Amplification of human platelet activation by surface pannexin-1 channels.

BACKGROUND: Pannexin-1 (Panx1) forms an anion-selective channel with a permeability up to ~1 kDa and represents a non-lytic, non-vesicular ATP release pathway in erythrocytes, leukocytes and neurons. Related connexin gap junction proteins have been reported in platelets; however, the expression and function of the pannexins remain unknown. OBJECTIVE: To determine the expression and function of pannexins in human plate-lets, using molecular, cellular and functional techniques. METHODS: Panx1 expression in human platelets was det-ermined using qPCR and antibody-based techniques. Contributions of Panx1 to agonist-evoked efflux of cytoplasmic calcein, Ca(2+) influx, ATP release and aggregation were assessed in washed platelets under conditions where the P2X1 receptor response was preserved (0.32 U mL(-1) apyrase). Thrombus formation in whole blood was assessed in vitro using a shear chamber assay. Two structurally unrelated and widely used Panx1 inhibitors, probenecid and carbenoxolone, were used throughout this study, at concentrations that do not affect connexin channels. RESULTS: PANX1, but not PANX2 or PANX3, mRNA was detected in human platelets. Furthermore, Panx1 protein is glycosylated and present on the plasma membrane of platelets, and displays weak physical association with P2X1 receptors. Panx1 inhibition blocked thrombin-evoked efflux of calcein, and reduced Ca(2+) influx, ATP release, platelet aggregation and thrombus formation under arterial shear rates in vitro. The Panx1-dependent contribution was not additive to that of P2X1 receptors. CONCLUSIONS: Panx1 is expressed on human platelets and amplifies Ca(2+) influx, ATP release and aggregation through the secondary activation of P2X1 receptors. We propose that Panx1 represents a novel target for the management of arterial thrombosis.

Two algorithms for fitting constrained marginal models.

The two main algorithms that have been considered for fitting constrained marginal models to discrete data, one based on Lagrange multipliers and the other on a regression model, are studied in detail. It is shown that the updates produced by the two methods are identical, but that the Lagrangian method is more efficient in the case of identically distributed observations. A generalization is given of the regression algorithm for modelling the effect of exogenous individual-level covariates, a context in which the use of the Lagrangian algorithm would be infeasible for even moderate sample sizes. An extension of the method to likelihood-based estimation under L1-penalties is also considered.

Discovery and characterization of LY2784544, a small-molecule tyrosine kinase inhibitor of JAK2V617F.

Owing to the prevalence of the JAK2V617F mutation in myeloproliferative neoplasms (MPNs), its constitutive activity, and ability to recapitulate the MPN phenotype in mouse models, JAK2V617F kinase is an attractive therapeutic target. We report the discovery and initial characterization of the orally bioavailable imidazopyridazine, LY2784544, a potent, selective and ATP-competitive inhibitor of janus kinase 2 (JAK2) tyrosine kinase. LY2784544 was discovered and characterized using a JAK2-inhibition screening assay in tandem with biochemical and cell-based assays. LY2784544 in vitro selectivity for JAK2 was found to be equal or superior to known JAK2 inhibitors. Further studies showed that LY2784544 effectively inhibited JAK2V617F-driven signaling and cell proliferation in Ba/F3 cells (IC50=20 and 55 nM, respectively). In comparison, LY2784544 was much less potent at inhibiting interleukin-3-stimulated wild-type JAK2-mediated signaling and cell proliferation (IC50=1183 and 1309 nM, respectively). In vivo, LY2784544 effectively inhibited STAT5 phosphorylation in Ba/F3-JAK2V617F-GFP (green fluorescent protein) ascitic tumor cells (TED50=12.7 mg/kg) and significantly reduced (P<0.05) Ba/F3-JAK2V617F-GFP tumor burden in the JAK2V617F-induced MPN model (TED50=13.7 mg/kg, twice daily). In contrast, LY2784544 showed no effect on erythroid progenitors, reticulocytes or platelets. These data suggest that LY2784544 has potential for development as a targeted agent against JAK2V617F and may have properties that allow suppression of JAK2V617F-induced MPN pathogenesis while minimizing effects on hematopoietic progenitor cells.

In vivo and in vitro models for the therapeutic targeting of Wnt signaling using a Tet-OΔN89ß-catenin system.

Although significant progress has been made in understanding the importance of Wnt signaling in the initiation of colorectal cancer, less is known about responses that accompany the reversal of oncogenic Wnt signaling. The aim of this study was to analyze in vivo and in vitro responses to an 'ideal' Wnt pathway inhibitor as a model for the therapeutic targeting of the pathway. A tetracycline-inducible transgenic mouse model expressing truncated ß-catenin (ΔN89ß-catenin) that exhibited a strong intestinal hyperplasia was analyzed during the removal of oncogenic ß-catenin expression both in 3D 'crypt culture' and in vivo. Oncogenic Wnt signaling was rapidly and completely reversed. The strongest inhibition of Wnt target gene expression occurred within 24 h of doxycycline removal at which time the target genes Ascl2, Axin2 and C-myc were downregulated to levels below that in the control intestine. In vitro, the small molecule Wnt inhibitor CCT036477 induced a response within 4 h of treatment. By 7 days following doxycycline withdrawal, gene expression, cell proliferation and tissue morphology were undistinguishable from control animals.In conclusion, these results demonstrate that the reversal of Wnt signaling by inhibitors should ideally be studied within hours of treatment. The reversible system described, involving medium throughput in vitro approaches and rapid in vivo responses, should allow the rapid advance of early stage compounds into efficacy models that are more usually considered later in the drug discovery pipeline.

The hot foot syndrome: Evans' sign and the old way.

BACKGROUND: Pelvic cancers such as cancer of the cervix can spread locally to involve adjacent structures such as the lumbosacral plexus and the sympathetic chain. When this happens the prognosis is usually poor. An early suspicion of recurrence may result in investigation leading to earlier and better treatment. A physical sign that may be an early and only sign of recurrence is described. OBJECTIVE: To report the late Dr Ramon Evans' unpublished case series of the hot foot syndrome due to (mostly malignant) retroperitoneal disease. This unique contribution is an opportunity to pay tribute to a man who was a meticulous recorder of the patient narrative and practitioner of a detailed and comprehensive physical examination. METHODS: A longitudinal, observational, retrospective, descriptive study is reported. Data were collected from a convenience sample of 86 patients, 75 of whom had retroperitoneal cancer and 11 of whom were diagnosed with other conditions in that area. Patients referred to the Smythe Pain Clinic were seen at both the Princess Margaret Hospital and Toronto General Hospital in Toronto, Ontario, in the 1970s. They were referred with intractable pain in the leg or back and often a history of a treated abdominal or pelvic cancer in the previous months or years. Baseline demographic data were collected including age, sex, diagnosis, pain location, characteristics and severity, physical findings, investigations and mortality. RESULTS: The 86 subjects comprised 27 men and 59 women. Carcinoma of the cervix was the most common tumour. Most had a presenting complaint of leg pain. Neurological physical signs were demonstrated in the lower extremities in 44%; however, 56% (48 patients) had only an ipsilateral, warm, dry 'hot foot' due to sympathetic deafferentation. The prognosis for the underlying illness was poor for the malignant group. DISCUSSION: Sympathetic interruption by cancer is well known in apical lung cancer as the tumour spreads upwards to involve the inferior brachial plexus. An analogous situation occurs as cancers, such as that of the cervix, spread laterally to invade the lumbosacral plexus and sympathetic chain. Signs of sympathetic deafferentation (the 'hot foot') may be the earliest and only sign in this situation. This sign may be missed unless it is anticipated and a thorough physical examination carried out. CONCLUSION: Evans' sign is important because it may be an early and solitary sign of retroperitoneal recurrence of pelvic (cervix, rectum, bladder, ovary and prostate) cancers. Recognition of this finding when intractable pain in the back and leg occurs with a history of this type of cancer could lead to earlier and more successful treatment.

ATP-gated P2X1 ion channels protect against endotoxemia by dampening neutrophil activation.

BACKGROUND: In sepsis, extracellular ATP, secreted by activated platelets and leukocytes, may contribute to the crosstalk between hemostasis and inflammation. Previously, we showed that, in addition to their role in platelet activation, ATP-gated P2X(1) ion channels are involved in promoting neutrophil chemotaxis. OBJECTIVES: To elucidate the contribution of P2X(1) ion channels to sepsis and the associated disturbance of hemostasis. METHODS: We used P2X(1) (-/-) mice in a model of lipopolysaccharide (LPS)-induced sepsis. Hemostasis and inflammation parameters were analyzed together with outcome. Mechanisms were further studied ex vivo with mouse and human blood or isolated neutrophils and monocytes. RESULTS: P2X(1) (-/-) mice were more susceptible to LPS-induced shock than wild-type mice, despite normal cytokine production. Plasma levels of thrombin-antithrombin complexes were higher, thrombocytopenia was worsened, and whole blood coagulation time was markedly reduced, pointing to aggravated hemostasis disturbance in the absence of P2X(1). However, whole blood platelet aggregation occurred normally, and P2X(1) (-/-) macrophages displayed normal levels of total tissue factor activity. We found that P2X(1) (-/-) neutrophils produced higher amounts of reactive oxygen species. Increased amounts of myeloperoxidase were released in the blood of LPS-treated P2X(1) (-/-) mice, and circulating neutrophils and monocytes expressed higher levels of CD11b. Neutrophil accumulation in the lungs was also significantly augmented, as was lipid peroxidation in the liver. Desensitization of P2X(1) ion channels led to increased activation of human neutrophils and enhanced formation of platelet-leukocyte aggregates. CONCLUSIONS: P2X(1) ion channels play a protective role in endotoxemia by negatively regulating systemic neutrophil activation, thereby limiting the oxidative response, coagulation, and organ damage.

Molecular basis of selective antagonism of the P2X1 receptor for ATP by NF449 and suramin: contribution of basic amino acids in the cysteine-rich loop.

BACKGROUND AND PURPOSE: The cysteine-rich head region, which is adjacent to the proposed ATP-binding pocket in the extracellular ligand-binding loop of P2X receptors for ATP, is absent in the antagonist-insensitive Dictyostelium receptors. In this study we have determined the contribution of the head region to the antagonist action of NF449 and suramin at the human P2X1 receptor. EXPERIMENTAL APPROACH: Chimeras and point mutations in the cysteine-rich head region were made between human P2X1 and P2X2 receptors. Mutant receptors were expressed in Xenopus oocytes and P2X receptor currents characterized using two-electrode voltage clamp. KEY RESULTS: The chimera replacing the region between the third and fourth conserved cysteine residues of the P2X1 receptor with the corresponding part of P2X2 reduced NF449 sensitivity a thousand fold from an IC(50) of ∼1 nM at the P2X1 receptor to that of the P2X2 receptor (IC(50) ∼1 µM). A similar decrease in sensitivity resulted from mutation of four positively charged P2X1 receptor residues in this region that are absent from the P2X2 receptor. These chimeras and mutations were also involved in determining sensitivity to the antagonist suramin. Reciprocal chimeras and mutations in the P2X2 receptor produced modest increases in antagonist sensitivity. CONCLUSIONS AND IMPLICATIONS: These data indicate that a cluster of positively charged residues at the base of the cysteine-rich head region can account for the highly selective antagonism of the P2X1 receptor by the suramin derivative NF449.

Proof of concept trial of tanezumab for the treatment of symptoms associated with interstitial cystitis.

PURPOSE: In this randomized, double-blind, placebo controlled phase 2 study we investigated tanezumab, a humanized monoclonal antibody that specifically inhibits nerve growth factor as a treatment for interstitial cystitis pain. MATERIALS AND METHODS: Patients with interstitial cystitis received a single intravenous dose of 200 µg/kg tanezumab or placebo. Patients recorded daily pain scores (on an 11-point numerical rating scale) 7 days before attending study visits and completed a urinary symptom diary for 3 of those days. Patients also completed the Interstitial Cystitis Symptom Index questionnaire and a global response assessment. The primary end point was change in average daily numerical rating scale pain score from baseline to week 6. Secondary end points included global response assessment, Interstitial Cystitis Symptom Index score, micturition and urgency episode frequency per 24 hours, and mean voided volume per micturition. The incidence of adverse events was also assessed. RESULTS: A total of 34 patients received tanezumab and 30 received placebo. At week 6 tanezumab produced a significant reduction from baseline in average daily pain score vs placebo (treatment difference [LS mean, 90% CI] was -1.4 [-2.2, -0.5]). A significantly higher proportion of patients on tanezumab responded as improved in the global response assessment and tanezumab also significantly reduced urgency episode frequency vs placebo. Tanezumab had no significant effect on Interstitial Cystitis Symptom Index score, micturition frequency or mean voided volume per micturition. The most common adverse events were headache (tanezumab 20.6%, placebo 16.7%) and paresthesia (tanezumab 17.6%, placebo 3.3%). CONCLUSIONS: Tanezumab has shown preliminary efficacy in the treatment of pain associated with interstitial cystitis.

Orthosteric and allosteric binding sites of P2X receptors.

P2X receptors for ATP comprise a distinct family of ligand gated ion channels with a range of properties. They have been shown to be involved in a variety of physiological processes including blood clotting, sensory perception, pain sensation, bone formation as well as inflammation and may provide a number of novel drug targets. In addition to the orthosteric site for ATP binding it has been suggested that there may be additional allosteric sites that regulate agonist action at the receptor. There is currently no crystal structure available for P2X receptors and the lack of sequence similarity to other ATP binding proteins has meant that a mutagenesis-based approach has been used primarily to investigate receptor structure-function. This review aims to provide an overview of recent work that gives an insight into residues involved in ATP action and allosteric regulation.

P2X1 and P2X4 receptor currents in mouse macrophages.

BACKGROUND AND PURPOSE: Activation of P2X receptors on macrophages is an important stimulus for cytokine release. This study seeks evidence for functional expression of P2X receptors in macrophages that had been only minimally activated. EXPERIMENTAL APPROACH: Whole-cell recordings were made from macrophages isolated 2-6 h before by lavage from mouse peritoneum, without further experimental activation. ATP (1-1000 muM) elicited inward currents in all cells (holding potential -60 mV). The properties of this current were compared among cells from wild type, P2X1 (-/-) and P2X4 (-/-) mice. KEY RESULTS: Immunoreactivity for P2X1 and P2X4 receptors was observed in wild type macrophages but was absent from the respective knock-out mice. In cells from wild type mice, ATP and alpha beta methyleneATP (alpha beta meATP) evoked inward currents rising in 10-30 ms and declining in 100-300 ms: these were blocked by pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 microM). ATP also elicited a second, smaller ( approximately 10% peak amplitude), more slowly decaying (1-3 s) at concentrations > or =10 microM: this was resistant to PPADS and prolonged by ivermectin. Macrophages from P2X1 (-/-) mice responded to ATP (>100 microM) but not alpha beta meATP: these small currents were prolonged by ivermectin. Macrophages from P2X4 (-/-) mice responded to ATP and alpha beta meATP as cells from wild type mice, except that ATP did not evoke the small, slowly decaying component: these currents were blocked by PPADS. CONCLUSION: Mouse peritoneal macrophages that are minimally activated demonstrate membrane currents in response to ATP and alpha beta meATP that have the predominate features of P2X1 receptors.

Purinergic 2X1 receptors mediate endothelial dependent vasodilation to ATP.

ATP is an important endogenous mediator in the cardiovascular system. It induces endothelium dependent vasodilation, but the precise receptor pathway activated in this response is currently under debate. We have used traditional bioassay techniques to show that ATP-induced vasodilation in mesenteric vessels is endothelium-dependent. Furthermore, ATP-induced vasodilation was inhibited by both suramin and 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP), consistent with a P2X(1)-, P2X(2)-, or P2X(3)-mediated event and was not potentiated by ivermectin, indicating that these responses were not P2X(4) receptor-mediated. ATP did not induce vasodilation in vessels from P2X (-/-)(1) mice, confirming an absolute requirement for this receptor. Finally, in pure cell populations of mouse mesenteric artery endothelial cells, we show that P2X(1) mRNA is specifically expressed. However, in line with observations in the brain, the P2X(1) present in endothelial cells does not seem to be recognized by conventional antibodies. Together, these results show that ATP-induced vasodilation is mediated by P2X(1) receptor activation on mesenteric arterial endothelial cells. These observations establish a critical role for P2X(1) receptors in the ATP vasodilator pathway.

P2X1 receptors mediate sympathetic postjunctional Ca2+ transients in mesenteric small arteries.

Brief, spatially localized Ca(2+) transients occur in the smooth muscle adjacent to perivascular nerves of small arteries during neurogenic contractions. We named these "junctional Ca(2+) transients" (jCaTs) and postulated that they arose from Ca(2+) entering smooth muscle cells through P2X(1) receptors activated by neurally released ATP. Nevertheless, the lack of potent, subtype-selective P2X-receptor antagonists made determining the exact molecular identity of the channels difficult. Here we used small, pressurized mesenteric arteries from P2X(1)-receptor-deficient mice (KO) to test the hypothesis that jCaTs arise from Ca(2+) entering the smooth muscle cell via P2X(1) receptors. In wild-type (WT) arteries, confocal microscopy of fluo-4 fluorescence during electrical field stimulation (EFS) of perivascular sympathetic nerves revealed jCaTs in the smooth muscle cells adjacent to the perivascular nerves, similar to those reported previously in rat arteries, and alpha-latrotoxin (2.5 nM) markedly increased the frequency of "spontaneous" jCaTs. In the KO arteries, however, neither EFS nor alpha-latrotoxin elicited any jCaTs. A potent P2X-receptor agonist, alpha,beta-methylene ATP (10.0 microM), elicited strong contractions and increased intracellular Ca(2+) concentration in WT arteries but elicited neither in KO arteries. A biphasic vasoconstriction in response to EFS was observed in WT arteries. In KO arteries, however, the initial rapid, transient component of the biphasic vasoconstriction was absent. The data support the hypothesis that jCaTs represent Ca(2+) that enters the smooth muscle cells through P2X(1) receptors activated by neurally released ATP and that this Ca(2+) is involved in the initial rapid component of the sympathetic neurogenic contraction.

Renal autoregulation in P2X1 knockout mice.

Autoregulation of renal blood flow is an established physiological phenomenon, however the signalling mechanisms involved remain elusive. Autoregulatory adjustments in preglomerular resistance involve myogenic and tubuloglomerular feedback (TGF) influences. While there is general agreement on the participation of these two regulatory pathways, the signalling molecules and effector mechanisms have not been identified. Currently, there are two major hypotheses being considered to explain the mechanism by which TGF signals are transmitted from the macula densa to the afferent arteriole. The adenosine hypothesis proposes that the released adenosine triphosphate (ATP) is hydrolysed to adenosine and this product stimulates preglomerular vasoconstriction by activation of A(1) receptors on the afferent arteriole. Alternatively, the P2 receptor hypothesis postulates that ATP released from the macula densa directly stimulates afferent arteriolar vasoconstriction by activation of ATP-sensitive P2X(1) receptors. This hypothesis has emerged from the realization that P2X(1) receptors are heavily expressed along the preglomerular vasculature. Inactivation of P2X(1) receptors impairs autoregulatory responses while afferent arteriolar responses to A(1) adenosine receptor activation are retained. Autoregulatory behaviour is markedly attenuated in mice lacking P2X(1) receptors but responses to adenosine A(1) receptor activation remain intact. More recent experiments suggest that P2X(1) receptors play an essential role in TGF-dependent vasoconstriction of the afferent arteriole. Interruption of TGF-dependent influences on afferent arteriolar diameter, by papillectomy or furosemide treatment, significantly attenuated pressure-mediated afferent arteriolar vasoconstriction in wild-type mice but had no effect on the response in P2X(1) knockout mice. Collectively, these observations support an essential role for P2X(1) receptors in TGF-mediated afferent arteriolar vasoconstriction.

Topical adrenaline and cocaine gel for anaesthetising children's lacerations. An audit of acceptability and safety.

OBJECTIVES: (1) To assess the acceptability of a gel solution of adrenaline (epinephrine) (1 in 2000) and cocaine (5%) for anaesthetising children's facial lacerations to the child, parent, and operator. (2) To assess the safety of the current protocol. SETTING: The emergency unit of a large university hospital. METHODS: All patients who were treated with topical adrenaline and cocaine (topAC) gel over a six month period were entered into a prospective audit (n = 75). Patient details, the nature and cause of the injury, and any treatment carried out were all recorded. The acceptability to children over 3 years of age, was assessed by the use of the Wong Baker face scale, in which 0 represents "no hurt" and 5 represents "hurts worst". The acceptability to both the parent and the operator was assessed by the use of a 0 to 9 Likert scale, where 0 represented "very acceptable" and 9 represented "not at all" acceptable. RESULTS: (1) Children aged 3 years or older graded their pain during the procedure as having a mean value of 1.17 on the Wong Baker (0 to 5) scale. Parents graded acceptability on the Likert scale (0 to 9) with a mean score of 1.13. Operators using the same grading system, recorded a mean score of 1.75. (2) No toxic side effects were seen but the protocol was updated in line with evidence. CONCLUSIONS: Topical adrenaline and cocaine is an effective anaesthetic for suturing children's facial lacerations and is acceptable to child, parent, and operator alike.

Platelets enhance endotoxin-induced monocyte tissue factor (TF) activity in the horse.

Endotoxaemia is the leading cause of death in horses. Disseminated intravascular coagulation (DIG), stimulated by induced monocyte proteins, is a prominent feature. Monocyte-platelet cellular interactions are central to the vascular dysfunction produced by circulating endotoxin and are implicated in many thrombotic diseases in the horse. This study reports that endotoxin (0.01-10 microg ml(-1)) and blood platelets (2.5 x 10(7) - 1 x 10(8) ml(-1)) are potent inducers of expression and activity of monocyte tissue factor (TF), the primary activator of the blood coagulation protease cascade. The co-incubation of endotoxin-stimulated monocytes with platelets resulted in greater production of this protein. Cycloheximide (1 mM) inhibited part of the stimulatory effect of endotoxin and/or platelets, the uninhibited part indicating de-encryption of cell-surface TF. Hence, platelets are considered to be an important component of the endotoxin-stimulated response of equine monocytes. The role of platelets as potent stimulators of endotoxin-stimulated monocyte proteins and mediators in vitro is likely to be of significance in vivo in the clinical manifestations and management of endotoxaemia in the horse.

Effects of a P2Y(12) receptor antagonist on the response of equine platelets to ADP. Comparison with human platelets.

Horses show susceptibility to platelet-related disorders. Equine platelets differ from human platelets in some of their responses, so information available about human platelets must be validated in the horse. Aggregation of platelets by ADP involves both P2Y(1) and P2Y(12) receptors on the platelet surface. We have compared the effect of the P2Y(12) antagonist, AR-C67085, on equine and human platelets in vitro using turbidimetric aggregometry to measure the rate and final extent of aggregation. Aggregation profiles, concentration-response curves and pA(2) values show that the rate of aggregation of equine platelets is much more susceptible to inhibition by AR-C67085 than that of human platelets. This species difference may reflect differences in the relative numbers of P2Y(1) and P2Y(12) receptors, or in intracellular signalling pathways, but will need to be considered by equine clinicians before using P2Y(12) antagonists in the treatment of thrombotic conditions.

Effects of P2Y(1) and P2Y(12) receptor antagonists on ADP-induced shape change of equine platelets: comparison with human platelets.

Platelet activation by adenosine 5' -diphosphate (ADP) is via both P2Y(1 )and P2Y(12) receptors and leads to shape change and aggregation. The effects on ADP-induced platelet shape change of two P2Y(1) antagonists, adenosine 3'-phosphate, 5'-phosphosulfate (A3P5PS) and 2-deoxy-N(6)-methyladenosine 3', 5'-diphosphate (MRS-2179) and a P2Y(12) antagonist 2-propylthio-D-beta,gamma-dichloromethylene-adenosine 5'-triphosphate (AR-C67085MX) were determined by turbidimetric aggregometry and scanning electron microscopy (SEM) on equine and human platelets. The platelet aggregation was inhibited during aggregometry by 4-[4-[4(aminoiminomethyl)phenyl]-1-piperazinyl]-1-piperidin acid hydrochloride trihydrate (GR 144053F), an inhibitor of fibrinogen binding. From aggregation profiles, concentration-response curves and SEM we conclude that the shape change of equine platelets was susceptible to inhibition by the P2Y(1) antagonists A3P5PS and MRS-2179, but less so than human platelets. The P2Y(12) antagonist AR-C67085 did not influence significantly the shape change of either equine or human platelets.

Use of pyrolysis molecular beam mass spectrometry (py-MBMS) to characterize forest soil carbon: method and preliminary results.

The components of soil organic matter (SOM) and their degradation dynamics in forest soils are difficult to study and thus poorly understood, due to time-consuming sample collection, preparation, and difficulty of analyzing and identifying major components. As a result, changes in soil organic matter chemical composition as a function of age, forest type, or disturbance have not been examined. We applied pyrolysis molecular beam mass spectrometry (py-MBMS), which provides rapid characterization of SOM of whole soil samples. to the Tionesta soil samples described by Hoover, C.M., Magrini, K.A., Evans, R.J., 2002. Soil carbon content and character in an old growth forest in northwestern Pennsylvania: a case study introducing molecular beam mass spectrometry (PY-MBMS). Environmental Pollution 116 (Supp. 1), S269-S278. Our goals in this work were to: (1) develop and demonstrate an advanced, rapid analytical method for characterizing SOM components in whole soils, and (2) provide data-based models to predict soil carbon content and residence time from py-MBMS analysis. Using py-MBMS and pattern recognition techniques we were able to statistically distinguish among four Tionesta sites and show an increase in pyrolysis products of more highly decomposed plant materials at increasing sample depth. For example, all four sites showed increasing amounts of older carbon (phenolic and aromatic species) at deeper depths and higher amounts of more recent carbon (carbohydrates and lignin products) at shallower depths. These results indicate that this type of analysis could be used to rapidly characterize SOM for the purpose of developing a model, which could be used in monitoring the effect of forest management practices on carbon uptake and storage.