The Adjuvant Activity and Mechanisms of Action for Mastoparan 7 Peptide After Intranasal Immunization in Mice

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Morphological characteristics of urban water bodies: mechanisms of change and implications for ecosystem function.

The size, shape, and connectivity of water bodies (lakes, ponds, and wetlands) can have important effects on ecological communities and ecosystem processes, but how these characteristics are influenced by land use and land cover change over broad spatial scales is not known. Intensive alteration of water bodies during urban development, including construction, burial, drainage, and reshaping, may select for certain morphometric characteristics and influence the types of water bodies present in cities. We used a database of over one million water bodies in 100 cities across the conterminous United States to compare the size distributions, connectivity (as intersection with surface flow lines), and shape (as measured by shoreline development factor) of water bodies in different land cover classes. Water bodies in all urban land covers were dominated by lakes and ponds, while reservoirs and wetlands comprised only a small fraction of the sample. In urban land covers, as compared to surrounding undeveloped land, water body size distributions converged on moderate sizes, shapes toward less tortuous shorelines, and the number and area of water bodies that intersected surface flow lines (i.e., streams and rivers) decreased. Potential mechanisms responsible for changing the characteristics of urban water bodies include: preferential removal, physical reshaping or addition of water bodies, and selection of locations for development. The relative contributions of each mechanism likely change as cities grow. The larger size and reduced surface connectivity of urban water bodies may affect the role of internal dynamics and sensitivity to catchment processes. More broadly, these results illustrate the complex nature of urban watersheds and highlight the need to develop a conceptual framework for urban water bodies.

Mechanisms of CD8+ T Cell Mediated Virus Inhibition in HIV-1 Virus Controllers

CD8+ T cells are associated with long term control of virus replication to low or undetectable levels in a population of HIV+ therapy-naïve individuals known as virus controllers (VCs; <5000 RNA copies/ml and CD4+ lymphocyte counts >400 cells/µl). These subjects' ability to control viremia in the absence of therapy makes them the gold standard for the type of CD8+ T-cell response that should be induced with a vaccine. Studying the regulation of CD8+ T cells responses in these VCs provides the opportunity to discover mechanisms of durable control of HIV-1. Previous research has shown that the CD8+ T cell population in VCs is heterogeneous in its ability to inhibit virus replication and distinct T cells are responsible for virus inhibition. Further defining both the functional properties and regulation of the specific features of the select CD8+ T cells responsible for potent control of viremia the in VCs would enable better evaluation of T cell-directed vaccine strategies and may inform the design of new therapies.

Here we discuss the progress made in elucidating the features and regulation of CD8+ T cell response in virus controllers. We first detail the development of assays to quantify CD8+ T cells' ability to inhibit virus replication. This includes the use of a multi-clade HIV-1 panel which can subsequently be used as a tool for evaluation of T cell directed vaccines. We used these assays to evaluate the CD8+ response among cohorts of HIV-1 seronegative, HIV-1 acutely infected, and HIV-1 chronically infected (both VC and chronic viremic) patients. Contact and soluble CD8+ T cell virus inhibition assays (VIAs) are able to distinguish these patient groups based on the presence and magnitude of the responses. When employed in conjunction with peptide stimulation, the soluble assay reveals peptide stimulation induces CD8+ T cell responses with a prevalence of Gag p24 and Nef specificity among the virus controllers tested. Given this prevalence, we aimed to determine the gene expression profile of Gag p24-, Nef-, and unstimulated CD8+ T cells. RNA was isolated from CD8+ T-cells from two virus controllers with strong virus inhibition and one seronegative donor after a 5.5 hour stimulation period then analyzed using the Illumina Human BeadChip platform (Duke Center for Human Genome Variation). Analysis revealed that 565 (242 Nef and 323 Gag) genes were differentially expressed in CD8+ T-cells that were able to inhibit virus replication compared to those that could not. We compared the differentially expressed genes to published data sets from other CD8+ T-cell effector function experiments focusing our analysis on the most recurring genes with immunological, gene regulatory, apoptotic or unknown functions. The most commonly identified gene in these studies was TNFRSF9. Using PCR in a larger cohort of virus controllers we confirmed the up-regulation of TNFRSF9 in Gag p24 and Nef-specific CD8+ T cell mediated virus inhibition. We also observed increase in the mRNA encoding antiviral cytokines macrophage inflammatory proteins (MIP-1α, MIP-1αP, MIP-1β), interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), and recently identified lymphotactin (XCL1).

Our previous work suggests the CD8+ T-cell response to HIV-1 can be regulated at the level of gene regulation. Because RNA abundance is modulated by transcription of new mRNAs and decay of new and existing RNA we aimed to evaluate the net rate of transcription and mRNA decay for the cytokines we identified as differentially regulated. To estimate rate of mRNA synthesis and decay, we stimulated isolated CD8+ T-cells with Gag p24 and Nef peptides adding 4-thiouridine (4SU) during the final hour of stimulation, allowing for separation of RNA made during the final hour of stimulation. Subsequent PCR of RNA isolated from these cells, allowed us to determine how much mRNA was made for our genes of interest during the final hour which we used to calculate rate of transcription. To assess if stimulation caused a change in RNA stability, we calculated the decay rates of these mRNA over time. In Gag p24 and Nef stimulated T cells , the abundance of the mRNA of many of the cytokines examined was dependent on changes in both transcription and mRNA decay with evidence for potential differences in the regulation of mRNA between Nef and Gag specific CD8+ T cells. The results were highly reproducible in that in one subject that was measured in three independent experiments the results were concordant.

This data suggests that mRNA stability, in addition to transcription, is key in regulating the direct anti-HIV-1 function of antigen-specific memory CD8+ T cells by enabling rapid recall of anti-HIV-1 effector functions, namely the production and increased stability of antiviral cytokines. We have started to uncover the mechanisms employed by CD8+ T cell subsets with antigen-specific anti-HIV-1 activity, in turn, enhancing our ability to inhibit virus replication by informing both cure strategies and HIV-1 vaccine designs that aim to reduce transmission and can aid in blocking HIV-1 acquisition.

Advances in understanding mechanisms of thalamic relays in cognition and behavior.

The main impetus for a mini-symposium on corticothalamic interrelationships was the recent number of studies highlighting the role of the thalamus in aspects of cognition beyond sensory processing. The thalamus contributes to a range of basic cognitive behaviors that include learning and memory, inhibitory control, decision-making, and the control of visual orienting responses. Its functions are deeply intertwined with those of the better studied cortex, although the principles governing its coordination with the cortex remain opaque, particularly in higher-level aspects of cognition. How should the thalamus be viewed in the context of the rest of the brain? Although its role extends well beyond relaying of sensory information from the periphery, the main function of many of its subdivisions does appear to be that of a relay station, transmitting neural signals primarily to the cerebral cortex from a number of brain areas. In cognition, its main contribution may thus be to coordinate signals between diverse regions of the telencephalon, including the neocortex, hippocampus, amygdala, and striatum. This central coordination is further subject to considerable extrinsic control, for example, inhibition from the basal ganglia, zona incerta, and pretectal regions, and chemical modulation from ascending neurotransmitter systems. What follows is a brief review on the role of the thalamus in aspects of cognition and behavior, focusing on a summary of the topics covered in a mini-symposium held at the Society for Neuroscience meeting, 2014.

Leptin metabolically licenses T cells for activation to link nutrition and immunity.

Immune responses are highly energy-dependent processes. Activated T cells increase glucose uptake and aerobic glycolysis to survive and function. Malnutrition and starvation limit nutrients and are associated with immune deficiency and increased susceptibility to infection. Although it is clear that immunity is suppressed in times of nutrient stress, mechanisms that link systemic nutrition to T cell function are poorly understood. We show in this study that fasting leads to persistent defects in T cell activation and metabolism, as T cells from fasted animals had low glucose uptake and decreased ability to produce inflammatory cytokines, even when stimulated in nutrient-rich media. To explore the mechanism of this long-lasting T cell metabolic defect, we examined leptin, an adipokine reduced in fasting that regulates systemic metabolism and promotes effector T cell function. We show that leptin is essential for activated T cells to upregulate glucose uptake and metabolism. This effect was cell intrinsic and specific to activated effector T cells, as naive T cells and regulatory T cells did not require leptin for metabolic regulation. Importantly, either leptin addition to cultured T cells from fasted animals or leptin injections to fasting animals was sufficient to rescue both T cell metabolic and functional defects. Leptin-mediated metabolic regulation was critical, as transgenic expression of the glucose transporter Glut1 rescued cytokine production of T cells from fasted mice. Together, these data demonstrate that induction of T cell metabolism upon activation is dependent on systemic nutritional status, and leptin links adipocytes to metabolically license activated T cells in states of nutritional sufficiency.

Statistical analysis of crystallization database links protein physico-chemical features with crystallization mechanisms.

X-ray crystallography is the predominant method for obtaining atomic-scale information about biological macromolecules. Despite the success of the technique, obtaining well diffracting crystals still critically limits going from protein to structure. In practice, the crystallization process proceeds through knowledge-informed empiricism. Better physico-chemical understanding remains elusive because of the large number of variables involved, hence little guidance is available to systematically identify solution conditions that promote crystallization. To help determine relationships between macromolecular properties and their crystallization propensity, we have trained statistical models on samples for 182 proteins supplied by the Northeast Structural Genomics consortium. Gaussian processes, which capture trends beyond the reach of linear statistical models, distinguish between two main physico-chemical mechanisms driving crystallization. One is characterized by low levels of side chain entropy and has been extensively reported in the literature. The other identifies specific electrostatic interactions not previously described in the crystallization context. Because evidence for two distinct mechanisms can be gleaned both from crystal contacts and from solution conditions leading to successful crystallization, the model offers future avenues for optimizing crystallization screens based on partial structural information. The availability of crystallization data coupled with structural outcomes analyzed through state-of-the-art statistical models may thus guide macromolecular crystallization toward a more rational basis.

Different Mechanisms Confer Gradual Control and Memory at Nutrient- and Stress-Regulated Genes in Yeast.

Cells respond to environmental stimuli by fine-tuned regulation of gene expression. Here we investigated the dose-dependent modulation of gene expression at high temporal resolution in response to nutrient and stress signals in yeast. The GAL1 activity in cell populations is modulated in a well-defined range of galactose concentrations, correlating with a dynamic change of histone remodeling and RNA polymerase II (RNAPII) association. This behavior is the result of a heterogeneous induction delay caused by decreasing inducer concentrations across the population. Chromatin remodeling appears to be the basis for the dynamic GAL1 expression, because mutants with impaired histone dynamics show severely truncated dose-response profiles. In contrast, the GRE2 promoter operates like a rapid off/on switch in response to increasing osmotic stress, with almost constant expression rates and exclusively temporal regulation of histone remodeling and RNAPII occupancy. The Gal3 inducer and the Hog1 mitogen-activated protein (MAP) kinase seem to determine the different dose-response strategies at the two promoters. Accordingly, GAL1 becomes highly sensitive and dose independent if previously stimulated because of residual Gal3 levels, whereas GRE2 expression diminishes upon repeated stimulation due to acquired stress resistance. Our analysis reveals important differences in the way dynamic signals create dose-sensitive gene expression outputs.

Adjunctive β2-agonist treatment reduces glycogen independently of receptor-mediated acid α-glucosidase uptake in the limb muscles of mice with Pompe disease.

Enzyme or gene replacement therapy with acid α-glucosidase (GAA) has achieved only partial efficacy in Pompe disease. We evaluated the effect of adjunctive clenbuterol treatment on cation-independent mannose-6-phosphate receptor (CI-MPR)-mediated uptake and intracellular trafficking of GAA during muscle-specific GAA expression with an adeno-associated virus (AAV) vector in GAA-knockout (KO) mice. Clenbuterol, which increases expression of CI-MPR in muscle, was administered with the AAV vector. This combination therapy increased latency during rotarod and wirehang testing at 12 wk, in comparison with vector alone. The mean urinary glucose tetrasaccharide (Glc4), a urinary biomarker, was lower in GAA-KO mice following combination therapy, compared with vector alone. Similarly, glycogen content was lower in cardiac and skeletal muscle following 12 wk of combination therapy in heart, quadriceps, diaphragm, and soleus, compared with vector alone. These data suggested that clenbuterol treatment enhanced trafficking of GAA to lysosomes, given that GAA was expressed within myofibers. The integral role of CI-MPR was demonstrated by the lack of effectiveness from clenbuterol in GAA-KO mice that lacked CI-MPR in muscle, where it failed to reverse the high glycogen content of the heart and diaphragm or impaired wirehang performance. However, the glycogen content of skeletal muscle was reduced by the addition of clenbuterol in the absence of CI-MPR, as was lysosomal vacuolation, which correlated with increased AKT signaling. In summary, β2-agonist treatment enhanced CI-MPR-mediated uptake and trafficking of GAA in mice with Pompe disease, and a similarly enhanced benefit might be expected in other lysosomal storage disorders.

Two Distinct Moral Mechanisms for Ascribing and Denying Intentionality.

Philosophers and legal scholars have long theorized about how intentionality serves as a critical input for morality and culpability, but the emerging field of experimental philosophy has revealed a puzzling asymmetry. People judge actions leading to negative consequences as being more intentional than those leading to positive ones. The implications of this asymmetry remain unclear because there is no consensus regarding the underlying mechanism. Based on converging behavioral and neural evidence, we demonstrate that there is no single underlying mechanism. Instead, two distinct mechanisms together generate the asymmetry. Emotion drives ascriptions of intentionality for negative consequences, while the consideration of statistical norms leads to the denial of intentionality for positive consequences. We employ this novel two-mechanism model to illustrate that morality can paradoxically shape judgments of intentionality. This is consequential for mens rea in legal practice and arguments in moral philosophy pertaining to terror bombing, abortion, and euthanasia among others.

Radiolabeled inhibitors as probes for imaging mutant IDH1 expression in gliomas: Synthesis and preliminary evaluation of labeled butyl-phenyl sulfonamide analogs.

INTRODUCTION: Malignant gliomas frequently harbor mutations in the isocitrate dehydrogenase 1 (IDH1) gene. Studies suggest that IDH mutation contributes to tumor pathogenesis through mechanisms that are mediated by the neomorphic metabolite of the mutant IDH1 enzyme, 2-hydroxyglutarate (2-HG). The aim of this work was to synthesize and evaluate radiolabeled compounds that bind to the mutant IDH1 enzyme with the goal of enabling noninvasive imaging of mutant IDH1 expression in gliomas by positron emission tomography (PET). METHODS: A small library of nonradioactive analogs were designed and synthesized based on the chemical structure of reported butyl-phenyl sulfonamide inhibitors of mutant IDH1. Enzyme inhibition assays were conducted using purified mutant IDH1 enzyme, IDH1-R132H, to determine the IC50 and the maximal inhibitory efficiency of the synthesized compounds. Selected compounds, 1 and 4, were labeled with radioiodine ((125)I) and/or (18)F using bromo- and phenol precursors, respectively. In vivo behavior of the labeled inhibitors was studied by conducting tissue distribution studies with [(125)I]1 in normal mice. Cell uptake studies were conducted using an isogenic astrocytoma cell line that carried a native IDH1-R132H mutation to evaluate the potential uptake of the labeled inhibitors in IDH1-mutated tumor cells. RESULTS: Enzyme inhibition assays showed good inhibitory potency for compounds that have iodine or a fluoroethoxy substituent at the ortho position of the phenyl ring in compounds 1 and 4 with IC50 values of 1.7 μM and 2.3 μM, respectively. Compounds 1 and 4 inhibited mutant IDH1 activity and decreased the production of 2-HG in an IDH1-mutated astrocytoma cell line. Radiolabeling of 1 and 4 was achieved with an average radiochemical yield of 56.6 ± 20.1% for [(125)I]1 (n = 4) and 67.5 ± 6.6% for [(18)F]4 (n = 3). [(125)I]1 exhibited favorable biodistribution characteristics in normal mice, with rapid clearance from the blood and elimination via the hepatobiliary system by 4 h after injection. The uptake of [(125)I]1 in tumor cells positive for IDH1-R132H was significantly higher compared to isogenic WT-IDH1 controls, with a maximal uptake ratio of 1.67 at 3 h post injection. Co-incubation of the labeled inhibitors with the corresponding nonradioactive analogs, and decreasing the normal concentrations of FBS (10%) in the incubation media substantially increased the uptake of the labeled inhibitors in both the IDH1-mutant and WT-IDH1 tumor cell lines, suggesting significant non-specific binding of the synthesized labeled butyl-phenyl sulfonamide inhibitors. CONCLUSIONS: These data demonstrate the feasibility of developing radiolabeled probes for the mutant IDH1 enzyme based on enzyme inhibitors. Further optimization of the labeled inhibitors by modifying the chemical structure to decrease the lipophilicity and to increase potency may yield compounds with improved characteristics as probes for imaging mutant IDH1 expression in tumors.

ROLE OF ICAM-1-MEDIATED ENDOCYTOSIS IN ENDOTHELIAL FUNCTION AND IMPLICATIONS FOR CARRIER-ASSISTED DRUG DELIVERY

Intercellular adhesion molecule 1 (ICAM-1) is a transmembrane protein found on the surface of vascular endothelial cells (ECs). Its expression is upregulated at inflammatory sites, allowing for targeted delivery of therapeutics using ICAM-1-binding drug carriers. Engagement of multiple copies of ICAM-1 by these drug carriers induces cell adhesion molecule (CAM)-mediated endocytosis, which results in trafficking of carriers to lysosomes and across ECs. Knowledge about the regulation behind CAM-mediated endocytosis can help improve drug delivery, but questions remain about these regulatory mechanisms. Furthermore, little is known about the natural function of this endocytic pathway. To address these gaps in knowledge, we focused on two natural binding partners of ICAM-1 that potentially elicit CAM-mediated endocytosis: leukocytes (which bind ICAM-1 via β₂ integrins) and fibrin polymers (a main component of blood clots which binds ICAM-1 via the γ3 sequence). First, inspired by properties of these natural binding partners, we varied the size and targeting moiety of model drug carriers to determine how these parameters affect CAM-mediated endocytosis. Increasing ICAM-1-targeted carrier size slowed carrier uptake kinetics, reduced carrier trafficking to lysosomes, and increased carrier transport across ECs. Changing targeting moieties from antibodies to peptides decreased particle binding and uptake, lowered trafficking to lysosomes, and increased transport across ECs. Second, using cell culture models of leukocyte/EC interactions, inhibiting regulatory elements of the CAM-mediated pathway disrupted leukocyte sampling, a process crucial to leukocyte crossing of endothelial layers (transmigration). This inhibition also decreased leukocyte transmigration across ECs, specifically through the transcellular route, which occurs through a single EC without disassembly of cell-cell junctions. Third, fibrin meshes, which mimic blood clot fragments/remnants, bound to ECs at ICAM-1-enriched sites and were internalized by the endothelium. Inhibiting the CAM-mediated pathway disrupted this uptake. Following endocytosis, fibrin meshes trafficked to lysosomes where they were degraded. In mouse models, CAM-mediated endocytosis of fibrin meshes appeared to remove fibrin remnants at the endothelial surface, preventing re-initiation of the coagulation cascade. Overall, these results support a link between CAM-mediated endocytosis and leukocyte transmigration as well as uptake of fibrin materials by ECs. Furthermore, these results will guide the future design of ICAM-1-targeted carrier-assisted therapies.

Effects of Gallium-Desferrioxamine Compounds on Bacteria

Over 70% of nosocomial infections in the United States are resistant to one or more traditional antibiotics, necessitating research for alternative treatment options. This study aims to chelate gallium (Ga) onto a bacterial siderophore, desferrioxamine (DFO), to retard bacterial growth. By exploiting natural bacterial pathways, metal-siderophore treatments are hypothesized to circumvent traditional resistance mechanisms. Additionally, the GaDFO complex will be tested against several bacterial species to determine the specificity of DFO uptake. This research aims to prove the feasibility of siderophore piracy as an alternative to antibiotics. In showing the feasibility of siderophore piracy mechanisms, this research will enable the development of future avenues for protecting against resistant nosocomial infections.

Evidence of various mechanisms of Cd sequestration in the hyperaccumulator Arabidopsis halleri, the non accumulator Arabidopsis lyrata and their progenies by combined synchrotron-based techniques

Arabidopsis halleri is a model plant for Zn and Cd hyperaccumulation. The objective of this study was to determine the relationship between the chemical forms of Cd, its distribution in leaves, and Cd accumulation and tolerance. An interspecific cross was carried out between A. halleri and the non-tolerant and non-hyperaccumulating relative A. lyrata providing progenies segregating for Cd tolerance and accumulation. Cd speciation and distribution were investigated using X-ray absorption spectroscopy and microfocused X-ray fluorescence. In A. lyrata and non-tolerant progenies, Cd was coordinated by S atoms only or with a small contribution of O groups. Interestingly, the proportion of O ligands increased in A. halleri and tolerant progenies, and they were predominant in most of them, while S ligands were still present. Therefore, the binding of Cd with O ligands was associated with Cd tolerance. In A. halleri, Cd was mainly located in the xylem, phloem, and mesophyll tissue, suggesting a reallocation process for Cd within the plant. The distribution of the metal at the cell level was further discussed. In A. lyrata, the vascular bundles were also Cd enriched, but the epidermis was richer in Cd as compared with the mesophyll. Cd was identified in trichomes of both species. This work demonstrated that both Cd speciation and localization were related to the tolerance character of the plant.

Use of a coupled mechanical-acoustic computational model to identify failure mechanisms in paper production

In this paper, a couple mechanical-acoustic system of equations is solved to determine the relationship between emitted sound and damage mechanisims in paper under controlled stress conditions. The simple classical expression describing the frequency of a plucked string to its material properties is used to generate a numberical representation of the microscopic structue of the paper, and the resulting numerical model is then used to simulate the vibration of a range of simple fibre structures when undergoing two distinct types of damange mechanisms: (a)fibre/fibre bond failure, (b) fibre failure. The numercial results are analysed to determine whether there is any detectable systematic difference between the resulting acoustic emissions of the two damage processes. Fourier techniques are then used to compare th computeed results against experimental measurements. Distinct frequency components identifying each type of damage are shown to exist, and in this respect theory and experiments show good correspondece. Hence, it is shown, that althrough the mathematical model represents a grossly-simplified view of the complex structure of the paper, it nevertheless provides a good understanding of the underlying micro-mechanisms characterising its proeperties as a stress-resisting structure. Use of the model and acoompanying software will enable operators to identify approaching failure conditions in the continuous production of paper from emitted sound signals and take preventative action.

Identifying the damage mechanisms in paper using the acoustic emission monitoring technique

This paper investigates the use of the acoustic emission (AE) monitoring technique for use in identifying the damage mechanisms present in paper associated with its production process. The microscopic structure of paper consists of a random mesh of paper fibres connected by hydrogen bonds. This implies the existence of two damage mechanisms, the failure of a fibre-fibre bond and the failure of a fibre. This paper describes a hybrid mathematical model which couples the mechanics of the mass-spring model to the acoustic wave propagation model for use in generating the acoustic signal emitted by complex structures of paper fibres under strain. The derivation of the mass-spring model can be found in [1,2], with details of the acoustic wave equation found in [3,4]. The numerical implementation of the vibro-acoustic model is discussed in detail with particular emphasis on the damping present in the numerical model. The hybrid model uses an implicit solver which intrinsically introduces artificial damping to the solution. The artificial damping is shown to affect the frequency response of the mass-spring model, therefore certain restrictions on the simulation time step must be enforced so that the model produces physically accurate results. The hybrid mathematical model is used to simulate small fibre networks to provide information on the acoustic response of each damage mechanism. The simulated AEs are then analysed using a continuous wavelet transform (CWT), described in [5], which provides a two dimensional time-frequency representation of the signal. The AEs from the two damage mechanisms show different characteristics in the CWT so that it is possible to define a fibre-fibre bond failure by the criteria listed below. The dominant frequency components of the AE must be at approximately 250 kHz or 750 kHz. The strongest frequency component may be at either approximately 250 kHz or 750 kHz. The duration of the frequency component at approximately 250 kHz is longer than that of the frequency component at approximately 750 kHz. Similarly, the criteria for identifying a fibre failure are given below. The dominant frequency component of the AE must be greater than 800 kHz. The duration of the dominant frequency component must be less than 5.00E-06 seconds. The dominant frequency component must be present at the front of the AE. Essentially, the failure of a fibre-fibre bond produces a low frequency wave and the failure of a fibre produces a high frequency pulse. Using this theoretical criteria, it is now possible to train an intelligent classifier such as the Self-Organising Map (SOM) [6] using the experimental data. First certain features must be extracted from the CWTs of the AEs for use in training the SOM. For this work, each CWT is divided into 200 windows of 5E-06s in duration covering a 100 kHz frequency range. The power ratio for each windows is then calculated and used as a feature. Having extracted the features from the AEs, the SOM can now be trained, but care is required so that the both damage mechanisms are adequately represented in the training set. This is an issue with paper as the failure of the fibre-fibre bonds is the prevalent damage mechanism. Once a suitable training set is found, the SOM can be trained and its performance analysed. For the SOM described in this work, there is a good chance that it will correctly classify the experimental AEs.