S. cerevisiae and E. coli. While these organisms may in many cases be more difficult to work with as “chassis” strains for synthetic biology than certain heterotrophs, the unique advantages of autotrophs in biotechnology applications as well as the scientific importance of improved understanding of photosynthesis warrant the development of these systems into something akin to a “green E. coli.” In this review, we highlight unique challenges and opportunities for development of synthetic biology approaches in cyanobacteria. We review classical and recently developed methods for constructing targeted mutants in various cyanobacterial strains, and offer perspective on what genetic tools might most greatly expand the ability to engineer new functions in such strains. Similarly, we review what genetic parts are most needed for the development of cyanobacterial synthetic biology. Finally, we highlight recent methods to construct genome-scale models of cyanobacterial metabolism and to use those models to measure properties of autotrophic metabolism. Throughout this paper, we discuss some of the unique challenges of a diurnal, autotrophic lifestyle along with how the development of synthetic biology and biotechnology in cyanobacteria must fit within those constraints.Photosynthetic organisms, and especially cyanobacteria, hold great promise as sources of renewably-produced fuels, bulk and specialty chemicals, and nutritional products. Synthetic biology tools can help unlock cyanobacteria's potential for these functions, but unfortunately tool development for these organisms has lagged behind that for  As compared with eukaryotic algae and plants, cyanobacteria are much easier to manipulate genetically and grow much faster. They have been engineered to produce a wide and ever-expanding range of products including fatty acids, long-chain alcohols, alkanes, ethylene, polyhydroxybutyrate, 2,3-Butanediol, ethanol, and hydrogen. These processes have been reviewed recently  or in trans (through plasmid addition) and synthetic biology experiments have used both approaches. We discuss advantages and disadvantages of each approach, as well as recent technical developments below. While even the best cyanobacterial model systems are still far from being a “green E. coli,” many tools are already available and more are being developed. The future holds great promise for this field.Several strains of cyanobacteria are known which are readily amenable to genetic modification  resistance cassette as well as any sequence of interest. Streptomycin-resistant, kanamycin-sensitive markerless mutants can be recovered in a second transformation .Until recently, it has been difficult to create mutants at high throughput in cyanobacterial strains, as transposon-based methods developed for use in other strains can work poorly in cyanobacterial hosts. However, libraries can be created in other strains and subsequently transferred to a cyanobacterial host via homologous recombination. A Tn7-based library containing ~10,000 lines was recently created to screen for strains with increased polyhydroxybutyrate (PHB) production . This move toward standardization of genetic parts is a critical aim for synthetic biology, independent of the chassis organism or method of transformation. However, a limited number of plasmids are available for expression in cyanobacterial hosts. Plasmid assembly for expression in cis or in trans in cyanobacterial hosts has generally been performed in E. coli because of the longer growth times that would be associated with assembling vectors in cyanobacterial hosts  means it is critical to have high-fidelity assembly methods to avoid a time-consuming screening process.Although transgene expression s Figure . This reSynechocystis sp. PCC 6803  for the iron-stress inducible protein, which when translated, forms a ring around trimers of photosystem I, preventing their activity and thus oxidative stress in the absence of sufficient iron  use 2-component systems, and even combine 2-component systems with non-coding RNA to control system dynamics  It will not be activated in the absence of inducer. (2) It will produce a predictable response to a given concentration of inducer or repressor. This response may be digital  or graded change with different concentrations of inducer/repressor. (3) The inducer at saturating concentrations should have no harmful effect on the host organism. (4) The inducer should be cheap and stable under the growth conditions of the host. Finally, (5) the inducible system should act orthogonally to the host cell's transcriptional program. Ideal transcriptional repressors should not bind to native promoters and if non-native transcriptional machinery is used (such as T7 RNA polymerase) it should not initiate transcription from native promoters. Promoters must perform as ideally as possible in order to be used in the construction of more complex genetic circuits  and PziaA (Cd2+ and Zn2+), both from Synechocystis sp. PCC 6803 and inducers can also activate multiple promoters, such as Cd2+ inducing ziaA and isiA  is structurally different between E. coli and cyanobacteria. In cyanobacteria the β ' subunit of the RNAP holoenzyme is split into two parts, as opposed to one in most eubacteria, creating a different DNA binding domain . The best performing promoter induced a 290-fold change in response to 1 μg/ml aTc .Success of FBA greatly depends on the quality of the metabolic network reconstruction as well as the availability of regulatory constraints under a given environmental or growth condition. For instance, constraints can be added that disable or limit fluxes due to known regulatory constraints or substrate availability  via this non-fermentative alcohol production pathway. These active areas of research will help to better define cyanobacterial metabolism and allow the generation of models that can more accurately predict cellular phenotypes. While newer fluxomics techniques can yield powerful results in well-characterized strains, developing a “green E. coli” will also require expanded knowledge of biochemistry that to date can only come from older methods of single gene or single protein analysis.The understanding of several key pathways in cyanobacteria has been recently revised. Zhang and Bryant  identifiin silico growth phenotype with available experimental data on the viability of single or multiple gene knockouts (Thiele and Palsson, in vivo gene essentiality data are available only for Synechocystis 6803 among the cyanobacteria in the CyanoMutants database (Nakamura et al., Synechocystis 6803, and the proportion is much less for any other strain. While a genome-wide library of knockout mutants has been created in Synechococcus 7942 (Chen et al., The quality or accuracy of any genome-scale metabolic model can be tested by contrasting the Cyanothece 51142 exhibits one of the highest rates of nitrogen fixation and is the first unicellular diazotrophic cyanobacterium to be completely sequenced (Welsh et al., Cyanothece 51142, iCce806, is recently developed (Vu et al., iCyt773 contains an additional 266 unique reactions spanning pathways such as lipid, pigment and alkane biosynthesis (Saha et al., iCyt773 also models diurnal metabolism by including flux regulation based on available day/night protein expression data (Stockel et al., Genome-scale models contain detailed Gene-Protein-Reaction associations, a stoichiometric representation of all possible reactions occurring in an organism, and a set of appropriate regulatory constraints on each reaction flux. They are differentiated from more basic FBA models simply by their completeness—they span all or nearly all of the metabolic reactions encoded in a genome. Thus, these models can have greater predictive value than those of only central metabolism. Among cyanobacteria, Synechocystis 6803 is a model cyanobacterial strain, it has long been the target for modeling of photosynthetic central metabolism (Yang et al., iSyn731, the latest of all Synechocystis 6803 models, integrates all recent developments and supplements them with improved metabolic capability and additional literature evidence. As many as 322 unique reactions are introduced in iSyn731 including reactions distributed in pathways such as heptadecane and fatty acid biosynthesis (Saha et al., iSyn731 is the first model for which both gene essentiality data (Nakamura et al., Synechococcus sp. PCC 7002 (Hamilton and Reed, Since in silico models are great tools for generating hypotheses on how to use synthetic biology interventions to alter metabolism, they need to be complemented by fluxomics methods that allow in vivo measurement of metabolic fluxes to assess these interventions. Such a suite of tools allows the closure of the design-build-test engineering cycle in synthetic biology (Figure 13CO3 and the labeling patterns of metabolic intermediates are followed over a time-course to determine relative rates of metabolic flux. Previous studies (Yang et al., 13C labeled glucose and metabolic fluxes are inferred from labeling patterns of proteinogenic amino acids. These studies have been extremely useful in identifying fluxes that exist in vivo, but have previously been regarded as wasteful or futile cycles, such as the oxidative pentose phosphate pathway and RuBP oxygenation. Comparisons between flux measurements (Young et al., Synechocystis 6803 have revealed the necessity of additional regulatory information for accurate in silico predictions of phenotype. These modeling and fluxomics efforts have resulted in deeper understanding of the metabolic capabilities of the modeled strains and of cyanobacteria in general.While y Figure . To thisy Figure  have devE. coli.” Metabolic modeling is a complementary tool that can help guide the creation of synthetic strains with desirable phenotypes. By developing the tools for strain manipulation and control, synthetic biologists can unlock a bright future for the biotechnological use of abundant light and CO2.Cyanobacterial synthetic biology offers great promise for enhancing efforts to produce biofuels and chemicals in photoautotrophic hosts. While several cyanobacterial chassis strains have been used in synthetic biology efforts, the tools for their manipulation and analysis need greater development to unlock this potential and develop a “green 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.


The development of depressive disorders had long been attributed to monoamine variations, and pharmacological treatment strategies likewise focused on methods of altering monoamine availability. However, the limited success achieved by treatments that altered these processes spurred the search for alternative mechanisms and treatments. Here we provide a brief overview concerning a possible role for pro-inflammatory cytokines and growth factors in major depression, as well as the possibility of targeting these factors in treating this disorder. The data suggest that focusing on one or another cytokine or growth factor might be counterproductive, especially as these factors may act sequentially or in parallel in affecting depressive disorders. It is also suggested that cytokines and growth factors might be useful biomarkers for individualized treatments of depressive illnesses. Major depression is not only the most common neuropsychiatric disorder, but it is also highly comorbid with other pathological conditions, including anxiety disorders, schizophrenia, heart diseases, auto-immune illnesses, metabolic syndrome, diabetes, neurodegenerative disorders, and post-stroke neuropsychiatric complications , monoamine oxidase inhibitors (MAOIs), selective serotonin reuptake inhibitors (SSRIs), or serotonin-norepinephrine reuptake inhibitors (SNRIs), attenuate depressive symptoms in only 50–60% of individuals, although combination drug therapies can yield appreciably improved success rates -6 and tumor necrosis factor (TNF)-α were elevated in non-medicated depressed patients, whereas variations of IL-1β were less consistently demonstrated  or with effects secondary to such changes, including variations of neurotransmitters, hormones, or growth factors. What circulating variations of cytokines in depressed individuals might reflect in relation to the actual disorder is still uncertain, especially as clinical improvements elicited by antidepressants are not uniformly accompanied by normalization of peripheral cytokine levels  or acute inflammatory conditions  commonly experienced comorbid depressive symptoms  receptor antagonists  or agonists  as well as other neurotoxins . These variations within the kynurenine pathway would then cause the activation of oxidative effects, elicit mitochondrial dysfunctions, and engender neurotoxic effects that culminate in depression  might stimulate the enzyme indoleamine 2,3-dioxygenase (IDO), which is responsible for the degradation of tryptophan within the kynurenine pathway  and the subventricular zone of the lateral ventricle (SVZ) and their functional integration to mature neural networks may occur in the adult brain. This process, adult neurogenesis, has received increasing attention owing to its important role in neuronal and synaptic plasticity and in promoting particular behavioral outcomes. As shown in Table Smaller hippocampal volumes, thought to reflect reduced neurogenesis and/or neuron survival, have been reported in patients with first episode depression as well as in those with recurrent depression . In line with this perspective, reduced serum levels and mRNA expression of BDNF were reported in drug-free depressed patients compared to those treated with antidepressants or healthy controls , post-mortem analyses indicated that hippocampal concentrations of the neurotrophin were elevated among depressed individuals who had been treated with antidepressants compared to those who had not been medicated  or chronic  stressors were accompanied by decreased hippocampal neurogenesis  and might thus also contribute to whether these responses culminate in depression. In fact, it was suggested that the impact of stressful-life events on the development of growth factor impairments and depressive illnesses might be modulated by gene polymorphisms that influence neuroplasticity in response to environmental challenges  of depressed individuals and that chronic antidepressants reduced CSF EPO levels  is involved in the pathogenesis of depressive symptoms as well as in the clinical outcomes of antidepressant treatments. Currently, however, the specific contributions of each growth factor to depression and their potential interactions in the course of the illness remain to be clarified.It will be recalled that increased activity of pro-inflammatory cytokines and impaired neurogenesis (including reduced BDNF functioning) are apparent in drug-free depressed patients and that clinical improvements resulting from antidepressant treatments have been associated with normalization of particular cytokines and growth factors. The question remains as to whether these systems act in parallel or interactively in relation to the promotion and/or the amelioration of depressive illnesses. It has been suggested that increased activity of pro-inflammatory cytokines might alter growth factors, and thus neurogenesis and plasticity, culminating in depression. It is, indeed, known that inflammatory responses elicited by immune challenges and/or stressors may damage microglia and neurons, although it is still uncertain which mechanisms are responsible for such outcomes.Treatment with the bacterial endotoxin LPS not only has peripheral effects on immune functioning, but also instigates several changes within the brain. Of particular significance to the present discussion is the finding that acute LPS treatment diminished the number of new neurons generated  or stroke, especially those variations that occur in the brain. In this regard, it has been suggested that pro-inflammatory cytokines released after brain trauma or injury could initially promote neural generation owing to the growth factor elevations and/or the neural stem cells that are recruited to the site of damage in response to the cytokine elevations or, under specific circumstances, impair neurogenesis and neuroplasticity and compromise clinical and functional recovery  differ from the key cytokines typically implicated in depressive illnesses that occur in other circumstances . Nevertheless, a prospective examination of cytokine changes during post-stroke recovery revealed that among those patients that expressed depressive symptoms one year after stroke, serum concentrations of IL-6, IL-10, TNF-α, and IFN-γ were concomitantly increased, supporting the supposition that these key cytokines might contribute to, or be an index of, post-stroke depressive symptoms  were associated with the presence of post-stroke depression  or of post-stroke or TBI recovery. It is of particular interest that growth factors, especially BDNF, and pro-inflammatory cytokines might have interactive effects on neurogenesis, depending on cytokine concentrations (and duration of their activation), although it is uncertain whether they act together or independently in the provocation or exacerbation of depressive illnesses and/or in contributing to the clinical outcomes of antidepressants. As depicted in Figure The moderate efficacy of SSRIs and SNRIs in the treatment of depression invigorated the search for the processes associated with depressive disorders as well as the hunt for biomarkers that would foretell the occurrence of depression and predict the efficacy of antidepressant strategies. The perspective that inflammatory and growth factors might serve in these capacities has received considerable support, although it appears likely that some instances of depression might not be related to them entirely. For example, as indicated earlier, it seems that those individuals with high inflammatory cytokine levels might be less responsive to SSRIs and SNRIs. Thus, there is the possibility that anti-inflammatory agents could be useful as an adjunctive treatment in attenuating depressive symptoms in these patients, although their efficacy in this regard has been variable.It seems that medication-free depressed patients may exhibit a unique profile in which activation of particular pro-inflammatory cytokines  and reductions of growth factors  are inversely related to the severity of depression. Yet, there are troubling findings that indicate that this supposition is too simplistic. It is possible that cytokine and/or BDNF contribute to particular symptoms of depression, or particular depressive subtypes. Despite the call for an endophenotypic approach to studying mental illnesses, few studies have focused on the link between specific inflammatory or growth factors and particular characteristics of depressive disorders. Ultimately, individualized treatment approaches might be most suitable in dealing with disorders that involve multiple, but varying symptoms and that seem biologically heterogeneous. At this time there is ample reason to suggest that particular cytokines  and growth factors  might serve as biomarkers in predicting who would be most prone to depression under various challenge conditions, and which treatment strategies would be most efficacious.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.


Tropical rainforests in Southeast Asia are facing increasing and ever more intense human disturbance that often negatively affects biodiversity. The aim of this study was to determine how tree species phylogenetic diversity is affected by traditional forest management types and to understand the change in community phylogenetic structure during succession. Four types of forests with different management histories were selected for this purpose: old growth forests, understorey planted old growth forests, old secondary forests (∼200-years after slash and burn), and young secondary forests (15–50-years after slash and burn). We found that tree phylogenetic community structure changed from clustering to over-dispersion from early to late successional forests and finally became random in old-growth forest. We also found that the phylogenetic structure of the tree overstorey and understorey responded differentially to change in environmental conditions during succession. In addition, we show that slash and burn agriculture (swidden cultivation) can increase landscape level plant community evolutionary information content. Here we apply this principle to assess the impact of human disturbance on species community assembly along a successional gradient in a traditionally managed forest landscape.Phylogenetic community structure analysis has proven to be an important new tool for differentiating the respective roles of environmental filtering and species interactions in shaping plant community structure Most of the tropics are now dominated by secondary forests Slash and burn (swidden) cultivation is a typical traditional forest use type in Southeast Asia Here we use phylogenetic community structure analysis to determine which process  is driving species co-existence during succession after slash and burn agriculture. The objectives of our study were (1) to understand the successional pathways after slash and burn agriculture in terms of abiotic  and biotic (species interactions) drivers, and (2) to determine the impact of traditional forest use on phylogenetic diversity and community phylogenetic structure.2 plots each) with different disturbance (management) histories: (i) old-growth forest that was open to understorey non-timber product collection, (ii) old-growth forests with understorey Amomum (Zingiberaceae) plantation, (iii) old secondary forests about 200-years after slash and burn, and (iv) young secondary forest about 15 to 50-years after slash and burn , Xishuangbanna, Yunnan province, China . Permission to study in the reserve was given by the Nabanhe National Nature Reserve Bureau, which is located within the reserve. We studied four types of forest (five 500 mand burn .Duabanga grandiflora (Lythraceae). Slash and burn was not repeated after abandonment. Slash and burn based agriculture usually only provides a food supply for the farmers family and very little extra income can be generated. The understorey planting of ginger Amomum villosum, a Chinese medicinal plant, was promoted by the government in the 1970s to improve the income of local people, and was then favored over slash and burn because of the relatively high income generated.Slash and burn is a typical land use/forest management type in tropical China and surrounding countries such as Myanmar, northern Laos, northern Vietnam and northern Thailand 2 plot. Vouchers were collected for plant species that could not be identified in the field and were deposited in the Herbarium of Xishuangbanna Botanical Garden (HITBC), CAS, Menglun, Yunnan, China.In the 20 plots , all trees with a diameter at breast height (dbh) ≥5 cm, and all lianas were identified and their diameter measured. Most previous analyses in Xishuangbanna used the 20×25 m plot shape Wood density, a good indicator of the successional stage and conservation value of tropical forest in Asia bladj algorithm of PHYLOCOM We constructed a phylogenetic tree , S2 withIn order to standardize plot comparisons all diversity analyses described below were based on a fixed number (based on the plot with the fewest stems) of randomly selected individuals from each plot. This rarefaction procedure reduces the impact of sample size differences between plots on the diversity analyses The phylogenetic similarity of the plant communities was assessed using Unifrac We calculated the abundance weighted net relatedness index (NRI) and nearest taxon index (NTI) of each plot to measure the phylogenetic dispersion (relatedness) of the co-occurring species by using PHYLOCOM random and MNTDrandom are the mean MPD and mean MNTD from 999 randomly generated assemblages. An independent swap null model was used to generate these 999 random assemblages. For both NRI and NTI, values close to zero indicate random phylogenetic structures, positive values indicate clustered, while negative values indicate overdispersed community phylogenetic structures.Where MPD is the mean pairwise phylogenetic distance between all individuals in each rarified sample and MNTD is the mean phylogenetic distance for each individual to its nearest relative within each rarified sample. The MPDSpecies-individual curves show the relationship between number of species and number of individuals and were generated with the program EstimateS (v. 7.5) PD-individual curves show the relationship between phylogenetic diversity and number of individuals. We obtained PD-individuals curves for each plot, for each of the four forest types, and for all the plots together. The curve was computed using R package “phylocurve” One-way ANOVA analysis with a post-hoc Least Significant Difference (LSD) test was used to test whether rarified tree species number per plot, PD, PSV, PSR, average wood density and liana abundance were different among the four forest types. When, even after data transformation, unequal variances were detected between the four forest types for a test variable (which violates the basic assumptions for running an ANOVA), a non-parametric Kruskal-Wallis test was used instead.Rarefied number of tree species, phylogenetic diversity (PD) and phylogenetic species evenness (PSE) were significantly lower in the young secondary forest than in the other forest types . HoweverNo significant difference was found in basal area weighted average wood density among different types of forest , but perThe PD and PSR accumulation curves of the forest types showed a similar pattern as the species accumulation curves . The youP<0.05). In addition, the level of similarity between underplanted forest and old growth forest was higher than that between old growth forest and old secondary forest, and between underplanted forest and old secondary forest  distinguished the young secondary forest from other forest types  of the tropical rainforest in China shifted from clustering in young secondary forests to over-dispersion in old secondary forests, and then to random (no pattern) in old growth forests. This change in phylogenetic structure (NRI) from clustering to over-dispersion along the successional gradient of this tropical rainforest in China generally agrees with findings from the few available other studies that estimated phylogenetic community structure in woody plants along succession gradients Amomum villosum (a ginger). Thinning may result in disappearance of some of the rarer species from the community, which is more likely to affect NTI than NRI simply because disappearance of species will immediately affect phylogenetic patterns within shallow nodes, but is unlikely to lead to changes in the older nodes .Interestingly, NTI showed an opposite pattern with NRI for the underplanted old growth forests in both the understorey and overstorey, signaling that although this forest type is characterized by a phylogenetically overdispersed tree community when deep (old) phylogenetic nodes are considered, this same tree community is clustered when only shallow (recent) nodes are considered. This means that within families and/or genera, species were more closely related than expected by chance, while at order and family level the forest was phylogenetically overdispersed. This may represent an artificial effect caused by the manual thinning that was carried out in this forest type to enhance growth conditions for the underplanted Young secondary forests are generally dominated by species that are adapted to fast colonization of open areas (pioneers), characterized by small and widely dispersed seeds, fast growth, low shade tolerance, high photosynthetic rates and large leaves with low length/width-ratios that are continuously shed and renewed (short leaf life-spans) In general, the life-spans of early successional species are short (∼30 years) and they usually fail to regenerate under their own canopy due to shade intolerance, leading to a gradual decline in abundance of early successional species during forest regeneration. Therefore, if seed limitation is no problem , the early succesional species will be replaced by late successional species which establish in the shady forest understorey. Most late successional tropical tree species are adapted to shade during their early establishment phase, so the number of phylogenetic lineages that can coexist during this phase is large and spread over almost the entire phylogeny. Therefore a decline in phylogenetic clustering is to be expected during later succesional stages. However, the strong phylogenetic over-dispersion that we detected during the later successional stage suggests that species traits linked to resource acquisition are phylogenetically conserved, i.e., species that are phylogenetically closely related will compete more intensely with each other for resources than they do with more distantly related species, eventually leading to the phylogenetically over-dispersed tree community that we observed Several old growth tropical forests have been reported to be phylogenetically over-dispersed Unlike overstorey trees, understorey tree seedlings showed a randomized phylogenetic pattern in young secondary forests (corresponding to findings from Costa Rica) Previously we found that traditional forest management has limited impact on plant diversity, and slash and burn agriculture even increased landscape level diversity In conclusion, we revealed a change in phylogenetic community structure in a seasonal rainforest in tropical China along a >200 year regeneration timescale that went from clustering to over-dispersion to randomization. We also found that the overstorey and understorey phylogenetic structure responded to change in environmental conditions with succession in different ways, i.e. less phylogenetic clustering in the understorey. In addition, we confirmed the beneficial effects of slash and burn (swidden) cultivation on landscape level phylogenetic variability and diversity.File S1Community phylogenetic tree used in the study.(PDF)Click here for additional data file.File S2Nexus format of the phylogenetic tree used in the study.(DOCX)Click here for additional data file.


Liver toxicity is a recognized adverse event associated with small molecule tyrosine kinase inhibitors (TKIs). Electronic Medical Record (EMR) databases offer the most precise data to investigate the rate of liver function test (LFT) elevations; however, they can be limited in sample size and costly to access and analyze. Health insurance claims databases often contain larger samples sizes but may lack key health information. We evaluated the feasibility of utilizing a large claims database to calculate incidence rates (IRs) of LFT elevations among a general cohort of cancer patients and a cohort of patients treated with TKIs by comparing the results to a “gold standard” oncology-specific EMR database. IRs for the TKI cohorts were very similar between the two databases; however, IRs were higher in the EMR database for the cancer cohorts. Possible explanations for these differences include lack of specificity when defining a cancer case, poor capture of laboratory data, or inaccurate assessment of person-time in the insurance claims database. This study suggests that insurance claims data may provide reliable results when investigating liver toxicities associated with oncology drug exposure; however, there are limitations when assessing laboratory outcomes for cohorts defined solely by disease status. Therapeutic agents that target cancer-specific molecules and signalling pathways have become increasingly integrated into cancer care in recent years. Activation of tyrosine kinases plays a critical role in modulation of growth factor signalling, such as increased cell proliferation and growth, induced antiapoptotic effects, and promotion of angiogenesis and metastasis, and as a result, these protein kinases are key targets for inhibition , 2. TyroThe liver plays a major part in metabolic and excretory functions, including a key role in the metabolism of a number of anticancer cytotoxics and biologic agents, causing drug inactivation or activation of a prodrug. In turn, chemotherapy and biological agents can induce liver injury or dysfunction, which can manifest in abnormal serum liver biochemistry . Drug-reIn order to benchmark the incidence of liver enzyme elevations observed in TKI clinical trials against expected rates, it is crucial to understand the background incidence rates of elevations in the general cancer patient population and in patients receiving TKIs. Population-based data sources commonly used to evaluate incidence rates include electronic medical record (EMR) databases and health insurance claims databases. Electronic medical records capture a complete patient record of diagnoses, treatments, hospitalizations, laboratory, and pathology results and thus serve as rich dataset for conducting research. EMRs, however, can be limited in the number of captured patients which may limit their usefulness for analyzing rare diseases and rare outcomes. In contrast, insurance claims databases often contain millions of patients, but the data captured can be limited since they are collected primarily for administrative and reimbursement purposes and not to create a clinical record. Furthermore, insurance claims databases may not capture detailed information regarding diagnoses , and although laboratory tests are covered, test results are not always available in the claims database. In this study, we evaluated the feasibility of characterizing liver enzyme elevations in cancer patients being treated in real-world, population-based settings by exploring two databases: an insurance claims database and an oncology-specific EMR database.The aims of this study were to (1) describe LFT availability in an insurance claims database and an EMR database; (2) calculate the incidence rate (IR) of LFT elevations in the insurance claims database and the EMR database; (3) compare the results obtained from the insurance claims database to the “gold standard” EMR database to describe the utility of using insurance claims databases to estimate incidence rates of laboratory abnormalities in an oncology setting. In order to address these aims, we utilized two patient cohorts: a general cohort of patients with selected solid tumors and a small molecule TKI drug treated cohort of patients with any cancer type. Although this study focused on these specific populations, we believe that the findings may be generally applicable to other pharmacoepidemiology studies utilizing laboratory-based claims data to investigate other laboratory events and classes of marketed oncology drugs.The Varian Medical Oncology database of outpatient oncology practices was considered the “gold standard” EMR database for this study. At the time the study was conducted (data extraction September 2008), this oncology-specific EMR system contained data on more than 185,000 cancer patients from 18 participating oncology practices in 15 states across the United States. During each patient visit, clinic staff enter data about the visit, including diagnoses, treatments, and other relevant information. Diagnoses are entered as ICD-9-CM codes, supplemented with International Classification of Diseases for Oncology (ICD-O) codes and staging information for a subset of patients. Treatment data include orders or prescriptions for medications, with specifics such as dose and route, as well as duration of supply of oral medications, and amount and timing of drugs administered in the clinic. Laboratory results are typically fed directly from the lab into the EMR system; data include the date of the test, lab test name, result, units, and normal range. The data used for the present study were deidentified, as required by the Health Insurance Portability and Accountability Act (HIPAA).We used the Clinformatics for Data Mart, a product of OptumInsight Life Sciences, as the insurance claims database for this study. This database is a comprehensive, deidentified US healthcare claims database that contains the aggregated health claims experience of over 24 million individuals covered by the health insurance program. It contains only those individuals covered for which there exists a combined benefit structure including medical and prescription coverage. Overall, it is representative of the nonelderly, insurance-carrying population in the United States, but it also contains information on several hundred thousand individuals enrolled in the government-sponsored Medicare Advantage program, a health and medical services program for persons 65 years and older, and the Managed Medicaid program, a health and medical services program for certain individuals and families with low incomes and few resources. The insurance claims database is geographically diverse, including data for members in all 50 states. It contains inpatient, outpatient, and pharmacy claims and integrates the outpatient test result values for lab tests processed by the two largest US national lab vendors. The data used for the present study were deidentified, as required by the Health Insurance Portability and Accountability Act (HIPAA).A summary of the cancer cohort study populations can be found in any cancer who were treated with one or more TKI agents including imatinib, gefitinib, erlotinib, dasatinib, lapatinib, and nilotinib. A small proportion of patients that had used two TKIs during the study period (4%) were classified according to the first TKI used. Patients in the EMR database with multiple primary cancer diagnoses were excluded as done in the EMR cancer cohort. However, the multiple primary exclusion was not similarly applied to the insurance claims TKI cohort, as a large proportion of eligible patients were lost when this was applied.Patients were followed from the index date  through the last visit in the database due to either death, drop out of the health plan, or through the end of the analysis period , whichever came first. Several additional eligibility criteria regarding availability of LFTs in the database and length of database enrollment around the index date were applied universally across both the cancer and TKI cohorts in both databases. In order to ensure that patients had laboratory data captured in their record and to exclude any patients with prevalent LFT elevations (prior to baseline), all included patients had to have (1) at least one LFT measured ≤30 days before index date and no abnormal elevations during that time and (2) at least one follow-up LFT result at any point in the record after the index date. Follow-up time criteria of at least one follow-up visit recorded (EMR databases) or at least one month of enrolment prior to and three months post index date (insurance claims database) were instituted to ensure that patients were continuously enrolled in their respective database during the analyses time period and to limit missing LFTs due to switching of insurance companies.Patients who qualified for more than one of the cohorts (cancer and TKI) were allowed to contribute to each, often with different index dates for each cohort. Demographics for ineligible patients were captured in order to make comparisons with the eligible populations.The outcome of interest is the incidence of elevated LFTs which was defined identically in all cohorts. Incidence rates (IRs) and 95% confidence intervals (CIs) were calculated for elevations of ALT, AST, ALP, and total bilirubin and were defined as a value measured after the index date that was above selected cut points of the upper limit of normal (ULN) for the test. Combinations of LFTs according to probable Hy's Law  which denote possible clinically significant liver abnormalities were also examined . For theχ2 statistics to determine whether significant differences in categorical variables were present between eligible and ineligible patients within each cohort from a single database. However, the comparisons between the insurance claims and EMR databases are only descriptive with no formal testing. All analyses were conducted using SAS version 9.1.For the cancer cohorts, patients were followed for elevations in LFTs from the index date  through the last LFT measurement before the database cutoff. For the TKI cohorts, LFT elevations were first calculated considering all follow-up time from initiation of TKI through the last LFT measurement before the database cutoff regardless of whether patients were actively taking the drug . Secondly, LFT elevations were calculated during distinct periods of drug exposure (and nonexposure) during followup. The duration of exposure to the oral TKI agents was determined by a combination of available variables for days supply, dispensed quantity, administration frequency (quantity/time), and number of refills. For example, an oral drug with a 30-day supply and 2 refills had a total duration of 90 days. We used a 45-day or more gap in refill or administration of treatment to define time “off” of treatment. We calculated Among the 38,940 patients who had an ICD-9-CM code for one of the solid tumors of interest, 11,452 (29%) met the additional LFT and follow-up time criteria . Among tThe most notable difference between eligible and ineligible patients in the EMR cancer cohort was the distribution in the type of health insurance. There was a lower proportion of eligible patients with private health insurance (14.9% versus 24.1%) and a greater proportion with other/unknown health insurance (53.1% versus 37.1%). The distribution of cancer type among the eligible and ineligible patients was fairly similar with breast, lung, and colorectal being the most common cancers among both groups.Of the 1,375 patients who had received one of the TKIs of interest, 537(39%) were eligible for the analyses . Among tAmong the 153,954 patients who met the case definition for cancer (two or more ICD-9-CM codes for the same cancer within six months), 6,343 (4%) met the additional LFT and follow-up criteria . Sixty-fAmong the 3,800 patients who met the enrolment criteria for the insurance claims database TKI cohort, 409 (11%) patients were eligible for the LFT analysis . As obseThe most notable differences observed between the eligible and ineligible groups in the insurance claims TKI cohort were insurance type (eligible population had lower proportion of IND and PPO coverage and higher EPO and POS coverage). This insurance type difference likely reflects the fact that certain insurers use commercial laboratory networks that automatically provide results back to the insurer, thus increasing LFT availability for patients with those insurance types.The insurance claims database was able to replicate the EMR eligible population with regard to gender distribution; however, eligible patients in the insurance claims database were slightly younger (mean 57 years versus 62 years). This age difference is probably because the insurance claims database represents primarily nonelderly, commercially insured adults whereas the EMR system may represent a broader patient population with both commercial insurance and Medicare coverage. While the most common cancers among the eligible population in the EMR database were breast (34.7%), lung (27.5%), and colorectal (17.9%), the insurance claims database commonest cancers were breast (48%), prostate (18%), and colorectal (12%). The higher prevalence of prostate cancer observed in the insurance claims database is likely an artifact of picking up ICD-9-CM codes for prostate cancer screening (PSA testing) rather than actual confirmed cases of prostate cancer.The insurance claims TKI eligible cohort was slightly more likely to be females (57%) compared to the EMR TKI eligible cohort (53%) and, as observed with the cancer cohort, was slightly younger (54 years versus 63 years). In addition, the insurance claims TKI cohort had fewer patients exposed to gefitinib , while slightly more patients were exposed to lapatinib (11.7% versus 0.4%), respectively, when compared to the TKI cohort in the EMR database.A comparison of IRs for selected LFT elevation thresholds for the two cancer cohorts is provided in Unlike the comparison of the cancer cohorts, the insurance claims TKI cohort had IRs of similar magnitude as those observed in the EMR TKI cohort . The EMRIn the analysis that stratified IRs by time “on” and “off” TKI, we observed very similar IRs for LFT elevations among patients currently “on” TKI drugs (data not shown). However, the sample size for these analyses was limited which decreases the ability to make firm conclusions with regard to these data.This study assessed the feasibility of using an insurance claims database to examine LFT elevations in cancer patients and patients receiving TKIs by comparing the patient population and IRs of LFT elevations to those obtained in a “gold standard” oncology-specific EMR database.After applying several inclusions criteria regarding availability of LFTs, only 5% and 13% of the insurance claims database cancer and TKI cohorts, respectively, were eligible to be included in the analysis. For the EMR database, the proportion of eligible patients was greater in both cohorts . It is important to recognize that these percentages do not reflect actual screening rates in medical practice (or more frequent screening in the clinics that participate in the EMR database), but rather how well laboratory results are captured within the respective databases. In addition, we required an LFT to be captured within a narrow window of only 30 days prior to the index date in order to exclude patients with prevalent LFT elevations immediately prior to cohort entry. A sensitivity analysis in the insurance claims database explored wider ranges of baseline time before and after the index date (data not shown). As expected, these wider time windows resulted in greater eligible sample sizes; however, the patient characteristics and LFT results were similar regardless of the baseline window utilised, and thus we utilised the 30 days prior window as this is in line with the design of the gold standard EMR cohorts. These eligibility data suggest, as expected, that the EMR cohort has better capture of laboratory test results. In addition, the higher availability of LFTs among the TKI cohorts regardless of database may reflect more frequent screening of patients who are prescribed these drugs, given the known class association with hepatotoxicity.The insurance claims cancer cohort produced IRs that were considerably lower than the EMR cancer cohort, whereas IRs for the TKI cohorts were generally comparable across databases. There are several potential explanations for why IR differences across the two databases might be observed in the cancer cohort but not the TKI cohort.As discussed earlier, we believe that the EMR database has more complete capture of LFTs, possibly resulting in greater numbers of incident LFT elevations being contributed to the IR numerator. However, it is unlikely that this would explain the observed IR disparities in only the cancer cohort, since the improved LFT capture was seen in both the cancer and the TKI cohorts.A second possibility is that the insurance claims eligible population differed from the eligible EMR population with regard to key characteristics that may influence the likelihood of having an LFT elevation. Indeed, some small differences in age and cancer site distribution were observed between the cancer cohort eligible populations; however, similar age differences were also noted in the TKI cohorts, and primary cancer site is not a strong predictor of LFT elevations, unless possibly through a pathway of enhance proclivity to metastasize to the liver. We were not able to investigate the influence of liver metastases as this variable was not available in the insurance claims database; however, in the EMR databases, this represented a very small proportion of the overall cancer cohort or TKI cohort populations.A third potential explanation is that the insurance claims database captures all patient follow-up time until a person has a change in health plan, whereas the EMR database reflects only the active cancer treatment period. Thus, the insurance claims database likely includes an inflated person-time denominator that includes “noncancer” time after patients have gone into remission/cure and are not likely to have elevated LFTs. This phenomenon may have less influence on the TKI cohort, where the index date of TKI initiation represents a later stage in the cancer continuum as these agents are mostly used in the metastatic setting when patients are closer to death/end of insurance plan enrolment.The most probable explanation relates to how the cohorts were defined within the insurance claims database. The EMR cancer cohort captured only patients actively undergoing cancer treatment, and thus we are certain that these patients have cancer. However, cancer cases in the insurance claims cancer cohort were identified using a case definition of two ICD-9-CM codes for the same cancer within six months. It is possible that this definition was not highly specific and resulted in overinflation of the IR denominator by including person-time of “false” cancer cases or people undergoing a screening or diagnostic workup for suspected cancer that was not deemed to be malignant. In an analysis examining the accuracy of cancer case identification in a claims database, Setoguchi and colleagues compared several increasingly detailed cancer case-definitions against confirmed cancer diagnoses from cancer registry data in the United States . They obHowever, these concerns are less of an issue in the insurance claims TKI cohort, as inclusion in that cohort required a prescription for a TKI, which are only used in oncology treatment. Furthermore, since we selected the date of the first TKI prescription as the index date, we have a higher confidence that these are true cancer patients who are actively in treatment (at least for the immediate time period after index date). Taken together, these arguments suggest that when patients are defined in an insurance claims database based upon receiving a particular anticancer treatment in addition to cancer codes, the claims database is more accurate compared to identifying cancer patients using only ICD-9-CM codes for diagnosis.A general limitation of using a claims database to study cancer patients is the lack of data on key cancer related variables that may be important factors in choice of treatment, cancer prognosis, or risk of adverse events. For example, the insurance claims database does not include information on the presence of liver metastasis, which may be contributing factor for liver function elevations. This data was available in the EMR cohorts and represented a very small proportion of either cohort; thus, the lack of this variable in insurance claims databases should perhaps not preclude the use of these databases for studying LFTs.Both databases primarily capture labs ordered on an outpatient basis and therefore may underestimate elevations recorded during inpatient hospital stays. Both databases utilized requirements of follow-up time (at least one further visit in the EMR database or at least three months of follow-up enrollment in the insurance claims database) which may result in immortal person-time. Since all patients in the cohorts have similar requirements, the only limitation this creates is that the included patients might not be representative of all cancer and TKI patients, since those who disenroll early or those who die shortly after initiating treatment may have had different LFT patterns than those seen in these cohorts. Finally, since all data are deidentified, we have no way of knowing if an individual who was treated at an oncology clinic in the Varian system also had an insurance plan captured in the insurance claims database and thus may have been included twice. We do not believe that there are a large number of individuals who would be included in both.The outcome of elevated liver function is considered to be an adverse event when the elevation can be directly attributed to the use of a particular medication. While neither the EMR nor insurance claims databases can be used to definitively associate a recorded elevation with a particular drug, we were able to measure LFT elevations by time “on” and “off” TKI in both cohorts (data not shown). The sample size for analysis in both of the cohorts was limited, thus decreasing the ability to draw conclusions from the analysis. However, the IRs for TKI elevations observed in the EMR database were similar in magnitude to those observed in the insurance claims database (data not shown).This study highlights the strengths and limitations of using an insurance claims databases to estimate the incidence rates of a laboratory abnormality in cancer patients in general, and those being treated with a particular class of antineoplastic drugs. The ability to compare the results derived from the insurance claims database to a “gold standard” oncology database is a key attribute of this study and illuminates some limitations to claims databases which may bias incidence results. These limitations include difficulty in accurately identifying “true” cancer patients in a claims database as well as possible underreporting of laboratory results. Interestingly, the results for the insurance claims database and EMR database were highly comparable for the TKI cohort, replicating other studies demonstrating that case definitions which incorporate both disease and treatment criteria are more accurate at identifying cancer patients under active treatment. While this study looked at only one claims database and one set of laboratory tests, we believe that the results may be widely applicable to other claims databases and other laboratory-defined events in the oncology setting.


Cancer is a devastating disease that alters the metabolism of a cell and the surrounding milieu. Metabolomics is a growing and powerful technology capable of detecting hundreds to thousands of metabolites in tissues and biofluids. The recent advances in metabolomics technologies have enabled a deeper investigation into the metabolism of cancer and a better understanding of how cancer cells use glycolysis, known as the “Warburg effect,” advantageously to produce the amino acids, nucleotides and lipids necessary for tumor proliferation and vascularization. Currently, metabolomics research is being used to discover diagnostic cancer biomarkers in the clinic, to better understand its complex heterogeneous nature, to discover pathways involved in cancer that could be used for new targets and to monitor metabolic biomarkers during therapeutic intervention. These metabolomics approaches may also provide clues to personalized cancer treatments by providing useful information to the clinician about the cancer patient’s response to medical interventions. Metabolomics is the latest of the omics technologies that employs state of the art analytical instrumentation in conjunction with pattern recognition techniques to monitor and discover metabolic changes in subjects related to disease status or in response to a medical or external intervention. Global metabolomics alterations reflect changes due to environmental factors, genetic variation and regulation, changes in gut microflora, and altered kinetic activity or levels of enzymes. Therefore, metabolomics alterations represent changes in the phenotype and molecular physiology ,2,3. MetOf the omics platforms, metabolomics has great potential to impact clinical health practices due to its ability to rapidly analyze tissue or biofluid samples with little sample preparation; metabolomics provides information that complements the genomic and proteomic profile of a patient. Global metabolic profiling has been referred to as either metabolomics or metabonomics where metabolomics refers to the measurable metabolite pool that exists within a cell or tissue under a particular set of environmental conditions  and meta13C labeled glucose) to define metabolic fluxes or biomarkers in disease states. This approach enables us to further our understanding of the metabolism in disease or drug responses by following the metabolism of labeled substrates into their pathway products within specific times. For example, glucose can undergo glycolysis to lactate or be shunted through the pentose phosphate pathway to form ribose, and the 13C labeled carbons in glucose can reveal how much goes into each pathway. This information provides a better understanding of the pathways that are upregulated or downregulated and can define metabolic phenotypes in disease states fluoro-2-deoxy-D-glucose (FDG), to detect glucose uptake by the tumor cells [X-ray, positron-emission tomography (PET), single photon-emission computed tomography (SPECT), magnetic resonance imaging (MRI), magnetic resonance spectroscopy imaging (MRSI), and ultrasound are e cancer . PET is or cells ,128. MRIor cells ,133,134.or cells . Diffusior cells ,137. MRSor cells , brain [or cells  and prosor cells . MRSI deor cells . The abior cells . Alusta or cells .De novo fatty acid synthesis is a cancer therapy target and the drug Orlistat and several other drugs target the fatty acid synthase (FASN) enzyme while other cancer drugs target ATP citrate lyase (ACLY). Lonidamine, 2-deoxyglucose, and 3-bromopyruvate are drugs being developed that target the glycolysis enzyme, hexokinase (HK), while another drug under development targets pyruvate kinase (PK). Each of these drugs should inhibit glycolysis, which is known to be upregulated in cancer. It is anticipated that cancer drugs that inhibit upregulated enzymes in cancer cells should reduce endogenous metabolites essential for cancer growth thus reducing cancer cell proliferation and may reduce transformation in pre-cancer cells. The levels of endogenous metabolites should start changing before the tumor reduces size, which is a common clinical end point for cancer therapy. Transporters are another cancer therapy target, where the drug phloretin targets the glucose transporter (GLUT) and the monocarboxylate transporter (MCT). There are several other drugs in clinical development that target MCT and cariporide is a drug in preclinical development that targets the Na+/H+ exchanger (NHE1). Compared with other omics, metabolomics is best suited to evaluate whether these cancer therapies ultimately cause changes to metabolic pathways while at the same time it can detect drug pharmacokinetics. Weiss [The first medical therapies for cancer used chemicals that were called “antimetabolites” . They wes. Weiss  describes. Weiss . In the s. Weiss . In the s. Weiss .Cancer is a disease that alters the metabolism of a cell and metabolomics approaches are being employed to better understand these changes in cancer metabolism. Both open and focused metabolomics approaches along with imaging techniques can be used to monitor metabolism in cancer for both diagnostic and prognostic biomarkers. The metabolomics biomarkers of cancer listed in the manuscript are just experimental and have not been validated. Some of these metabolomics biomarkers could be submitted to the FDA or Biomarker Consortium ,150. Met


Trauma, in the form of pressure and/or friction from footwear, is a common cause of foot ulceration in people with diabetes. These practical recommendations regarding the provision of footwear for people with diabetes were agreed upon following review of existing position statements and clinical guidelines. The aim of this process was not to re-invent existing guidelines but to provide practical guidance for health professionals on how they can best deliver these recommendations within the Australian health system. Where information was lacking or inconsistent, a consensus was reached following discussion by all authors. Appropriately prescribed footwear, used alone or in conjunction with custom-made foot orthoses, can reduce pedal pressures and reduce the risk of foot ulceration. It is important for all health professionals involved in the care of people with diabetes to both assess and make recommendations on the footwear needs of their clients or to refer to health professionals with such skills and knowledge. Individuals with more complex footwear needs  should be referred to health professionals with experience in the prescription of these modalities and who are able to provide appropriate and timely follow-up. Where financial disadvantage is a barrier to individuals acquiring appropriate footwear, health care professionals should be aware of state and territory based equipment funding schemes that can provide financial assistance. Aboriginal and Torres Strait Islanders and people living in rural and remote areas are likely to have limited access to a broad range of footwear. Provision of appropriate footwear to people with diabetes in these communities needs be addressed as part of a comprehensive national strategy to reduce the burden of diabetes and its complications on the health system. Diabetes related foot ulceration is a serious complication of diabetes and a significant risk factor for lower limb amputation-3. ThereWhilst the presence of peripheral neuropathy is the leading risk factor for diabetes related foot ulceration, a pivotal event, such as trauma from footwear, is also needed for most ulcers to occur. RubbingThe purchase and wearing of appropriate footwear is therefore an important process for all individuals with diabetes, especially for those who demonstrate loss of protective sensation from peripheral neuropathy. This patient group, who are unable to feel pressure and/or pain caused by inappropriate or ill-fitting shoes are more likely to develop blisters, callus and corns. These early complications are pathological and are warning signs that require prompt intervention if ulceration and potential amputation are to be avoided. The simple measure of wearing appropriately fitted or prescribed footwear has been shown to significantly reduce plantar foot pressures, therefore decreasing the likelihood of developing callus and ultimately ulceration,11.Health professionals involved in the care of people with diabetes need to define the individuals’ level of risk for developing foot complications and thus tailor footwear advice accordingly. Risk stratification is determined following a basic foot assessment, which includes evaluation for the presence of peripheral neuropathy, peripheral arterial disease and foot deformity-14. In aGuidelines from the National Health and Medical Research Council (NHMRC) cite appropriate footwear as one of the key strategies in preventing foot ulceration,14. ThisIndividuals with no identifiable risk factors  on foot assessment are at low risk for developing foot ulceration Table . A commoIndividuals with peripheral neuropathy and/or peripheral arterial disease on foot assessment are ‘at risk’ of developing foot ulceration Table 15]. Co. Co15]. Individuals assessed as having an abnormal foot shape, including previous amputation, are at high risk of developing foot ulceration Table 16]. Th. Th16]. Individuals with a previous foot ulcer are at high risk of developing further ulcers in the future. Even thFoot orthoses may be ‘non-moulded’ and designed to provide cushioning or ‘custom-moulded’ to additionally accommodate the shape of the foot and reduce plantar pressures. Custom Individuals who require medical grade footwear may find the cost prohibitive. Those who cannot afford medical grade footwear may be eligible for government funding through one of the state/territory based schemes. Health care professionals need to be aware of what footwear schemes exist in their area and refer appropriate individuals for consideration. Websites for state based schemes for the provision of footwear and orthoses are provided in Table Individuals in rural and remote areas may have a limited range of footwear options available to them. Many general shoe stores servicing these communities do not have foot measuring devices or skilled staff to assist with fitting. Inequities also exist in the availability of medical grade and custom-made medical grade footwear providers in rural and remote areas. Often smaller communities will only have an occasional visiting service.Aboriginal and Torres Strait Islanders who live in remote locations may have no access to footwear and potentially may not understand the health benefits of wearing particular footwear even when they have diabetes. Recent pilot programs in remote locations in Queensland have demonstrated that Aboriginal and Torres Strait Islanders can be encouraged to wear footwear to protect their feet . These multi-disciplinary programs provide footcare education, diabetic foot screening, podiatric interventions and appropriately fitted footwear. Different environmental and cultural circumstances may require a compromise to be reached, whereby footwear such as reef sandals, that are not generally recommended for individuals with diabetes, may be considered better than no footwear at all.Individuals with diabetes need consistent and ongoing education from health care providers regarding the role of footwear and the type most appropriate to their level of risk for ulceration. Access to government funding for footwear is variable across the States and Territories and long waiting times can be experienced. Individuals who cannot afford medical grade footwear, custom-made medical grade footwear and/or foot orthoses, despite being assessed as medically requiring these devices, should be provided with assistance from health professionals to apply for funding via government subsidised schemes.Abnormal foot shape: An abnormal foot shape is one that cannot be accommodated in regular, off the shelf footwear. This includes, but is not limited to feet with: hallux valgus, clawed/hammer toes, severe pes-planus or cavus foot type, abnormally wide feet, partial foot amputation or chronic Charcot deformity.Custom-made medical grade footwear: Custom-made medical grade footwear (or bespoke footwear) has the features of medical grade footwear but is manufactured specifically for one individual, as the individual cannot be safely accommodated in non custom-made footwear. Multiple measurements, impressions or a mould is generally required for manufacture.Custom moulded foot orthoses / insoles: Custom moulded foot orthoses or insoles are manufactured using an impression of the foot and are generally comprised of multiple layers of different materials. They are designed to conform to the shape of the foot, providing cushioning and redistribution of plantar pressure.Medical grade footwear: Medical grade (or therapeutic) footwear is footwear that meets the specific needs of the client, on the basis that they provide extra depth, multiple width fittings and features designed to accommodate a broader range of foot types. Other features may include modified soles, fastenings and smooth internal linings. This type of footwear is usually available at specialty shoe shops.Off the shelf footwear: Readily available comfort or sports shoes that have not been modified.Peripheral Arterial Disease (PAD): Absence of both pedal pulses, past intervention for PAD or established history of PAD.Peripheral neuropathy: Sensory loss that prevents the individual feeling a 10 g monofilament.The authors have no relevant conflict of interest to disclose.This manuscript was conceived, developed and approved by the Australian Diabetes Foot Network (ADFN). The ADFN is a working group of the Australian Diabetes Society and members include health professionals from endocrinology, podiatry, nursing/diabetes education, indigenous health, orthopaedic surgery and vascular surgery. Each member of the ADFN is an author of this manuscript, contributed to its development, agrees with its recommendations and approved the final manuscript.


Cough is a common symptom in people who develop interstitial lung diseases (ILD). The pathological features of the ILDs are many and varied suggesting that the cause of cough may also vary with each disease. This article reviews what is currently known about cough in sarcoidosis and idiopathic pulmonary fibrosis; two of the commonest ILDs. It also outlines some of the theories which have been proposed to explain why cough develops in these conditions and describes what little is known about how to treat it. Sarcoidosis is a multi-system disorder of unknown aetiology which is characterised histologically by the infiltration of affected tissue by non-caseating granulomatous inflammation. Whilst any part of the body can be involved, sarcoidosis typically affects hilar and mediastinal lymph nodes, the lung interstitium and bronchial epithelum. Less commonly, the upper respiratory tract, pleura and pulmonary vessels may be involved. A number of epidemiological studies have investigated the prevalence of cough in people with sarcoidosis and whilst it appears rare in Japan, it is common in other parts of the world where the prevalence is between 30 and 50% Table  1-3].-3.1-3].Whilst little is known about the mechanisms by which sarcoidosis causes cough , clinicaOne retrospective study from China supports the link between granulomatous inflammation of the airways and cough in sarcoidosis. This study compared respiratory symptoms in 87 patients who had undergone tracheal biopsy and found that significantly more (92%) of those who had granulomatous tracheitis complained of cough compared with those (49%) who had normal tracheal biopsies . This stSarcoidosis of the upper respiratory tract (SURT) occurs in approximately 5% of cases and may variably affect the larynx, epiglottis, arytenoids, the aryepiglottic folds and vocal chords as well as the nasal mucosa and sinuses . There aThe enhanced cough reflex observed in people with saroidosis is likely initiated by sensory fibres of the vagus nerve that innervate the airways and larynx which have become sensitised by granulomatous inflammation. The precise mechanism by which this occurs is uncertain. However, it is interesting that Ricci and co-workers demonstrated that a number of non-neuronal cell types can express neurotrophins and their receptors in normal human lung . These iOccasional, unusual manifestations of sarcoidosis have been described which cause patients to present with the symptom of cough. These include granulomatous infiltration of the pulmonary veins causing pulmonary veno-occlusive disease with engorgement of airway epithelium and sarcoidosis affecting the vagus nerve at the level of the jugular foramen causing vocal cord palsy and cough ,13.There have been no randomised, placebo-controlled clinical trials which have investigated the effect of any pharmacological treatment specifically for cough in sarcoidosis. However, clinical observation suggests that when used in an appropriate clinical setting such as progressive interstitial lung disease, oral corticosteroid therapy has a beneficial effect on cough when it is a troublesome accompanying symptom. One double-blind, placebo-controlled study of inhaled corticosteroids in newly diagnosed patients with pulmonary sarcoidosis reported an improvement in global symptom score which included cough . HoweverIdiopathic pulmonary fibrosis (IPF) is the commonest form of idiopathic interstitial pneumonia. It is a disease of unknown aetiology which develops in the sub-pleural aspects of the posterior, basal parts of the lungs and becomes more extensive over time. On CT scan of the thorax the lungs show reticular opacities, often associated with ‘traction bronchiectasis’ and honeycombing Figure . On histlater, with severe and extensive fibrosis cough, unproductive or with scanty mucoid expectoration, may be troublesome’[It has been known for many years that cough is a clinical feature of IPF. In 1974, Scadding observed: ‘ublesome’. Howeverublesome’. More reublesome’. Interesublesome’. There iublesome’.Superficially it appears anomalous that an interstitial lung disease, which predominantly affects the alveolar walls in the peripheral lung where sensory innervation is relatively sparse , should silent dysphagia’ was a possible cause of ‘pulmonary lesions of obscure origin’. This hypothesis has been given further support by recent objective studies which used 24-hour oesophageal pH monitoring and oesophageal manometry to demonstrate that up to 87% of patients with IPF have evidence of acid reflux which is often asymptomatic [A significant barrier to the methodical study of cough in IPF is that patients frequently have confounding co-morbidities which can cause or exacerbate cough. These include COPD, sinusitis, hypertension treated by angiotensin converting enzyme inhibitors and, most importantly, gasto-oesophageal reflux disease (GORD). The latter is a known cause of chronic cough  and it hptomatic ,27. Inteptomatic .A recent, preliminary study applied the technique of combined oesophageal pH and impedance monitoring, which characterises all reflux episodes regardless of pH, to show that non-acid reflux also occurs in people with IPF . When coThe hypothesis that aspiration of gastric contents is an explanation for both the pathogenesis of IPF and its associated symptom of cough, has been confounded by a number of recent clinical and epidemiological observations. Firstly, that obstructive sleep apnoea (OSA) may be prevalent in people with IPF ,33; secoA different theory for the pathogenesis of cough in IPF proposes that mechanical distortion of the larger airways by progressive interstitial fibrosis, so-called ‘traction bronchiectasis’ Figure  increaseAs with sarcoidosis, there is some evidence for increased expression of neurotrophins in the lungs of people with IPF. It has been demonstrated that induced sputum from such people contains higher levels of NGF and BDNF compared with normal controls  and BALFTreatment of cough in IPF remains problematic for both patients and physicians, particularly in the later stages of the condition when it may be associated with increasingly severe breathlessness. In such cases, palliative therapy using conventional anti-tussive agents such as opiate-derived preparations often proves to be of limited benefit, as does optimal pharmacological treatment of acid reflux. One small, uncontrolled, open-labeled study found that cough sensitivity to capsaicin and cough symptom score was reduced after one month of high-dose oral corticosteroids suggesting that cough in IPF should be amenable to pharmacological therapy . In an oA recent double-blind, two-treatment, two-period crossover trial comparing thalidomide with placebo demonstrated a beneficial treatment effect on cough and quality of life as determined by questionnaires and visual analogue score . Whilst At the present time, physicians are all too often reduced to treating the debilitating cough of IPF with opiates as part of an end-of-life pathway. There is thus an urgent need for better treatments which can only be developed through our better understanding of the cough reflex in IPF.The author declares that he has no competing interests.


Based on the knowledge that a cured infection protects the individual from re-infection, the development of a vaccine to prevent leishmaniasis has been a goal for nearly a century. Indeed, it is generally believed that after healing of leishmaniasis, sterile cure is never achieved and that few residual living parasites will remain sequestered within some host cells that offer them a safe shelter and hence maintain anti-parasite immune memory. This statement is mainly supported by data from experimental leishmaniasis in mice of susceptible or resistant phenotype, in which, live parasites could be recovered from lesions even after healing, and in which disease reactivation can be obtained by immune manipulation even after apparent complete cure.Leishmania major parasite persistence vs. sterile healing in zoonotic cutaneous leishmaniasis (ZCL) by analyzing biopsies of scars from healed volunteers.Whether maintenance of a long-term immune effector memory in humans will also require persistence of live parasites is presently unknown but is very important in the perspective of a vaccine development. Our aim herein was to address the issue of i) the first sample was processed for quantitative real time PCR, (ii) the second was cultured in vitro in enriched medium and (iii) the third was inoculated into the footpad of susceptible BALB/c mice which were kept under observation for five months.Skin-punch scars’ biopsies  have been obtained from volunteers (18–55 years old) living in two ZCL endemic foci, who had a confirmed past history, are clinically cured of ZCL and who gave their written consent. The whole protocol was approved by the local IRB. The specimens were taken under sterile conditions and local anaesthesia. Each specimen was divided into three parts: (in vitro isolation and after microscopic observation for at least 8 weeks, all cultures were found negative. For in vivo isolation, biopsy-inoculated mice were killed five months later, and skin fragments, draining lymph nodes and spleen were inoculated into culture medium, observed carefully for at least 8 weeks before being also designated as negative. PCR results were found negative for the majority of biopsies. These results indicate that any potential persistent living parasites after cure are unlikely to be sequestered in ZCL scars. This issue is of importance in strategies for control of leishmaniasis and requires further discussion.For


Deception is a complex social behavior which involves a set of higher cognitive functions. Studying this common phenomenon in humans has in all epochs been driven not merely by the wish to understand the underlying framework of cognitive functioning but rather by the ambition to detect deceptive behavior in criminal suspects. Thus, identifying valid indicators of deceptive behavior has always been in the focus of deception research. Such indicators can be defined in terms of specific behavior, physiological correlates, or content of verbal reports. The question of how validly each indicator allows for differentiating truthful and deceptive accounts is inherent in the majority of research efforts in this domain.Another important aspect concerns the development of deception theory. According to current opinion, deception is not characterized by a single cognitive process but rather involves the combination of a variety of basic cognitive processes such as working memory, response monitoring and inhibition. Identifying these processes, modeling their interplay and their modulation by personality and situational factors is still one major challenge in deception research. Furthermore, deception is no unitary phenomenon. Correspondingly, researchers need to examine and describe different variants of this phenomenon occurring in distinct contexts, which entails a variety of experimental and theoretical approaches that largely differ in scope and methods.One major field in deception research concerns the use of psychophysiological methods to detect deceptive behavior. Over time, the traditional physiological measures  have been supplemented by electroencephalographic, functional imaging, and other innovative procedures. Finding measures that validly discriminate between truth and lie, and the wish to optimize their use, have received new impetus from recent technological development. Neuroimaging techniques, for example, yield new promises and deserve a deep evaluation. Thermal imaging and eyetracking are other innovative methods which might provide additional information about the mental processes involved in generating deceptive responses. However, even “classic” behavioral measures such as response times are still frequently used in this domain for theoretical as well as applied purposes.per se but rather aims at detecting whether a suspect has concealed knowledge of specific  details. A different approach is the so-called differentiation-of-deception paradigm  as compared to neutral items . More recently, comparable differences were reported for behavioral responses, specific components of event-related brain potentials, and neurovascular changes in specific brain regions measured by neuroimaging techniques (for a comprehensive review see Verschuere et al., Several studies focused on event-related brain potentials and demonstrated that ERP components were susceptible to contextual factors such as the personal involvement in a misdeed during encoding (Jang et al., Besides using new measures or combining different behavioral and physiological indices, it has been suggested to increase cognitive load during the examination to facilitate the detection of liars. Walczyk and colleagues provided a review and a detailed theoretical outline of this approach (Walczyk et al., One major problem of current techniques is their susceptibility to countermeasures. Thus, guilty examinees might be able to deliberately alter their pattern of responses to appear innocent. Similarly, certain groups of suspects might have less difficulty in lying as compared to others because of frequent lying in general. Two studies in the current Research Topic explored these issues using variants of the differentiation-of-deception paradigm. Increasing the proportion of deceptive as compared to truthful responses led to reduced reaction time differences between truth and lie, which might indicate that lies require less cognitive effort in frequent liars and are therefore more difficult to detect (Van Bockstaele et al., Besides exploring specific cues of deceptive behavior in highly standardized situations and with highly standardized interrogation techniques, it also seems interesting to examine deception in more naturalistic settings. For example, Spence and colleagues asked participants to provide relatively unrestricted honest and deceptive accounts of their opinion regarding social issues. For these accounts, speech parameters were extracted and the authors found a significantly reduced speech rate along with increased response latency during deception compared with truth-telling (Spence et al., Research on deception has a long tradition in psychology and related fields. On the one hand, the drive for detecting deception has inspired research, teaching, and application over many decades. On the other hand, research on deception as a process or phenomenon is characterized by manifold interactions with other areas of psychological research such as attention, memory, executive control, or motor behavior. It remains to be debated whether deception and its detection should be studied as a key topic which entails addressing these other fields, or rather as a particular, illustrative manifestation of them. We regard the present Research Topic as clearly underlining the scientific benefits arising from the broad and multidisciplinary perspective that characterizes deception research today and we hope that it will enrich and inspire future research in this domain.


Bone pain is the most common type of pain in cancer. Bony metastases are common in advanced cancers, particularly in multiple myeloma, breast, prostate or lung cancer. Current pain-relieving strategies include the use of opioid-based analgesia, bisphosphonates and radiotherapy. Although patients experience some pain relief, these interventions may produce unacceptable side-effects which inevitably affect the quality of life. Acupuncture may represent a potentially valuable adjunct to existing strategies for pain relief and it is known to be relatively free of harmful side-effects. Although acupuncture is used in palliative care settings for all types of cancer pain the evidence-base is sparse and inconclusive and there is very little evidence to show its effectiveness in relieving cancer-induced bone pain (CIBP). The aim of this critical review is to consider the known physiological effects of acupuncture and discuss these in the context of the pathophysiology of malignant bone pain. The aim of future research should be to produce an effective protocol for treating CIBP with acupuncture based on a sound, evidence-based rationale. The physiological mechanisms presented in this review suggest that this is a realistic objective. Bone pain is the most common type of pain in cancer. Bony metastases are common in advanced cancers, particularly in multiple myeloma, breast, prostate or lung cancer and they occur more commonly on the axial skeleton , 2. NowaCurrent pain-relieving strategies include the use of opioid-based analgesia, bisphosphonates and radiotherapy. Although patients experience some pain relief, these interventions may produce unacceptable side-effects. In particular, the use of opioid-based analgesia commonly results in side-effects including constipation, nausea and drowsiness. A common symptom experienced by patients with cancer-induced bone pain (CIBP) is spontaneous breakthrough pain, which is severe and difficult to control because the levels of medication required for pain relief would be unacceptable , 5.Acupuncture may represent a potentially valuable adjunct to existing strategies for pain relief and is known to be relatively free of harmful side-effects –8; howevThe physiological mechanisms of bone pain are not fully understood , but theThe release of chemical mediators, such as cytokines, from tumor cells.Increased pressure within the bone.Microfractures.Periosteal stretching.Muscle spasm.Nerve root infiltration.Nerve compression .It is likely that a combination of peripheral and central mechanisms contributes to pain associated with bony metastases and these are described below and illustrated in periosteum is well innervated and the sensory free nerve endings present are thought to be involved in the mediation of pain. Although the precise mechanisms are unclear, it is thought that excessive osteoclast-induced bone destruction leads to pain through stimulation of mechanoreceptors in the periosteum . SensoryN-methyl-d-aspartate (NMDA) glutamate receptor and is a feature of many chronic pain states.It has been suggested that CIBP is a unique pain state showing physiological characteristics of both inflammatory and neuropathic pain and changes in dorsal horn cell phenotype –24. WherStudies using rat models of CIBP have also shown that the proportion of WDR neurons almost doubles in the presence of bony metastases. The proportion of nociceptive-specific (NS) to WDR neurons in normal controls is 74 : 26%, and changes to 53 : 47%, respectively, during CIBP . This meμ-opioid receptors in dorsal root ganglia and this may explain the decreased response to opiate drugs such as morphine. This response was not shown in inflammatory models, where morphine was effective in reducing pain [It has been shown that CIBP causes a decrease in ing pain . CIBP-ining pain , for exaThe multifactorial mechanisms underlying CIBP probably explain why the level of pain experienced by patients does not necessarily correspond with the extent of the bony metastases , 19. CouMany of the studies investigating the physiological effects of acupuncture have been conducted in the laboratory and a significant number have used animal subjects , 28–30. δ fibers in muscle and skin which synapse with interneurones in the substantia gelatinosa (SG) of the spinal cord resulting in the release of inhibitory neuromodulators such as enkephalin that reduce activity in NS and WDR neurons [in situ for 10–20 min to ensure the release of enkephalin [δ input (long-term depression) has been shown to outlast the stimulation by hours [Acupuncture stimulates small myelinated A neurons . It is ckephalin . Reducedby hours , 34.δ fibers using electrical currents has been shown to reduce sensitization of postsynaptic receptors and/or a decrease in neurotransmitter release in the dorsal horn in normal rats [β expression was measured in rats with CIBP-related hyperalgesia, as this is related to the maintenance of persistent pain [β expression it would therefore be an effective treatment for the treatment of CIBP. Another recent animal study used a neuropathic cancer pain model in mice and found that electroacupuncture decreased levels of substance P in the dorsal horn but increased levels of β-endorphin in the blood by 51.5% and in the brain by 12.6%, suggesting that it may be useful as an alternative treatment for this type of cancer-related pain [Continuous or repetitious stimulation of Amal rats . Other smal rats , 15. Daient pain . The datted pain . In all δ fibers using manual acupuncture alone and studies using rats have shown that this depression may last for days or weeks [Further animal studies have shown that acupuncture –35, elecor weeks , 35. Recor weeks , 38. Howδ fibers to achieve long-lasting analgesia is borne out by anecdotal evidence, which suggests that patients with cancer pain need to be treated two or three times per week initially for optimal effects, particularly in cases of CIBP where pain relief is often achieved for a very short time [The need for regular and repetitive stimulation of afferent Aort time . For treδ fiber input resulting from acupuncture and β-endorphin is released, which has an effect on the PAG via the descending pain inhibitory system. This is similar to the effect of opioid drugs in stimulating the release of serotonin and met-enkephalin that inhibit the SG cells, and is supported by clinical trials which showed that naxolone reverses some of the pain-relieving effects of acupuncture and electroacupuncture [The cumulative response of acupuncture may also be demonstrated by permanent changes in the opioid peptide mechanism which enhances gene expression. The arcuate nucleus of the hypothalamus is activated by peripheral Apuncture , 40–42.δ fibers also results in increased amounts of peptides being manufactured and stored, therefore prolonging the analgesic response to acupuncture. Furthermore, up-regulation of the opioid peptide mechanism as a cumulative result of Aδ stimulation has been found to activate μ-opioid receptors and increase their binding potential, thus overriding central changes to the endogenous opioid system in CIBP [The relatively sustained effects of acupuncture analgesia might be due to an up-regulation in analgesic gene expression, which demonstrates the importance of regular “top-ups”. It has also been suggested that sustained analgesia may be maintained by a positive feedback loop in the meso-limbic system which results in a continuous outflow from descending inhibitory pathways , 43. Cum in CIBP .Non-opioid systems also play an important part in acupuncture analgesia. The release of serotonin in the brainstem activates descending inhibitory systems resulting in the release of more serotonin and noradrenaline in the dorsal horn. Noradrenaline modulates pain transmission within the dorsal horn by inhibiting the post-synaptic membrane of SG cells, thus reinforcing the effects of the opioid-peptide mechanism .An immediate and powerful sympathetic response occurs within a segmental distribution when needles are inserted. In the long-term, in addition to depressing pain transmission within the dorsal horn it reduces pain perception and produces an autonomic blockade by interrupting autonomic reflexes . This isFunctional brain imaging studies have demonstrated that the application of acupuncture affects the limbic system, which modulates emotional (affective) responses to pain –54. The The effect of acupuncture on the limbic system might also explain why some patients (strong reactors) experience a euphoric reaction to acupuncture. The phenotypic changes in lamina I neurons in the dorsal horn mentioned earlier are also likely to contribute to the affective and autonomic responses associated with CIBP . The cenAnother manifestation of chronic pain is the presence of reactive muscle spasm which in turn causes additional pain and shortening of the muscle, affecting function . The useThe effect of acupuncture on MTPs in normalizing muscle tone, restoring muscle balance and thus improving mobility and function has been noted in cancer patients and is therefore an important outcome for patients suffering from CIBP .There is evidence to suggest that acupuncture is widely used in palliative cancer care , 66, 67,Although studies have shown acupuncture to be relatively safe from serious adverse effects , 8 the uPatients undergoing cancer treatment such as chemotherapy can become severely immunosuppressed and potentially more vulnerable to infection and bleeding due to low platelet counts. This represents a known risk but is regarded as an unlikely occurrence provided safety guidelines are followed and practitioners liaise closely with oncology staff and maintain a close scrutiny of blood counts , 71.The insertion of needles causes tissue damage and the resulting inflammation increases local blood flow which is normally a desired effect because it promotes tissue healing. However, in cancer an increase in blood flow might not be desirable, particularly around the site of a tumor because it could encourage proliferation or metastatic spread . Caution must also be used when lymphedema is present due to a risk of infection and cellulitis  and it iThe effect of acupuncture on MTPs and muscle spasm must be borne in mind when needling around the spine. Muscle spasm may be acting to protect spinal instability due to metastatic growth and the reduction of this spasm by acupuncture may result in spinal collapse and cord transaction, though we have found no published evidence of this , 71. Altn = 28) of four groups of palliative care patients, including patients with neuropathic pain, benign soft tissue pain, benign bone pain and malignant bone pain, significant pain relief was achieved in all groups using auricular stud acupuncture [The extent of the use of acupuncture in CIBP is not known although anecdotal evidence and preliminary clinical data are available. In a small, uncontrolled trial (puncture . Clinicapuncture . Filshiepuncture  collecteThe most recent systematic review on acupuncture for cancer-related pain was inconclusive and none of the included studies used CIBP populations . RecentlCIBP is a unique pain state and is characterized by central sensitization and an “up-regulated” nociceptive system. Acupuncture is known to reduce central sensitization in the dorsal horn and to reduce transmission of nociceptive information. It is likely that regular treatments reduce ongoing nociceptive transmission and sensitization and up-regulate the opioid peptide system. The release of MTPs may also help to lower central sensation. Additionally, the effect of acupuncture on the limbic system in the brain may modulate emotional responses to pain and render it more tolerable. Future research must focus upon well-designed randomized controlled clinical trials using human subjects to specifically investigate the effect of acupuncture on CIBP and focus in particular upon aspects of needle placement and dosage for optimal results. The aim of such research should be to produce an effective protocol for treating CIBP based on a sound, evidence-based rationale. The physiological mechanisms presented here suggest that this is a realistic objective.