HIV vaccine development must address the genetic diversity and plasticity of the virus that permits the presentation of diverse genetic forms to the immune system and subsequent escape from immune pressure. Assessment of potential HIV strain coverage by candidate T cell-based vaccines  is now a critical component in interpreting candidate vaccine suitability.We have utilized an N-mer identity algorithm to represent T cell epitopes and explore potential coverage of the global HIV pandemic using natural sequences derived from candidate HIV vaccines. Breadth (the number of T cell epitopes generated) and depth (the variant coverage within a T cell epitope) analyses have been incorporated into the model to explore vaccine coverage requirements in terms of the number of discrete T cell epitopes generated.We show that when multiple epitope generation by a vaccine product is considered a far more nuanced appraisal of the potential HIV strain coverage of the vaccine product emerges. By considering epitope breadth and depth several important observations were made: (1) epitope breadth requirements to reach particular levels of vaccine coverage, even for natural sequence-based vaccine products is not necessarily an intractable problem for the immune system; (2) increasing the valency (number of T cell epitope variants present) of vaccine products dramatically decreases the epitope requirements to reach particular coverage levels for any epidemic; (3) considering multiple-hit models (more than one exact epitope match with an incoming HIV strain) places a significantly higher requirement upon epitope breadth in order to reach a given level of coverage, to the point where low valency natural sequence based products would not practically be able to generate sufficient epitopes.When HIV vaccine sequences are compared against datasets of potential incoming viruses important metrics such as the minimum epitope count required to reach a desired level of coverage can be easily calculated. We propose that such analyses can be applied early in the planning stages and during the execution phase of a vaccine trial to explore theoretical and empirical suitability of a vaccine product to a particular epidemic setting. It is tstrategy -8. Such strategy -13. Whilstrategy -16. The strategy .T cells recognize virally infected target cells or antigen-presenting cells via T cell receptor binding to short peptides  presented in the context of HLA class I or class II alleles -21. It iMounting data suggests that the breadth (number of discrete epitopes) and depth (amount of epitope variants recognized) of the T cell response is an important determinant of both HIV pathogenesis and primate model SIV vaccine efficacy. Multiple studies have recently indicated that it is the breadth of the T cell response rather than the magnitude, which correlates best with viral load set point in chronic HIV infection -50. In agag/pol open reading frame and a native env open reading frame with a C-terminal truncation of gp41 to maximize expression. The products are based upon isolates representing subtype A (MVA-KEA), subtype C (MVA-TZC), subtype D (MVA-UGD), and CRF01_AE (MVA-CMDR). Additional File http://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html using the 2009 alignments for HIV-1. The final sequence sets were filtered to remove all N- and O-group and Cpz origin viruses and retain all M-group sequences to form a dataset without bias for subtype or recombinant form inclusion. The final Gag and Env query datasets consisted of 3585 and 2258 sequences respectively.The source of all natural HIV-derived Gag and Env protein query sequences used in the study were a series of recombinant modified vaccinia Ankara (rMVA) constructs that have been developed at the Military HIV Research Program (MHRP) . Each pr+ T cell epitope length (9-10 amino acids) and the minimal core of a CD4+ T cell epitope (10-12 amino acids). The Epicover algorithm  into all possible 10-mers (the vaccine "epitome"), then walking each 10-mer along the test sequences in one amino acid "windows" and scoring each test sequence for a hit (1), or no hit (0). The coverage potential of the vaccine is then expressed as a percentage (or fraction) of sequences in the test dataset for which an exact 10-mer match is made. Epicover calculates and expresses coverage as the mean coverage of N-mers from all test sequences and is not a strict "hit/no-hit" model of N-mer matching for each individual sequence. As shown in Additional File To quantify the coverage that a particular HIV vaccine insert may provide in terms of T cell recognition an N-mer identity approach was used to scan for epitope matches between vaccine product test sequences and a query dataset of global M-Group and subtype-specific HIV Gag and Env sequences. For the purposes of this study epitopes are defined as 10 amino acids in length since this represents a reasonable compromise between the median CD8nt tool; ,52) as iThe above calculation of vaccine coverage assumes that each vaccine is capable of generating only a single T cell epitope, which in both theoretical and practical terms is not the case. Incorporation breadth (number of discrete epitopes) and depth (amount of epitope variants recognized) of epitope recognition into the vaccine coverage analysis is performed at two stages. Depth was included in the epitope coverage analysis above by considering multivalent vaccines and each of the different epitope variants they might present. Analysis of breadth was included by considering the total number of epitopes that may be generated by a given vaccine modality. To integrate the capacity of vaccines to generate multiple epitopes into the analysis, the following calculation was used:n is the number of epitopes generated by a vaccineWhere: p is the probability of an exact hit on the query datasetp(n-1) value for the formula is derived from the Epicover calculation for single epitope coverage. The resulting coverage potential of a vaccine should therefore increase as the breadth and depth of the T cell response increases. Positional coverage analysis was performed by aligning the LANL 2009 Gag dataset to the HXB2 reference sequence and performing gap stripping where necessary to maintain the consensus sequence. Coverage provided at each of 493 10-mer windows across the 502 amino acid Gag HXB2 was plotted on a frequency histogram and examples "low" and "high" depth of coverage epitopes were annotated.This calculation can be iterated for as many epitopes as considered practically possible for a vaccine. The initial The prior analyses all work on the implicit assumption that a single exact epitope match (or "hit") on a potential incoming strain of HIV-1 will be sufficient for vaccine efficacy. This assumption may be flawed if we consider that more than one exact match with a potential incoming strain of virus may be required. Such a requirement can be thought of in terms of a "multiple hit" model where two or more exact epitope matches are required with any incoming strain of virus for killing of the infected cell or inhibition of virus replication and preventing viral escape. This model is best described using the exact binomial probability calculation:k = the number of exact epitope matches required (or hits)Where: N = the actual number of epitopes generated by a vaccinep = probability that a single exact epitope match occursq = probability that a single exact epitope match does not occurk equal to or smaller than N and summing the values yields the probability of getting at least the required number of exact matches (hits) for any given epitope count. For example, when considering a 3-hit model, the probability of at least 3 exact matches with a vaccine that generates 10 epitopes would be calculated by:Applying this calculation to all values of For the purpose of simplicity the two-hit and three-hit models are considered for this study, however the models could be extended to four-hit or five-hits if empirical data supported this requirement.in vitro stimulated PBMC cultures were established as previously described to generate effector cells for assessing HIV-specific cytotoxic activity [To demonstrate the application of breadth and depth analysis in the setting of a vaccine trial, a responder subject from a recently completed trial of the MVA-CMDR vaccine product  was assactivity . The effactivity ,52). TheInitially the global HIV-1 group M coverage that would be provided by the Gag and Env natural sequence inserts in the MHRP rMVA product development pipeline was determined  were first assessed for coverage of the homologous subtype (88 Gag sequences and 112 Env sequences from the 2010 LANL database). As shown in Figure The prior analysis is predicated on the notion that a single T cell epitope match with an incoming strain of HIV would be sufficient for recognition of an infected target cell and prevention of productive infection. While conceptually valid this assumption may be an over simplification of the complexities of antigen processing and presentation and the differential kinetics of viral protein expression, viral epitope entropy and the TCR repertoire. Hence, some epitopes may provide more effective protection or control of HIV infection than others and so the chances of eliciting a response against a "good" epitope increases as the breadth of the response increases. To incorporate this potential constraint into the assessment of epitope requirements for T cell-based vaccines, a multiple hit model was incorporated into the analysis. Using such a model, two or more exact epitope matches would be required with any incoming strain of virus for vaccine efficacy. As described in the Methods section this model is a generalized "Analysis of epitope requirements and incorporation of multiple hit requirements demonstrates that it is possible to set standards for HIV vaccine trials in terms of the mean number of epitopes generated per vaccinated subject as a metric of potential vaccine coverage in different settings of HIV diversity and for different vaccine insert formulations. Comparative analyses considering all possible mono-, di-, tri- and tetra-valent formulations for all four MHRP vaccine products considered in this study were performed and are included in Additional File While use of the mean 10-mer coverage that any given vaccine insert sequence provides is a practical solution to rapidly determining the theoretical epitope requirements, there are potential further refinements that can be made when considering vaccine coverage analysis. Figure As an example of how to practically assess the theoretical coverage that may be provided by a vaccine, data is presented from epitope mapping studies performed during a recently completed vaccine trial RV158) involving one of the native sequence constructs (MVA-CMDR) considered in the prior analyses . Epitope8 involviAs a final refinement to the coverage analysis the sequence of the peptide(s) recognized from the vaccine product(s) can be used to directly query the database for depth of coverage independently. This represents both the most stringent form of coverage analysis and the most technically demanding and laboratory intensive analysis. Once the T cell epitopes have been enumerated, further studies can be performed to identify and confirm the peptides and/or peptide overlaps that are recognized. These short sequences can be used to screen databases and datasets for a more precise determination of the coverage provided. This affords a much greater precision for the coverage analysis calculations because as shown in Figure + T cell epitope length (9-10 amino acids) and the minimal core of a CD4+ T cell epitope (10-12 amino acids). However, shorter or longer epitope windows  and more relaxed matching requirements (1 or 2 tolerated amino acid mismatches) could be incorporated as desired into this analysis. It was felt that the requirements used here represent a more conservative approach and hence may in fact over-estimate the epitope requirements since no mismatches are tolerated. The studies of Korber et al. [In this study it has been demonstrated that measurement and analysis of the breadth of the T cell response - the number of discrete T cell epitopes - generated by a vaccine provides an important metric for HIV vaccine assessment. Analysis of the breadth of T cell coverage not only illustrates the problem that HIV genetic diversity presents to the immune system, but it also demonstrates that increasing the breadth of coverage can amplify the capacity of even natural sequence-based vaccine products to cope with viral diversity. Practical metrics can be set prior to the commencement of a vaccine trial that take into account both the HIV sequence content in the vaccine product and the diversity of HIV in particular target settings. Importantly, the relative capacities of different multi-valent formulations to generate sufficient epitope coverage can be compared directly. While the analyses presented here are limited to exact 10-mer matches as epitope hits this was considered to be a reasonable compromise between the median CD8r et al. , using cr et al. ,32. Impor et al. ,56.+ T cell epitopes is associated with reduced viral load in linked subjects, and if multiple epitopes are targeted the likelihood of epitope reversion is diminished [Empirical data has begun to emerge that implicates T cell epitope breadth as a critical parameter in both the natural control of HIV-1 infection and in pre-clinical testing of different vaccine strategies and modalities. In cross-sectional studies of naturally HIV-1 infected, treatment naïve human subjects, investigators have identified the breadth of the cellular immune response, rather than the magnitude of the response, as the primary discriminator of effective immune control as measured by set-point viral load ,50,57,58minished ,50,59-61minished . The semminished  demonstrminished ,56. In nminished ,54. Wheta priori knowledge of epitopes that should be targeted by any given individual, the multi-hit models of vaccine assessment presented here provides a potential alternative. While the single-hit model assumes that all epitopes may be equally effective at mediating infected cell recognition, the 2-hit and 3-hit models assume that only one-half and one-third of all possible T cell epitopes respectively are actually effective in mediating T cell recognition of virally infected target cells and inhibiting viral replication. The data presented here show how the consideration of multiple hit models places a greater onus upon any given vaccine product to provide appropriate coverage of all possible incoming strains of HIV-1. Using the natural sequence based vaccine constructs developed in the MHRP it is shown that if used in mono-valent formulations there is sufficient coverage of the homologous subtype, even within the Env protein if sufficient breadth of the immune response (4-6 epitopes) is generated. Furthermore, multi-valent formulation of the products potentially provides ample coverage (> 90% of strains) even for the global HIV-1 group M epidemic for both Gag and Env when considering the single-hit model. The greater diversity within the Env protein compared with Gag is reflected in the generalized requirement for nearly twice the number of epitopes in Env to reach any level of equivalent coverage. However, if a multi-hit model proves to be an accurate description of T cell requirements for an effective vaccine, then the requirements to meet any level of coverage increase dramatically. For mono-valent formulations both the 2- and 3-hit models place such high requirements upon both Gag (8-12 epitopes for 75% coverage using a 2-hit model) and Env (17-19 epitopes for 75% coverage using a 2-hit model), that generating sufficient breadth would be a difficult proposition. Considering a 3-hit model and 90% coverage of the global epidemic the epitope breadth requirements present what is most likely an insurmountable challenge for the immune system (> 20 epitopes required per protein). Multi-valent formulation of the natural sequences does provide increased benefit in terms of generating potentially more cross-reactive cells, especially when considering subtype-specific vaccine coverage. However, as far as global group M coverage is concerned multi-valent formulation and delivery of the natural sequence based products is the most realistic means of ensuring sufficient coverage if multiple exact T cell epitope matches are required for vaccine efficacy.It is therefore not surprising that increasing the breadth of T cell recognition might be associated with better control of viremia, since the more epitopes recognized the more difficult it would be for the virus to find multiple escape mutations of relatively low fitness costs. A less obvious and perhaps more biologically cogent reason behind the breadth association with viral control is that certain T cell epitopes may be more effective than others. Prior studies have implicated T cells targeting certain gene products of the virus (e.g. Gag) as being associated with better control of viremia ,48-50,62While quite sobering, these data serve to underscore the practical requirements for a T cell-based vaccine to be efficacious in a broader setting and also provides meaningful tools for assessing those requirements. This kind of analysis could also be directly applied to any kind of computationally derived vaccine insert sequences such as consensus, center-of-tree, mosaic, conserved element or string epitope concatenated sequences ,29-41. SThe T cell epitope breadth analysis presented here also highlights the issue of formulation and delivery of multi-valent vaccine products. While the simplest approach for delivery would be co-formulation for both priming and boosting vaccinations, a multi-valent product may benefit from priming with a single valency product and sequential boosting with the heterologous products. Such an approach might focus the T cell response upon the boosting of cross-reactive T cell responses at the expense of boosting potentially immunodominant responses with limited cross-reactivity. The heterologous prime-boost approach has already been shown to be effective in generating high magnitude T cell responses in several human vaccine trials -66. The + versus CD8+ T cell responses. CD8+ T cells are generally considered the effector cells responsible for viral control and clearance and therefore the specific contribution of these cells to the overall should be specifically assessed. The most robust and practical assay for epitope enumeration and identification in a vaccine trial is the IFNγ Elispot assay. However, this assay is not readily adaptable to discerning the phenotype of the responding cells without significant sample usage. Therefore, high throughput flow cytometry-based assays that can measure epitope depth and breadth as well as provide high content phenotypic analysis of the responding cell populations are required.There are several further refinements and caveats that need to be kept in mind when such analysis is performed. An implicitly important variable in this analysis is the query database. This study has made use of the LANL HIV sequence database without bias for the prevalence of any given subtype. However, this database is heavily biased towards subtypes B and C in terms of raw sequence constituency. Hence, more nuanced analyses for vaccines targeted for East Africa, West Africa, Asia and eastern Europe, would benefit from more expansive query datasets that include better sampling of subtypes A, D, G, CRF01_AE and CRF01_AG. This highlights the importance of sequence sampling and the dataset used as the basis for the coverage requirement calculations. Experimentally, there is also a need to discern the relative contributions of CD4In summary, we have shown here that appraisal of epitope breadth and depth of recognition should be an important metric that is considered when laboratory assessment of HIV vaccine trials is performed. Furthermore, the incorporation of a multi-hit model into the theoretical breadth analysis helps to set the metrics for broadly specific T cell responses in the absence of mechanistic data that explains the biological underpinnings of why high breadth responses may be advantageous. The suitability for a particular vaccine and multi-valent formulation can be also assessed during the design phase of a clinical trial as well as during the execution phase. Moreover, this study shows that multi-valent formulation, or heterologous prime-boost strategies, may extend the utility of vaccine products based upon natural sequences. While it is clearly shown that mono-valent natural sequence based products face a potentially intractable problem for providing appropriate coverage for the global HIV-1 pandemic, multi-valent formulation can improve the utility of these extant vaccine products, especially in an epidemic consisting of a single subtype. Given that computationally designed vaccine products optimized for maximizing coverage, such as mosaic products, will soon be tested in human clinical trials it remains paramount that robust and practical breadth assessment is included in the testing schema for comparing and contrasting these and other approaches for successful HIV vaccine implementation.The authors declare that they have no competing interests.JRC: Conceived and designed the study, developed the model and wrote the manuscript. MAM: Wrote the manuscript and provided important intellectual input into the overall concept of the study. MLR and NLM: Provided support for the study and participated in interpretation of the data and its implication for study vaccine design and assessment. All authors read and approved the final manuscript.Subtype and isolate sequence of origin of the natural sequence-based HIV vaccine products assessed in the study.Click here for fileComparison of Epicover algorithm with a "hit/no-hit" model of N-mer epitope identity matching. A stringent "hit/no-hit" model of 10-mer coverage was applied to a dataset of 2285 Gag sequences using a tetra-valent ACDE natural sequence vaccine formulation as the query set. As shown below a near-Gaussian distribution of the percent coverage of all isolates in the dataset was obtained. Near identical mean coverage results were obtained using the Epicover algorithm (as implemented in the LANL HIV Sequence Database) and a larger dataset of 3585 Gag sequences. While a broad distribution of "hits per isolate" was obtained (range = 13%-75%), the near identical mean coverage results for the Epicover algorithm and the "hit/no-hit" model resulted in no difference between the two methods for the calculation of epitope coverage requirements and the multiple epitope hit requirement model.Click here for fileGlobal group M Gag coverage analysis. Potential HIV isolate coverage provided by mono-valent , di-valent  and multi-valent  formulations of the four natural sequence based products is shown. Theoretical coverage (90% in the examples shown here) is profoundly dependent upon the number of epitopes generated and the number of exact epitope (10-mer) matches that are required for infected cell recognition. For example if a 1-Hit model is considered, then the tetra-valent product would reach 90% coverage by generating four epitopes per subject on average . In contrast, if a 3-Hit model is considered the mono-valent subtype A product does not reach 90% coverage even if 20 epitopes are generated on average per subject .Click here for fileGlobal group M Env coverage analysis. Potential HIV isolate coverage provided by mono-valent , di-valent  and multi-valent  formulations of the four natural sequence based products is shown. Theoretical coverage (90% in the examples shown here) is again dependent upon the number of epitopes generated but there is a much greater epitope requirement than for Gag. For example if a 1-Hit model is considered, then the tetra-valent product would reach 90% coverage by generating 7 epitopes per subject on average . If a 3-Hit model is considered the mono-valent subtype products face an intractable problem with extreme epitope requirements for 90% global coverage (Panel C). Even the multi-valent products have stringent epitope requirements (17-20) for reaching 90% coverage (Panel I).Click here for filein vitro stimulated cells from a vaccine trial responder subjectGag epitope mapping for post-. PBMC from a vaccinee in the RV158 trial (received 3x rMVA-CMDR) were subjected two rounds of in vitro stimulation, first with rMVA-CMDR for 14 days, then with autologous, irradiated BLCL pulsed with peptide pools matching the Gag (CM240) insert in the vaccine. Each stimulation cycle was 14 days, with rIL-7 added during the first week and rIL-2 added during the second week. Effector cells were tested for responses to a matrix of peptides (11 × 11 peptide pools) matching the Gag insert sequence. Epitopes were counted and identified from the de-convoluted peptide matrix (Panels A and B) in an IFN-γ Elispot assay. Two epitopes were identified within the Gag-specific effector cells and are shown in Panel C.Click here for filein vitro stimulated cells from a vaccine trial responder subjectEnv epitope mapping for post-. PBMC from a vaccinee in the RV158 trial (received 3x rMVA-CMDR) were subjected two rounds of in vitro stimulation, first with rMVA-CMDR for 14 days, then with autologous, irradiated BLCL pulsed with peptide pools matching the Env (CM235) insert in the vaccine. Each stimulation cycle was 14 days, with rIL-7 added during the first week and rIL-2 added during the second week. Effector cells were tested for responses to a matrix of peptides (14 × 13 peptide pools) matching the Env insert sequence. Epitopes were counted and identified from the de-convoluted peptide matrix (Panels A and B) in an IFN-g Elispot assay. Two epitopes were identified within the Env-specific effector cells and are shown in Panel C.Click here for file


Cardiovascular disease is the leading cause of death. The disease is due to atherosclerosis which is characterized by lipid and fat accumulation in arterial blood vessel walls. A key causative event is the accumulation of oxidised low density lipoprotein particles within vascular cells, and this is mediated by scavenger receptors. One such molecule is the LOX-1 scavenger receptor that is expressed on endothelial, vascular smooth muscle, and lymphoid cells including macrophages. LOX-1 interaction with OxLDL particles stimulates atherosclerosis. LOX-1 mediates OxLDL endocytosis via a clathrin-independent internalization pathway. Transgenic animal model studies show that LOX-1 plays a significant role in atherosclerotic plaque initiation and progression. Administration of LOX-1 antibodies in cellular and animal models suggest that such intervention inhibits atherosclerosis. Antiatherogenic strategies that target LOX-1 function using gene therapy or small molecule inhibitors would be new ways to address the increasing incidence of vascular disease in many countries. Their work led to the now widely accepted view that the internalisation of a modified form of LDL rather than native LDL is responsible for the development of atherosclerotic plaque, and that this occurs via membrane-bound receptors distinct to that of the native LDL receptor. Basu et al. first demonstrated this in an experiment in which an iodine-labelled acetylated form of LDL could induce massively increased cholesterol accumulation in macrophages in vitro [in vitro has shown that the oxidative modification of LDL increases the cellular content of cholesterol. Today, several receptors have been identified that bind and internalise oxidised LDL (OxLDL). This review focuses on one of those receptors, the lectin-like oxidised low-density lipoprotein receptor-1 (LOX-1) and explores its potential as a therapeutic target.Cholesterol is a key cellular molecule that is vital to growth and repair and is also a major component of the mammalian plasma membrane bilayer. It is also required for the synthesis of steroid hormones and bile salts. However, an elevated level of serum cholesterol and in particular, the low-density lipoprotein (LDL) fraction are well-established risk factors for the development of atherosclerosis. Atherosclerosis is a systemic disease, commonly affecting multiple vascular beds  and is tin vitro . Furtherin vitro and inhibits the vasodilation normally induced by nitric oxide [The oxidative modification of LDL results in a particle with greatly increased proatherogenic and proinflammatory properties than that of native LDL. It also becomes a ligand for scavenger receptors but is not recognised by the native LDL receptor, thought to be due to the greater net negative charge . The “avic oxide .Modified LDL is internalized or endocytosed by membrane-bound scavenger receptors, and as it stands today, numerous scavenger receptors have been identified that recognize OxLDL. Scavenger receptors are membrane-bound proteins that are capable of binding a wide variety of ligands. The scavenger receptor family is subdivided into 8 subclasses. The class A scavenger receptors all bind modified LDL and are primarily expressed on macrophages. Class B includes the CD36 receptor, which has been shown to bind and internalize modified LDL . The affκB activation, and apoptosis [LOX-1 (Genbank designation OLR1) was first cloned as a major receptor for OxLDL in 1997 by Sawamura and colleagues . LOX-1 ipoptosis , 25. OxLDL is rapidly internalised into cells upon binding LOX-1 and can be observed in punctate perinuclear structures less than an hour after exposure to a LOX-1 expressing cell . This internalisation is blocked by the LOX-1-blocking-antibody JTX92 , which pThe main form of receptor-mediated endocytosis is clathrin-mediated endocytosis, but it has become apparent that clathrin-independent pathways may represent up to 50% of cellular uptake . ReverseIn eukaryote cells, receptor-mediated endocytosis is regulated by the recognition of cytoplasmic motifs by cellular machinery which promotes the selection of “cargo” for transport within transport intermediates such as membrane-bound vesicles . The cytAfter internalization at the plasma membrane, LOX-1 has been shown to be separated from OxLDL in endosomes . ImmunofLOX-1 appears to be essential for the phagocytosis of aged and apoptotic cells in endothelial cells . LOX-1 wLOX-1 endocytosis is also potentially important in immune surveillance as it has been shown to regulate antigen presentation by MHC class I and II molecules on dendritic cells  and B ceScavenger receptors are heavily implicated in the process of atherosclerotic plaque formation . This reSignificant recent progress has been made in the elucidation of the pathways of internalization of the OxLDL-LOX-1 complex; however, there remains much to be done to further characterise the role of this multiligand scavenger receptor in regulating human health and disease. The existence of several different subclasses of scavenger receptor capable of internalising OxLDL makes the development of targeted therapies a more complex issue. The molecular study of scavenger receptors remains an exciting avenue in the search for therapeutic agents to attenuate the atherosclerotic process in humans.


The U.S. study, entitled, ‘Integrated Telehealth and Care Management Program for Medicare Beneficiaries with Chronic Disease Linked to Savings’ , explored the economic impact of using content-based remote patient monitoring combined with physician led patient-centred care management for high cost patients with congestive heart failure, chronic obstructive pulmonary disease and/or diabetes mellitus. Researchers conducted an analysis evaluating changes in healthcare spending resulting from physician led patient-centred care management supported by remote patient monitoring (n=1767) and demonstrated spending reductions of 7.7–13.3% (£197.17–£342.52) per intervention patient in acute hospital cost per quarter over the two-year period studied. In addition, significant mortality differences between the treatment and control groups were noted, which suggest that the intervention may have produced noticeable Improvements/changes in health outcomes. Findings from the analysis of the two-year study that could guide the design of future innovative physician led telehealth-enabled care coordination projects in the UK.Parallels between the proposed UK local health system model as described by the Health and social care bill and the Medicare system in the US in the context of an innovative community-based physician led telehealth enabled integrated system of care.Design and deployment of a system of care utilizing content-based telehealth technology to enable physician led integrated care coordination.the presentation will outline the:In an intent-to-treat population of 1725 patients, a reduction in critical aspects in utilization including hospitalization and A and E resulted in improved quality, satisfaction and cost outcomes for the patients and providers in the local region.Innovative reimbursement models drove physician led telehealth enabled care coordination that generated reductions in key utilization and cost while optimizing health status. The intervention also generated substantive efficiency improvements that led to substantive cost savings.


Escherichia coli (EPEC). Nucleolin also exists at low levels at the cell surface of eukaryotic cells and here we show that upon infection of an intestinal cell model, EPEC recruits and subsequently sequesters cell-surface EGFP-nucleolin into extracellularly located bacterial microcolonies. The recruitment of nucleolin was evident around bacteria within the centre of the microcolonies that were not directly associated with actin-based pedestals. Incubation of host intestinal cells with different ligands that specifically bind nucleolin impaired the ability of EPEC to disrupt epithelial barrier function but did not inhibit bacterial attachment or other effector-driven processes such as pedestal formation or microvilli effacement. Taken together, this work suggests that EPEC exploits two spatially distinct pools of nucleolin during the infection process.Nucleolin is a prominent nucleolar protein that is mobilized into the cytoplasm during infection by enteropathogenic  Escherichia coli (EPEC) is a non-invasive pathogen that binds to human small intestinal enterocytes and delivers multiple effector proteins into host cells via its type III secretion system (Enteropathogenic n system . These en system . EPEC pon system . AlthougE. coli (EHEC) and Citrobacter rodentium. These pathogens all share the LEE (locus of enterocyte effacement) genomic pathogenicity island, which encodes several important virulence factors including the type III secretion system, six effectors and the outer-membrane protein intimin. The effector Tir (translocated intimin receptor) is the best-studied LEE effector; it inserts into the host plasma membrane, where it acts as a receptor for intimin and facilitates bacterial attachment -nucleolin in the small intestinal Caco-2 model. We reveal that nucleolin is not only recruited to the EPEC infection site but is also sequestered transiently inside extracellularly located bacterial microcolonies. We present data to suggest that cell-surface nucleolin is involved in the EPEC infection process but does not significantly contribute to bacterial attachment or effector delivery. Taken together, the work shows that EPEC exploits two spatially distinct pools of nucleolin: the main nucleolar pool during late-stage infection and a cell-surface pool at earlier infection times.Caco-2 cells were cultured and transiently transfected with pEGFP-nucleolin as described previously  were infected with EPEC E2348/69 at an m.o.i. of 1 : 50 using standard infection procedures  and procDifferentiated Caco-2 cells (15 days post-confluence) were exposed to different concentrations of midkine or pleiotrophin (R and D systems) for 3 h prior to infection with wild-type EPEC as described by t-test using the statistical package SPSS, with P-values below 0.01 taken as significant.Means were compared by ANOVA or Student’s in vivo infection  that were transiently transfected with EGFP-nucleolin revealed that in agreement with the IF data , nucleolAs EPEC infection caused highly localized recruitment of EGFP-nucleolin at the cell surface, we tested whether native nucleolin could be visualized at the cell surface of infected cells by IF of infected cells. A small increase in native cell-surface nucleolin signal was detected that colocalized with attached microcolonies but the staining was weak and diffuse (data not shown). Because of this, we continued to use EGFP-nucleolin to study nucleolin’s behaviour during EPEC infection.z-axis of infected cells. Remarkably, confocal sectioning through attached bacterial microcolonies revealed that the recruited EGFP-nucleolin was found, not beneath the bacteria, but throughout the microcolony, sequestered around individual bacteria . Further analysis using MK revealed no significant defects in the ability of EPEC to attach to host cells  or cause microvilli effacement  and the growth factor pleiotrophin (PTN), which bind cell-surface nucleolin . We assefacement  at the sin vivo (Nucleolin is a well-studied eukaryotic protein that is important for ribosome biogenesis and represents the most abundant nucleolar protein in dividing cells (Cell-surface nucleolin was previously shown to be an adhesin for EHEC but this may not be the case for EPEC as (a) nucleolin was found inside EPEC microcolonies and not at the interface between host cells and bacteria and (b) MK or PTN had no significant effect on bacterial attachment. Both MK and PTN were used at high concentrations that have previous been shown to saturate the nucleolin-binding sites on a range of cell types  and thatThe positive correlation between MK exposure and EPEC’s inability to disrupt epithelial barrier function suggests that nucleolin may play a role in this process, and indeed several studies have shown that cell-surface nucleolin mediates signalling pathways  that mayIn this study, and in agreement with a recent report (


Brugia malayi.Developing intervention strategies for the control of parasitic nematodes continues to be a significant challenge. Genomic and post-genomic approaches play an increasingly important role for providing fundamental molecular information about these parasites, thus enhancing basic as well as translational research. Here we report a comprehensive genome-wide survey of the developmental transcriptome of the human filarial parasite Using deep sequencing, we profiled the transcriptome of eggs and embryos, immature (≤3 days of age) and mature microfilariae (MF), third- and fourth-stage larvae (L3 and L4), and adult male and female worms. Comparative analysis across these stages provided a detailed overview of the molecular repertoires that define and differentiate distinct lifecycle stages of the parasite. Genome-wide assessment of the overall transcriptional variability indicated that the cuticle collagen family and those implicated in molting exhibit noticeably dynamic stage-dependent patterns. Of particular interest was the identification of genes displaying sex-biased or germline-enriched profiles due to their potential involvement in reproductive processes. The study also revealed discrete transcriptional changes during larval development, namely those accompanying the maturation of MF and the L3 to L4 transition that are vital in establishing successful infection in mosquito vectors and vertebrate hosts, respectively.Characterization of the transcriptional program of the parasite's lifecycle is an important step toward understanding the developmental processes required for the infectious cycle. We find that the transcriptional program has a number of stage-specific pathways activated during worm development. In addition to advancing our understanding of transcriptome dynamics, these data will aid in the study of genome structure and organization by facilitating the identification of novel transcribed elements and splice variants. Brugia malayi in different lifecyle stages using deep sequencing technology. The analysis revealed major transitions in RNA expression from eggs through larval stages to adults. Using statistical approaches, the authors identified groups of genes with distinct life stage dependent transcriptional patterns, with particular emphasis on genes displaying sex-biased or germline-enriched patterns and those displaying significant changes during larval development. This study presents a first comprehensive analysis of the lifecycle transcriptome of B. malayi, providing fundamental molecular information that should help researchers better understand parasite biology and could provide clues for the development of more effective interventions.Lymphatic filariasis, also known as elephantiasis, is a tropical disease affecting over 120 million people worldwide. More than 40 million people live with painful, disfiguring symptoms that can cause severe debilitation and social stigma. The disease is caused by infection with thread-like filarial nematodes (roundworms) that have a complex parasitic lifecycle involving both human and mosquito hosts. In the study, the authors profiled the transcriptome  of the human filarial worm  Wuchereria bancrofti, Brugia malayi and Brugia timori are mosquito-borne filarial nematode parasites that cause the tropical disease lymphatic filariasis (LF). The manifestation of the disease ranges from swelling of the lymph nodes to elephantiasis and hydrocele. LF is a major cause of clinical morbidity and disability, leading to significant psychosocial and psychosexual burden in endemic countries. B. malayi is the primary organism for the study of LF because it has a tractable lifecycle that can be replicated in a laboratory setting. Like other filarial nematodes it is a heteroxenous parasite alternating between arthropod vectors and vertebrate hosts. Filarial nematodes are dioecious and reproduce sexually via copulation. Inseminated adult female worms are ovoviviparous and release live larvae (microfilariae) into the lymph, where they eventually circulate in the bloodstream to be taken up by mosquitoes during blood feeding. After a microfilaria (MF) successfully penetrates the midgut of a susceptible vector, it migrates to the thoracic muscles, and develops intracellularly through two molts to achieve the developmentally arrested third-stage larva (L3) that exits the mosquito proboscis during bloodfeeding and subsequently penetrates the mammalian host. Once L3s enter the definitive host, they undergo two additional molts and mature to adults in the lymphatics.B. malayiB. malayi lifecycle stages: eggs & embryos, immature MF (of less than 3 days of age), mature MF, L3, L4, adult male and adult female. We find that the transcriptional program has a number of stage-specific pathways activated during worm development and that a number of these are potential targets for drugs or vaccines.Characterization of the transcriptional program over the complete lifecycle is necessary to clearly understand the development of the parasite and could help devise better target strategies for control. From the standpoint of possibly designing drug-based or vaccine interventions that prevent infection or curtail parasite transmission, there is particular interest in understanding the biology of the L3 to L4 transition in the mammalian host, and the reproductive biology of filarial worms. The completion of the draft genome of All animal work was conducted according to relevant national and international guidelines outlined by the National Institutes of Health Office of Laboratory Animal Welfare, and was approved under UWO Institutional Animal Care and Use Protocol 0-03-0026-000246-4-6-11; and UWM Research Animal Resource Center Protocol V00846-0-10-09.Brugia malayi adults and MF were obtained from the peritoneal cavities of patently infected dark-clawed Mongolian gerbils (Meriones uguiculatus) by peritoneal flush with prewarmed RPMI media . MF were purified by centrifugation through Ficoll-Paque® lymphocyte isolation media , and washed in PBS three times prior to flash freezing at −80°C. Adult worms were separated by gender, washed three times in RPMI, and flash frozen. Egg and embryo preparations were made by repeated cutting of 10 female worms with a scalpel to release eggs and embryos into a small volume of cold RPMI. The sample was examined microscopically and pieces of uterine tissue were removed using watchmaker's forceps. The sample was washed three times in cold RPMI prior to flash freezing. Immature MF (≤3 days old) were generated and purified as previously described B. malayi GPX gene [GenBank:X69128] (data not shown). Isolation of RNA from L3s often produces low yields therefore we used a modified protocol employing homogenization of tissue combined with organic extraction in RNAzol Total RNA was isolated from the majority of samples using a previously described protocol Polyadenylated mRNA was purified from total RNA using oligo-dT dynabead selection followed by metal ion hydrolysis fragmentation with the Ambion RNA fragmentation kit. First strand synthesis, primed using random oligonucleotides, was followed by 2nd strand synthesis with RNaseH and DNApolI to produce double-stranded cDNA using the Illumina mRNA Seq kit. Template DNA fragments were end-repaired with T4 and Klenow DNA polymerases and blunt-ended with T4 polynucleotide kinase. A single 3′ adenosine was added to the repaired ends using Klenow exo- and dATP to reduce template concatemerization and adapter dimer formation, and to increase the efficiency of adapter ligation. Adapters (containing primer sites for sequencing) were then ligated and fragments size-selected (200–275 bp) by agarose gel electrophoresis. DNA was extracted using a Qiagen gel extraction kit protocol but with dissolution of gel slices at room temperature (rather than 50°C) to avoid heat induced bias. Libraries were then amplified by PCR to enrich for properly ligated template strands, to generate enough DNA, and to add primers for flowcell surface annealing. AMPure SPRI beads were used to purify amplified templates before quantification using an Agilent Bioanalyser chip and Kapa Illumina SYBR Fast qPCR kit.in situ, the linearization, blocking and hybridization step was repeated to regenerate clusters, release the 2nd strand for sequencing and to hybridize the R2 sequencing primer followed by another 54 cycles of sequencing to produce paired end reads. These steps were performed using proprietary reagents according to manufacturer's recommended protocol (https://icom.illumina.com/). Data were analyzed using the RTA1.6 or RTA1.8 Illumina pipeline and submitted to Array Express (http://www.ebi.ac.uk/arrayexpress/) under the accession number E-MTAB-811.Libraries were denatured with 0.1 M sodium hydroxide and diluted to 6 pM in a hybridization buffer to allow the template strands to hybridize to adapters attached to the flowcell surface. Cluster amplification was performed on the Illumina cluster station or the Illumina cBOT using the V4 cluster generation kit following the manufacturer's protocol. A SYBRGreen QC was performed to measure cluster density and to determine whether to pass or fail the flowcell for sequencing. This was followed by linearization, blocking and hybridization of the R1 sequencing primer. The hybridized flowcells were loaded onto the Illumina Genome Analyser IIx for 54 cycles of sequencing-by-synthesis using Illumina's v4 or v5 SBS sequencing kit then, Each lane of Illumina sequence was assessed for quality based on %GC content, average base quality and Illumina adapter contamination. To assess the quality of the lane, the mean base quality at each base position in the read was computed over all reads from the lane. To assess %GC content of the reads a frequency distribution of values was plotted. For a single sample in a lane, a GC plot with a normal distribution around the expected GC for the organism would be expected. Any lanes containing a contamination could therefore be identified by the presence of multiple peaks in the %GC plot. To screen for adapter contamination, the sequence reads were aligned to the set of Illumina adapter sequences using BLAT v.34 with default parameters de novo junction mapping algorithm. Default parameters were used except for the following: minimum intron length was set to 50; minimum isoform fraction filter was disabled; closure-search, coverage-search, microexon-search and butterfly-search were enabled for maximum sensitivity. The resulting alignment files were converted to BAM format and low quality alignments with mapping quality scores less than 5 were removed before downstream analyses www-huber.embl.de/users/anders/HTSeq). Reads overlapping more than one gene model were counted as ambiguous with the mode parameter set as “union”. Following Mortazavi et al. Sequence reads from each lifecycle stage were aligned to the genome assembly [GenBank:DS236884–DS264093] using TopHat v1.0.14, a splice junction mapper built upon the short read aligner Bowtie Differential expression analysis was performed in edgeR by fitting a negative binomial model to the sequence count data. Using the quantile-adjusted conditional maximum likelihood method, dispersion parameters were estimated for each gene as a measure of the overall stage-to-stage variability to facilitate between-gene comparisons. All hypothesis testing was carried out using exact test for the negative binomial distribution with a common dispersion term for all genes. P-values less than 0.01 were considered significant. Dispersion parameters were estimated directly from the count data for comparisons contrasting a single stage or two related stages relative to all other stages. For comparisons between pairs of lifecycle stages, a common dispersion value of 0.2 was used, which is equivalent to allowing within-stage variations in expression levels of up to 45%. This value was chosen based on the level of variability observed between the immature and mature MF samples. Because longer transcripts give more statistical power for detecting differential expression between samples In total, 104 million paired-end reads (2×54 bp) were generated from polyA-tailed mRNA using the Illumina Genome Analyser IIx . SequencWolbachia genome [GenBank:AE017321]. However, the study was not adequately designed such that one could quantitatively analyze these reads in a biologically meaningful way. Abundance estimates (inferred from read counts) of these transcripts most likely deviate substantially from their true in vivo levels. Poly-A selection directly affects the relative abundance of non-poly-A Wolbachia transcripts with respect to B. malayi transcripts. Moreover, the nature and extent of the biases introduced by oligo-dT method to the relative abundance levels among the non-poly-A species (with respect to each other) is not well understood, and one cannot assume that these biases would remain uniform among different sample preparations. Another layer of uncertainty stems from the possibility that these “Wolbachia” sequences were transcribed from the B. malayi nuclear genome rather than the endosymbiont as a consequence of the past horizontal gene transfer events, leading to a differential capture of (presumably) poly-A tailed “Wolbachia” transcripts of the B. malayi nuclear origin. However, given the incomplete draft nature of the B. malayi genome assembly and the inherent difficulty in mapping short reads originating from multiple loci that are similar in sequences, it remains challenging to rigorously test this hypothesis in silico.Our sequencing libraries contained reads that map to the To investigate the global transcriptional differences between stages and between genes during development, a negative binomial (NB) based model Next, we made between-gene comparisons in terms of overall transcriptional variability across stages. It is generally hypothesized that while some genes are expressed constitutively, genes with specific developmental functions are expressed at specific stages. To quantify the level of transcriptional variation for each gene across the seven lifecycle stages, the NB dispersion parameters were estimated for each gene, and used as a measure of the extra-Poisson, stage-to-stage variability. Genome-wide distribution of the dispersion parameter estimates suggests that the level of transcriptional variation is not uniform across all genes . AlthougTo examine genes displaying life stage dependent transcriptional patterns in greater detail, the top 25% most variable genes according to the NB dispersion  were subjected to an unsupervised hierarchical clustering . The resWe identified and compared statistically overrepresented GO terms in groups of genes that differ in their level of transcriptional variation over the lifecycle  to investigate specific gene sets and functional categories distinctly associated with high levels of transcriptional variation . This anAlthough transcript levels of 990 genes are significantly higher during larval stages, 886 and 554 genes display elevated transcript abundance in adult male, and adult female and/or eggs & embryos, respectively . A direcIn an effort to elucidate female germline-enriched transcripts and gain insight into intrauterine reproductive processes, the transcriptome profile of a library enriched for eggs and embryos was compared with that of whole adult female . However2H2-type zinc finger domains. There are 195 zinc finger protein genes annotated in the B. malayi draft genome, some of which have high transcript levels in stages other than MF . In a similarly biased manner, 3 out of 4 endochitinase genes identified in the current B. malayi genome show transcriptional increase during MF stages. Diverse classes of proteases are also represented in this gene set . Direct comparison of immature and mature MF (IM and MM) indicates that 126 genes show differential transcript abundance between the two samples  are prominently represented in this gene set, a pattern similarly found in the MF transcriptome. A pairwise comparison of the transcriptomes of late larval stages indicates that 342 genes have elevated transcript levels in L3s, and 155 in L4s. At the level of functional categories, cysteine-type peptidase activity  and serpin activity are overrepresented among L3-enriched transcripts, whereas structural constituents of the cuticle and cellular component organization are overrepresented among L4-enriched transcripts  show strong statistical bias towards GO categories related to cytoskeleton, structural molecule activity, protein phosphorylation and dephosphorylation . Many offab-1 (Bm1_33050), an embryonic fatty acid binding protein transcript previously found to be female-associated by differential display PCR and whose protein localizes to embryos In filarial nematodes, fertilization is internal and gravid females hold oocytes, sperm, zygotes, developing embryos, and MF in their uteri. Structural constituents of cuticle, transcription factor activity, DNA binding, and regulation of transcription emerged as notable themes in our analysis of overrepresented functional categories among genes with increased transcript levels in adult female and/or eggs & embryos . These aB. malayi genes, and a growing body of research on nematode neurobiology and extracellular signaling lends support to the idea that some gender-associated genes can be expressed in non-reproductive tissues. For example, free-living and parasitic nematodes use gender-specific receptors to sense environmental signals, as demonstrated by the presence of anterior chemosensors in male worms that specifically bind female pheromones Much of our current information on molecular aspects of filarial reproduction comes from microarray and PCR-based transcriptome comparisons between whole adult male and female worms. These studies were based on the assumption that gender-associated transcripts arise from the reproductive organs and their contents. Our data suggest that such an assumption is not wholly unreasonable but may not always hold true. Out of 651 female-enriched transcripts we identified , 82 display somatic tissue-enriched expression patterns, and it is likely that some of these transcripts are truly not derived from the germline tissues. Spatial expression patterns have not been confirmed for the majority of gender-associated Brugia are detectable by in situ hybridization only in adult female tissues and not in eggs or embryos in utero sheathed MF but not in maternal tissues C. elegans, nas-4 and nas-9 are found in pharyngeal marginal cells, and in the hypodermis and reproductive tract, respectively On the other hand, these observations are not inconsistent with the possibility that some somatic tissue derived transcripts play an essential role in embryonic development or intrauterine reproductive processes. The current study suggests that components incorporated into the embryonic cuticle and the eggshell membrane may be in some part maternal in origin. This interpretation is supported in at least one case where MF sheath protein transcripts in Brugia MF must undergo a maturation process within the mammalian host to become infective to the mosquito vector Brugia MF are sheathed in a remnant of the eggshell membrane that is acellular and insoluble, and is composed of chitin and a variety of cross-linking proteins, lipids and polysaccharides After expulsion from females, developmentally arrested Onchocerca volvulus activation associated secreted protein (Ov-ASP-1) B. malayi glutathione-s-transferase Following the introduction of L3s into the peritoneal cavity of gerbils, the L3 to L4 transition requires no migration and occurs approximately 8 days post infection (unpublished). This particular lifecycle transition is of great interest to researchers trying to identify parasite molecules that mediate interactions with the host immune system, and that could be exploited with vaccines to confer protective immunity, or with drugs to prevent infection. Antigens that historically have been of interest in this regard are the ALT  family of potentially secreted larval acidic proteins found predominately in L2 and L3 stages col-2, cystatin, microfilarial surface associated protein, metabolic proteins and BmSERPIN. The differential expression of cathepsins Bm-cpl-1, 4, and 5 in vector stage L3s is supported by EST sequences and these genes are grouped phylogenetically into a distinct clade (Ia) separate from other nematode cathepsin-like proteases cpl-1 gene in O. volvulus by RNAi decreased the rate of molting B. malayi retarded worm growth, disrupted development and resulted in cuticular sloughing In the present study, the transcriptome of developmentally arrested, vector-derived L3s was compared to that of peritoneally-derived L4s at 12–13 days post infection. Comparing our RNA-seq data to a recent microarray experiment B. malayi. It is important to emphasize, however, that this is just an overview of observations and that these data will be mined by the community to provide specific information on particular gene sets to bring these deep sequencing data into more complete biological context.In this study we provide a detailed overview of the molecular repertoires that define and differentiate distinct lifecycle stages of the parasite, extending and complementing previously published work on stage-specific gene expression Because expression dynamics is an important consideration in the genome-wide assessment of candidate targets for control Figure S1Histogram of raw read counts per gene model and scaling-normalized RPKM values. To facilitate transcript abundance comparisons between genes and stages, read counts were RPKM-transformed and TMM-normalized (PDF)Click here for additional data file.Figure S2Genome-wide distribution of the dispersion parameter estimating stage-to-stage variability in transcript abundance. Based on the NB dispersion parameter, genes were ordered from the most variable to the least variable, and partitioned into four equal-sized groups as indicated by the horizontal dotted lines. GO term enrichment tests were performed on each of the four groups .(PDF)Click here for additional data file.Figure S3Venn diagram analysis to identify genes with female somatic tissue- or germline-enriched expression pattern. (1) genes with enriched expression in adult female relative to eggs & embryos; (2) genes with enriched expression in eggs & embryos relative to adult male; (3) genes with enriched expression in adult female and/or eggs & embryos relative to all other stages.(PDF)Click here for additional data file.Table S1Total number of reads sequenced and mapped to the genome.(PDF)Click here for additional data file.Table S2List of over-represented GO terms in groups of genes with different levels of overall variability in their transcript abundance across lifecycle stages. Genes were partitioned into four equal-sized groups designated as Q1 to 4 in the order of decreasing variability . Over-re(PDF)Click here for additional data file.Table S3List of over-represented GO terms and their corresponding p-values among genes that show relative transcriptional enrichment in pairwise comparisons . Female (PDF)Click here for additional data file.Dataset S1Brugia malayi lifecycle transcriptome.Gene-level RNA-seq read counts and RPKM values for (XLS)Click here for additional data file.


The entanglement of lignin polymers with cellulose and hemicellulose in plant cell walls is a major biological barrier to the economically viable production of biofuels from woody biomass. Recent efforts of reducing this recalcitrance with transgenic techniques have been showing promise for ameliorating or even obviating the need for costly pretreatments that are otherwise required to remove lignin from cellulose and hemicelluloses. At the same time, genetic manipulations of lignin biosynthetic enzymes have sometimes yielded unforeseen consequences on lignin composition, thus raising the question of whether the current understanding of the pathway is indeed correct. To address this question systemically, we developed and applied a novel modeling approach that, instead of analyzing the pathway within a single target context, permits a comprehensive, simultaneous investigation of different datasets in wild type and transgenic plants. Specifically, the proposed approach combines static flux-based analysis with a Monte Carlo simulation in which very many randomly chosen sets of parameter values are evaluated against kinetic models of lignin biosynthesis in different stem internodes of wild type and lignin-modified alfalfa plants. In addition to four new postulates that address the reversibility of some key reactions, the modeling effort led to two novel postulates regarding the control of the lignin biosynthetic pathway. The first posits functionally independent pathways toward the synthesis of different lignin monomers, while the second postulate proposes a novel feedforward regulatory mechanism. Subsequent laboratory experiments have identified the signaling molecule salicylic acid as a potential mediator of the postulated control mechanism. Overall, the results demonstrate that mathematical modeling can be a valuable complement to conventional transgenic approaches and that it can provide biological insights that are otherwise difficult to obtain. Cellulose-based biofuels presently offer the most environmentally attractive and technologically promising alternative to fossil fuels. To be viable, biofuels must be derived from non-food crops, such as grasses, wood, bark, and plant residues. Techniques for releasing the energy stored in these renewable materials must first untangle a very recalcitrant scaffold of interlinking molecules inside the plant cell walls, which is very costly. Much of the recalcitrance is due to the natural polymer lignin, which hardens the cell walls and is composed of three different building blocks, called monolignols. Modern transgenic techniques have yielded plant lines whose cell walls are easier to break down, but some of these modified plants have exhibited unexplained and undesired features. Here, we present new computational methods for analyzing monolignol biosynthesis in unprecedented detail. The analysis simultaneously accounts for lignin biosynthesis in various transgenic lines and different developmental stages and yields six novel, testable postulates regarding the metabolic control of the pathway. The results suggest new, targeted experiments towards a better understanding of monolignol biosynthesis and issues of recalcitrance reduction. More generally, the results highlight the genuine benefits of using computational methods as companions and complements to experimental studies. The complex, interwoven structure of lignin, cellulose, and hemicellulose polymers in plant cell walls is the main cause for the recalcitrance of lignocellulosic feedstocks to microbial and enzymatic deconstruction towards fermentable sugars. This recalcitrance, in turn, accounts for the high cost of biofuel production from renewable sources in situ. Most pertinent genes have been identified in model species with complete sequence information, and knowledge from these genomes is currently being used for homology searches in species where sequencing efforts are ongoing. Such homology investigations are often effective, but caution is necessary, because multiple genes with similar sequences and annotations pointing to the same enzyme may possess distinct expression patterns and substrate preferences in vivo might be strikingly different among species and depend not only on gene sequences, but also on the tissue or even cell type of interest. Thus, before genetic modification strategies that had proven effective in some species are implemented in another species, it is prudent to consider and account for contextual differences. As a case in point, alfalfa (Medicago sativa L.), the organism used for our analysis, exhibits substantial differences in cell wall composition among the different internodes of young plants.Reflecting the substantial impact of lignin on forage digestibility p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) monolignols  plants by independently modifying the activities of seven key enzymes of monolignol biosynthesis. While many of the results were easily explained, down-regulation of caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) had little effect on S lignin, an observation that is conceptually inconsistent with the commonly accepted pathway structure (Medicago truncatulaO-methyltransferase (COMT) in alfalfa In most woody plants, the biochemical pathway of monolignol biosynthesis leads to three building blocks of lignin, which are known as olignols . In poteinternodes. During maturation, all internodes grow asynchronously and thus independently represent different developmental stages. This phenomenon suggests a customized modeling approach: Instead of studying the pathway within a single developmental context, it seems advantageous to launch a systematic investigation that simultaneously encompasses dozens of internodes from seven wild-type or transgenic plants. This comprehensive approach supports the fact that lignin biosynthesis is tightly coordinated by a hierarchy of transcription factors during secondary wall thickening In dicotyledonous plants like alfalfa, the stem consists of many segments, called With a comprehensive analysis of several datasets as the target, we propose here a novel modeling approach that integrates the data in a semi-dynamic fashion. First, flux balance analysis (FBA) This combined modeling approach represents, to the best of our knowledge, the first computational study of lignin biosynthesis in angiosperm stem tissues and, more generally, of secondary plant metabolism in angiosperms. As we will discuss later, the model analysis resulted in six postulates concerning the metabolic control of monolignol biosynthesis that had not been considered at all or at least not in detail. These postulates address the reversibility of some enzymatic reactions, shed light on the hypothesis of independent pathways for the synthesis of G and S monolignols, and suggest a novel feedforward regulatory mechanism exerted by a cinnamic acid-derived compound. Of note is the fact that evidence in support of this last postulate has subsequently been obtained in laboratory experiments. By critically evaluating the transgenic data against a revised pathway structure in alfalfa, we hope these postulates will not only serve as guidelines for directing future experiments, but also provide mechanistic insights that will aid the design of combined genetic modification strategies toward the generation of bioenergy crops with reduced recalcitrance.Accounting for recent experimental observations, we adopted a revised pathway structure of monolignol biosynthesis in alfalfa stems that includes the CCR2-catalyzed reduction of caffeoyl-CoA to caffeyl aldehyde and the subsequent synthesis of coniferyl aldehyde by COMT  towards the synthesis of S lignin, one would expect to see its expression being up-regulated in mature versus young internodes, which has recently been validated by microarray analysis  permitted feasible solutions in all cases tested.A very interesting result is the fact that no feasible solution exists for four of the six transgenic plants, if the revised metabolic map is correct  within the network. Arguably the simplest explanation is that HCT  is reversible p-coumaroyl-shikimate accumulates due to a reduced C3H activity, HCT converts it back to p-coumaroyl-CoA in the presence of free CoA, thereby allowing the cell to escalate the production of H lignin beyond the wild-type level. The catalytic efficiency of HCT acting on p-coumaroyl-shikimate as substrate remains to be experimentally determined, along with the possible competition for CoA between two shikimate esters .In transgenic plants where C3H is down-regulated, the proportion of H lignin among total monomer yields is significantly increased over control plants, especially in mature internodes . This fip-coumaroyl-CoA and subsequently channeled towards H lignin, a scenario which seems unlikely based on the known catalysis by cytochrome P450 enzymes. However, the amounts of H lignin determined by thioacidolysis appear to be unaffected by the low CCoAOMT activity despite a noticeable decrease in total lignin content , because manipulation of any downstream enzyme, such as COMT and F5H, has no substantial effect on H lignin  are organized into a functional complex through which the intermediates are channeled without much leakage. Similarly, we postulate that v13 and v14 form a corresponding complex without much leakage. This dual postulate for crossing channels is supported indirectly by literature information and by findings from our flux analysis, as outlined below.As an explanation for the altered S/G ratios in cases of CCoAOMT or COMT down-regulation, we postulate that the enzymes controlling cf. CCOMT antisense line ACC305 in Table 1 of First, an analysis of mature stems (internodes 6–9) collected from CCoAOMT down-regulated transgenic lines indicated that the levels of G lignin were greatly reduced, whereas those of S lignin were nearly unaffected (14C]-labeled S-adenosyl L-methionine (a co-substrate necessary for COMT-mediated O-methylation) and NADPH (the reducing agent for F5H), is converted to coniferyl aldehyde, 5-hydroxyconiferyl aldehyde, and a small amount of sinapyl aldehyde.Second, one of the constituent enzymes, F5H, is localized to the external surface of the endoplasmic reticulum v13 and v14 for all but the CCoAOMT-deficient plants   buffering changes in earlier precursors.Finally, our flux distribution analysis reveals a strong correlation between the computed flux values of e<0.001) . This coribution  and the ribution  indicatev14 and v16 at steady state could depend on whether the respective enzyme works within the linear or saturation region of its kinetic profile.An alternative explanation for an increased S/G ratio upon modifications of the CCoAOMT-CCR1 pathway could be that the kinetic features of the enzymes that catalyze coniferyl aldehyde and coniferyl alcohol are fine-tuned such that they could permit the adjustment of fluxes leading to G and S lignin and thus change the S/G ratio. For instance, given that down-regulation of CCoAOMT or CCR1 may alter the intracellular level of coniferyl aldehyde, the relative values of To investigate this alternate hypothesis, we designed and analyzed a kinetic Michaelis-Menten model that contains the two alternative pathways from caffeoyl-CoA to coniferyl aldehyde as well as the two principal branch points where the fluxes leading to G and S lignin diverge see . The modWe first examined the case where CCoAOMT is down-regulated. Only ∼5% of all admissible systems  when either PAL or C4H is down-regulated. It seems that these alterations should not differentially affect monolignol biosynthesis, because both occur before the first branch point, but they do. Closer inspection of the data from different internodes reveals that the S/G ratio is consistently increased in PAL-deficient plants but decreased in C4H-deficient plants . While ev22 in In accordance with the proposition of separate metabolic channels for G and S lignin, we postulate that the different effects of PAL or C4H down-regulation on the S/G ratio are due to feedforward regulation. Specifically, we suggest that this regulation is mediated by a downstream product of the cinnamic acid degradation pathway, which is represented collectively as Consistent with the observation of all transgenic experiments, an appropriate control strategy by this unknown compound X is summarized in v22 is likely to increase because the consumption of cinnamic acid through a competing branch v2 is not as effective as in wild-type plants. Thus, an accumulation of X could in turn activate the expression of CCoAOMT/CCR1/CAD and/or reduce the expression of CCR2/COMT/F5H, leading to a smaller S/G ratio.In the case of C4H deficiency, however, the production of X through e.g. HCT Salicylic acid (SA) is a notable endogenous signaling molecule that is known to be derived from cinnamic acid MedicagoFunctional genomics is a premier tool for identifying metabolic pathways in sequenced model species and for pinpointing genes involved in them Specifically, the new integrative modeling approach proposed here combines static flux-based models and a Monte Carlo simulation of randomly parameterized kinetic models. This approach has the advantage that it allows the collective analysis of many experimental results and sheds light on pathway features that are particularly important for functionality under normal and altered conditions. The analysis here revealed a quantitative trend of flux patterns during development, which in turn allowed the identification of principal branch-point metabolites at which internode-specific flux partitioning patterns control the observed mode of lignification. While it is relatively easy to single out principal metabolites in linear or slightly branched pathways, the system studied here is confounded by the plant's employment of the same enzymes, such as CCR and CAD, in different key positions. Due to this multiple use, manipulating the flux partitioning pattern towards a desired mode of lignification may incur undesired “side effects.”The computational analysis indicates that a single flux analysis just for wild-type plants is insufficient for understanding because even a seemingly simple pathway like monolignol biosynthesis requires relatively minor, yet important, extensions to account for the overflow of some intermediate metabolites that only occurs in transgenic plants. At the same time, the analysis also demonstrates that the simultaneous analysis of several independent datasets, in this case transgenic lines and sequential internodes, can lead to insights that otherwise would have been difficult to obtain. Here, it led to several postulates that are specific enough for experimental validation or refutation.Some model-free postulates refer to the need for reversibility or committedness of key reactions, which might not be too surprising. Two further postulates are more intriguing. They refer to the functional channeling within the pathway and its mechanistic control. Based on the observation of an increased S/G ratio in CCoAOMT or CCR1 down-regulated lines, the computational results suggest an S lignin-specific channel capable of converting caffeyl aldehyde directly into 5-hydroxyconiferyl aldehyde or sinapyl aldehyde. Different experiments in the literature suggested the co-localization of COMT and F5H in lignifying alfalfa stems O-methyltransferases (OMTs) are involved in converting caffeyl aldehyde to coniferyl aldehyde. These OMTs may have distinct sub-cellular localization (to cytoplasm or endomembrane) and therefore a different affinity to F5H. Thus, it could be that the cytosolic OMT in the transgenic lines with reduced CCoAOMT expression is up-regulated and helps consume extra caffeyl aldehyde outside the proposed channel. A corresponding labeling experiment in alfalfa While the proposed membrane-bound channel for synthesizing S lignin could constitute an important control mechanism, it may only have comparatively limited capacity because even in CCoAOMT down-regulated lines G lignin is generated in a higher proportion of total monomer yields than S lignin  being complexed or co-localized on internal membranes.The observed decrease in the S/G ratio of COMT down-regulated lines alone is not sufficient to prove the existence of a G lignin-specific channel, because a reduced COMT activity affects all fluxes that are specific for the synthesis of S lignin, thus leading to a smaller S/G ratio. Nevertheless, the strong correlation between M. truncatula, a species closely related to alfalfa, leads to an increased S/G ratio, whereas M. truncatula CCR1 knock-out mutants show a reduction in the S/G ratio M. sativa L.) and diploid M. truncatula. For instance, the S/G ratio in wild-type alfalfa stems  is approximately twice as large as that in wild-type M. truncatula stems . Consequently, further experimental work is required to validate or reject the postulate that a G lignin-specific channel is operational in alfalfa.One option for testing this postulate would be to down-regulate CCR2 and record if the strain exhibits a greater decrease in S lignin than in G lignin, giving a smaller S/G ratio. Surprisingly, knocking out CCR2 in Compound X. Supporting this hypothesis, the transgenic experiments used here have shown that down-regulation of CCoAOMT, which we postulate to be involved in the G lignin-specific channel, yields similar proportions of G and S lignin among total monomers as does the down-regulation of PAL, which is postulated to inhibit and/or activate the functioning of the G lignin- and S lignin-specific channels, respectively  and thus become available to make SA. In other words, the shikimate recycling facilitated by HCT enables the shikimate pool to work as a sensor of the flux into lignin. Future in-depth studies, whether they are experimental or computational, are required to justify this hypothesis. It is noteworthy, however, that the reason why plants shuttle monolignol pathway intermediates between Coenyme A and shikimate esters has yet to be explained.Salicylic acid (SA), a phenolic phytohormone derived from phenylalanine, was proposed as a potential candidate for this unknown Compound X. Intriguingly, post-hoc experiments showed that the intracellular levels of SA are indeed highly proportional to the extent of lignin reduction in transgenics where different pathway genes are down-regulated . This reIn conclusion, our analysis shows that a combined modeling effort can uniquely and effectively complement experimental studies of the type used here. In contrast to analyzing one dataset at a time, it allowed us to integrate all results from a comprehensive experimental investigation of various transgenic lines and internodes. This integration, in turn, revealed dynamic, developmental patterns and their dependence on key enzymes. Together, the analyses uncovered elusive control of monolignol biosynthesis and led to testable hypotheses regarding various pathway aspects that should be clarified before one attempts to generate and optimize viable, productive “designer” crops with minimal recalcitrance.cf.O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), and caffeic acid 3-O-methyltransferase (COMT). Each transgenic plant was cultivated to early flowering stage, and the mature stem consisting of eight internodes was harvested and divided into individual segments; all internodes were numbered according to their maturity, with internodes 1–2 representing the pooling of the two uppermost stem segments. The lignin content and monomer composition for each internode were determined for each transgenic line via established protocols in vitro (Table 2c of In a previous study Acinetobacter sp. ADPWH_lux as described previously Acinetobacter sp. ADPWH_lux was diluted in LB (1∶20) and grown at 37°C for ∼2 hrs to an OD600 of 0.4. Sixty µl of LB medium, 50 µl of salicylate biosensor culture and 20 µl of each crude extract were mixed in a 96-well cell culture plate. The plate was incubated at 37°C for 1 h without shaking and bioluminescence and OD600 of negative controls  were read using a Glomax Multi detection system . SA standard and negative controls were read in parallel with the experimental samples and every sample was replicated five times. Relative bioluminescence was obtained by subtracting bioluminescence OD600 of negative controls, and SA concentration was estimate according to the SA standard curve.Salicylic acid levels in stems from the same plant lines (excepting PAL down-regulated plants) as well as from CAD down-regulated lines Lignin content was determined by the thioacidolysis method as described previously N is the stoichiometric matrix of the pathway system and v is a vector of fluxes. Other commonly used constraints are the upper and lower bounds on individual fluxesi. The only exceptions are the three overflow fluxes  j: If cf. Table S3 in For lignin-modified lines, where a particular enzyme is genetically down-regulated, we use the per se are straightforward. Importantly, this Monte Carlo-type simulation allowed us to examine thousands of combinations of kinetic parameters without limiting ourselves to a few particular cases of manually tuned, ill-characterized parameter values.A major surprise emerging from the experiments with lignin-modified alfalfa lines was the differential effect of CCoAOMT down-regulation on G and S lignin production. It is conceivable that these results could be due to the kinetic features of the participating enzymes. To analyze this possibility, we designed a kinetic model of the involved reactions and tested this model with a large-scale simulation. The details of this model can be found in Figure S1Developmental evolution of the steady-state flux distribution in PAL-deficient plants versus wild-type plants. Please refer to (TIF)Click here for additional data file.Figure S2Developmental evolution of the steady-state flux distribution in C4H-deficient plants versus wild-type plants. Please refer to (TIF)Click here for additional data file.Figure S3Developmental evolution of the steady-state flux distribution in HCT-deficient plants versus wild-type plants. Please refer to (TIF)Click here for additional data file.Figure S4Developmental evolution of the steady-state flux distribution in C3H-deficient plants versus wild-type plants. Please refer to (TIF)Click here for additional data file.Figure S5Developmental evolution of the steady-state flux distribution in COMT-deficient plants versus wild-type plants. Please refer to (TIF)Click here for additional data file.Text S1Supplementary materials. This supplementary text includes two main sections. In the first section, we present the formulation of FBA and MOMA, and identify equivalent pathways that underlie the occurrence of alternate FBA solutions. In the second section, we present a kinetic model for the analysis of pathway operation at the critical branch point of coniferyl aldehyde.(DOC)Click here for additional data file.


Sensory deafferentation produces extensive reorganization of the corresponding deafferented cortex. Little is known, however, about the role of the adjacent intact cortex in this reorganization. Here we show that a complete thoracic transection of the spinal cord immediately increases the responses of the intact forepaw cortex to forepaw stimuli (above the level of the lesion) in anesthetized rats. These increased forepaw responses were independent of the global changes in cortical state induced by the spinal cord transection described in our previous work , as the responses increased both when the cortex was in a silent state (down-state) or in an active state (up-state). The increased responses in the intact forepaw cortex correlated with increased responses in the deafferented hindpaw cortex, suggesting that they could represent different points of view of the same immediate state-independent functional reorganization of the primary somatosensory cortex after spinal cord injury. Collectively, the results of the present study and of our previous study suggest that both state-dependent and state-independent mechanisms can jointly contribute to cortical reorganization immediately after spinal cord injury. When a major lesion of the nervous system – such as stroke, amputation or spinal cord injury - interrupts the normal flow of sensory inputs from the body to the brain, the corresponding sensory function is obviously dramatically affected. In addition to the direct loss of function, sensory deafferentation produces extensive long-term reorganization of brain structures up to the cortex –5. Long-Cortical reorganization after deafferentation is typically described in terms of the deafferented cortex becoming more responsive to stimulation of the surrounding intact body regions. In a recent study , we indiTo disentangle possible contributions of state-dependent vs state-independent mechanisms, we performed electrophysiological recordings in both the forepaw and hindpaw cortex in anesthetized rats, recording cortical responses to forepaw stimuli under three conditions: (1) intact rats at the same level of anesthesia as in our previous study ; (2) aftExperiments were performed following the rules of the International Council for Laboratory Animal Science, European Union regulation 2010/63/EU and approved by the Ethical Committee for Animal Research of the Hospital Nacional de Parapléjicos . A total of 20 male Wistar rats were used, divided in 2 experimental groups: 1) in the main experiment in 12 animals we performed extracellular recordings from the intact forepaw cortex and the deafferented hindpaw cortex before and immediately after complete thoracic transection of the spinal cord ; 2 in a Animals were anesthetized with urethane (1.5g/kg i.p.). The body temperature was kept constant (36.5 ºC) using an automatically controlled heating pad. Animals were placed in a stereotaxic frame . A laminectomy was performed at thoracic level (T9-T10) keeping the dura mater intact and covered with saline to protect the area. The skull was exposed and a craniotomy was performed on the right side of the midline over the primary somatosensory cortex  and theRecordings were obtained using tungsten electrodes with 4-5 MΩ impedance at 1000 Hz . Two electrodes were stereotaxically lowered in the infragranular somatosensory cortex, one in the forepaw area  and the other one in the hindpaw area  following the coordinates of . In 10 oElectrical pulses were applied using bipolar needle electrodes located subcutaneously in the wrist of the forepaw and of the hindpaw, one pole in each side of the paw. The rationale for this stimulation was to activate all types of somatosensory fibers originating within the paws, including tactile, proprioceptive and nociceptive fibers. The protocol consisted of a total of 100 pulse stimuli with duration of 1ms and frequency of 0.5Hz. Two different intensities were applied: low-intensity (0.5mA) and high-intensity (5mA). Low-intensity stimuli were intended to activate only a fraction of the available fibers, mainly low-threshold primary fibers running through the lemniscal pathway, from the dorsal columns to the brainstem ,37. HighWe also performed a set of “sham” experiments (n=8) in which the spinal cord remained intact after the laminectomy for the entire duration of the experiment. Besides the absence of spinal cord lesion, the experimental protocol was the same as in the transected animals, but sham animals were studied only under deep anesthesia.Spontaneous activity was studied in recordings at least 150-s long, performed immediately after the low-intensity stimulation and immediately before the high-intensity stimulation. To quantitatively evaluate the level of cortical spontaneous activity, we extracted the rectified multi-unit activity (rMUA) by band-pass filtering the raw signals at high frequencies (300-3000 Hz) and rectifying the resulting signal. The rMUA is a good measure of cortical state, as it correlates with the membrane potential of adjacent intracellularly recorded neurons . Under lLocal field potential (LFP) responses were obtained by averaging across stimuli the raw signals recorded from the electrodes. The amplitude of LFP responses was evaluated as the absolute value of the negative peak in the average response. Single-trial analyses were performed by quantifying the average rectified MUA in the 50ms before each stimulus and the peak-to-peak amplitude of the LFP response to the subsequent stimulus. Furthermore, stimuli were visually classified as delivered during DOWN or UP cortical states based on both LFP and MUA signals on a trial-by-trial basis according to the following criteria: (1) if both LFP and MUA signals showed no activity for at least 100 ms before the stimulus, the stimulus was classified as delivered during a DOWN state; (2) if both LFP and MUA showed activity immediately before the stimulus, the stimulus was classified as delivered during an UP state (3) if neither of the above criteria was verified, the stimulus remained unclassified.Multi-unit responses were obtained by band-pass filtering the raw LFP signals at high frequencies (300-3000Hz), rectifying the resulting signal, and averaging across stimuli. The magnitude of rMUA responses was evaluated as the area of the response in the first 50 ms post-stimulus, after subtracting the background. The duration of post-response inhibition was measured in the rMUA response as the time interval from the end of the first excitatory response to the onset of the subsequent activation, using only stimuli occurring during silent states.Changes in cortical spontaneous activity were evaluated separately entering the mean and the mode of the rMUA into two-way repeated-measures ANOVAs, with TIME as first factor with three levels  and ELECTRODE as second factor with two levels (forepaw cortex and hindpaw cortex). The same two-way ANOVA was used for the peak of the rMUA spectrum, but because this measure is often ambiguous during lighter anesthesia, only two levels in the TIME factor were considered . Note that one animal was recorded only under deeper anesthesia and after spinal cord transection, so it was treated as missing value under lighter anesthesia.Changes in LFP responses in the forepaw cortex to forepaw stimuli were evaluated with two-way repeated-measures ANOVAs, with TIME as first factor with three levels  and STIMULUS as second factor with two levels (low-intensity and high-intensity). Changes in the duration of post-response inhibition to high-intensity forepaw stimuli were evaluated with a one-way repeated-measures ANOVA on the TIME factor. Changes in cortical LFP responses separating stimuli as delivered during UP or DOWN cortical states were evaluated with a two-way repeated-measures ANOVA, with TIME as first factor with two levels  and STATE as second factor with two levels (UP and DOWN), using only high-intensity stimuli.In the experiments with the third electrode, changes in LFP responses in the forepaw cortex to high-intensity forepaw stimuli were also evaluated with two-way repeated-measures ANOVA, with TIME as first factor with two levels  and ELECTRODE as second factor with two levels (forepaw cortex 1 and forepaw cortex 2).In the control experiments with sham animals, we performed the same two-way ANOVAs as is the main experiments, but with only two levels in the TIME factor .Changes in rMUA responses in the hindpaw cortex to high-intensity forepaw stimuli were evaluated with a one-way repeated-measures ANOVA on the TIME factor.Tukey Honest Significant Difference test was used for all post-hoc comparisons. Additional comparisons between means, percentages and correlations were performed with t-tests, two-proportion test and Pearson’s correlation coefficient, as indicated in the text. All results were considered significant at p<0.05.We simultaneously recorded local field potentials (LFPs) and multiunit activity (MUA) from the hindpaw representation and the forepaw representation of the primary somatosensory cortex (n=12 rats) in response to somatosensory stimuli delivered to the contralateral forepaw and hindpaw, under three experimental conditions in the same animals: (1) intact animals under relatively light anesthesia =8.3, p=0.0028; Tukey: p=0.0063), whereas the state-independent increase of LFP response amplitude after spinal cord transection was mirrored by an increase in the duration of the post-response inhibition , as evident in the rMUA responses  increase even in the absence of global changes in cortical state, reflecting a state-independent immediate cortical reorganization – in addition to the state-dependent reorganization we previously described – after spinal cord injury.Once established that state-independent functional reorganization occurs in the intact forepaw cortex immediately after spinal cord transection, we investigated whether state-independent reorganization also occurred in the deafferented hindpaw cortex. To this end, we used the rectified multi-unit activity (rMUA) – which reflects the overall spiking activity around the electrode – to quantify the responses evoked in the deafferented hindpaw cortex by forepaw stimuli in the same animals for which the forepaw responses were analyzed above (n=12).As expected, we observed two types of “responses”: (1) short-latency responses (<50 ms) and (2) long-latency activations (>50 ms). Short-latency responses are typical somatosensory responses – of the same type as the “non-homologous responses” we described in  – observLong-latency activations are instead due to active states triggered by the stimuli . As expeThese results suggest that long-latency activations depend exclusively on the global state of the somatosesory cortex, whereas the increased short-latency responses represent state-independent functional reorganization of the deafferented hindpaw cortex immediately after spinal cord transection. Interestingly, reorganization of the deafferented hindpaw cortex (as measured by the percent increase of short-latency rMUA responses evoked in the hindpaw cortex by forepaw stimuli) and reorganization of the intact forepaw cortex (as measured by the percent increase of LFP responses evoked in the forepaw cortex by forepaw stimuli) were positively correlated across animals , suggesting that they represent two complementary views of the same immediate cortical reorganization after spinal cord injury  in anesthetized rats. These increased forepaw responses are independent of the global changes in cortical state induced by the spinal cord transection described in our previous work , as theyWe previously showed that thoracic spinal cord transection immediately changes the state of the brain – slowing it down – and that this state change is at least partly responsible for the increased responses evoked in the intact forepaw cortex by forepaw stimuli . In our The experimental strategy of bringing animals into a state of deep anesthesia before the spinal cord injury was critical to disentangle the contribution of state-independent mechanisms to the increased responses in the intact forepaw cortex immediately after the spinal injury. Even though we cannot exclude more subtle changes in the local state of the somatosensory cortex, several complementary observations support the existence of state-independent reorganization : (1) with the cortex already in slow-wave activity, the overall level of cortical spontaneous activity did not change after spinal transection  shotonic intra-cortical inhibition is ruled out here, because the responses in the intact forepaw cortex increased even if the stimuli were delivered during silent cortical states, when there is no tonic inhibition to be reduced [increased noradrenergic neuromodulation [Several non-exclusive mechanisms can contribute to cortical reorganization after deafferentation, including growth of new connections due to axonal sprouting , unmaski reduced ,54,55. Odulation ,57. Thisdulation . Heterogdulation . It is tOne might wonder about the possible mechanistic role of urethane anesthesia in our results. On the one hand urethane anesthesia is considered a good model of natural sleep . Slow-waFinally, we cannot exclude that at least part of the reorganization we observed at cortical level could reflect reorganization occurring at subcortical level. The fact that our results were significant only with high intensity stimuli – which likely activate both the dorsal columns and the spinothalamic tract ,37 – andIndependently of the exact cortical/subcortical mechanisms, the increased cortical responsiveness reported here represents the overall state-independent reorganization of the somatosensory system immediately after thoracic spinal cord transection.The great majority of studies investigating reorganization in the somatosensory system after peripheral or spinal cord injuries implicitly assume that intact structures do not undergo any reorganization. Our results clarify that this is not the case. Several previous findings conceptually support the possible long-term pathophysiological relevance of the immediate reorganization of the intact forepaw cortex reported here: (1) careful a posteriori examination of the figures/results of previous works on long-term cortical reorganization after thoracic spinal cord injury in rats ,45 suggeIn conclusion, the results of the present study and of our previous study  collecti