryland, Baltimore .Hyper scrapie brain homogenate was kindly provided by Richard Bessen ; 263K and 10% normal brain homogenate (NBH) were kindly provided by Robert Rohwer . SSLOW and LOTSS scrapie brain homogenates were prepared using animals from the third passage of SSLOW and LOTSS 2+-free and Mg2+-free PBS, pH 7.5, supplemented with 0.15 M NaCl, 1.0% Triton and 1 tablet of Complete protease inhibitors cocktail  per 50 ml of conversion buffer. The resulting 10% NBH in conversion buffer was used as the substrate in PMCAb reactions. To prepare seeds, 10% scrapie brain homogenates in PBS were serially diluted in conversion buffer.PMCAb procedures have been described in detail elsewhere To prepare PMCAb-derived seeds, six standard sPMCAb rounds were conducted for each strain. The standard round consisted of 30 sec sonication pulses delivered at 50% or 60% efficiency applied every 30 min for 24 hours, which is 48 cycles. 10-fold dilution between serial rounds was used for all strains. To seed the kinetic experiment, the products of the 6th round of standard sPMCAb reactions were diluted 100-fold for 263K or HY and 30-fold for SSLOW or LOTSS.Sc replication, the sonication programs consisted of 30 sec sonication pulses delivered at 50% to 60% efficiency applied every 30 min, 10 min or 5 min in experiments with 30 min, 10 min or 5 min incubation time intervals, respectively. Aliquots were taken after 8, 16, 24, 48 or 72 cycles. To analyze PMCAb products, 10 µl of each sample were supplemented with 2.5 µμl SDS and 2.5 µl proteinase K (PK), to a final concentration of SDS and PK of 0.25% and 50 µg/ml, respectively, followed by incubation at 37°C for 1 hour. The digestion was terminated by addition of SDS-sample buffer and incubated for 10 min at 100°C. Samples were loaded onto NuPAGE 12% BisTris gels, transferred to PVDF membrane, stained with 3F4 antibody, and the Western blot was developed with Supersignal West Pico Chemiluminescent Substrate kit .For all PMCAb reactions, samples in 0.2 ml thin-wall PCR tubes  supplemented with 3 Teflon beads  were placed in a floating rack inside a Misonix S-4000 microplate horn filled with 350 ml water and maintained at 37°C inside the sonicator chamber. To monitor kinetics of PrPSc amplification yield, the signal intensity of PrPSc seeds measured at PMCAb cycle #0 was subtracted from the signal intensity measured as a function of PMCAb cycle number, and the resulting signal intensity were normalized relative to undigested BH.Western blot signal intensity was assessed by densitometry and quantified using ImageJ software . To calculate PrPFigure S1Western blot of 263K brain material (0.3%) treated with 50 μg/ml PK. After PK digestion, increasing concentrations of inactivated PK  were added to samples prior to SDS-PAGE. Arrows marks a blind spot from PK that cuts dyglycosylated PrPSc band in half.(TIF)Click here for additional data file.


Myosin Va was localized in nNOS-positive nerve terminals and was co-immunoprecipitated with nNOS in both GF and CCP. In comparison to C57BL/6J wild type (WT) mice, electrical field stimulation (EFS) of precontracted smooth muscles of GF and CCP from DBA animals showed significant impairment of nitrergic relaxation. An NO donor, Sodium nitroprusside (SNP), caused comparable levels of relaxation in smooth muscles of WT and DBA mice. These normal postjunctional responses to SNP in DBA tissues suggest that impairment of smooth muscle relaxation resulted from inhibition of NO synthesis in prejunctional nerve terminals. Our results suggest that normal physiological processes of relaxation of gastric and cavernosal smooth muscles that facilitate food accommodation and penile erection, respectively, may be disrupted under conditions of myosin Va deficiency, resulting in complications like gastroparesis and erectile dysfunction.The intracellular motor protein myosin Va is involved in nitrergic neurotransmission possibly by trafficking of neuronal nitric oxide synthase (nNOS) within the nerve terminals. In this study, we examined the role of myosin Va in the stomach and penis, proto-typical smooth muscle organs in which nitric oxide (NO) mediated relaxation is critical for function. We used confocal microscopy and co-immunoprecipitation of tissue from the gastric fundus (GF) and penile corpus cavernosum (CCP) to localize myosin Va with nNOS and demonstrate their molecular interaction. We utilized  Nitric oxide (NO) produced by splice variants of the enzyme neuronal nitric oxide synthase (nNOS) has been shown to be a major inhibitory neurotransmitter at smooth muscle neuromuscular junctions. Smooth muscle relaxation facilitated by NO released from nerve varicosities subserves essential physiological functions in many organ systems that are as diverse as gastric motility and penile erection de novo and released on demand from nitrergic varicosities During nitrergic neurotransmission, NO is synthesized in vitro NO production as well as nitrergic inhibitory junction potentials in the stomach are significantly diminished While nNOS tethering to the membrane has been well studied, the mechanisms involved in the translocation of nNOSα from the cytosol to the varicosity membrane have not been fully clarified. It was reported that PIN (protein inhibitor of nNOS), also identified as LC8 (light chain 8 kDa), may be involved in the transport of cytosolic nNOSα to varicosity membranes Analogous to its role in the stomach, nitrergic neurotransmission provokes smooth muscle relaxation in the corpus cavernosum of the penis (CCP). Relaxation of cavernosal smooth muscle permits filling of the CCP with blood leading to penile erection The purpose of the present study was to determine the functional role of myosin Va in nitrergic neurotransmission in representative, yet distinct smooth muscle systems. Therefore, nitrergic nerve mediated smooth muscle relaxation was compared in GF and CCP of normal and myosin Va-deficient mice.The study protocol was conducted according to the recommendations in the Guide for the Care and Use of Laboratory Animals of the NIH and approved by the Institutional Animal Care and Use Committee of VA Boston Healthcare System (#101W).Male DBA mice  were used as models of myosin Va deficiency. DBA mice lack cellular myosin Va due to an inherited frameshift mutation in the MYO5A gene, induced by a proviral/inactivating insertion Tissue samples from the GF of the stomach and the corpora cavernosa of the penis (CCP) were harvested from WT mice immediately after euthanasia, embedded in cryoprotective compound and quickly frozen in dry ice. Tissue sections were cut on a cryostat (12 µm), fixed in cold acetone (10 minutes), and incubated for 1 hour in blocking solution . After blocking, sections were first incubated overnight at 4°C with rabbit anti-myosin Va primary antibody . For detection of primary antibodies from the same host species, tissue sections were incubated with excess Fab fragment goat anti-rabbit IgG  to present the first antibody as a different species. A fluorescent-conjugated donkey anti-goat secondary antibody  was used to detect the first primary antibody. After extensive washing, the same sections were incubated overnight with anti-rabbit nNOS, (1∶100 Santa Cruz), followed by fluorescent-conjugated secondary antibody (Alexa Fluor donkey anti-rabbit 568) for 1 hour at RT. Control slides were prepared as described above with the omission of primary antibodies. Sections were examined using a laser scanning confocal microscope (Zeiss LSM 710). High resolution images were acquired sequentially from separate channels using Zen Imaging software.The molecular interaction between Myosin Va and nNOS in WT fundus and CCP was determined by co-immunoprecipitation (IP) experiments. The mucosa was removed from the GF by microdissection. Tissues were lysed in IP extraction buffer  supplemented with 100 mM NaCl, 2 mM MgCl2, 1 mM DTT, and protease inhibitors for 15 min at 4°C. After incubation, homogenates were centrifuged at 3000×g for 5 minutes and the supernatant representing the total protein lysate was collected. Protein concentrations were determined using the bicinchoninic acid protein assay (BCA) by measuring the absorbance at 280 nm with a biophotometer (Eppendorf). Anti-nNOS antibody, was covalently immobilized onto the surface of M-270 epoxy magnetic beads  by incubating the antibody-bead complex for 24 hrs under agitation at 37°C. Equal amounts of the antibody-coupled beads (1.5 mg per reaction) were added to protein lysates from both WT fundus (1.7 mg/ml) and CCP (3.4 mg/ml) and incubated under agitation for 1 hour at 4°C. After incubation, the beads with immobilized proteins were collected by placing the tubes in a magnetic field  and the remaining supernatants were stored. Immunoprecipitated complexes were eluted from beads by incubation in EB-buffer (Invitrogen) for 5 minutes at RT and separated from the antibody-coupled beads by magnet. Total lysates (15 µg), purified protein complexes and supernatants from the fundus and CCP were resuspended in sample loading buffer, boiled for 5 minutes, loaded onto a tris-acetate SDS polyacrylamide gel and separated by electrophoresis. Proteins were then transferred to a 0.45 µm pore nitrocellulose membrane (Invitrogen). Non-specific binding was inhibited by incubating membranes in Tris Buffered Saline (TBS) with 5% dry milk for 1 hour. Membranes were incubated overnight at 4°C with primary antibodies for myosin Va or nNOS. Unbound antibody was removed by extensive washing with TBS containing 0.05%Tween 20. Membranes were incubated for 1 hour at RT with horseradish peroxidase-conjugated secondary antibody (Santa Cruz). After washing, the membrane was incubated with chemiluminescence substrate , and immunoreactive bands were visualized by exposure of membrane to radiographic film (Kodak Biomax).2 asphyxiation, animals were weighed and the stomach and the penis were excised and placed in cold Kreb’s solution . The stomach was opened by a longitudinal incision along the greater curvature, and the gastric mucosa was removed from the fundic portion of the organ by stereoscopic micro-dissection. Four longitudinal smooth muscle strips from the fundus were obtained from each animal. The penis was isolated from the mid-shaft to the base of the crura. After removing the fascia and corpus spongiosum, the septum was cut longitudinally to separate the two corpora cavernosa. Cavernosal smooth muscle was exposed by removing the tunica albuginea. At the end of the experiment, the weight of each tissue from the GF and CCP was recorded after blotting on filter paper.After euthanasia induced by CO2+5%CO2). One end of each strip was attached to a fixed hook, and the other end to a force transducer measuring isometric tension (Grass Technologies). Smooth muscle strips from both GF and CCP were stretched under 0.5 grams of tension and equilibrated for 1 hour. Force measurements were displayed on a strip chart recorder (Gould) and digitally acquired by computer (WinDaq Software). After equilibration, tissue strips from the fundus were contracted by addition of carbachol  to the organ bath and contractile responses in WT and DBA animals were compared. Contractile responses of muscle strips from WT and DBA mice to high extracellular potassium (KCl 120 mM) were also compared. In the CCP, pre-contraction of smooth muscle was achieved by addition of phenylephrine . Guanethidine (1 µM) was added to inhibit noradrenergic transmission Fundus and CCP smooth muscle tissues were isolated from DBA and WT mice. Each end of the tissue was tied with silk, and transferred to an organ bath containing Kreb’s solution maintained at 37°C and continuously gassed with carbogen  delivered by platinum electrodes connected to a stimulator (Harvard Apparatus). In smooth muscle tissues obtained from WT, the effect of EFS-induced relaxation in the GF and CCP was investigated in the presence of NOS inhibitor L-nitro-L-Arginine Methyl Ester . The direct effect of nitric oxide on smooth muscle relaxation was examined in both GF and CCP by exposing pre-contracted tissue to submaximal concentrations of NO donor sodium nitroprusside .The amplitude of contractile and relaxation responses were reported as the difference in tension (expressed in Newton) before and after the stimulus and normalized by the weight of the tissue strip (expressed in grams). EFS induced relaxation responses from the fundus were also expressed as a percent change from the active tension achieved by CCh pre-contraction. Data are reported as mean ± SEM. Differences in means for the contractile or relaxation responses to agonists and EFS before and after L-NAME were analyzed by paired student’s t test. Comparison of contractile or relaxation responses between WT and DBA were analyzed by unpaired t-test followed by Holm-Sidak post-hoc analysis. P<0.05 was considered significant.s).Myosin Va immunoreactivity was detected in both gastric and penile tissues. Myosin Va staining was specifically localized within nNOS immunoreactive nerve varicosities coursing through smooth muscle bundles in both tissues figure 1Smooth muscle strips from the fundus of both WT and DBA contracted after the addition of either KCl or CCh. The contractile responses induced by KCl were similar . However, the contractile response to CCh was significantly lower in DBA (2.14±0.18 N/g) compared to WT fundus . This reduced carbachol response was not further investigated; however, it is possible that it may be due to impaired membrane localization of cholinergic receptors in the smooth muscle since myosin Va has been shown to regulate membrane localization of cholinergic (nicotinic) receptors in striated muscle In WT animals, carbachol pre-contracted tissue relaxed in response to EFS in a frequency dependent manner, reaching maximal relaxation at frequencies between 20 and 30 Hz. Administration of L-NAME (10 µM), a non-selective NOS inhibitor, had no effect on the amplitude of CCh-induced pre-contraction (maximum amplitude 3.206±0.36N/g) but significantly reduced the relaxation induced by EFS. As shown in In the DBA mice, EFS also produced frequency dependent relaxation; however, this relaxation response was significantly lower than that in WT at all frequencies of stimulation figure 3The relaxation response induced by EFS in the presence of L-NAME treatment in WT mice was compared with the nerve-mediated relaxation in DBA mice. The suppression of nerve mediated relaxation by L-NAME in WT mice at 20 Hz was not significantly different from the decreased relaxation response in DBA relative to WT (68.9±4.8% vs 67.3±6.5%). These observations suggest that nerve mediated nitrergic relaxation in GF was largely abolished in myosin Va deficient DBA mice.The relaxation responses in the fundus elicited by the addition of the NO donor SNP in CCh pre-contracted strips were not different between tissues of WT and DBA mice figure 4In penile cavernosal smooth muscle tissue, phenylephrine (PE) caused a stable, tonic contraction. The amplitude of contractile responses induced by PE was not significantly different between strains (0.45±0.09 N/g and 0.32±0.02 N/g respectively in WT and DBA).Pre-contracted CCP muscle strips from WT mice relaxed in response to EFS in a frequency dependent manner. Administration of L-NAME did not affect the amplitude of PE-induced pre-contraction in WT (0.33±0.05 N/g) but significantly reduced nerve-mediated relaxations at all the frequencies tested Figure 5EFS-induced relaxation responses in DBA CCP tissue were significantly lower than those in WT CCP tissue at all frequencies Figure 6Cavernosal tissue from both WT and DBA mice, pre-contracted with PE, relaxed to the same degree in response to the NO donor SNP (1 µM) Figure 7To determine whether myosin Va differentially affected nitrergic relaxation in GF relative to CCP, we compared the NO-mediated relaxation response with the myosin Va-dependent relaxation response. The L-NAME sensitive relaxation response in WT mice relative to the untreated response was similar in GF compared to CCP . The myosin Va dependent relaxation response, determined from the difference in relaxation between DBA and WT as a percent of the WT response, was significantly lower in CCP (38.9±5.8%) compared to GF . Since the total NO mediated relaxation response in CCP was not different than in GF, this finding suggests that nitrergic relaxation may be more sensitive to loss of myosin Va in GF than in CCP.The present study demonstrates an important role for myosin Va in nitrergic neurotransmission. We showed, using disparate smooth muscle neuromuscular junctions, that: 1) myosin Va localizes in nitrergic nerve fibers in GF and CCP; 2) nNOS interacts with myosin Va in both GF and CCP; 3) smooth muscle relaxation in response to nitrergic nerve stimulation is suppressed in DBA mice in both the GF and CCP due to impaired synthesis of NO rather than defective post-junctional NO signaling.The loss of smooth muscle relaxation in response to neuronal stimulation in DBA mice is consistent with our previous electrophysiological studies showing loss of inhibitory junction potentials in these animals, despite normal hyperpolarization responses to exogenous NO donors In the penis, neurogenic NO is fundamental to relaxation of cavernosal smooth muscle and initiation of erection The myosin Va dependent relaxation response induced by nerve stimulation was a smaller fraction of the WT response in CCP tissue compared to the relative responses in the GF. This difference was not due to a disparity in the overall extent of nitrergic relaxation in gastric and penile smooth muscle, as the nerve-mediated relaxation in the two tissues was similarly and effectively suppressed by a nonselective NOS inhibitor, L-NAME. These observations suggest that a portion of nerve-mediated nitrergic transmission in CCP may be independent of myosin Va. The larger residual nitrergic relaxation in CCP likely results from acetylcholine mediated eNOS activation The equivalent postjunctional responses of WT and DBA stomach and penis to exogenous NO suggest that the impaired nitrergic relaxation induced by EFS in DBA mice is due to defective synthesis of NO in nerve terminals, rather than a defect in NO signaling in smooth muscle. This deficit in NO synthesis may be related to inadequate localization of nNOS in the appropriate signaling compartment at the membrane that is required for its activation. Thus, the present study suggests that deficiency of the intracellular motor protein, myosin Va, may be involved in the pathogenesis of gastroparesis and erectile dysfunction.In many disorders of gastrointestinal motility, impaired nitrergic function with near normal or only slightly reduced nNOS has been reported. For example, nNOS content was normal in 60 and 80% of gastric biopsies in idiopathic and diabetic gastroparesis, respectively in vivo phenotypes of the gut and penis in DBA mice are not well characterized. DBA mice have lower levels of physical activity and lower food intake than WT litters If the function of nNOSα and pnNOS is regulated by myosin Va, the phenotype of DBA mice might be expected to resemble nNOS−/− mutants and other animal models of functional nNOS deficiency. DBA mice, an inbred strain of myosin Va deficiency, have a near normal life span, though they have been reported to be difficult breeders In summary, our immunofluorescence and mechanical studies showed that myosin Va plays an important role in nitrergic neurotransmission in smooth muscle of the gastric fundus and penile corpora cavernosa as evidenced by the functional impairment of nitrergic relaxation demonstrated in an animal model of myosin Va deficiency. These studies suggest that loss of myosin Va contributes to functional abnormalities, including gastric motility disorders and erectile dysfunction.


Common variants in genes related to inflammation, innate immunity, epithelial cell function, and angiogenesis have been reported to be associated with risks for Acute Lung Injury (ALI) and related outcomes. We tested whether previously-reported associations can be validated in an independent cohort at risk for ALI.We identified 37 genetic variants in 27 genes previously associated with ALI and related outcomes. We prepared allelic discrimination assays for 12 SNPs from 11 genes with MAF>0.05 and genotyped these SNPs in Caucasian subjects from a cohort of critically ill patients meeting criteria for the systemic inflammatory response syndrome (SIRS) followed for development of ALI, duration of mechanical ventilation, and in-hospital death. We tested for associations using additive and recessive genetic models.IL6 and ALI susceptibility . In a sensitivity analysis limiting ALI cases to those who qualified for the Acute Respiratory Distress Syndrome (ARDS), rs61330082 in NAMPT was nominally associated with risk for ARDS. In terms of ALI outcomes, SNPs in MBL2 (rs1800450) and IL8 (rs4073) were nominally associated with fewer ventilator-free days (VFDs), and SNPs in NFE2L2 (rs6721961) and NAMPT (rs61330082) were nominally associated with 28-day mortality. The directions of effect for these nominal associations were in the same direction as previously reported but none of the associations survived correction for multiple hypothesis testing.Among Caucasian subjects with SIRS (n = 750), we identified a nominal association between rs2069832 in IL6 and NAMPT and risk for development of lung injury and for SNPs in IL8, MBL2, NFE2L2 and NAMPT with severity in ALI outcomes. These associations provide further evidence that genetic factors in genes related to immunity and inflammation contribute to ALI pathogenesis.Although our primary analyses failed to statistically validate prior associations, our results provide some support for associations between SNPs in  Acute Lung Injury (ALI) and it's more severe manifestation, Acute Respiratory Distress Syndrome (ARDS) are associated with high mortality rates among intensive care unit patients Candidate gene studies in ALI have focused on variation within pathways thought to be important in ALI pathogenesis including genes involved in inflammation, immunity, oxidation, coagulation, angiogenesis, cell growth, endothelial barrier, surfactant function, and transcription regulation In this study, we sought to determine whether previously-reported associations between variants in genes of immunity, inflammation, angiogenesis and oxidative stress and risk for development of ALI or related outcomes, are robust. We used a nested case-control study design in a prospective cohort of critically ill patients with SIRS followed for development of ALI. Secondary analyses looked at ALI related outcomes in a case-only design.www.ncbi.nlm.nih.gov/pubmed/) for publications reporting associations between genetic variants and development of ALI and/or ALI related outcomes as of August 2010. A panel of ALI investigators  selected variants based on strength of prior studies, biological relevance, and HapMap 2 = 0.97).We searched PubMed  criteria for ARDS; presence of a proximal risk factor for ALI, presence of a PaO2/FiO2  <300, absence of overt congestive heart failure, and a chest radiograph with bilateral parenchymal opacities as adjudicated by three critical care physicians The cohort used for this study has been previously described Genomic DNA was extracted from whole blood samples using the Puregene DNA Isolation Kit . All SNPs were genotyped using Taqman-based allelic discrimination assays, , on a multichannel microfluidics chip . Genotyping was carried out per the vendor's protocol in a blinded fashion.t-test for continuous variables. Observed genotype frequencies were compared with expected frequencies to test for deviations from Hardy-Weinberg equilibrium using Fisher's exact test. To reduce possible confounding from population-stratification and racial differences, all analyses were restricted to Caucasian subjects. As a screen for major population substructure, we performed principal component analysis (PCA) using SNP genotypes from 67 SNPs available on our samples and which were also publically available for HapMap 3 subjects We identified differences in demographic variables between ALI and control subjects using Fisher's exact test for categorical variables and Student's a priori based on known effects on risk for ALI development and mortality and included age, sex, APACHE III score, comorbidities, smoking status and alcohol abuse. A two sided P value <0.05 was considered to be nominally significant in all analyses. To control for type I error from multiple hypotheses testing, we used a false discovery rate (FDR) approach setting a threshold of 0.1 as indicative of corrected significance. FDR was calculated separately for each association test using the total number of SNPs tested.We used multivariate logistic regression to estimate the genotype-specific odds ratio (OR) and 95% confidence interval (CI) for ALI susceptibility. The genotype associations were analyzed in both additive and recessive models. Covariates were chosen We conducted a sensitivity analysis restricting case and control subjects to avoid phenotype misclassification. We restricted cases of ALI to only those qualifying for moderate and severe ARDS by oxygenation criteria (PaO2/FiO2 <200) and we restricted at-risk controls to only subjects who did not have an episode of hypoxemia (PaO2/FiO2 <300).In secondary analyses we tested for associations between genotypes and ventilator free days (VFDs) using linear regression and mortality within 28 days using logistic regression, adjusting for covariates as above.In a post hoc analysis, and for SNPs with adequate genotyping data available in prior publications, we completed unadjusted meta-analyses for genotype associations with ALI risk or mortality in ALI subjects by combining our results with the published data.We estimated that, under an additive model, our study would have adequate statistical power (1-β>0.8) to detect associations of moderate strength with ALI (relative risk (RR) >1.5) for SNPs with MAF>0.2. Assuming a recessive model, we would have adequate power to detect a strong association  with SNPs having a MAF>0.25. See Statistical analyses were run using the R statistical package and SNP and Variation Suite 7™ .We genotyped 879 subjects; 238 subjects who developed ALI and 641 at-risk control subjects See . These sWe searched PubMed for gene association studies of ALI and ARDS susceptibility and related outcomes. At the time of the search , we identified 37 genetic variants in 27 genes including SNPs, insertion/deletions, and SNP haplotypes . The set2 test.We achieved genotype call rates across all subjects of over 99% for each of the 12 SNPs. One of the SNPs, rs4444903, had genotype frequencies for the control subjects that deviated from predicted frequency under Hardy Weinberg equilibrium (HWE). The remaining SNPs had genotype results for the control subjects that were not significantly different from HWE predictions using the χWe did not observe strong evidence of population stratification by PCA comparing 67 shared genotypes among our subjects and HapMap3 subjects. When plotting PC1 vs. PC2, our subjects clustered with HapMap CEU and TSI subjects but not with Asian or African clusters as expected. No subject outliers or associations between phenotype and eigenvalue were observed. See Genotype frequencies for control and case subjects are presented in IL6 by recessive modeling   . The dir = 0.03)  or unadj = 0.03) .NAMPT, VEGF, IL8, MBL2 and TNF. In unadjusted genotype association meta-analyses using additive modeling, we observed an association between rs59744560 in NAMPT and increased risk for ALI (OR 1.33 (95% CI 1.14–1.56) p value <0.0003) and an association between rs61330082 in NAMPT and decreased risk for ALI (OR 0.86 (95% CI 0.74–1.0) p value 0.049). We did not observe associations between SNPs in VEGF, IL8, MBL2 or TNF and ALI risk in this meta-analysis. See We evaluated whether we could complete meta-analyses by combining our genotype results with the previously published results but found that the many of the publications lacked adequate genotyping results details. However, we were able to complete meta-analyses for several of the SNPs including in NAMPT and reduced ARDS susceptibility  which replicates the direction of effect seen previously for this SNP NFE2L2 , and rs2069832 in IL6   . None ofIL8 and rs1800450 in MBL2 and decreased VFD in adjusted analyses , p value 0.001) and rs1800450 inMBL2 displayed a trend toward increased risk (OR 3.07 (95% CI 0.99–9.5), p value 0.07). See Several of the SNPs selected for this study were previously associated with ALI-related outcomes analyses . We alsove model . NeitherIL6  and risk for ALI. In analyses restricting case and control definitions to minimize misclassification, we observed a nominal association between rs61330082 in NAMPT and reduced risk for moderate and severe ARDS consistent with previous reports IL8 and rs1800450 in MBL2 and decreased VFDs. We observed nominal associations between rs61330082 in NAMPT and rs6721961 in NFE2L2 and increased 28-day mortality. None of the associations observed in the secondary analyses survived correction for multiple hypothesis testing.At the time of our study, candidate gene association studies had identified more than 30 genetic variants in over 27 genes that associate with risk for ALI or ALI-related outcomes. These associations provide evidence that genetic factors contribute to ALI and strengthen the link between these specific genes in disease pathogenesis. In this study, we sought to identify robust associations between previously-associated genetic variants and risk for ALI and related outcomes. Using a nested case-control study in a well-phenotyped ICU cohort, we compared SIRS subjects who remained at-risk for ALI to those who developed ALI. Although, in the primary multivariable analyses, the significance levels for the associations with ALI did not survive correction for multiple comparisons, our nominal association results provide some support for prior findings. We observed a nominal association between a SNP in IL6 and IL8 have been extensively studied as important proinflammatory mediators in ALI. Elevated levels of IL-6 and IL-8 are associated with development of ALI, and persistence of elevated levels has been associated with poor outcomes IL8 is a promoter SNP that affects gene transcription and has multiple published disease associations IL6 which results in lower promoter activity was previously associated with reduced mortality in ARDS but not with risk for ALI IL6 that included the C allele of rs1800795 was actually associated with reduced risk of ALI IL6 tagging all common linkage disequilibrium bins and found that none of these SNPs demonstrated an association with ALI. However, they did find that carriage of two copies of a 6-SNP haplotype that included the G allele at rs1800795 was associated with an OR of 2.73  for development of ALI IL6 haplotype clade that included the G allele of rs1800795 was associated with increased risk of death and fewer days alive and free of ALI 2 = 0.98), is associated with increased risk for ALI and, similar to the studies by Flores and Sutherland, the effect was best modeled assuming a recessive effect. The SNP in IL8 was previously associated with increased days on mechanical ventilation IL8 was similarly associated with reduced days alive and free of ventilation. These data suggest that common variants in inflammatory cytokines contribute to disease pathogenesis among subjects at risk for or who have ALI.NAMPT (PBEF1) has been associated with lung injury pathogenesis in recent translational and genetic association studies. Pre-B-cell colony-enhancing factor (PBEF) is a cytokine encoded by the gene NAMPT and that has been shown to be present at higher concentrations in serum and BALF from patients with ALI NAMPT and development of ALI. Ye et al. identified 11 SNPs in NAMPT and, in a comparison between ALI cases and healthy controls, demonstrated that two of the SNPs in the promoter region, T-1001G (rs59744560) and C-1543T (rs61330082) were associated with risk for sepsis and ALI NAMPT in ALI pathogenesis and suggest that NAMPT promoter variants are associated with risk for disease development.NFE2L2 is a gene that has been identified as a potential mediator of hyperoxia-induced lung damage via linkage analysis and positional cloning in mice NFE2L2 is a transcription factor that targets antioxidant response elements (AREs) leading to gene regulation that is protective in the setting of oxidative stress. Resequencing of NFE2L2 identified a variant (rs6721961) that resulted in diminished promoter activity in an in vitro transfection experiment IL6, NAMPT and NFE2L2. While these findings did not achieve statistical significance after correction for type I error they provide support for the contention that phenotypic misclassification is a major issue reducing power and precision in case-control studies of ALI.Misclassification of ALI, particularly the inclusion of subjects without true ALI among cases, can lead to significant reductions in statistical power to detect true genetic associations Overall, our study failed to demonstrate robust associations between the SNPs studied and risk for ALI and related outcomes. Reasons for lack of validation include inadequate statistical power, misclassification of subject phenotypes, sample population differences compared to the prior studies, population substructure, and absence of true association with ALI. As power was limiting to identify small effect sizes, especially in the recessive model, we cannot exclude the possibility that we failed to observe true associations between the SNPs and ALI susceptibility and related outcomes. A limited analysis on 67 genotypes demonstrated that subjects from our cohort clustered with HapMap CEU and TSI subjects suggesting that there was not obvious evidence for cryptic admixture. However, more subtle differences in population substructure could have also have affected our ability to detect true associations. Finally, most of the SNPs included in this study have not been definitively shown to have functional significance and may merely serve as markers for causal variants. Future work will require more dense genotyping or sequencing at these loci to gain further insight into potential mechanisms.IL6 and NAMPT, and risk for ALI and ARDS, but these associations failed to meet significance after adjusting for multiple comparisons. Nominal associations were also identified between SNPs in IL8, MBL2, NAMPT and NFE2L2 and ALI severity outcomes, namely VFDs and 28-day mortality. The failure to replicate the majority of ALI risk and outcome associations with statistical significance emphasizes the challenges of genetic association studies in critical care populations including sample size and disease classification. Our results also highlight the importance of independent confirmatory studies of associations with ALI susceptibility and related outcomes in order to support a better understanding of ALI pathogenesis and prognosis.This study used a well-phenotyped cohort of ICU subjects with SIRS and a nested-case control design to replicate ALI risk associations for several previously identified genetic variants. We observed nominal associations for between SNPS in Figure S1Genotype principal component analysis plot demonstrating overlap of our subjects with HapMap ethnic populations. Population stratification was assessed by PCA for 67 shared genotypes among our subjects and HapMap 3 subjects (TIF)Click here for additional data file.Figure S2Plot of power estimates based on minor allele frequency and genotype effect size. Using CaTS (TIF)Click here for additional data file.Table S1Genetic variants associated with acute lung injury and associated outcomes in peer reviewed publications.(DOCX)Click here for additional data file.Table S2Genotype frequencies and unadjusted frequency analysis for ALI survivors vs. non-survivors at 28 days.(DOCX)Click here for additional data file.Table S3Meta-analysis for ALI risk: genotype frequencies used in meta-analysis calculations.(DOCX)Click here for additional data file.Table S4Meta-analysis for ALI-related mortality: genotype frequencies used in meta-analysis calculations.(DOCX)Click here for additional data file.


GAD1 risk allele associations were restricted to females.Panic disorder is common  and females are twice as likely to be affected as males. The heritable component of panic disorder is estimated at 48%. Glutamic acid dehydrogenase GAD1, the key enzyme for the synthesis of the inhibitory and anxiolytic neurotransmitter GABA, is supposed to influence various mental disorders, including mood and anxiety disorders. In a recent association study in depression, which is highly comorbid with panic disorder, GAD1 were genotyped in two independent gender and age matched case-control samples . Thirteen SNPs passed quality control and were examined for gender-specific enrichment of risk alleles associated with panic disorder by using logistic regression including a genotype×gender interaction term. The latter was found to be nominally significant for four SNPs  in the discovery sample; of note, the respective minor/risk alleles were associated with panic disorder only in females. These findings were not confirmed in the replication sample; however, the genotype×gender interaction of rs3749034 remained significant in the combined sample. Furthermore, this polymorphism showed a nominally significant association with the Agoraphobic Cognitions Questionnaire sum score.Nineteen single nucleotide polymorphisms (SNPs) tagging the common variation in GAD1. Our tentative results provide a possible explanation for the higher susceptibility of females to panic disorder.The present study represents the first systematic evaluation of gender-specific enrichment of risk alleles of the common SNP variation in the panic disorder candidate gene  GAD1, located at 2q31.1 and GAD2 (10p11.23), respectively). Intriguingly, brain GAD expression was shown to be influenced by sex hormones in female rats and rhesus macaques GAD expression in the amygdala and the hippocampus (which both are involved in regulating fear), which provides a link between hormone levels and anxiety as well as mood attacks during menstruation in humans The lifetime prevalence of anxiety disorders has been estimated to be as high as 30% with a spectrum that ranges from 2% for agoraphobia up to 12% for specific phobia; with the prevalence of panic disorder being estimated at 5% GAD1) and 15q GAD genes may serve as plausible candidates to influence neuroticism, the examination of allelic variation in these genes revealed associations of GAD1 single nucleotide polymorphisms (SNPs) rs2241165, rs2058725 and rs3791850 in a mixed anxiety (including panic) and mood disorder sample GAD1 with mood disorders  GAD1 alleles were shown to convey genetic risk  GAD1 SNPs examined in GAD1 SNPs that were not (rs12185692) or only marginally (rs769407) associated with neuroticism GAD1 variants are also detectable in panic disorder. In the present study we used two independent gender-matched case/control samples with a total size of ncase = 531 to test this hypothesis. Nominally significant gender differences and associations with disease were found in the discovery sample, which were however not supported by the replication sample; in the analysis of the combined sample, only one SNP displayed a nominally significant gender-specific risk allele enrichment, the reasons for which are discussed in depth below. Studies in larger samples and subsequent meta-analysis are thus needed to unequivocally prove a possible female-specific contribution of the GAD1 gene in panic disorder.A recent twin study estimated the heritability of panic disorder at 48% GAD1 and approximately 10 kb of the gene's up- and downstream flanking regions, we chose a representative set of 19 SNPs (based on HapMap CEU data) for genotyping. Thereof, six SNPs did not pass our stringent quality criteria in the discovery sample; hence, the remaining set of 13 SNPs  derived from this interaction term represents the ratio of effect sizes (i.e. ORs) from genetic associations in males versus those in females, thus quantifying differential risk allele enrichment between genders. In the discovery sample, four SNPs displayed nominally significant genotype×gender interactions; particularly the respective minor alleles (assumed to convey the genetic risk) of rs3762555, rs3749034 and rs2241165 had effects that were almost twice as large in males as in females (ORs close to 2) and vice versa for rs1978340 , however, associations reached nominal significance only in females  deletes a putative transcription factor binding site (TFBS) for the E2F4-TFDP2 dimer, whereas the minor alleles of rs3762555 (upstream) and rs3749034 (5′ untranslated region) create predicted TFBSs for SRF and ZEB1/ZFHEP/AREB6, respectively. For the intronic SNP rs2241165 no allele-specific functional consequences (affecting e.g. splicing) were evident, nor is linkage disequilibrium data available for nearby SNPs with putative structural impact mediated by amino acid exchanges . The restriction of this analysis to our candidate SNPs imposes the limit that true causal variants may be missed, since each of our SNPs examined in this study is representative for the variation of several polymorphisms. Nevertheless, it appears conceivable that the mode of action of gender-wise differentially enriched GAD1 risk alleles affects promoter function and thus transcriptional efficiency.In addition to genetic association testing we tried to predict functional consequences of our candidate SNPs using a bioinformatic approach. We found that SNPs close to GAD1 with panic disorder. However, the initial findings from our clinical discovery sample were not supported by our replication sample originating from a cognitive behavioral psychotherapy (CBT) trial. Of four genotype associations, no replicated association was found at the categorical level and in the combined sample only the interaction of rs3749034 with gender became significant. Interestingly though, rs3749034 was also among the SNPs associated with the dimensional sum score of ACQ in the replication sample, but at first sight this dimensional association seems to contradict the categorical results: although the latter revealed significant gender-differences, rs3749034 had a protective effect on all females in the discovery sample and on male panic patients in the replication sample as well. However, since the ACQ quantifies agoraphobia In this paper, we present the first systematic evaluation of gender-dependent associations of common SNP variation in A major limitation of this study is imposed by the restricted sample size and the high female to male ratio. Due to the high LD within the examined region, we used nominal p-values for the assessment of significance to avoid the (in this case) overly strict Bonferroni correction, which revealed associations in the discovery sample that would otherwise have been negative. This measure decreases type-II error, while increasing type-I error, which has to be taken into account when interpreting the data. Due to the lower prevalence of panic disorder in males, especially the male subsamples were rather limited in size, which may hinder the realistic estimation of genetic effect sizes because of insufficient sampling and therefore confound possible gender differences in risk allele enrichment. This point is more relevant to the replication sample, which features an even smaller male sub-sample as compared to the discovery sample, and which did not support the findings from the discovery sample. As the combined sample did not reflect our associations from the discovery sample either, it is tempting to speculate that lack of replication may also be explained by differences in the composition of our samples see : FirstlyGAD1 and its gene product are implicated in the pathophysiology of panic disorder. GABA levels in various brain regions are reduced in panic patients GAD1 SNPs are associated with neuroticism  and anxiety  GAD1 polymorphism rs1978340 was found to influence brain GABA levels: healthy individuals that are homozygous for the minor allele display higher concentrations than major allele carriers COMT promoter polymorphism rs2075507 In conjunction with the present genetic data, convergent lines of evidence suggest that GAD1 risk alleles in panic disorder. In the latter, a linkage analysis of an US American sample revealed that both chromosome 2 microsatellite marker loci, which flank the GAD1 gene at positions 169 and 178 cM, display moderate differences in male and female associations GAD1 polymorphisms rs12185692 (upstream) and rs769407 (in the sixth intron) to go along with disease only in females GAD1 SNPs GAD1 in mixed gender samples of depression GAD1 SNP associations even without considering gender effects Further genetic studies on psychiatric disorders are in line with our tentative finding of gender-specific enrichment of Gad1 promoter in the rat hippocampus, thus silencing gene expression GAD1 SNPs and childhood adversities has very recently been examined in the context of anxiety disorders, however failed to detect significant effects GAD1 expression Since the heritability for panic disorder is somewhat lower than that for other mental disorders GAD1 variation for the pathogenesis of panic disorder, but is far from conclusive. Future studies in larger gender-balanced samples well characterized for panic disorder and agoraphobia as well as comorbid psychopathology  and life events will be necessary to evaluate the relevance of our tentative findings.In summary, our study provides supporting evidence for gender differences in the role of All SNPs examined in this study were genotyped in panic disorder (with or without agoraphobia) patients which were matched by gender to an equal number of healthy controls. Details on case/control pairs of the discovery and the replication sample are specified below. All cases as well as controls were unrelated and of self-reported Western European descent. Patients with comorbid schizoaffective or other psychotic disorders, substance abuse disorders, mental retardation, neurological or neurodegenerative disorders were excluded. Only patients and volunteers who gave written informed consent were enrolled in the study. The present study complied with the Declaration of Helsinki and was specifically approved by the Ethics Committee of the University Hospital, University of Würzburg. A demographic overview of the discovery and the replication samples can be found in The discovery sample consisted of 239 panic disorder patients, collected in Bonn, Würzburg, Münster and Göttingen , from out- and inpatients of the respective centers treated there as part of the regular clinical care. The diagnosis of panic disorder and absence or presence of comorbid agoraphobia  was ascertained by experienced clinicians on the basis of medical records and structured or standardized clinical interviews (Schedule for Affective Disorders and Schizophrenia (lifetime version), SADS-LA; Structured Clinical Interview for DSM IV, SCID; and Composite Inernational Diagnostic Interview, CIDI) according to the criteria of DSM-  III-R or DSM-IV, respectively Panic-Net” multicenter psychotherapy treatment study; 15 of these patients did not enter the study yet fulfilled the respective inclusion criteria. Patients were enrolled from specialized outpatient treatment units within the framework of the Panic-Net study as described in detail The replication sample's patients  were enrolled from the BMBF “GAD1 gene, 19 tag SNPs were derived from HapMap CEU data Tagger function as implemented in Haploview V4.2 In order to capture allelic variation in the GAD1 gene was analyzed with logistic regression, assuming the minor alleles to convey the genetic risk in an additive manner. SNP associations with dimensional anxiety traits were analyzed with univariate linear regressions modeling the ACQ and the ASI sum scores in each gender separately. Each risk allele's effect is presented as OR for categorical outcomes or as linear increase for dimensional phenotypes. The respective p-values represent the probabilities for the estimated effects being indistinguishable from zero. P-values <0.05 are termed nominally significant. Although conducting repeated statistical tests, p-values were not corrected for multiple testing, which was – despite large sample size - due to the limited power of the study. Given a SNP with MAF = 5% conveying a moderate relative risk of 1.5 to develop panic disorder in an additive model, the power to find a nominal association (p<0.05) is 65% for the mixed gender combined sample (n = 1062), 48% for its female (n = 718) and 26% for its male (n = 344) subset. In the same analysis setting, but split up in discovery and replication sample, the mixed gender discovery sample (n = 478) and its female (n = 286) and male (n = 192) subsets provide 35%, 24% and 18% power, respectively; the mixed gender replication sample (n = 584) and its female (n = 432) and male (n = 152) subsets provide 42%, 32% and 15% power, respectively. The analyses were performed with Haploview V4.2 Prior to statistical analysis, SNPs had to fulfill several quality control criteria. The minimal call rate threshold was set to 90%, SNPs with a call rate below this threshold  where not further analyzed. Deviations from Hardy-Weinberg equilibrium (HWE) were considered to be indicative for the presence of genotyping errors; SNPs rs769393 and rs769395 yielded a HWE p-value below the threshold of 0.01 and were thus excluded from further analysis. Also, rare variants with a minor allele frequency (MAF) below 5%  were not further examined. The resulting set of 13 SNPs tagging the common allelic variation in the Analyses of SNPs and their sequence contexts were performed with tools that are contained in the GenEpi toolbox Figure S1Linkage disequilibrium (LD) structure of GAD1 single nucleotide polymorphisms examined in the discovery sample. LD analysis was performed with Haploview v4.2 using default settings, i.e. D′ was used as measure for LD and haplotype blocks were defined with the method “confidence intervals”.(DOC)Click here for additional data file.Figure S2Linkage disequilibrium (LD) structure of GAD1 single nucleotide polymorphisms examined in the replication sample. LD analysis was performed with Haploview v4.2 using default settings, i.e. D′ was used as measure for LD and haplotype blocks were defined with the method “confidence intervals”.(DOC)Click here for additional data file.Table S1Single nucleotide polymorphisms (SNPs) examined in the study. rs4439928 did not meet the quality criteria in the replication sample and was therefore not included in the replication and combined sample analysis.(DOC)Click here for additional data file.Table S2Demographic overview of the discovery and the replication sample. Abbreviations: AG, agoraphobia; CIDI, Composite International Diagnostic Interview; DSM, Diagnostic and Statistical Manual of Mental Disorders; MINI, Mini International Neuropsychiatric Interview; SADS-LA, Schedule for Affective Disorders and Schizophrenia (lifetime version); SCID, Structured Clinical Interview for DSM IV.(DOC)Click here for additional data file.Table S3Gender-specific associations of GAD1 polymorphisms in swapped samples. Analysis of gender subsamples was performed only for polymorphisms that displayed significant gender differences in the discovery case-control sample Click here for additional data file.Table S4Gender-specific associations of GAD1 polymorphisms in samples with and without co-morbid agoraphobia. Analysis of gender subsamples was performed only for polymorphisms that displayed significant gender differences in the discovery case-control sample Click here for additional data file.Table S5Published gender-specific associations of GAD1 single nucleotide polymorphisms (SNP).(DOC)Click here for additional data file.Table S6Associations of GAD1 polymorphisms with panic disorder in mixed-gender samples. rs4439928 did not meet the quality criteria in the replication sample and was therefore not included in the replication and combined mixed-gender sample analysis. Abbreviations: OR, odds ratio; p, p-value; SNP, single nucleotide polymorphism.(DOC)Click here for additional data file.Table S7Primer sequences used in this study.(DOC)Click here for additional data file.