Chlamydia. One exit strategy, extrusion, is mediated by host signaling pathways involved with actin polymerization. Here, we show that actin is recruited to the chlamydial inclusion as a late event, occurring after 20 hours post-infection (hpi) and only within a subpopulation of cells. This event increases significantly in prevalence and extent from 20 to 68 hpi, and actin coats strongly correlated with extrusions. In contrast to what has been reported for other intracellular pathogens, actin nucleation on Chlamydia inclusions did not ‘flash’, but rather exhibited moderate depolymerization dynamics. By using small molecule agents to selectively disrupt host signaling pathways involved with actin nucleation, modulate actin polymerization dynamics and also to disable the synthesis and secretion of chlamydial proteins, we further show that host and bacterial proteins are required for actin coat formation. Transient disruption of either host or bacterial signaling pathways resulted in rapid loss of coats in all infected cells and a reduction in extrusion formation. Inhibition of Chlamydia type III secretion also resulted in rapid loss of actin association on inclusions, thus implicating chlamydial effector proteins(s) as being central factors for engaging with host actin nucleating factors, such as formins. In conclusion, our data illuminate the host and bacterial driven process by which a dense actin matrix is dynamically nucleated and maintained on the Chlamydia inclusion. This late stage event is not ubiquitous for all infected cells in a population, and escalates in prevalence and extent throughout the developmental cycle of Chlamydia, culminating with their exit from the host cell by extrusion. The initiation of actin recruitment by Chlamydia appears to be novel, and may serve as an upstream determinant of the extrusion mechanism.The ability to exit host cells at the end of their developmental growth is a critical step for the intracellular bacterium  Chlamydia spp. continue to have a major burden on global public health. C. trachomatis is the leading cause of sexually transmitted infection, responsible for an estimated 90 million new cases annually worldwide Chlamydia infections can lead to ectopic pregnancy and pelvic inflammatory disease Chlamydia elementary bodies (EB), the infectious and metabolically inert form of the bacteria, with columnar epithelial cells. Chlamydia EB attach to and internalize into epithelial cells, and take residence in a vacuole known as the inclusion. Within this protective intracellular niche, Chlamydia convert into the larger, metabolically active reticulate bodies (RB) and undergo successive rounds of replication and division. This proceeds until the bacteria number in the hundreds and the vacuole has swollen to fill the host cell; during this time Chlamydia asynchronously convert back into EB and are released from the host cell.Chlamydia have evolved two non-redundant strategies for accomplishing this task In accordance with the fundamental importance of exiting host cells at the end of their intracellular growth, Chlamydia exit, it is likely that these pathways are engaged by Chlamydia from within the inclusion, or from the inclusion membrane itself. This hypothesis is consistent with the emerging theme that the inclusion represents the keystone of Chlamydia pathogenesis. Chlamydia uniquely modify and interact with this compartment in order to avoid endolysosomal trafficking pathways, acquire lipids from the host cell, activate Src family kinases, induce cytoskeletal rearrangements and limit detection by immune surveillance pathways Extrusion is critically dependent on actin polymerization and cellular pathways that regulate actin dynamics, including N-WASP Chlamydia to secrete effector proteins by type III secretion (TTS) and manipulate actin at early steps of infection is well established. The Chlamydia effector protein CT456 (TARP) is secreted during bacteria entry into host cells and initiates actin polymerization at attachment foci by a mechanism that involves Rac, WAVE2 and Arp2/3 Chlamydia effector protein CT166 may also play a role in actin nucleation during bacteria entry Chlamydia inclusions The ability of Chlamydia direct the recruitment of actin to the mature inclusion membrane as an initial step in the extrusion exit pathway. We report that actin structures are dynamically recruited to the Chlamydia inclusion beginning at 20 hpi for C. trachomatis, and the prevalence of this event increases throughout the chlamydial developmental cycle. Importantly, this mechanism requires both host signaling pathways associated with actin polymerization and Chlamydia type III secretion products. Actin coat formation is critical for extrusion formation.Based on preliminary data, we hypothesized that at late times of infection, Chlamydia spp. by the extrusion mechanism Chlamydia vacuole (inclusion) has been reported Chlamydia-infected HeLa cells using the LifeAct-TagGFP reporter , which consists of a 17 amino acid peptide that binds actin, fused to TagGFP Cryptococcus phagosomes Actin polymerization and nucleation factors have been shown to mediate cellular release of Chlamydia inclusions C. trachomatis LGV serovar L2. Chlamydia inclusions were readily detected in tandem with actin-GFP by live cell fluorescence microscopy. Representative examples are illustrated in HeLa cells were co-transfected with cytosolic DsRed—to enable detailed visualization of Chlamydia inclusions was quantitatively measured by enumerating inclusions that were positive for significant actin recruitment. The data revealed a dramatic increase in the prevalence of inclusions with actin recruitment over the developmental cycle , an established method for dissolving membrane bilayers while preserving cytoskeletal structures Chlamydia is that it enables resolution of the respective spatial distributions of bacteria and actin structures with inclusion and plasma membranes removed.We next sought to characterize the precise ultrastructural details of actin deposition on the C. trachomatis, cells were processed and imaged by SEM. In uninfected cells, this technique revealed cells with their plasma membranes removed, unmasking a dense network of actin in the cell body and fibrous structures at the cell periphery  recruited actin, although not to the same extent as for C. trachomatis L2. C. muridarum inclusions exhibited only minimal actin recruitment .To demonstrate that actin recruitment to the Chlamydia inclusion may be similar to the rapid, transient actin ‘flashes’ that have been described for other pathogenic vacuoles and intracellular bacteria Chlamydia inclusions, cells at 44 hpi were mounted on a fluorescence microscope for live cell time-lapse imaging. The data revealed that actin complexes on inclusions were not stable structures, nor did they exhibit rapid flashing. The average duration of actin association on inclusions at 44 hpi was 45 minutes (see Video S3), and we were unable to successfully capture events of de novo actin coat formation. Infected cells at 20 hpi were also examined, and revealed small foci and dense pockets of actin-GFP that were frequently adjacent to inclusions and highly dynamic in character (see Video S4).The dynamic spatial and temporal recruitment of actin to the Chlamydia inclusion would be: (i) entirely due to chlamydial effector proteins secreted onto the inclusion membrane and/or into the host cytosol; (ii) exclusively host driven; or (iii) an outcome of chlamydial and host factors working together. Previous data support the notion that host factors are involved in this phenomenon Chlamydia secretes proteins that interact with actin polymerization pathways at early steps of infection Chlamydia potentially secrete different effector proteins into the host cytosol, or as the vacuole is modified differently over time.We rationalized that the mechanism for actin recruitment to the Chlamydia infection, such as entry Our experimental design overall was to quantify the effects of host and bacterial protein perturbation on actin cage formation using actin-GFP in living cells. The most direct approach was by short-term treatment of cells with specific inhibitors of discrete cellular targets C. trachomatis inclusions was mediated by Arp2/3 or formins; polymerization by these mechanisms typically result in y-branched or unbranched filamentous actin structures, respectively We first investigated whether the mechanism of actin nucleation on Chlamydia inclusions at 44 hpi , Rho GTPases, Rho kinase (ROCK) and Src family kinases (SFK). Of these signaling pathways, only myosin II and SFK played significant roles in actin recruitment to chlamydial inclusions . CollectChlamydia might also play a central role in initiating actin polymerization on the inclusion membrane surface, and we tested this by treating cells for 4 h at 44 hpi with chloramphenicol, rifampicin or the type III secretion (TTS) inhibitor C1 compound Chlamydiae can activate actin polymerization in host cells by at least one effector protein that is secreted into the host cytosol during entry Chlamydia protein(s) are necessary to initiate and maintain actin polymerization on the inclusion.We conducted a time analysis of chloramphenicol and rifampicin treatment and determined that as little as one hour treatment of infected cells with chloramphenicol was sufficient to disrupt the actin association phenotype ; lengthiChlamydia-infected cells to determine the resultant changes in actin ultrastructure on the inclusion. Cells that were treated with chloramphenicol  and prevalence throughout a cell monolayer over the chlamydial developmental cycle—from 20–68 hpi for C. trachomatis LGV, in agreement with the literature Chlamydia extrusion Here, using a combination of real-time imaging of GFP labeled actin, immunofluorescence microscopy and SEM, we describe the detailed manner in which host-derived actin complexes are recruited and polymerized to the cytosolic surface of the Chlamydia inclusion consisted of discrete steps. At 20 hpi, very little actin association with inclusions was detected. The only meaningful associations we observed were actin clouds in close proximity to the inclusion and puncta on the cytosolic face of the inclusion. The origin of these foci, and why they are first manifest in this manner , are not clear; however, we propose that these are actin nucleation sites induced by Chlamydia type III secreted proteins.Actin recruitment to the As infection proceeds to 44 hpi, actin recruitment dramatically progressed to form arc  and partial cage structures (in 3-dimensions) on the regions of inclusion membranes distal to the nucleus. This particular spatial distribution was curious because it did not correlate with the location of 20 hpi actin clouds—which were typically adjacent to nuclei. At the ultrastructural level, actin deposition on inclusions at 44 hpi consisted of a rich layered actin network that was continuous with the actin matrix of the host cell.At 68 hpi, and especially for extrusions, actin recruitment was much fuller, more deeply layered and frequently enwrapped the entire inclusion. When not completely circumscribing the inclusion, the regions of strongest actin recruitment were areas of the inclusion distal to the nucleus and where the inclusion protruded or bulged out of the swollen cell. We therefore speculate that these enriched areas of actin may mark the locations for imminent extrusions.Chlamydia inclusion is markedly different than what has been reported for other intracellular bacteria and pathogenic vacuoles, for example Listeria, Rickettsia spp., Burkholderia pseudomallei and Mycobacterium marinumy-branched actin polymerization via the Arp2/3 complex and promote intracellular motility. Actin polymerization on these bacteria is rapid and appears as so-called flashes in live cells. Transient flashes of actin polymerization have also been demonstrated on the surface of parasitophorous vacuoles containing Cryptococcus, using a Arp2/3 and WASP based mechanism Chlamydia inclusions were dynamic yet did not exhibit rapid flash kinetics. Short term perturbation of host and bacterial signaling pathways in Chlamydia infected cells was sufficient to remove actin coats from inclusions, thus implicating their need for dynamic maintenance.Actin recruitment to the Chlamydia interact with host signaling pathways in order to promote infection, growth and survival. The mechanism of actin coat formation required cooption of host signaling pathways, most notably formins and septins. The strong role of formins argues for a filamentous nature of actin assembly, and this notion was corroborated by the presence of filamentous structures by SEM and the lack of an involvement of the Arp2/3 complex and N-WASP—which promote y-branched actin polymerization.Resolution of the molecular pathways that give rise to this phenomenon are expected to inform new insights on how intracellular Chlamydia inclusions that interact with four Inc proteins and centrosomes Chlamydia and host proteins, and play important roles in coordinating a number of signaling pathways in the cytosol: trafficking of nascent inclusions to the MTOC and processes that regulate C. trachomatis growth. Some Chlamydia species, for example C. muridarum, do not display active SFK microdomains on their inclusion surface and also do not associate with the MTOC The involvement of Src family kinases in actin recruitment is particularly intriguing in the context of recent data showing the existence of microdomains of active SFK on Myosin, Rho GTPase and ROCK function are all essential for extrusion formation Chlamydia pathogenesis. Septins are a conserved family of GTP-binding proteins that form nonpolar filamentous structures and associate with cellular membranes and the actin and microtubule cytoskeletons Chlamydia inclusion, and that they may serve as a bridge between actin filaments and bacteria-derived proteins on the inclusion membrane. We additionally investigated the spatial distribution of the prominent septin Sept2 during Chlamydia infection; however, Sept2 distribution was not changed significantly with respect to infection or the Chlamydia inclusion (not shown).The participation of septins was unexpected, and represents the first description of a role for septins in Chlamydia type III secreted proteins in actively controlling actin coat assembly, and that extrusion was also dependent on chlamydial type III secreted product(s). We do not think Tarp is involved with this process, since Tarp staining did not colocalize with actin rings or foci, nor was Tarp found in the cytosol of cells at 20–68 hpi (not shown). Although their identity is unknown, the chlamydial proteins responsible exhibited a rapid turnover inside the host cell—as little as 1–2 hour treatment with chloramphenicol or rifampicin resulted in complete loss of actin-coated inclusions. Similar results were obtained by short-term treatment of cells with the C1 type III secretion inhibitor compound, which has been shown to effectively disrupt chlamydial type III secretion Perhaps the salient finding of this work was the central role of Chlamydia biology and their interactions with the host cell.Two emergent themes of this study are the striking heterogeneity of the phenomenon from cell-to-cell and the functional connection between actin coats and extrusion formation. Both of these themes inform new insight into Chlamydia. This could be due to: (i) differential timing of Chlamydia effector secretion into the host cytosol or onto the inclusion membrane, (ii) threshold levels of effector proteins that need to be met in balance with cognate host factors, (iii) differences among inclusions in the location of key secreted chlamydial proteins on the inclusion or in relation to cellular organelles. The subcellular distribution of actin coats showed clear patterns of organization—perinuclear actin foci for 20 hpi inclusions and arcs distal to nuclei for 44 hpi inclusions. These data are consistent with a spatial bias for actin nucleation on the inclusion. Moreover, there is a precedent for heterogeneous modifications of the Chlamydia inclusion by Inc proteins and their colocalization with Src family kinase microdomains Actin coat formation exhibited little to no partiality—inclusions either exhibited it fully (for their respective time of infection) or not at all, and this contrast was frequently observed for neighboring cells in a field. The simplest interpretation of this is that not all inclusions are modified equivalently and concurrently by Chlamydia extrusion in vitro. Both of these processes are directed by chlamydial proteins, and future identification of these proteins will reveal how intracellular Chlamydia induce their escape from host cells by the novel extrusion mechanism.Finally, our data demonstrates that actin coating of inclusions is a key determining factor for extrusion; disruption of coat formation led to a marked reduction in the number of extrusions produced. This process may be functionally similar to a proposed mechanism for exocytosis of large secretory vesicles and organelles Unless otherwise specified, all reagents and chemicals were obtained from Sigma . Additional reagents and concentrations used include: chloramphenicol (100 µg/ml), rifampicin (100 µg/ml), C1 compound N′--4-nitrobenzohydrazide (50 µM) , jasplakinolide (200 nM) , latrunculin B (500 nM) (Invitrogen), CK-548 (10 µM) , CK-666 (10 µM) , blebbistatin (10 µM) (EMD Millipore), phalloidin (Invitrogen), wiskostatin (10 µM) (EMD Millipore), small molecule inhibitor of formin homology 2 domain (10 µM) , LY294002 (50 µM) (Enzo), cell permeable C3 transferase CT04 (2.5 µg/ml) , Y27632 (20 µM) (Enzo), Src Kinase Inhibitor I (25 µM) (EMD Millipore), nocodazole (10 µM).2. For all experiments, cells were subcultured and plated onto chambered coverglass slides , glass bottom culture dishes  or 24-well plates .HeLa 229 and L929 cells Chlamydia trachomatis serovars L2 and D, and C. muridarum were grown for 48 h in L929 cells as described previously Chlamydia EB diluted in HBSS to a multiplicity of infection ≤1, for 2 h at 25°C. Following static incubation, cells were rinsed twice with HBSS, reimmersed in growth media and incubated at 37°C.Infections were performed by washing cells with Hank's balanced saline solution  and incubating cells with Chlamydia, and transfections were performed using the Effectene reagent (Qiagen), per manufacturer's protocol.Cells with transient expression of cytosolic DsRed and LifeAct-GFP were generated by co-transfecting freshly plated cells with 0.1 µg each of DsRed-Express (Clontech) and LifeAct–TagGFP2 . This was routinely performed 6 hours prior to infection with z-slices were acquired for each cell or field, using a z-focus motor drive  under control of Volocity Acquisition software v6  on an Apple iMac . Fields of cells were selected at random and imaged if they contained identifiable inclusions by DsRed fluorescence Chlamydia inclusions were scored as positive for actin recruitment if any of the z-slices for a particular cell exhibited significant association of fluorescent actin on the inclusion; the minimum criteria for significance was an arc of actin that circumscribed at least ∼30% of the inclusion boundary.Experiments using infected, DsRed- and LifeAct-GFP-expressing cells were performed in triplicate or wells of 4-well or 8-well chambered coverglass slides. At times hpi indicated, cells were washed with RPMI without phenol red and mounted live on a Zeiss Axiovert 200 inverted microscope with a Orca-R2 cooled digital CCD camera , 40× oil objective (1.3NA Plan-NEOFLUAR) and Sedat Quad filter set . Three xy stage  and z focus , a humidified incubation chamber for maintaining environmental control of 37°C temperature and 5% CO2 (In Vivo Scientific), and using NIS elements software (Nikon). Images were captured every 5 minutes for 6–8 hours. Time-lapse recordings were exported into Volocity for further analysis, and movies were compiled using Volocity and QuickTime (Apple).Infected cells for real-time imaging were grown and prepared in 4-well chambered coverglass slides, and rinsed and incubated with serum-free RPMI media lacking phenol red (HyClone) prior to mounting on the microscope. Slide chambers were secured and imaged on a Nikon TE2000-E inverted microscope , with a CoolSnap HQ cooled CCD camera , 40× oil objective (1.3NA Plan Fluor), with computer control of an C. trachomatis as described above. Samples undergoing detergent extraction were rinsed with PBS and incubated for 5 min at 25°C in an extraction solution consisting of 1% Triton X-100, 2% PEG 35 kD and 2 µM phalloidin that was prepared in 1X PEM buffer: 100 mM PIPES, pH 6.9, 1 mM MgCl2, 1 mM EGTA. All samples were then rinsed three times with PEM and fixed with 2.5% glutaraldehyde  for at least 20 minutes at 25°C. Samples were stored in fixative at 4°C until ready for further processing using techniques described Cells were grown to subconfluency on 10 mm square silicon wafers  and infected with z dimension) and deconvolution (25 iterations). Individual xy and xz slices were obtained from image stacks for figure assembly. Three dimensional opacity renderings of fluorescent image stacks were generated in Volocity. Minor retouching of all micrographs—for example, color assignment, contrast adjustment, RGB merges and cropping—were performed with Volocity and Photoshop CS5 (Adobe). Illustrator CS5 (Adobe) was used to assemble all figures into their final form: add scale bars, arrows, labels, etc.In some instances, three dimensional image stacks were further processed in Volocity by performing illumination correction . Extrusions were subsequently enriched by centrifugation (10 min at 1200 rpm); pellets containing extrusions were resuspended in 1–5 mL growth media. To enumerate the number of extrusions obtained from a cell monolayer, resuspended extrusions were stained with SYTOX Green  and Hoechst  for 5 minutes at 25°C, plated as 5–10 µl drops onto glass slides and imaged immediately on an inverted fluorescence microscope. Intact extrusions were identified as having chlamydial inclusions, lacking nuclei and being the appropriate size.t-test. P-values <0.01 (*) were considered statistically significant. P-values of <0.001 (**) and <0.0001 (***) were marked as indicated. Calculations were performed in Numbers (Apple) and Microsoft Excel.Statistical evaluation of data was performed by calculating the standard error of the mean (SEM) or using a two-tailed student Figure S1Three dimensional distribution of LifeAct-GFP in Chlamydia infected cells. HeLa cells expressing LifeAct-GFP (green) and cytosolic DsRed (magenta) were infected with C. trachomatis L2 and imaged at 20 hpi (A), 44 hpi (B) and 68 hpi (C) by live microscopy. Over 55 z-stacks were acquired at 0.5 µm intervals. Image stacks were processed by deconvolution. Representative fields of cells for each time point are depicted. The cell in (B) and the leftmost cell in (C) were also rendered in three dimensions to show the extensiveness of actin coating on the inclusion. In each case, views were rotated slightly to yield the most informative perspective. Insets in (A) are magnifications of the inclusion in the lower left cell. Asterisks (*) mark the locations where orthogonal planes in xz were taken. Arrows in (A) mark the inclusions. Scale bars = 20 µm.(TIF)Click here for additional data file.Figure S2Three dimensional distribution of endogenous actin filaments in Chlamydia infected cells. HeLa cells were infected with C. trachomatis L2, and fixed and processed for immunofluorescence at 20 hpi (A), 44 hpi (B) and 68 hpi (C). Immunofluorescence staining was performed using phalloidin conjugated to Alexa633 (purple), a specific antibody to Chlamydia MOMP (green) and the nucleic acid dye DAPI (blue) for staining nuclei and, to a lesser extent, bacteria. The partial staining of nuclei and bacteria in the actin channel are likely artifact, and due to bleedthrough of Evans blue dye . A total of 17, 28 and 56 z-stacks were acquired at 0.5 µm intervals for (A), (B) and (C), respectively. Image stacks were processed by deconvolution. Representative fields of cells for each time point are depicted. Asterisks (*) mark the locations where orthogonal planes in xz were taken. Arrows in (A) and (B) mark the inclusions. Scale bars = 20 µm.(TIF)Click here for additional data file.Video S1Three dimensional rendering of GFP-actin recruitment to a C. trachomatis L2 inclusion at 48 hpi. Cell is labeled with LifeAct-TagGFP (green) and cytosolic DsRed (magenta). Three dimensional rendering and rotation are from the cell depicted in (MOV)Click here for additional data file.Video S2Three dimensional rendering of GFP-actin recruitment to a C. trachomatis L2 inclusion at 72 hpi. Cell is labeled with LifeAct-TagGFP (green) and cytosolic DsRed (magenta). Three dimensional rendering and rotation are from the leftmost cell depicted in (MOV)Click here for additional data file.Video S3Time-lapse videomicroscopy of GFP-actin in C. trachomatis L2 infected cell at 48 hpi. Cell is labeled with LifeAct-TagGFP (green) and cytosolic DsRed (magenta). Frames were captured every 5 minutes.(MOV)Click here for additional data file.Video S4Time-lapse videomicroscopy of GFP-actin in C. trachomatis L2 infected cell at 24 hpi. Cell is labeled with LifeAct-TagGFP (green) and cytosolic DsRed (magenta). Frames were captured every 5 minutes.(MOV)Click here for additional data file.


Anesthetic management of laboring parturients with Arnold-Chiari type I malformation poses a difficult challenge for the anesthesiologist. The increase in intracranial pressure during uterine contractions, coughing, valsalva maneuvers, and expulsion of the fetus can be detrimental to the mother during the process of labor and delivery. No concrete evidence has implicated high cerebral spinal fluid pressure on maternal and fetal complications. The literature on the use of neuraxial techniques for managing parturients with Arnold-Chiari is extremely scarce. While most anesthesiologists advocate epidural analgesia for management of labor pain and spinal anesthesia for cesarean section, we are the first to report the use of combined spinal-epidural analgesia for managing labor pain in a pregnant woman with Arnold-Chiari type I malformation. Also, we have reviewed the literature and presented information from case reports and case series to support the safe usage of neuraxial techniques in these patients. Arnold-Chiari type I malformation (ACM-I) is a congenital neurological anomaly associated with prolapse of the cerebellar tonsils into the magnum foramen , 2. ApprA combined spinal-epidural (CSE) technique was used to provide labor analgesia for our parturient with ACM-I. We also conducted a literature search for our case presentation using a public accessible medical database MEDLINE. Individual key words were entered into the query: “Arnold-Chiari,” “vaginal delivery,” “pregnancy,” “combined spinal-epidural analgesia,” “epidural analgesia,” “spinal analgesia,” “cesarean section,” “perioperative outcomes,” and their combinations. Only articles in English language were selected. The database search yielded limited number of articles, mainly case reports and case series .A 17-year-old female, G1P0, with history of hypothyroidism and ACM-I diagnosed during childhood, presented with symptoms of occasional headache and neck pain. She denied any visual disturbances or abnormal pain and temperature sensation in both upper extremities. She was consulted by a multidisciplinary team, including the anesthesiologist, perinatologist, and neurologist, for a planned labor induction with instrument-assisted vaginal delivery. MRI of the brain showed a 7 mm cerebellar tonsil herniation into the foramen magnum without syringomyelia .9/L. With a single attempt, CSE was achieved using a 17-gauge Tuohy needle and a 5-inch 27-gauge Whitacre spinal needle at the midline of the L3-L4 interspinous space while the patient was in a sitting position. Analgesia was obtained with fentanyl 15 μg and bupivacaine 1.5 mg intrathecally. Aspiration of the epidural catheter and test dose of lidocaine 1.5% with epinephrine 1 : 200,000 were negative. A continuous epidural infusion of bupivacaine 0.1% and fentanyl 0.0002% was initiated at the rate of 10 mL/h. A 5 mL bolus of bupivacaine 0.25% was injected epidurally 90 minutes before the onset of fetal expulsion and subsequently augmented with another bolus to provide a denser block to minimize the urge to push. Physical examination showed a 62 kg afebrile woman, in mild distress from uterine contractions, with a blood pressure of 134/89 mmHg, pulse of 62/min, respiratory rate of 12/min, and pulse oximetry saturation of 99%. Baseline laboratory values were hemoglobin 11.9 g/dL and platelets 206 × 10Fetal heart rate (FHR) and uterine contractions were continuously monitored by an external cardiotocograph. Category I FHR tracing was noted throughout the first and second stages of labor. Maternal and fetal hemodynamics were stable during the entire labor and delivery process. Labor progressed smoothly and lasted for 9 hours. The patient gave birth to a 2,995 g healthy girl using vacuum-assisted extraction. Apgar scores at 1 and 5 minutes were 9 and 9, respectively. Estimated blood loss was 200 mL. The patient had an uneventful postpartum course without any neurological sequelae. She was discharged home three days later. Attempts to demonstrate the efficacy and safety of neuraxial technique  in a pregnant woman with ACM-I have been the subject of controversy. The risks of accidental dural puncture with the epidural needle can lead to tentorial herniation, decreased cerebral perfusion pressure, and brain shifts. Intentional intrathecal puncture using spinal needle can also present with similar manifestations but the magnitude of the effect and incidence is relatively less than the epidural needle-induced dural puncture due to the larger size of the dural puncture. Selection of smaller size epidural and spinal needles is an important factor to improve safety, but, ultimately, the danger can be significantly minimized with an experienced and trained anesthesiologist to avoid inadvertent dural puncture as well as multiple needle attempts.The safety of providing intrathecal analgesia for immediate pain relief during labor and anesthesia for cesarean section (CS) can be effectively implemented provided that there are no acute worsening of clinical signs and symptoms of intracranial pressure (ICP). In our case presentation, we selected the use of CSE to provide immediate pain relief intrathecally for our patient and the epidural catheter to administer intermittent extradural boluses for analgesia during the course of labor and delivery as well as for anesthesia for emergent CS due to obstetrical and fetal concerns. Had our patient developed severe or new onset of neurological symptoms during pregnancy, neuraxial technique would be contraindicated. Even without any absolute contraindications, there are currently no firm guidelines to suggest preference for general anesthesia over neuraxial techniques except many believe that the patients with ACM-I have inherent high ICP; therefore, neuraxial techniques are unsuitable choice for analgesia and anesthesia –17. GeneThe choice for the mode of delivery  is also a controversial issue. The contractile force of the uterus on cerebral spinal fluid (CSF) can cause an increase in ICP and unsuspected herniation. The hydrodynamic effect on CSF pressure during labor was investigated by several researchers in the 1960s –21. ChanKey points in the anesthetic management of laboring parturients with ACM-I include (1) early CSE analgesia to decrease painful uterine contractions to limit intra-abdominal and intrathoracic excursions to dampen elevated CSF pressure; (2) slow titration of bolus through the epidural to prevent undue extradural pressure; (3) vacuum-assisted vaginal delivery in the second stage of labor to minimize increase in ICP during fetal expulsion and maternal valsalva maneuvers; and (4) minimization of wide variations of maternal hemodynamics to preserve adequate cerebral perfusion pressure. In summary, CSE labor analgesia can provide safe and effective pain relief to parturient with ACM-I. We emphasize the importance of multidisciplinary approach to tailor an individualized care plan for favorable maternal and fetal outcomes.


Uncovering the functional properties of individual synaptic inputs on single neurons is critical for understanding the computational role of synapses and dendrites. Previous studies combined whole-cell patch recording to load neurons with a fluorescent calcium indicator and two-photon imaging to map subcellular changes in fluorescence upon sensory stimulation. By hyperpolarizing the neuron below spike threshold, the patch electrode ensured that changes in fluorescence associated with synaptic events were isolated from those caused by back-propagating action potentials. This technique holds promise for determining whether the existence of unique cortical feature maps across different species may be associated with distinct wiring diagrams. However, the use of whole-cell patch for mapping inputs on dendrites is challenging in large mammals, due to brain pulsations and the accumulation of fluorescent dye in the extracellular milieu. Alternatively, sharp intracellular electrodes have been used to label neurons with fluorescent dyes, but the current passing capabilities of these high impedance electrodes may be insufficient to prevent spiking. In this study, we tested whether sharp electrode recording is suitable for mapping functional inputs on dendrites in the cat visual cortex. We compared three different strategies for suppressing visually evoked spikes: (1) hyperpolarization by intracellular current injection, (2) pharmacological blockade of voltage-gated sodium channels by intracellular QX-314, and (3) GABA iontophoresis from a perisomatic electrode glued to the intracellular electrode. We found that functional inputs on dendrites could be successfully imaged using all three strategies. However, the best method for preventing spikes was GABA iontophoresis with low currents (5–10 nA), which minimally affected the local circuit. Our methods advance the possibility of determining functional connectivity in preparations where whole-cell patch may be impractical. By gaining access to the subthreshold membrane potential, in vivo intracellular recordings partially bridged this gap and greatly improved our understanding of the synaptic underpinnings of sensory receptive fields  is organized into precise maps for a variety of stimulus attributes, e.g., orientation, direction, ocular dominance, and binocular disparity  hyperpolarization by injection of negative current, (2) pharmacological intracellular blockade of voltage-gated sodium channels with QX-314, and (3) near-somatic ejection of GABA from an iontophoresis electrode glued to the sharp intracellular electrode. The last strategy currently appears to be the most promising tool for mapping the functional connectivity in the neocortex of non-rodent mammals.2 was maintained between 3.5% and 4.5%. A heating pad maintained the rectal temperature between 37.0 and 38.0°C. Dexamethasone  and atropine  were injected to prevent inflammatory reactions and to reduce secretions, respectively. A headplate was cemented to the skull over the primary visual cortex, a 1.5 mm × 3 mm craniotomy was performed and the dura was resected. To reduce heart and respiratory induced pulsations, the underlying cortex was covered with warm agarose (2.5% in ACSF) and the craniotomy was sealed by a coverglass  . All procedures were approved by the Institutional Animal Care and Use Committee of the Medical University of South Carolina and were based on those previously published  square- or sine-wave gratings were presented at a variety of orientations, directions of motion, spatial frequencies, spatial positions, binocular phase disparities, and monocularly through the left or right eye. For ocular dominance or binocular disparity assays, each animal viewed monoptic and dichoptic stimuli displayed on a CRT monitor (160 Hz) through custom ultra-fast ferroelectric liquid crystal shutters. All other tests were conducted monocularly through the dominant eye. Visual stimuli were presented for 4–8 s interspersed with equal duration blank periods and the stimulus sequence was repeated 2–10 times.Sharp microelectrodes were pulled from filamented borosilicate glass capillaries  using a horizontal micropipette puller . The resistance of the microelectrodes was ~90 MΩ (range 70–110 MΩ) when filled with 2 M potassium methyl sulfate (Acros Organics) and 2–4 mM Oregon Green BAPTA-1 (OGB-1) hexapotassium salt (Life Technologies) at pH = 7.3–7.5. In some cases the pipette solution also contained 20–100 mM QX-314 (Alomone Labs) to block voltage gated sodium channels Figure  and 200 Iontophoresis electrodes were pulled using the same type of glass capillaries as for sharp electrodes. The puller program was modified so that the shank length of the iontophoretic electrode was comparable to the sharp electrode (~8–9 mm) and the tip diameter was larger for the iontophoresis electrode (~1 μm) than for the sharp electrode (<0.2 μm). The iontophoresis electrode was bent to an angle of ~30° by pressing on the shank (~5 mm above the tip) with a 16G needle during exposure to the heating filament of a vertical puller .OGB-1 fluorescence was monitored using a custom-built microscope (Prairie Technologies) coupled with a Mai Tai Deep See (Newport Spectra-Physics) mode-locked Ti:sapphire laser (810 nm or 920 nm). Excitation light was focused by a water-immersion objective . The average power delivered to the brain was kept below 40 mW. Two non-descanned photomultiplier tube (PMT) detector channels (Hamamatsu R3896) were used with a primary dichroic  and IR blocking filter , and barrier filters (Chroma) customized for the fluorophores of interest: ET 525/50m-2p for OGB-1, ET 665/40m-2p for Alexa 633, and HQ 645/70m-2p for Alexa 594  hexapotassium salt, as described above .Our procedure was adapted from Stosiek et al.  and descXY-T images of OGB labeled neurons were analyzed using customized Matlab (Mathworks) software. Regions of cortex were imaged at 364 × 353 pixels (42 × 41 μm) to 512 × 512 pixels (300 × 300 μm) at 0.64–1.64 s per frame. Images were realigned by maximizing the correlation between frames.For OGB-1 AM bulk-loading experiments, cell bodies were identified automatically through a series of morphological filters that defined the contours of cell bodies based on intensity, size and shape. Dendritic segments were initially ~2 μm in size, selected manually. Based on the similarity of response selectivity in adjacent dendritic segments, in subsequent iterations of the analysis we increased the size of each segment up to 25 μm. In a given iteration, all segments had the same size. For the purposes of comparing the three methods of spike suppression, only a small representative subset of visually responsive dendritic segments are shown in Figures n visual stimuli (p < 0.01). Cell bodies or dendritic segments selective for particular stimuli were defined by ANOVA across n stimulus periods (p < 0.01).Time courses of calcium transients were extracted by calculating mean pixel values within cell bodies or dendritic segments. The fluorescence during visual stimulation was divided by the average fluorescence during the 4–8 s baseline period prior to each stimulus to obtain the visually evoked change in fluorescence (ΔF/F). Visually responsive cell bodies or dendritic segments were defined by ANOVA across blank and GABA/Rcontrol) × 100, where Rcontrol and RGABA are the responses before and during GABA iontophoresis, respectively. This ratio is sensitive to noise in very weakly responsive cells. Thus, when plotting the residual response as a function of distance from the GABA electrode  were scored 100 (no effect). In a second control, eight directions of drifting gratings were presented to assess the orientation selectivity index (OSI) of each cell in the presence and absence of GABA  is the response at stimulus orientation θ.An iontophoresis electrode was positioned in the center of a region of layer 2/3 cortex that was bulk-loaded with OGB-1 AM. In the first control, somatic calcium indicator responses to gratings drifting in one direction were measured before, during and after GABA iontophoresis at varying current intensities Figure . The rese Figure , visuallIntrinsic signals were recorded as described elsewhere ; (2) blockade of voltage-sensitive sodium channels by including QX-314 in the electrode solution (n = 7 neurons); or (3) GABA iontophoresis near the soma of the intracellularly recorded neuron from an iontophoresis electrode glued to the sharp electrode (n = 4 neurons).Sharp electrodes were used to record the membrane potential and infuse OGB-1 salt in 31 pyramidal neurons in layer 2/3 of the cat visual cortex. In 16 of these neurons, recording durations were long enough to measure the membrane potential while imaging visually evoked responses on dendrites . In a given neuron, visually evoked spikes were suppressed by one of the following strategies: (1) hyperpolarization by injection of a constant negative current  would blur the functional differences between synaptic inputs to distinct dendrites and could make a dendrite appear more broadly tuned than the local synaptic drive. QX-insensitive spikelets were a common feature in neurons when using 50–100 mM QX-314  was necessary to suppress the responses to optimal stimuli Figure . The larn = 2 cats). First, we presented a stimulus whose orientation was near optimal for all the imaged neurons and measured the somatic calcium response while GABA was delivered from an iontophoresis electrode identical to those used for making double-barrel electrodes . Orientation selectivity across the population was significantly decreased by GABA iontophoresis at +20 nA, but not at +5 nA and +10 nA .In two separate control experiments, we labeled populations of layer 2/3 neurons with OGB-1 AM (s Figure . As expes Figure . Second,s Figure . The preWe have shown that the location and selectivity of sensory-evoked synaptic inputs can be measured by changes in calcium indicator fluorescence on dendrites in the cat visual cortex. Subcellular mapping of functional inputs was possible only when action potentials in the recorded neuron were prevented, which we verified by intracellular sharp electrode recording. We compared different strategies to prevent spiking, and found that GABA ejection from a perisomatic iontophoresis electrode provided the best control. To our knowledge this is the first time that functional inputs have been mapped on the dendritic tree of cortical neurons in a non-rodent mammal.in vivo holds great promise for the study of cortical functional connectivity (see Grienberger and Konnerth, in vitro: Jaffe et al., in vivo: Svoboda et al., in vivo relied on whole-cell recording through patch electrodes (Jia et al., The development of dendritic imaging using fluorescent calcium indicator proteins and synthetic dyes Compared to patch electrodes, sharp electrodes are much thinner, and therefore much more flexible. This flexibility provided good intracellular recording stability in the cat, after we dampened brain pulsations by covering the craniotomy with agarose and a coverglass. The time required for labeling neurons was comparable to the loading times reported previously in sharp and patch intracellular recordings (Svoboda et al., Hyperpolarization by current injection through the sharp electrode is the most straightforward strategy to control spiking in the recorded neuron. In practice however, the maximum negative current our sharp electrodes can deliver is approximately −1 nA. This is seldom sufficient to completely prevent spikes in response to optimal binocular stimuli, which evoke a large excitatory drive in cat cortical neurons (Freeman and Ohzawa, QX-314 is an appealing pharmacological alternative to current injection for blocking spikes (also see Takahashi et al., extracellular responses to sensory stimuli during the iontophoretic administration of various pharmacological agents (Stone, intracellular recording and local iontophoresis in vivo are much less common (but see Douglas and Martin, in vivo were not more difficult to obtain with the double-barrel electrode, compared to the conventional single sharp electrode. Moreover, of the three spike suppression methods tested, local GABA iontophoresis offered the finest control such that the spiking responses to optimal binocular stimuli could be completely prevented with +5 to +10 nA of iontophoretic current. Our results and an earlier study (Ferron et al., A receptors on the cell body and axon initial segment of cortical pyramidal neurons (Douglas et al., Multi-barrel electrodes are typically used to study the s Stone, . Double-Our double-barrel electrodes were fabricated such that the tip of the iontophoresis electrode was located close to the soma of the recorded neuron, enabling GABAergic inhibition of spikes at low current intensities. Delivering additional hyperpolarizing current intracellularly through the sharp electrode further minimized the GABA iontophoretic current that was needed to prevent all spikes. Most of the neurons suppressed by +5 to +10 nA GABA iontophoretic currents were located less than 50 μm away from the electrode tip. This corresponds to approximately 30 neurons (Beaulieu and Colonnier, As a future development of our technique, intracellular recordings may not be required for dendritic functional imaging provided that the iontophoretic current has been calibrated for spike suppression in a particular preparation. Neurons could be sparsely labeled by electroporation or by a genetically encoded calcium indicator, e.g., GCaMP6 (Chen et al., in vivo while simultaneously imaging calcium responses in dendrites, double-barrel electrodes could advance our understanding of the role of synaptic plasticity and dendritic excitability during sensory processing.In conclusion, the choice of patch vs. sharp electrodes for dendritic functional imaging with synthetic dyes depends on the experimental model being used. In small animals such as rodents, patch electrodes are preferable, due to their lower impedance. In large mammals such as the cat, sharp electrodes may be more suitable, because they provide long-duration intracellular recordings and because the dye does not accumulate in the extracellular space as the pipette advances to the neuron. More generally, by providing a means to apply pharmacological agents such as neuromodulators or channel blockers The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Both transcriptional subtype and signaling network analyses have proved useful in cancer genomics research. However, these two approaches are usually applied in isolation in existing studies. We reason that deciphering genomic alterations based on cancer transcriptional subtypes may help reveal subtype-specific driver networks and provide insights for the development of personalized therapeutic strategies. In this study, we defined transcriptional subtypes for colorectal cancer (CRC) and identified driver networks/pathways for each subtype. Applying consensus clustering to a patient cohort with 1173 samples identified three transcriptional subtypes, which were validated in an independent cohort with 485 samples. The three subtypes were characterized by different transcriptional programs related to normal adult colon, early colon embryonic development, and epithelial mesenchymal transition, respectively. They also showed statistically different clinical outcomes. For each subtype, we mapped somatic mutation and copy number variation data onto an integrated signaling network and identified subtype-specific driver networks using a random walk-based strategy. We found that genomic alterations in the Wnt signaling pathway were common among all three subtypes; however, unique combinations of pathway alterations including Wnt, VEGF and Notch drove distinct molecular and clinical phenotypes in different CRC subtypes. Our results provide a coherent and integrated picture of human CRC that links genomic alterations to molecular and clinical consequences, and which provides insights for the development of personalized therapeutic strategies for different CRC subtypes. Colorectal cancer (CRC) is a major cause of global cancer morbidity Because signaling pathways and networks rather than individual genes govern the course of tumorigenesis and progression Network-based methods have started to provide a systems level understanding of complex genomic variations. However, because existing studies usually consider all tumor samples together in contrast to normal controls, they tend to identify signaling networks common to all tumor samples and may fail to address the heterogeneity among cancer genomes.Transcriptional subtype analysis has provided great insights into disease biology, prognosis and personalized therapeutics for different cancer types For CRC, the TCGA (The Cancer Genome Atlas) network recently reported a classification of three transcriptional subtypes, which were named as “MSI/CIMP”, “Invasive”, and “CIN”, respectively In this study, we hypothesize that highly heterogeneous genomic alterations observed in CRC may converge to a limited number of distinct mechanisms that drive unique gene expression patterns in different transcriptional subtypes. First, we extended the TCGA findings by performing subtype discovery based on gene expression data from 1173 CRC tumor samples accumulated during the past decade, validated identified subtypes in an independent cohort with 485 samples, and associated each subtype with unique biology and clinical outcome. Next, we mapped somatic mutation and copy number variation (CNV) data onto an integrated signaling network and identified a driver network for each subtype. The inferred networks and associated pathways correlated perfectly with downstream transcriptional programs characteristic for each subtype, providing strong circumstantial evidence for the effectiveness of our approach and the validity of our inference. Based on the unique combinations of pathway alterations and clinical outcomes, we have proposed specific therapeutic strategies for different CRC subtypes.As shown in To investigate gene expression changes in CRC samples relative to normal mucosa samples, gene expression data for these 182 samples were normalized together by the RMA algorithm http://array.mc.vanderbilt.edu), where RNA was purified with the use of the RNeasy kit  and hybridized to the Affymetrix Mouse Genome 430 2.0 GeneChip Expression Arrays  according to manufacturer’s instructions. The RMA algorithm was used for data normalization. Mouse gene symbols were mapped to human gene symbols by the Human and Mouse Orthology list available from the Mouse Genome Informatics (http://www.informatics.jax.org/).To characterize the embryonic development of colon, we conducted a time course microarray study using the inbred C57BL/6  mice . This study was carried out in strict accordance with animal care and use guidelines and approval of the Vanderbilt Institutional Animal Care and Use Committee (IACUC). Mice were monitored throughout the experiment for signs of distress during their normal life cycle, although no experimental manipulations of these mice were carried out besides breeding. If signs of distress were seen during weekly monitoring, mice were euthanized by CO2 asphyxiation followed by cervical dislocation to reduce animal suffering. Seven samples corresponding to the mouse colonic development from E13.5 to E18.5 and adult  were collected. Embryonic colon collection and RNA preparation were performed as previously described CNV data and somatic mutation data for TCGA samples with matched gene expression data were downloaded from the TCGA website.Signaling pathways curated by NCI-Nature, Cancer Cell Map, and REACTOME were downloaded from the Pathway Commons database . BioCarta signaling pathways were downloaded from the NCI Pathway Interaction Database . Integrating pathways from all the above sources resulted in a signaling network containing 3152 genes and 47,833 edges. Its largest component contained 3078 genes and 47,772 edges, which was used for the inference of the upstream driver subnetworks.Based on the gene expression matrix with 10,481 genes and 1173 samples for the discovery cohort, we calculated the Pearson’s correlation coefficients for all the 54,920,440 gene pairs. The construction of a co-expression network requires an appropriate selection of a threshold for the pair-wise correlation coefficients. To ensure the biological relevance of the constructed network, we used a knowledge-guided method for threshold selection i, we computed a(i) as the average distance between i and all other samples from the subgroup where i belongs. Then, the average distance between i and all samples from each of the other subgroups was computed respectively, and the smallest average distance, b(i), was identified. Next, we calculated the silhouette width s(i) as defined by: s(i) = (b(i) − a(i) )/max(a(i), b(i) ) For subtype discovery, we performed the consensus average linkage hierarchical clustering p<0.05) in one subtype compared to all other subtypes were defined as up-regulated genes for this subtype. Next, remaining genes that were significantly down regulated in one subtype compared to all other subtypes were defined as down-regulated genes for this subtype. For each subtype, both the up-regulated genes and the down-regulated genes were considered as signature genes.We used a nearest shrunken centroid classification method, Prediction Analysis of Microarrays (PAM) We employed the Netwalker algorithm n as the total number of genes and m as the total number of samples. The somatic mutation status of gene i is defined as:To assign higher weight to genomic alterations observed in samples with fewer total number of alterations and alterations observed in multiple samples, we performed column-wise normalization followed by row-wise summarization for each binary matrix, and thus transformed each matrix into a vector. For a subtype, let’s denote i in sample j in the somatic mutation matrix. Similarly, the CNV status of gene i is defined as: i in sample j in the CNV matrix. Next, i  was reported as the driver subnetwork.To assess the statistical significance of the scores for each gene, we constructed 1000 sets of randomly permuted start probabilities and generated 1000 sets of random scores. For each gene in the network, a local Standard Kaplan–Meier survival curves were generated for CRC subgroups, and the survival difference between groups was statistically evaluated using the log-rank test. The univariate and multivariate Cox proportional hazard regression analyses were used to evaluate potential independent prognostic factors associated with survival. All these analyses were performed using the survival package in R.GO and KEGG pathway enrichment analyses were performed using WebGestalt, in which the hypergeometric test was used for enrichment analysis and the Benjamini-Hochberg procedure was used to control the False Discovery Rate (FDR) Networks were visualized using Cytoscape We used a well-established method, Consensus Clustering According to the consensus matrices and the empirical cumulative distribution function (CDF) plots in Next, we used PAM to build a classifier for the above defined subtypes. The shrinkage in PAM performs automatic gene selection and can potentially make the classifier more accurate by reducing the effect of noisy genes. The smallest average cross-validation error of 0.5% was achieved using all the 1472 genes based on 100 times of 10-fold cross validation, suggesting that noisy genes might have already been removed in our co-expression module-based gene selection procedure. With relaxed error rate requirement, PAM was able to further reduce the number of genes in the classifier. For example, when the error rate increased to 9%, a classifier with 853 genes was reported. Classifiers with reduced gene numbers are usually preferred in classification tasks; however, because an important goal in this study was to understand the biology underlying different subtypes, we selected the 1472-gene classifier to facilitate downstream GO enrichment analysis.Using the method described in Materials and Methods, we found 449 signature genes for subtype 1  with the largest median absolute deviation across the 1173 samples. Compared to our method, the single-gene based method generated larger average cross-validation error in the PAM analysis (2% vs 0.5%). Moreover, most of the subtype-specific signatures produced by the single-gene based method showed no significant functional coherence compared to random gene lists of the same size.To validate the CRC subtypes discovered above, we compiled an independent gene expression dataset with 485 CRC samples from six additional resources . The subFor subtype identification, we focused on the relative gene expression changes across all tumor samples. To further clarify the absolute direction of gene expression changes, we compared the expression of signature genes in each CRC subtype to their expression in normal colon mucosal samples. As shown in It has been suggested that CRC tumorigenesis and progression recapitulates embryonic development and epithelial mesenchymal transition (EMT) programs −24), DNA metabolic process (FDR = 9.18×10−12) and mRNA metabolic process (FDR = 2.63×10−7) (−4) and neurological system process (FDR = 1.56×10−14). These genes are repressed in undifferentiated embryonic cells First, we generated a gene expression dataset  of normal mouse colon development (E13.5–E18.5 and adult) and defined development-related genes as the top1000 genes with the largest median absolute deviation across different time points among those with a high correlation to developmental time points (absolute Spearman correlation coefficient >0.9). Based on the development-related genes, we evaluated the correlation between the expression patterns of different CRC subtypes and different developmental time points. Specifically, for each pair of CRC subtype and developmental time point, we calculated the Pearson’s correlation coefficient between the subtype centroids of the development-related genes and the expression levels of the same genes at the time point. As shown in 63×10−7) . It is wp-value <0.01 in t-test were used in our analysis. These genes had a much higher level of expression in subtype 1 tumors compared with the other two subtypes (−4) and blood vessel morphogenesis (FDR = 7.49×10−5), biological processes closely related to EMT Next, we examined the expression pattern of a previously published EMT signature subtypes . GO enriUsing overall survival information available for samples from the Moffitt Cancer Center (GSE17536), the Vanderbilt Medical Center (GSE17537) and the Max Planck Institute (GSE12945), we performed a survival analysis to compare clinical outcomes for different CRC subtypes. Combining all the three datasets created a cohort with a total of 251 samples. This cohort included 161 samples from CRC stage II and III, where molecular stratification of these intermediate stage tumors could greatly facilitate treatment decision making p = 0.001 for all the patients and p = 0.002 for stage II and III patients only, log-rank tests with H0: hazard_subtype1 =  hazard_subtype2 =  hazard_subtype3). Subtype 1 with a highly activated EMT program had the poorest overall survival among the three subtypes , which was in agreement with previous reports on the association between EMT and CRC patient survival As depicted in p = 0.0041 for subtype 2 vs 1 and p = 0.036 for subtype 3 vs 1). Thus, the prognostic value of the subtype classification is independent of the AJCC stage.To evaluate the prognostic value of the subtype classification in combination with clinical variables including patient age at diagnosis, gender and AJCC stage, we performed univariate and multivariate Cox proportional hazards regression analyses based on the cohort of 251 samples . In the We compared our subtype classification with the TCGA classification During our manuscript preparation, two alternative CRC subtype classification schemes have been proposed and applied to the TCGA samples A contradictory observation was that the TCGA invasive subtype showed the highest overlap with our subtype 2, which was found to have the best clinical outcome among the three subtypes . We furtBased on the hypothesis that highly heterogeneous genomic alterations of CRC may converge to a limited number of distinct mechanisms that drive unique gene expression patterns in different CRC subtypes, we attempted to elucidate these distinct mechanisms by inferring upstream driver subnetworks for the unique gene expression patterns and their associated tumor physiologies in different CRC subtypes.This analysis was based on a subset of The Cancer Genome Atlas (TCGA) samples from the validation dataset that had matched somatic mutation and CNV data . After filtering for samples with both somatic mutation and CNV information, we identified 30 subtype 1 samples, 15 subtype 2 samples, and 22 subtype 3 samples.To infer upstream driver subnetworks that are enriched with CNVs and/or somatic mutations, we used the Netwalker algorithm p<0.05 and global p<0.05, see methods) for subtypes 1, 2 and 3, respectively . In addition, the driver subnetwork for subtype 1 was enriched with genes in the VEGF signaling pathway (FDR = 1.0×10−4), and that for subtype 2 was enriched with genes in the Notch signaling pathway (FDR = 1.7×10−3). We further performed the Fisher’s exact test to examine the subtype-specificity of the VEGF and NOTCH pathways. The results showed that genes from the VEGF pathway were significantly over-represented in the driver subnetwork specific to subtype 1 compared to the other two subtypes  and genes from the NOTCH pathway were significantly over-represented in the driver subnetwork specific to subtype 2 .To associate these driver subnetworks with known pathways, we performed functional enrichment analysis based on the signaling pathways curated in the KEGG database . Consistp<0.02), suggesting an active status of the WNT signaling in all three subtypes. PIK3CA was only up-regulated in subtype 1 , suggesting an active status of the VEGF signaling in subtype 1. NOTCH1 was up-regulated in all three subtypes compared to normal samples , suggesting an active status of Notch signaling in all three subtypes. However, RBPJ was only up-regulated in subtypes 1 and 3 , suggesting a relatively less-active status of Notch signaling in subtype 2 compared to the other two subtypes.Because our approach cannot distinguish activating mutations from deactivating mutations, we checked the expression change of some critical genes in the identified signaling pathways using the gene expression dataset GSE17536 with 177 CRC samples and five normal samples. We found that CTNNB1, DKK2, LEF1, LRP5, MYC and RUVBL1 were all up-regulated in all three subtypes compared to normal samples , where the c-Myc target list were downloaded from the MsigDB (version 2.5). The VEGF signaling pathway was inferred specifically for subtype 1. It has been reported that alterations in the VEGF signaling pathway among Wnt-activated cells may activate the expression of genes involved in cell migration, angiogenesis, and the EMT program A well-recognized challenge in computational network and pathway inference is the lack of gold standard for an objective evaluation. In this study, because subtype-specific gene expression signatures and driver networks and pathways were independently identified from gene expression data and genomic data respectively, we assessed our pathway inference by checking the consistency between the inferred pathways and observed gene expression changes for each subtype. Correlating upstream genomic alterations to downstream transcriptional changes also allowed us to further assess whether the genomic alterations had resulted in pathway activation or inhibition. The c-Myc (MYC) transcription factor is a well-known downstream effector of the Wnt signaling pathway Rapid advancement in high-throughput sequencing technologies has shifted our focus from data acquisition to data interpretation. For cancer genomic studies, pinpointing the genomic alterations underlying tumor initiation and progression and determining their downstream functional effects are among the most critical and challenging questions. In this study, we have developed a novel transcriptional subtype-based network inference strategy for deciphering genomic alterations. Many existing studies performed pathway and/or network analysis in tumor cohorts without considering transcriptional subtype classification vs 220 samples in the TCGA study) and our module-based gene selection method. (ii) Subtype characterization requires associating the subtypes with clinical outcome and known cancer biology. The TCGA study was limited by the lack of survival data with enough follow up time for establishing clinical relevance of the subtypes and according to our analysis, the naming of the invasive subtype in the TCGA classification is misleading. (iii) Subtype analysis can be strengthened by associating the subtypes with genomic data. The TCGA study linked genomic features such as MSI/CIMP, hypermutation, and CIN with the transcriptional subtypes, and we also found statistically significant enrichment of CIMP-high tumors in our subtype 1. These are interesting findings, however, it remains unclear how these global genomic features drive downstream distinct expression patterns in different transcriptional subtypes. Our transcriptional subtype-based signaling network analysis provides a more direct means to link genomic alterations to subtype-specific gene expression changes. The TCGA paper also identified frequently altered signaling pathways in CRC, however, these alterations were not associated with transcriptional subtypes.Both the TCGA and our studies have identified three CRC subtypes, and a moderate but statistically significant overlap between two TCGA subtypes and our subtypes was observed . A carefIn addition to a more comprehensive understanding of human CRC, our findings also provide guidance on possible personalized therapeutic strategies for different subtypes. The poor outcome for subtype 1 patients calls for an emphasis on the development of efficient therapeutic strategies for this subtype, and targeting the VEGF pathway or simultaneously targeting the VEGF and Notch pathways seem like rational choices. Interestingly, clinical trials on the anti-VEGF therapy for colorectal cancer patients have reached inconsistent conclusions To test the possible targeted therapeutic strategies, we also tried to identify cell line models for the subtype by applying our classifier to CRC cell lines with publicly available gene expression data. However, representative cell line models were not identified for subtype 1 with the poor overall survial, possibly due to the lack of highly expressed cell migration and blood vessel morphogenesis signatures related to EMT program in CRC cell lines. We are working on the development and use of xenograft models for this subtype.File S1This file contains Tables S1, S2, S5, S7, S8 and S9.(DOCX)Click here for additional data file.File S2This file contains all supplementary figures (S1–S8).(PDF)Click here for additional data file.File S3This file contains Tables S3, S4, S6, and S10.(XLSX)Click here for additional data file.


Pinus albicaulis) ecosystems of western North America have not been previously investigated. Mountain pine beetle attack has driven widespread WbP mortality, which could drive shifts in both the pools and fluxes of nitrogen (N) within these ecosystems. Because N availability can limit forest regrowth, understanding how beetle-induced mortality affects N cycling in WbP stands may be critical to understanding the trajectory of ecosystem recovery. Thus, we measured above- and belowground N pools and fluxes for trees representing three different times since beetle attack, including unattacked trees. Litterfall N inputs were more than ten times higher under recently attacked trees compared to unattacked trees. Soil inorganic N concentrations also increased following beetle attack, potentially driven by a more than two-fold increase in ammonium (NH4+) concentrations in the surface soil organic horizon. However, there were no significant differences in mineral soil inorganic N or soil microbial biomass N concentrations between attacked and unattacked trees, implying that short-term changes in N cycling in response to the initial stages of WbP attack were restricted to the organic horizon. Our results suggest that while mountain pine beetle attack drives a pulse of N from the canopy to the forest floor, changes in litterfall quality and quantity do not have profound effects on soil biogeochemical cycling, at least in the short-term. However, continuous observation of these important ecosystems will be crucial to determining the long-term biogeochemical effects of mountain pine beetle outbreaks.Ecological disturbances can significantly affect biogeochemical cycles in terrestrial ecosystems, but the biogeochemical consequences of the extensive mountain pine beetle outbreak in high elevation whitebark pine (WbP) ( Pinus albicaulis; hereafter referred to as WbP) – a coniferous tree species common in western North America – is considered a keystone species in subalpine ecosystems Dendroctonus ponderosae, MPB) outbreak, unprecedented in terms of its extent, particularly in WbP ecosystems Whitebark pine  and organic N concentrations would be higher under red and gray stage trees compared to green trees. An increase in available soil N could have many possible fates, including microbial immobilization, uptake by understory plants, and/or conversion of NH4+ to NO3− with the potential for loss from the ecosystem through leaching We took advantage of an ongoing “natural experiment” initiated by the current beetle outbreak to investigate the effects of tree mortality as a result of beetle attack on N cycling in a WbP pine ecosystem. We measured a suite of N cycling metrics under WbP trees occupying three different stages of beetle attack in the Pioneer Mountains of southwestern Montana: ‘green’ (unattacked) stage, ‘red’ stage, and ‘gray’ stage. Given observed changes in N cycling following beetle disturbance in other types of forest ecosystems http://websoilsurvey.nrcs.usda.gov/app/HomePage.htm). The site contains an open canopy forest with whitebark pine  as the dominant canopy tree species co-occurring with occasional lodgepole (Pinus contorta) and limber pine (Pinus flexilis). The understory is sparse, consisting primarily of perennial grasses and forbs. The current mountain pine beetle outbreak at Vipond Park was first observed in 2005 and, at the time of the study, had progressed to the point where over 70% of the WbP trees are at some stage of beetle infestation .The study was conducted at Vipond Park  in the Pioneer Mountains in the Beaverhead-Deerlodge National Forest of southwestern Montana, USA . All nec2) plots centering around individual “focal trees” green’, uninfested WbP; 2) ‘red’, recently infested WbP (within 2 years) with needles that had turned red but had not fallen; and 3) ‘gray’, WbP trees infested more than two years ago and that lacked needles. These three categories reflect approximate times since tree death and depict significant changes in terms of potential N fluxes. Ten WbP pine individuals at each of the three infestation stages  were used as focal trees. Focal tree locations were selected based on where patches of trees of a particular beetle infestation stage occurred . Each focal tree was at least 30 m from every other focal tree. In addition to beetle infestation stage, focal trees were chosen based on a >15 cm minimum diameter at breast height (DBH) threshold, as this is considered to be the cutoff size that is conducive to beetle attack. In addition, all focal trees were relatively similar in size, ranging from 15–40 cm DBH.WbP at the study site does not grow in a stand per se, but in “clumps” or “patches” At each focal tree, soil samples were collected at four different points between the bole and the crown drip line of the tree, in each of four cardinal directions (90° apart) starting 0.5 m from the base of the tree and rotating outward in 0.5 m increments. Soil organic horizon samples were collected to a depth of approximately 5 cm at each sampling point. Mineral soil samples were collected directly below the organic horizon cores to 15 cm depth using an eight cm diameter hand corer. In cases where rocks obstructed the mineral cores, samples were taken adjacent to the organic sampling point after removing the organic material from the mineral soil surface. During each sampling event, soil samples from each of the two depths were composited by tree , placed in coolers and transported to the laboratory at the University of Montana for analysis. Within 48 h, soil samples were sieved (to 2 mm) and subsampled for physical and chemical analyses including gravimetric moisture content, inorganic N concentrations and microbial biomass analysis (see below), and the remaining soil was air-dried for pH and dried at 105°C for total C and N analyses. Soil pH was determined at 2∶1 (water: soil) .Focal tree, soil, and understory plant sampling were done in three phases. First, WbP focal tree foliage, litterfall and standing litter assessments were performed in July 2010. Soil samples were collected in September and October 2010; in September, soil inorganic N concentrations and potential N mineralization rates were assayed, and in October soil microbial biomass and total soil C and N concentrations were assessed. Understory cover and foliar chemistry was determined in July of 2011.2 litter trap was placed 0.5 m from the bole of each focal tree in July 2010. Litter was collected after one month to obtain an index of relative litterfall among beetle infestation stages and to perform litter C and nutrient analyses. Following collection, litter was dried at 70°C for 48 hours and subsequently weighed. Oven-dried subsamples from each litter trap were ground with a Wiley Mill (20-mesh screen), weighed into tin capsules (approximately 4 mg sample), and combusted on a CHNS-O elemental analyzer  to determine total litter C and N concentrations .One 0.25 m2 sampling quadrat was placed 0.5 m away from each tree and all intact standing litter in the quadrat was collected down to the organic horizon, dried at 70°C for 48 hours and weighed. Subsamples were ground, weighed into tin capsules (approximately 4 mg sample), and combusted on a CHNS-O elemental analyzer (CE Instruments) for total standing litter C and N.Canopy foliage was collected in July 2010 from green and red stage trees. Samples were taken approximately 2.5 m from the ground at various points around each tree. All green and red foliage was collected near the branch tips in order to maintain consistency with respect to needle cohort. Foliage was analyzed for total C and N concentration using the above protocols. Standing litter was sampled from below each focal tree in July 2010. A 0.25 m2 quadrats were placed 0.5 m from the bole of each tree oriented toward the north and south. Percent ground cover for all understory species in each quadrat was assessed using ocular estimation according to plant functional group . Foliar samples from two common understory species, Viola praemorsa (Canary violet) and Oxytropis sericea (Silky locoweed), were collected from the quadrats and composited by focal tree and species. Samples were oven-dried at 70°C for 48 hours, ground as described above, weighed into tin capsules (approximately 4 mg sample), and combusted on a Carlo Erba elemental analyzer for total C and N concentration.Understory vegetation under each focal tree was measured in July 2011. Two 0.25 m4+] and nitrate [NO3−]) was extracted from the September 2010 soil samples. Ten grams of each soil sample were extracted in 2 M KCl for 18 hours, vacuum filtered through 11 µm Whatman (Grade 1) filter paper, and stored at 4°C until analysis. Extracts were analyzed colorimetrically for inorganic N (NH4+ and NO3−) using a Synergy 2 Microplate Reader  after Weatherburn 4+ and NO3−, respectively.Within 24 hours of collection, soil inorganic N (ammonium [NH2SO4 for one hour and vacuum-filtered through 11 µm Whatman (Grade 1) filter paper. Organic C and total N in extracts were analyzed using a Shimadzu TOC-V CPN/TNM-1 analyzer . Microbial biomass C was determined as the difference between extractable organic C in fumigated and unfumigated samples using a proportionality constant (Kc) of 0.45 n) of 0.54 Soil microbial biomass C and N concentrations were analyzed using the chloroform fumigation-extraction method 4+ and NO3− as described above (after Weatherburn 3− concentrations instead of inorganic N (NH4++NO3−) concentrations.We conducted a 28-day laboratory incubation of soil samples collected in September 2010  to assess net N mineralization rates. Ten grams of field moist, sieved soil were mixed and weighed into plastic vials, covered with perforated plastic wrap, and incubated in the dark at room temperature (22°C) for 28 days. Vials were reweighed weekly and deionized water was added to maintain field moisture of each sample. After 28 days, samples were analyzed for NH4++NO3−) concentrations were determined following extraction in 2 M KCl and colorimetric analysis.Soil N fluxes were assessed using ion-exchange resin capsules . Capsules were deployed in the field twice, first capsules remained in soil from July – October 2010, and second capsules remained in soil from October – July 2011, following snowmelt. Resin capsules were inserted in soil beneath each focal tree to 10–15 cm depth by carefully creating a slit in the soil with a hand trowel, inserting the capsule, and carefully removing the blade to minimize disturbance. Once the capsules were collected from the field, resin-exchanged inorganic N .All statistical analyses were conducted using SPSS v. 19 . Analysis of variance (ANOVA) was used to determine significant differences between the three beetle infestation stages for litterfall mass, standing litter mass, litter C and N concentrations, soil C and N pools and fluxes, and understory vegetation cover and nutrient concentrations. The assumptions of normality and homogeneity of variances were met for all data. Repeated measures ANOVA was used to test for the effects of both time and beetle infestation stage on soil moisture. The normality, homogeneity of variance, and sphericity assumptions were all satisfied. For Soil pH did not vary significantly with beetle infestation stage , but soi2) than under either green stage or gray trees . However, July 2010 litterfall inputs did not vary significantly between the gray and green stages  were higher (P<0.001) in the organic horizon compared to the mineral horizon for all stages. Across infestation stages, the organic horizon under red stage trees had higher extractable inorganic N concentrations than the organic horizon of either the green or gray stage trees   and highly variable for all mineral soils measured. There were no differences in NO3− levels across infestation stages in either soil horizon, and there were no significant differences in either microbial biomass N concentrations or total soil N concentrations across infestation stages in either the organic or mineral soil horizons (Soil inorganic N concentrations (NH= 0.071) . These dn stages  and soilhorizons  and 3. Hhorizons .Net soil N mineralization rates were higher in both organic and mineral soils under gray trees, followed by red and then green trees , but theViola praemorsa and Oxytropis sericea) exhibited significantly different C:N values across the three stages of beetle infestation . Neither of the two understory forb species  did not translate into differences in standing litter biomass under infested trees. Griffin Litterfall nutrient chemistry varied with infestation stage. Litterfall from gray stage trees had significantly lower C:N ratios than green stage trees, largely driven by higher N concentrations in the needles falling from the infested trees. This result matches the findings of studies conducted in both MPB-attacked lodgepole and ponderosa pine ecosystems Carex geyerii, in beetle-infested stands compared to uninfested stands, but did not measure higher sedge productivity in infested stands. Other studies of post-beetle stands have observed increases in understory growth in a number of forest types Viola praemorsa and Oxytropis sericea). There was also no measurable increase in understory ground cover under beetle-attacked trees. Taken together, these data suggest that biogeochemical cycles in different forests may respond differently to attack.Once increased litterfall N inputs reach the forest floor, the N has multiple possible fates, one of which is uptake by surviving understory vegetation. This could manifest itself through increases in understory net primary productivity, or increases in foliar N concentration of understory plants after the disturbance 4+ concentrations, not to changes in NO3− concentrations. The increase in soil inorganic N under red stage trees could be explained by one or a combination of factors. For example, higher N in litterfall, reduced plant uptake 4+ than uninfested stands As hypothesized, the highest soil inorganic N values were observed in the organic horizon under red stage trees, and the differences in inorganic N concentrations observed were due to shifts in soil NHIn contrast to the differences in inorganic N in the organic (surface) soil horizons, inorganic N concentrations in the mineral soil horizons did not vary among infestation stages. This may suggest that sufficient time has not elapsed for the increased inputs of N to be transported into the lower mineral horizons. Leaching of inorganic N from the organic horizon and litter layers requires time and movement of water through the soil profile 3− concentrations in both the organic and mineral horizons were consistently very low, highly variable and did not differ among beetle infestation stages. Low soil NO3− levels are often measured in high elevation, N-limited ecosystems, as available N is often rapidly taken up by plants or immobilized by microbes 3− may also have been lost from the ecosystem quickly through leaching during rain events or snowmelt 3− have been observed, but usually involved nearly 100% vegetation mortality, or took place in systems with substantial N deposition 3− in hemlock stands that had been infested by the insect defoliator, hemlock woody adelgid, and observed significantly higher levels in attacked vs. unattacked stands. These forests were nearly pure hemlock and experienced almost complete mortality as the outbreak moved through the system. Soil texture and the timing of snowmelt or rain events also influence the magnitude of N losses from an ecosystem While we predicted that the relatively low C:N ratios in litter from beetle-infested trees would drive increases in soil N pools, not all forms of inorganic N shifted following beetle attack. Soil NOMicrobial biomass C:N ratios were lower under red stage trees compared to gray stage trees, but surprisingly red stage trees did not differ from green stage trees. Microbial immobilization of N is one pathway mineralized N may take after a disturbance, particularly if N is limiting 3− levels have been observed in a number of insect defoliation studies  also did not vary with beetle infestation stage in our site. Nitrogen losses are one of the most commonly measured biogeochemical variables in disturbance studies, and increased NOOverall, the results of this study suggest that, from an N cycling perspective, WbP ecosystems experience relatively subtle changes in the years immediately following beetle attack. While N inputs under focal trees changed significantly following tree mortality, they were accompanied by few shifts in internal cycling and pools of N, at least for the variables we measured. In addition, the internal N cycling characteristics that did change with beetle infestation stage varied in the upper, organic soil horizon, but did not appear in the mineral soil horizon, and there was no evidence suggesting significant shifts in N losses from the system. We would expect gaseous N losses to be very low at this site given the coarse texture of the soil and the dry environment Considering the extent of WbP mortality at the site, the lack of many significant biogeochemical responses to the increased litter C and N inputs was unexpected. The characteristics of WbP ecosystems, however, provide some insight into potential lags in response time to the disturbance, which may ultimately allow regeneration to occur before any longer-term nutrient shifts occur. For example, the very short growing season and harsh climatic conditions that exist in WbP ecosystems most likely cause many microbial processes to progress more slowly than in other ecosystems. From a biogeochemical perspective, climatic constraints on N cycling could have some benefit. Following this large-scale, high mortality disturbance, major shifts in C and N do not appear to occur over the timeline of a few years, but instead may play out over longer timescales that ultimately do not lead to dramatic N losses. Longer-term monitoring at a number of sites is required to determine whether biogeochemical changes are indeed more subtle in WbP ecosystems, or whether they merely take longer to manifest themselves. Thus far the data suggest that, from an N cycling perspective, the prospects for the future regeneration of WbP may be relatively promising.