Vice versa, several metals are needed in trace amounts for carrying out vital functions and their deficiency due to rapid growth, pregnancy, excessive blood loss, and insufficient nutritional or digestive uptake results in organic and systemic shortcomings. Established in situ techniques, such as EDX-ray spectroscopy, are not sensitive enough to analyze trace metal distribution and the quantification of metal images is difficult.Hereditary disorders associated with metal overload or unwanted toxic accumulation of heavy metals can lead to morbidity and mortality. Patients with hereditary hemochromatosis or Wilson disease for example may develop severe hepatic pathology including fibrosis, cirrhosis or hepatocellular carcinoma. While relevant disease genes are identified and genetic testing is applicable, liver biopsy in combination with metal detecting techniques such as energy-dispersive X-ray spectroscopy (EDX) is still applied for accurate diagnosis of metals. In this study, we developed a quantitative biometal imaging technique of human liver tissue by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in order to compare the distribution of selected metals in cryo-sections of healthy and fibrotic/cirrhotic livers.Most of the metals are homogeneous distributed within the normal tissue, while they are redirected within fibrotic livers resulting in significant metal deposits. Moreover, total iron and copper concentrations in diseased liver were found about 3-5 times higher than in normal liver samples.via LA-ICP-MS is a sensitive innovative diagnostic tool that will impact clinical practice in identification and evaluation of hepatic metal disorders and to detect subtle metal variations during ongoing hepatic fibrogenesis.Biometal imaging  HFE gene that is quite common among white people resulting in an amino acid exchange (i.e. C282Y). Homozygous carriers usually have increased serum transferrin saturation levels and increased serum ferritin levels. In addition, non-HFE hepatic iron overload diseases originate from mutation in other genes such as transferin receptor 2, hepcidin, hemojuvelin, and ferroportin Hepatic fibrosis, cirrhosis and hepatocellular carcinoma are characterized by a drastic increase of extracellular matrix proteins comprising collagens, glycoproteins, proteoglycans and hyaluronan via the ATP-dependent pumps ATP7B and ATP7A ATP7B resulting in copper accumulation in different tissues. In liver, ATP7B is mainly expressed in hepatocytes and its loss is the basis for reduced hepatic biliary copper excretion and reduced incorporation of copper into ceruloplasmin Atp7b-/- mutant mice display a gradual accumulation of hepatic copper inducing gross anatomic liver abnormalities in older mice with an irregular liver surface with protruding regenerative nodules of different sizes indicative for cirrhosis Copper is a unique essential trace element playing an important function as a cofactor for a number of cellular processes in situ metal content in combination with scanning electron microscopy with energy-dispersive X-ray analysis (SEM-EDX) is traditionally the confirmatory test in cases in which the non-invasive markers suggest elevated metal concentrations. Unfortunately SEM-EDX is often not sensitive enough for trace element imaging. However, liver heterogeneity can result in wrong analytical data). Therefore, novel imaging techniques that provide information about the regional distribution of individual metals or groups of metals combined with a diagnostic score system further providing information about the extent of hepatic insult are urgent required.These examples underline that body metal homeostasis is crucial and metal deficiencies or dramatic build-ups should be prevented to avoid hepatic or other organic impairments. Therefore, indicative, non-invasive serum parameters  and genetic testing of causative genes is widely propagated in diagnosis to identify metal disorders or predisposition for respective diseases. Moreover, the performance of liver biopsy to measure or stain Although potentially not causative, the outcome of hepatic fibrosis is also significantly modulated by variations in hepatic metal distribution and concentrations. Hepatic iron overload for example has been described in chronic hepatitis C as a cofactor affecting fibrosis progression and hepatic iron deposits were found in well-compensated chronic hepatitis B infection, especially in patients that were co-infected with hepatitis D virus Magnetic resonance imaging (MRI) allows the mapping only of selected single metals (such as iron) in organs staphylococcal aureus liver abscesses from normal mice are enriched in Ca2+, while these abscesses are devoid of detectable Zn2+ and Mn2+Recently, LA-ICP-MS imaging technique has been applied as a reference standard method to validate the results from quantitative gadolinium enhanced MRI technique The aim of the present study is to develop a novel quantitative imaging technique for trace metals and to characterize the distribution of various metals in cryo-sections of normal, fibrotic and cirrhotic human liver tissues. We demonstrate that most of the metals analyzed are regularly distributed within normal liver tissue, while they are redirected within fibrotic and cirrhotic livers resulting in defined deposits suggesting that this method is not only valuable in diagnosis of common metal disorders but also adequate to determine subtle changes during ongoing liver fibrogenesis.Liver samples were taken from internal tissue banks of the RWTH University Hospital Aachen or the University Hospital Regensburg in which liver tissue was used for experimental purposes that was acquired either from biopsies for routine clinical purposes or explants of cirrhotic livers that were obtained during liver transplantation. Each patient from which material is stored provided written informed consent and the study was approved by the local ethics committee at the RWTH Aachen University and the University of Regensburg. Liver fibrosis/cirrhosis was diagnosed mainly on the grounds of liver routine histology. Fibrotic/cirrhotic tissue samples used in this study were obtained from five independent patients (D1 – D5). Secluded tissue from tumour-free margins of resected hepatic metastasis within normal liver parenchyma (N1 – N5) served as controls.After being fixed in 4% buffered formalin for 24 h, the liver tissue was embedded into paraffin wax. A histological semi-quantitative examination of the liver was performed on sections after standard Sirius Red and H & E stains. Liver sections were stained with an anti-human α-SMA antibody using standard procedures. To assess the degree of fibrosis, a standard Ladewig staining was performed and the severity of liver biopsy lesions and overall immunological activity were graded and staged according to routine scoring guidelines of Desmet and Scheurer Slices of 20 to 50 µm thickness were obtained from paraffin-embedded liver tissue, sputtered with a conductive silver paint to make contact on the surface, carbon-coated in a rotary evaporator, and then maintained in desiccators to prevent air-contact before analysis. Analysis of element content by EDX was rapidly performed within two days of preparation using an environmental scanning electron microscope  in a high vacuum environment operating at 15 to 20 kV. The slices were imaged by back-scattered electrons and analysed for the elemental composition of the element present. For quantitative analysis of the element concretions, the contributions of the C-coating and the embedding resin containing C, O and trace of Cl were computer-based subtracted from the quantitative data of each spectrum.-1, laser fluency 0.24 J cm−2). The ablated material was transported by argon gas (as carrier gas) into the inductively coupled plasma (ICP). The ions formed in the atmospheric pressure ICP were extracted in the ultrahigh vacuum mass spectrometer via a differential pumped interface, separated in the quadrupole mass analyzer according to their mass-to-charge ratios and detected by an ion detector. No reference standard materials for quantification of metals in human liver were available. Therefore, SRM NIST 1577b (bovine liver) was used as standard reference material. The trace metal concentrations in the samples were determined by single point calibration using this SRM. Moreover, the selection of internal standard element is the important part for LA-ICP-MS analyzing. The appropriate internal standard element was chosen to correct for plasma instabilities and sample-to-sample variations in the ion signal intensity. In this work, sulphur was used as an internal standard element for all the analyses because sulphur in human livers present rather a homogeneous distribution and provide constant concentration (see below). The human liver tissue and the NIST standard reference material deposited on glass slide were mounted in the laser ablation chamber to perform LA-ICP-MS imaging of sample and standard reference material under identical experimental conditions. Mass spectrometric measurements by LA-ICP-MS for imaging of liver tissue were performed by line scanning ablation (line by line) with a focused laser beam under the optimized experimental parameters given in 63Cu+ using a SRM 1577b bovine liver standard. To validate the metal ion images, two isotopes of the same element were analyzed, whenever possible. From the continuous list of raw pixel values elemental images were reconstructed using the IMAGENA LA-ICP-MS Image Generation software created at Forschungszentrum Juelich www.pmod.com).A quadrupole-based inductively coupled plasma mass spectrometer  coupled to a laser ablation system  was used to study elemental distributions in tissue sections of human livers (30 µm thickness). Laser ablation of biological tissue was performed using a focused Nd:YAG laser beam in the scanning mode . It relies on the investigation of an interaction of some source of X-ray excitation and the sample resulting in a unique set of peaks on the X-ray spectrum that is characteristic for each element. In brief, the necessary equipment for this technique is based on an excitation source that transmits an electron beam, an X-ray detector that converts X-ray energy into voltage signals, a pulse processor and an analyzer that processed the signals and matches them to individual atoms. Based on the fact that this technique allows discriminating of metals, this technique is used to corroborate or even score metal disorder in organ tissue.SEM-EDX is used for chemical analysis and determination of the elemental composition of microscopic particles or regions within a sample by When we applied this method to non-WD patients suffering from liver fibrosis or cirrhosis, we noticed that although it allows the identification of individual metal deposits , it was The introduction of LA-ICP-MS technology has recently been successfully applied as a powerful imaging (mapping) technique to produce quantitative images of detailed regionally specific element distributions in thin tissue sections of human or rodent brain In an attempt to use this methodology for analysis of liver tissue, we established a protocol that allows quantitative metal imaging of liver tissue. In the final workflow, liver tissue is cryo-sected into specimen of 30 µM thickness and mounted on glass slides . Mass spTo evaluate the LA-ICP-MS imaging technique in liver specimen and to further analyse if the concentration or distribution of individual metals is altered in fibrotic or cirrhotic liver tissue, we thought to apply this imaging technique in a small set of histological well-characterized samples from normal, fibrotic and cirrhotic liver tissue .The reproducibility of the LA-ICP-MS imaging technique for the analysis of 20 µm thin cross section of human brain tissues is already well studied Quantitative bioimages of Mn, Fe, Cu, Zn, and Cd of control and diseased (fibrotic and cirrhotic) human liver samples are illustrated in The bioimaging approach of metals in fibrotic/cirrhotic liver tissue sections by LA-ICP-MS revealed inhomogeneous distribution of Mn, Fe, Cu, Zn, and Cd. Very low concentrations of metals were observed in fibrous septa areas when compared to nodules areas. Moreover, the concentrations of Fe and Cu in all disease liver  were sig-1 in control; were 4.9, 576, 35, 80, and 0.5 in disease (fibrotic/cirrhotic) liver samples. In the case of Mn and Cd, the different concentrations between control and disease are not significant. On the other hand, the concentration of Fe, Cu, and Zn are quite different between the control and disease liver. Fe and Cu concentrations in diseased liver are higher than in normal liver samples, while Zn concentrations are decreased in diseased samples suggesting that these changes can be used to predict the changing of liver state from healthy to diseased liver.per se in that way that inhomogeneous samples result in inadequate or dissimilar excitations.Hereditary liver diseases resulting in metal intoxication are reasons for morbidity and mortality worldwide X-rays can be generated by any atom in the sample that is sufficiently excited by the incoming beam. These X-rays are emitted in any direction, and so they may not all escape the sample. The likelihood of an X-ray escaping the specimen, and thus being available to detect and measure depends on the energy of the X-ray and the amount and density of material it has to pass through. This can result in reduced accuracy in inhomogeneous and rough samples.All these limitations of SEM-EDX measurements become evident in our experiment that is depicted in LA-ICP-MS on the other side is one of the most powerful and sensitive techniques for the analysis of a variety of solid samples because it provides good sensitivity for major, minor, trace, and ultra-trace elements with high sample throughput and without sample preparation which generates a large amount of waste as occurring in solution-based techniques The use of matrix matched reference material for quantification of analytical data is important because the ablation yield varies with the sample matrix. However, an appropriate matrix-matched standard reference material that for metal in human liver is not available. The most common quantification method for tissue in LA-ICP-MS is external calibration utilizing matrix-matched standards Human liver consists of two main lobes, left and right, which overlap slightly. Each lobe contains lobules that are the building blocks of the liver that are six-sided structures or hexagonal shapes. As shown in 2+ to the powerful oxidizing agent Mn3+3+ has been suggested as a cause of the toxic effects of it. For our results, in fibrotic/cirrhotic tissue samples , fibrosis (223 µg g-1), and cirrhosis (192 µg g-1), respectively. Data obtained from our and previous studies suggest that ongoing liver disease correlates with reduced Zn levels. These findings fit quite well with recent experimental observations showing that Fe and Cu act in an antagonistic fashion and iron is an important factor triggering copper deficiency in liver Zn is necessary for normal liver function and, the liver plays a central role in Zn homeostasis -1 in all tissue samples.Cadmium is a highly toxic element present in food and water. It can deposit in the liver and kidney and strongly binds to macromolecules in the intracellular compartment and influences the metabolism of Fe, Ca, Zn, Mn and Cu Considering all known hepatic diseases that are caused, promoted or modulated by accumulation of metals, there is a mandatory need for the establishment and evaluation of precise non-invasive metal imaging methods for diagnostics. In recent years, many studies have demonstrated the possibility of performing hepatic iron quantification with magnetic resonance imaging technology. However, a consensus has not been reached yet regarding the technique or the possibility to reproduce the same method of calculus in different equipments and intensive discussion for the establishment of consensus guidelines for clinical practice of hepatic iron quantification by magnetic resonance imaging techniques are conducted It would be an absolute goal to further analyze the sub-lobular and sub-cellular distribution of signals regarding different metals in both normal and disease conditions. In our study we noticed that the signals of several metals are more punctuated than others. However, the precise mapping of individual metals to cellular subpopulation of the liver is a complex venture and must be addressed in future studies. There are however already pioneering studies available that have already precisely co-localized aggregates and mercury deposits within cellular subpopulations of the liver In summary, all these findings indicate that LA-ICP-MS is a novel powerful and innovative analytical technique that will have tremendous impact on diagnostics of metal disorders in liver. The fact that the obtained metal concentrations in liver tissue were the same in specimen that were obtained from two different hospitals (Aachen and Regensburg) further demonstrates that the results obtained in LA-ICP-MS are mostly independent from potential pre-analytical differences that may arise from sample collection. Although we presently do not know how individual hepatic disorders impact the distribution and location of individual metals or groups, it is known that metal overload or lack of individual metals may induce or contribute to the pathogenesis of liver disease. Therefore, LA-ICP-MS allowing quasi-simultaneous measurement of all metals and selected non-metals will provide an important add-on to routine diagnosis of liver sections or biopsies.


The disease mainly affects children under five in endemic areas and is the leading cause of death. Red blood cells (RBCs) infected with P. falciparum express parasite-derived molecules on the surface, which bind to host adhesion molecules expressed on vascular endothelial cells, leading to sequestration of infected RBCs in capillaries and microvessels. Pathogenesis of CM was previously thought to derive from the occlusion of brain microvessels induced by the sequestration of infected RBCs, but recent studies show that the etiology of CM is more complex and involves host immune responses  infection of susceptible mouse strains, such as C57BL/6 and CBA is the most widely used model where animals, develop the fatal disease  is one of the most serious complications of » Pathogenesis of CM was previously thought to derive from the occlusion of brain microvessels induced by the sequestration of infected RBCs, but recent studies show that the etiology of CM is more complex and involves host immune responses«+ T cells for its pathogenesis  cross-presenting the malaria antigen during infection  and a MHC-tetramer, which can identify CD8+ T cells, was generated. Using this MHC-tetramer, the authors showed that a substantial proportion of effector CD8+ T cells in the infected mice recognize this PbGAP50. Interestingly, this epitope was also expressed in non-ECM inducing malaria parasites. A surprise came when they examined tetramer-positive cells in mice infected with the parasites; PbGAP50-specific CD8+ T cells were present in high proportions in the brain of mice infected with both PbA and non-ECM inducing parasites. In search for the mechanisms of ECM development in PbA-infected mice, they found that microvessel preparations from PbA-infected mice presented PbGAP50 peptide but not those from mice infected with non-ECM inducing strains (+ T cells are critical for the pathogenesis of ECM. One caveat however remains, as the cell type that cross-presented malaria antigen has not been defined. Microvessels contain multiple cell types including endothelial cells, pericytes and astrocytes. While vascular endothelial cells are the primary candidates, purification and functional evaluation of each cell-type should be done.In this issue, Howland et al shows that brain microvessels indeed cross-present  strains . This st» … microvessel preparations from PbA-infected mice presented PbGAP50 peptide but not those from mice infected with non-ECM inducing strains.«Plasmodium-specific CD8+ T cells by DCs in periphery is not directly linked to ECM, and cross-presentation of malaria antigens by microvessel target cells, is critical for the pathogenesis. The authors explain that the difference between ECM-inducing and non-inducing parasites in microvessel cross-presentation comes from the amount of available antigen. The levels of infected RBCs attached to the brain microvessels of PbA-infected mice were much higher than those infected with non-ECM inducing strain. It remains to be answered, however, why PbA-infected RBCs preferentially accumulate in the brain. Is it due to the intrinsic properties of PbA or due to the immune response of the host? In addition, PbGAP50-specific CD8+ T cells alone did not reproduce ECM-like disease, which was induced only after treatment with enhancing reagent. Perhaps, multiple CD8 epitopes are involved in the pathogenesis of ECM, and it would be interesting to find other PbA antigens recognized by CD8+ T cells and ask whether their sum can induce an ECM-like disease. The other remaining question is why certain mouse strains are more or less susceptible to ECM, with C57BL/6 and CBA being highly susceptible (de Souza et al, This study also indicates that activation of Plasmodium-specific CD8+ T cells are involved in the pathogenesis of human CM. It has been shown that malaria antigens can be transferred to human endothelial cells (Jambou et al, Plasmodium-specific CD8+ T cells are activated in malaria patients, and identify antigens of human malaria species that are recognized by CD8+ T cells. Identification of parasite antigen cross-presented by brain microvessels would be a significant advance towards understanding the pathogenesis of CM. Furthermore, it would be intriguing to speculate that mechanisms similar to ECM might underlie the pathogenesis of other human neurological encephalopathy.Finally, an important question is whether


Conventional immunosuppressive therapies have radically transformed patient survival in systemic lupus erythematosus (SLE), but their use is associated with considerable toxicity and a substantial proportion of patients remain refractory to treatment. A more comprehensive understanding of the complexity of SLE immunopathogenesis has evolved over the past decade and has led to the testing of several biologic agents in clinical trials. There is a clear need for new therapeutic agents that overcome these issues, and biologic agents offer exciting prospects as future SLE therapies.An array of promising new therapies are currently emerging or are under development including B-cell depletion therapies, agents targeting B-cell survival factors, blockade of T-cell co-stimulation and anti-cytokine therapies, such as monoclonal antibodies against interleukin-6 and interferon-α. Systemic lupus erythematosus (SLE) is a complex autoimmune rheumatic disease, characterized by unpredictable exacerbations and remissions. Clinical manifestations are variable ranging from arthralgia, photosensitivity and the classic ‘butterfly’ rash to internal organ involvement, most notably renal and central nervous system disease. The preLupus nephritis remains a major cause of morbidity and mortality in SLE. There have been major improvements in the risk of premature mortality in patients with lupus nephritis. HoweverA retrospective review of lupus nephritis patients over a 30-year period 1975 to 2005) from a single center showed that five-year mortality decreased by 60% between the first and second decades of the study but remained unchanged over the third decade with rates of 17.2, 7.7 and 4.7%, respectively, after the diagnosis of renal disease[5 to 2005Over the last two decades, there have been tremendous advances in the understanding of the immunopathology of this autoimmune disorder. A variety of novel therapeutic targets have been identified and there have been many clinical trials in patients with SLE in an attempt to translate these new treatments into clinical practice. The results of these studies have been very mixed and there has been a steep learning curve for everyone involved in designing and executing these trials. SLE is a particularly challenging disease to study due to the broad spectrum of clinical manifestations and varying patterns of disease activity. Furthermore, disease specific outcome measures that were developed for use in observational clinical studies were exposed as inadequate when used in therapeutic clinical trials. This has led to the development of a composite outcome measure, the Systemic Lupus Erythematosus Responder Index (SRI), which has become the industry standard for lupus trials. AnotherGiven that autoantibody production is the hallmark of SLE, it is not surprising that B cell depletion therapy is a promising therapeutic option in the management of SLE. The main drug in current clinical practice is rituximab, with other drugs in development including epratuzumab. B cells, including the populations that interact with T cells, play an integral part in the autoimmune pathogenesis of SLE, and it is thought that after B cell depletion, disease activity may be modified and durable disease remission achieved, minimizing the use of other immunosuppressive agents and corticosteroids.+ B lymphocytes which results in complete B cell depletion[Rituximab is a chimeric anti-CD20 monoclonal antibody that has been used off-license in the management of severe refractory SLE since 2002. The mechanism of action of rituximab involves antibody-dependent cell toxicity (ADCC), complement-dependent cell toxicity (CDC) and direct apoptosis of CD20epletion. Plasma A recent review of the efficacy of rituximab in the management of SLE patients with biopsy-proven severe lupus nephritis from pooled data in European cohorts (n = 164) reported the clinical efficacy of rituximab in clinical practice. This opThe Study to Evaluate the Efficacy and Safety of Rituximab in Patients With Severe Systemic Lupus Erythematosus (EXPLORER) included patients with moderate to severe SLE but excluded lupus nephritis patients (n = 257). The EXPThe Study to Evaluate the Efficacy and Safety of Rituximab in Subjects with ISN/RPS Class III or IV Lupus Nephritis (LUNAR) trial compared rituximab plus MMF to MMF alone for the management of severe proliferative lupus nephritis class III and class IV. The published results did not show superiority of the rituximab combination therapy. As withThere are many possible explanations for the failure of the EXPLORER and LUNAR trials such as the relatively short trial duration and high doses of concomitant corticosteroids. Rituximab continues to be used off-label in a select group of patients with severe refractory SLE. This off-license use of rituximab takes into account the potential benefits reported from clinical practice and the possible complications of biologic therapy, such as severe or recurrent infections, adverse drug reactions and the few case reports of progressive multi-focal leuco-encephalopathy (PML),15.An additional benefit of rituximab induction therapy followed by MMF maintenance therapy for the management of severe proliferative lupus nephritis class III and class IV, is the ability to reduce and eventually withdraw corticosteroid therapy in patients who respond to treatment.A new treatment strategy termed the Rituxilup regimen has been pioneered in a center in the United Kingdom. The Rituxilup regimen avoids the use of concomitant oral corticosteroid therapy after rituximab induction therapy, thereby minimizing the duration of corticosteroid exposure and steroid side-effects. A propohttp://www.clinicaltrials.gov). This study is still at the development stage.RING – Rituximab for Lupus Nephritis With Remission as a Goal, an investigator-initiated randomized international open multicenter study, aims to determine the clinical effectiveness of rituximab in achieving complete renal remission in lupus nephritis patients with persistent proteinuria (≥1 grams/day) despite a minimum of six months of standard immunosuppressive therapy  n = 39, epratuzumab 800 mg cumulative dose  n = 38, epratuzumab 2,400 mg cumulative dose (600 mg weekly) n = 37, epratuzumab 2,400 mg cumulative dose  n = 37, epratuzumab 3,600 mg cumulative dose  n = 38.Epratuzumab at a cumulative dose of 2,400 mg was clinically effective and demonstrated a significant reduction in disease activity as measured by a composite disease activity score. Epratuzumab 600 mg weekly was associated with the greatest improvement in British Isles Lupus Assessment Group (BILAG)-2004 scores (from A/B to C/D) than placebo in all organ domains included in the study. Overall epratuzumab was well tolerated.Two randomized controlled trials evaluating the efficacy of epratuzumab in severe SLE as determined by the presence of BILAG A (RCT SL0003) and/or moderate patients with BILAG B in at least two systems (RCT SL0004) were discontinued due to irregularities in the manufacture of epratuzumab. The results of patients recruited in these trials were pooled and indicate the potential benefit of epratuzumab in facilitating a reduction in prescribed corticosteroid dose.http://www.clinicaltrials.gov). A phase III, multicenter, open-label, extension study to assess the safety and tolerability of epratuzumab treatment in SLE subjects EMBODY™4 started recruiting in July 2011 and is aiming to recruit 1,400 patients with a completion date of February 2016 .Two Phase III, randomized, double-blind, placebo-controlled, multicenter studies of the efficacy and safety of four 12-week treatment cycles  of epratuzumab in SLE subjects with moderate to severe disease EMBODY™1 & EMBODY™2 have an expected completion date of February 2014 with a recruitment of 780 patients. The main aim is to evaluate the efficacy, safety, tolerability and immunogenicity of epratuzumab in patients with moderate and severe SLE  trial was suspended[The BELONG study had recruited 381 lupus nephritis class III and class IV patients to study the clinical efficacy and safety of ocrelizumab 400 mg or ocrelizumab 1,000 mg administered at baseline, a fortnight later, then every four months thereafter. All lupus nephritis patients enrolled in the study were treated with either intravenous cyclophosphamide using the EuroLupus regimen or MMF and high-dose corticosteroids concomitantly. Week 42 data from 221 patients who had enrolled at least 32 weeks prior to study termination have been reported in abstract form and, although ocrelizumab is clinically effective in reducing lupus nephritis disease activity, the data have not demonstrated superiority to standard immunosuppression.Belimumab is a human immunoglobulin G1λ monoclonal antibody which blocks the binding of the soluble form of the cytokine B-lymphocyte stimulator (B-Lys), also known as B cell activating factor (BAFF), to the transmembrane activator/calcium modulator/cyclophilin ligand interactor (TACI) receptor, B-cell maturation (BCMA) receptor and BAFF receptor 3 (BR3) on B cells and thus interrupts the B cell survival role of B-Lys.BAFF/BLys is expressed by several cells including dendritic cells, monocytes, activated neutrophils and T cells. It is vital in facilitating the maturation and survival of B cells via signaling through the BAFF-R, BCMA and TACI receptors with high, intermediate and low affinity respectively. APRIL, a BAFF homologue proliferation-inducing ligand binds with higher affinity to the TACI receptor than BAFF. DimerizIn the presence of an excess amount of BAFF/BLys, low-affinity self-reactive B cells may survive and mature into self-reactive auto-antibody secreting plasma cells implicated in autoimmune disease pathogenesis. As a result, it has been deduced that the inhibition of BAFF/BLys by belimumab has therapeutic implications in SLE.In March 2011 the United States Food and Drug Administration (FDA) and the European Medicines Evaluation Agency (EMEA) licensed belimumab as the first new drug in over 50 years for SLE. Belimumab was licensed as a biologic agent to be prescribed with standard therapy for autoantibody-positive adult SLE patients excluding those with active lupus nephritis and central nervous system manifestations of SLE.Belimumab is administered on a weight-based dosing schedule of belimumab 10 mg/kg as an hour long intravenous infusion fortnightly for three infusions then monthly thereafter.A phase III randomized placebo-controlled trial Belimumab International SLE Study (BLISS-52) conducted between May 2007 and July 2009 included 865 SLE patients enrolled in Central and Eastern Europe, Latin America, and Asia Pacific. A phaseP = 0.013 and P = 0.0006, respectively)[The trial outcome at 52 weeks in BLISS-52 reported positive clinical response in 44% of those treated with placebo with standard therapy, 51% of those treated with belimumab 1 mg/kg with standard therapy and 58% of those treated with belimumab 10 mg/kg with standard therapy (ctively).P = 0.10 and P = 0.021, respectively)[The trial outcome at 52 weeks in BLISS-76 reported positive clinical response in 34% of those treated with placebo with standard therapy, 41% of those treated with belimumab 1 mg/kg with standard therapy and 43% of those treated with belimumab 10 mg/kg with standard therapy (ctively). Howeverhttp://www.clinicaltrials.gov).The BLISS-52 and BLISS-76 clinical trials both excluded patients with active lupus nephritis. BLISS-LN is a phase III, randomized, double-blind, placebo-controlled study to evaluate the efficacy and safety of belimumab plus standard of care versus placebo plus standard of care in adult subjects with active lupus nephritis which will provide clinically relevant information about the use of belimumab in lupus nephritis NCT01639339  reported lower clinical effectiveness in this group as compared to other ethnic groups.http://www.clinicaltrials.gov).A phase III/IV multi-center, randomized, double-blind, placebo-controlled, 52-week study to evaluate the efficacy and safety of belimumab in adult subjects of black race with SLE is planned as a future study NCT01632241 .In 2012, fatal anaphylaxis was reported in a patient treated with belimumab and it is now known that there is a risk of a delayed acute hypersensitivity reaction to belimumab, especially in patients with multiple drug allergies. Long-term observational data will provide further safety and tolerability data on belimumab. At present the FDA Center for Drug Evaluation and Research has reviewed the safety labeling for belimumab .Although belimumab received regulatory approval from the US FDA and the EMEA, its use in some countries has been restricted until approval by national drug evaluation organizations. The German Institute for Quality and Efficiency in Health Care (IQWiG) has recommended evaluation of belimumab for additional benefit over optimized immune-suppression rather than over standard therapy prior to full approval (http://www.nice.org.uk).In 2012 The National Institute for Health and Clinical Excellence (NICE) provided a draft national guidance on the use of belimumab for SLE in the United Kingdom. NICE did not recommend belimumab within its licensed indication as add-on therapy to standard immune-suppressive drugs in adult patients with active auto-antibody positive SLE. In making this decision, NICE considered the clinical trial evidence, clinical specialist and patient opinions. NICE concluded that the use of belimumab was not sufficiently cost-effective to the National Health Service (NHS) in relation to its reported clinical effectiveness. A final decision will be expected after the appeals process has been concluded .In 2012 approval was granted by the EMEA and FDA for phase III clinical trials of blisibimod, CHABLIS-SC1 and CHABLIS-SC2. These international multicenter, randomized, double-blind trials aim to evaluate the efficacy, safety, tolerability and immunogenicity of blisibimod in patients with severe active SLE (SELENA-SLEDAI >10) despite high-dose corticosteroids NCT01395745 .Tabalumab (LY2127399) is a human IgG4 monoclonal antibody targeting membrane-bound and soluble BAFF. A phase III, multicenter, randomized, double blind, placebo-controlled study to evaluate the efficacy and safety of subcutaneous LY2127399 in patients with SLE is expected to be completed in May 2015 (NCT01196091). Tabalumab is administered subcutaneously in addition to standard of care therapy for active SLE  and B7-2 (CD86). CTLA4 (cytotoxic T-lymphocyte antigen) is expressed on activated T cells and interacts with B7 with higher affinity than CD28 resulting in a negative feedback mechanism that inhibits T cell activation-32. AbatA 12-month Phase II/III double-blind placebo controlled trial in proliferative lupus nephritis failed to meet its primary end-point of time to complete renal response as defined as glomerular filtration rate within 10% of pre-flare/screening value, urinary protein creatinine ratio <0.26 mg/mg and inactive urinary sediment. HoweverCD40 ligand (CD40L) is a transmembrane glycoprotein belonging to the tumor necrosis factor (TNF) super family which binds with CD40 on the surface of B-cells and macrophages. The interaction between CD40/CD40L plays a pivotal role in B-cell class switching. CD40L iIL-6 is a pleiotropic cytokine with both pro-inflammatory and anti-inflammatory properties and has been implicated in the pathogenesis of lupus nephritis. Exogenous IL-6 increases autoantibody production and accelerates progression of nephritis in both the NZB/NZW and BXSB lupus mouse models,44. Treahttp://www.clinicaltrials.gov).Tocilizumab is a fully humanized monoclonal antibody against the IL-6 receptor and prevents binding of IL-6 to both membrane bound and soluble IL-6 receptor. A phase I trial over a 12 week period has demonstrated the safety and tolerability of tocilizumab in SLE patients. While active urinary sediment and anti-dsDNA antibody titres were reduced, proteinuria remained unchanged. The shoRecent studies of SLE patients and data from murine models of lupus, suggest that inappropriate activation of type I IFNs play an essential role in SLE pathogenesis. Microarray gene expression analysis has shown widespread activation of IFN-inducible genes in SLE patients which correlates with disease activity,53. In ahttp://www.clinicaltrials.gov). A Phase II clinical trial evaluating rontalizumab, a recombinant humanized monoclonal antibody to IFN-α for SLE is also ongoing (NCT00962832) .Sifalimumab, a fully human anti-IFN-α monoclonal antibody, induced a dose-dependent inhibition of type I IFN-induced mRNAs (type I IFN signature) in whole blood in a phase I study. No increase in viral infections was noted and a general trend towards improvement in disease activity was seen. Furtheret al. entitled ‘Efficacy and Safety of Rontalizumab (Anti-Interferon Alpha) in SLE Subjects with Restricted Immunosuppressant Use: Results of a Randomized, Double Blind, Placebo-Controlled Phase 2 Study’ was presented at the American College of Rheumatology Annual Scientific Conference in November 2012.The efficacy and safety of rontalizumab, a recombinant humanized monoclonal antibody to IFN-α was recently assessed in a randomized, double-blind, placebo-controlled phase II trial in adults with moderate to severe non-renal SLE. An abstract by Kalunian K In the initial part of the study, SLE patients received either 750 mg intravenously of rontalizumab or placebo for four weeks. In the second part of the study, SLE patients received either 300 mg subcutaneously of rontalizumab or placebo for two weeks. Overall, response rates at 24 weeks as measured by BILAG and SRI were similar between rontalizumab and placebo. However, in patients taking >10 mg/kg of steroids daily, rontalizumab was more effective in reducing lupus disease activity than placebo. Patients were further analyzed as per their IFN gene expression signature, which showed that rontalizumab was more effective in those with a more elevated IFN signature.The complement system plays an important role in the pathophysiology of SLE although individual complement components have distinct and varied functions in the disease process. Early components of the complement cascade are critical in the clearance of immune complexes and apoptotic material. Their absence in congenital C3 or C4 deficiency predisposes individuals to development of SLE. Activation of terminal complement components is associated with exacerbations of disease, particularly in lupus nephritis.Monoclonal antibodies that specifically inhibit terminal complement activation while preserving early complement function have been developed. Eculizumab, a monoclonal antibody directed against the complement protein C5, inhibits the cleavage of C5 to C5a and C5b and thus blocks the formation of the terminal membrane attack complex C5b-9. Anti-C5Fcγ receptors are a heterogeneous group of hematopoietic cell surface glycoproteins that recognize the Fc portion of specific Ig isotypes, facilitating antibody-antigen interactions with effector cells and, thus, play a key role in the clearance of immune complexes. Fcγ rechttp://www.clinicaltrials.gov).Laquinimod is an oral quinoline-3-carboxamide small molecule which to date has been mainly investigated in the context of relapsing-remitting multiple sclerosis (MS). In MS, laquinimod biases the CD4+ phenotype in favor of Th2/Th3 cytokine production and inhibits disease development and infiltration of inflammatory cells into the CNS,59. LaquTofacitinib is a Janus kinase (JAK) selective inhibitor which has been approved as the first oral biologic for the management of rheumatoid arthritis. JAKs are essential for signal transduction of cytokines and contribute to inflammatory responses. TargetiSpleen tyrosine kinase (Syk) is implicated in the B cell immunopathogenesis of SLE and is a potential therapeutic target. Syk inhibitors have been shown to prevent the onset of skin and renal disease in lupus-prone mice. In addition, Syk inhibitors reduce inflammatory arthritis. Fostamatinib is an oral Syk inhibitor being evaluated for the management of autoimmune rheumatic diseases.The management of SLE is likely to change significantly with the introduction of new biological therapies and the discovery of other therapeutic targets. The exact role of these drugs will be determined after completion of the trials and with clinical experience. It is envisioned that the majority of the biological therapies will initially be reserved for patients who have failed to respond satisfactorily to optimal conventional immunosuppressive drugs. The new biological drugs will need to be used appropriately to target disease remission; reduction of the disease severity; frequency of lupus flares and the subsequent high morbidity associated with lupus.Conventional immunosuppressive therapies have radically transformed patient survival in SLE, but their use is associated with considerable toxicity and a substantial proportion of patients remain refractory to treatment. A more comprehensive understanding of the complexity of SLE immunopathogenesis has evolved over the past decade and has led to the testing of several biologic agents in clinical trials. An array of promising new therapies are yet to emerge or are under development. There is a clear need for new therapeutic strategies that overcome these issues, and biologic agents offer exciting prospects as future SLE therapies. The role of new therapeutic agents to date has chiefly centered on SLE patients who have been refractory to conventional therapies. There are few clinical trials examining their role as first line induction or maintenance therapy. Questions remain as to how these therapies can potentially be combined with existing proven treatments and indeed with one another to achieve maximum clinical benefit while minimizing toxicity. Although so far many biologics have been generally well tolerated, we must not be complacent regarding potential toxicity of these new agents, as we do not yet know the long-term effects of these medications on the immune system. Rituximab is currently used off-license for the management of severe refractory SLE and is likely to continue to be used for this indication due to overall positive clinical experience.Based on the clinical trial and extension study data, belimumab has a modest level of clinical effectiveness when used in combination with standard immunosuppressive drugs in autoantibody-positive SLE patients. The BILAG data at week 52 of the BLISS trials suggested more favorable outcomes in the mucocutaneous, musculoskeletal domains. The SELENA-SLEDAI cutaneous, musculoskeletal, immunologic, vascular and CNS components significantly improved at week 52 in the BLISS trials. Physicians will, therefore, be inclined to closely monitor patients on belimumab and switch to alternate therapeutic regimens if the clinical response is inadequate after six months. SLE patients of black ethnicity are to be studied in greater numbers than in the original BLISS trials in order to ascertain whether or not belimumab is beneficial in this group of patients. As belimumab use becomes more prevalent and the results of the on-going belimumab clinical trials are published, the group of SLE patients likely to benefit the most from this drug may be identified and this will guide future use of this medication.The place for other therapeutic agents in development for the management of SLE, such as epratuzumab, blisibimod, tabalumab and atacicept, as induction or maintenance therapies will be determined after robust reviews of the clinical trial data which are expected upon completion of the studies. It is anticipated that only drugs which show long-term clinical effectiveness, benefit as steroid-sparing agents and satisfactory safety profiles in SLE will gain approval for clinical use.Some novel biologic therapies have been associated with significant toxicity leading to premature discontinuation of clinical trials such as the association of anti-CD40L and thrombo-embolic events and the high frequency of reported severe and opportunistic infections associated with ocrelizumab. Although some drugs have not progressed to phase II or III clinical trials after phase I studies, research into cytokine therapies, drugs targeting FcγRIIB and small molecule targets is on-going and may yield important results for the future of SLE management.Health economic studies will be essential in determining the future use of the new therapeutic agents in SLE and may influence the international use of these drugs.A number of key questions remain. How can these therapies be potentially combined with existing proven treatments and indeed with one another to achieve maximum clinical benefit with minimal side-effects, such as increased risk of serious infection. As is clear to all physicians involved in the day to day management of SLE patients, this is a heterogeneous disease and there is not one therapeutic regimen suitable for all. With a deeper understanding of the pathophysiology of SLE particularly from a genetic perspective, the era of personalized therapy may represent the greatest advance that is yet to come in optimizing treatment of SLE.Conventional immunosuppressive therapies have radically transformed patient survival in SLE, but their use is associated with considerable toxicity and a substantial proportion of patients remain refractory to treatment. A more comprehensive understanding of the complexity of SLE immunopathogenesis has evolved over the past decade and has led to the testing of several biologic agents directed against new molecular targets in clinical trials. An array of promising new therapies is yet to emerge or is under development. There is a clear need for new therapeutic agents that overcome these issues, and biologic agents offer exciting prospects as future SLE therapies. Several challenges still remain in designing clinical trials in SLE. One of the main issues is that conventional therapies have been optimized and are effective in the majority of patients. There is, therefore, quite a high bar for new therapies to demonstrate a significant benefit over conventional approaches and progress is likely to be incremental rather than revolutionary.The role of new therapeutic agents has chiefly centered on SLE patients who have been refractory to conventional therapies. There are few clinical trials examining their role as first line induction or maintenance therapy. Questions remain around how these therapies can potentially be combined with existing proven treatments and indeed with one another to achieve maximum clinical benefit while minimizing toxicity. As is clear to all physicians involved in the day to day management of SLE patients, this is a heterogeneous disease and there is no single therapeutic regimen suitable for all. With a deeper understanding of the pathophysiology of SLE particularly from a genetic perspective, the era of personalized therapy may represent the greatest advance that is yet to come in optimizing treatment of SLE.ADCC: Antibody-dependent cell toxicity; ANA: Anti-nuclear antibody; BAFF: B cell activating factor; BCMA: B cell maturation; BLys: B lymphocyte stimulator; BILAG: British Isles Lupus Assessment Group; CDC: Complement-dependent cell toxicity; CNS: Central nervous system; CTLA4: Cytotoxic T-lymphocyte antigen; dsDNA: Anti-double stranded DNA antibody; EMEA: European Medicines Evaluation Agency; ESRD: End-stage renal disease; FDA: United States Food and Drug Administration; IFN: Interferon; Ig: Immunoglobulin; IL-6: Interleukin-6; IL-10: Interleukin-10; IV: Intravenous; JAK: Janus kinase; MMF: Mycophenolate mofetil; PML: Progressive multifocal leuco-encephalopathy; RCT: Randomized controlled trials; SC: Subcutaneous; SELENA-SLEDAI: Safety of estrogens in lupus erythematosus national assessment trial systemic lupus erythematosus disease activity index; SF-36: Short form 36; SLE: Systemic lupus erythematosus; Syk: Spleen tyrosine kinase; SRI: Systemic lupus erythematosus responder index; TACI: Transmembrane activator/calcium modulator/cyclophilin ligand interactor; TNF: Tumor necrosis factor.NJ and PL have no competing interests. DDC has received consulting fees and/or has participated in clinical trials for Glaxo-SmithKline, Bristol Myers Squibb, Roche and Eli-Lily.NJ joined the lupus team at St Thomas’ Hospital in 2009 and was subsequently awarded an Arthritis Research UK clinical research fellowship to undertake a PhD focusing on lupus nephritis. Her research is based at the Centre for Molecular and Cellular Biology of Inflammation at King’s College London and she continues to work as a clinician at the Louise Coote Lupus Unit primarily in the areas of lupus nephritis and vasculitis. PL joined the lupus team at St Thomas’ Hospital in 2010 as a specialist registrar in Rheumatology and is currently a clinical research fellow at the Peter Gorer Department of Immunobiology King’s College London and Lupus Unit St Thomas’ Hospital studying the effects of B cell depletion therapy on lymphocyte subsets in lupus and vasculitis. DDC is clinical team lead for the Louise Coote Lupus Unit and is Professor of Lupus Biology and Consultant Rheumatologist.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/11/120/prepub


Drosophila species. DNA structure is associated with the binding of chromatin remodelers and histone modifiers to DNA sequences in coding regions, which influence RNA polymerase II occupancy that controls gene expression level. We also found that genes with similar DNA structures are involved in the same biological process and function. These results reveal the previously unappreciated roles of DNA structure as cis-effects in gene expression.Sequence changes in coding region and regulatory region of the gene itself (cis) determine most of gene expression divergence between closely related species. But gene expression divergence between yeast species is not correlated with evolution of primary nucleotide sequence. This indicates that other factors in cis direct gene expression divergence. Here, we studied the contribution of DNA three-dimensional structural evolution as cis to gene expression divergence. We found that the evolution of DNA structure in coding regions and gene expression divergence are correlated in yeast. Similar result was also observed between  The unique phenotype of each organism is partly determined by gene expression. Changes in gene expression are an important source of phenotypic variation, and can be caused by changes in regulatory and coding sequences of the gene itself (cis) and changes in regulatory factors (trans). The contribution of cis regulation to gene expression divergence between closely related species is much greater than that of trans regulation. However, evolution of primary nucleotide sequences is not correlated with gene expression divergence in yeast, suggesting that other factors in cis drive gene expression divergence. Here, we found that evolution of DNA structure in coding regions is coupled to gene expression divergence in yeast. We also found that DNA structure is associated with specific gene characteristics. Genes with similar DNA structures are involved in the same biological process and function. These results demonstrate the important roles of DNA structure in directing gene expression. Proper control of gene expression is central for the unique phenotype of each organism. Phenotypic diversity can be generated through changes in gene expression. Divergence in gene expression of a specific gene between closely related species can result from sequence changes in its coding region and regulatory region (cis), or from changes in sequences or expression of its direct or indirect upstream regulators (trans). The binding of transcription factors (TFs) to sequence-specific sites in gene upstream regions plays a very important role in regulation of gene expression. Changes in TF-binding sequences and changes in abundance and binding domains of TFs can influence TF binding, which may cause variation in gene expression. The divergence of gene expression is also coupled to that of gene sequences in multicellular organisms Drosophila species is much greater than that of trans regulation The effects of cis and trans regulation on gene expression divergence can be measured by comparison of different strains of the same species The three-dimensional structure of DNA, which reflects the physicochemical and conformational properties of DNA, is critical for the packaging of DNA in the cell A recent study has found that DNA structure in the human genome is more evolutionary constrained than the primary nucleotide sequence alone Drosophila species. The relationships of various types of structural evolution with gene expression divergence are conserved between yeast and Drosophila. We next investigated whether DNA structure is associated with gene characteristics. Genes that differ in DNA structure are distinguished by chromatin remodeler occupancy and histone modification levels, indicating that DNA structure influences gene expression by regulating the binding of chromatin regulators to DNA. Genes with similar DNA structures tend to belong to the same biological process and function.We evaluated DNA structure in terms of various physicochemical and conformational properties. We found that high levels of cis-driven gene expression divergence between yeast species correspond to high evolution rates of DNA structure in coding regions. This result also holds true between Saccharomyces cerevisiae and Saccharomyces paradoxusWe examined the role of primary nucleotide sequence evolution in cis-driven gene expression divergence. Although a previous study has already found that gene expression divergence is not correlated with evolution of gene sequence in yeast First, we tested the relationship between sequence evolution in upstream regulatory regions and cis-effects to gene expression divergence. TF-binding sequences in promoter regions are the best-characterized elements that regulate gene expression. A previous study has analyzed the conservation of TF-binding sequences in promoters of closely related yeast species and identified the loss of TF-binding sites Second, we investigated into the contribution of sequence evolution in 3′ untranslated regions (UTR) to cis-driven gene expression divergence. Cis-regulatory elements in 3′ UTR are crucial for controlling RNA stability and expression. A previous study has calculated the evolutionary conservation of 3′ UTR cis-regulatory elements between closely related yeast species S. cerevisiae and S. paradoxus, differences in their expression reflect cis effects on gene expression divergence S. cerevisiae and S. paradoxus than the other genes, the statistical significance is rather weak  and clustering analysis. As most (32 out of 35) of the structural scales are based on dinucleotide, we performed the two analyses above on the dinucleotide structural scales. Considering that the dinucleotide and its reverse complement have the same parametric value for a single structural property, there are only 10 unique dinucleotides. We first performed a PCA calculating the 32 principal components for the 10 dinucleotides. Only the first 9 principal components (PCs) carry relevant information, roughly indicating that about this low number of scales is needed to represent all information of the complete set of 32 scales. As the first 5 PCs carry ∼88% of information , we next clustered the 32 scales into 5 classes using K-means clustering . Each scS. cerevisiae and S. paradoxus, we calculated DNA structural evolution rate for each of the 35 DNA structural scales  in randomized experiments are weaker than that on the realistic profile , roll (free DNA) and slide (DNA-protein complex), show significant positive correlation with gene expression levels. Following the definitions of the structural parameters in the EMBO workshop We further investigated into how DNA structure influences gene expression. As chromatin remodeler occupancy and histone modification levels in coding regions influence gene expression, we examined the relationship of DNA structural levels with these two chromosomal features. First, we used genome-wide occupancy data for chromatin remodelers in vitro nucleosome occupancy that is determined only by the intrinsic DNA sequence in vitro nucleosome occupancy have high sequence preference for nucleosome formation, while sequences covered by low in vitro nucleosome occupancy inhibit nucleosome formation. We found that DNA structural levels are significantly correlated with in vitro nucleosome occupancy in coding regions: some structural scales facilitate nucleosome formation, while others inhibit nucleosome formation . Yeast transcript coordinate data were taken from David et al. S. cerevisiae and S. paradoxus were taken from Wapinski et al. D. melanogaster and D. simulans were taken from Heger et al. S. cerevisiae and S. paradoxus were taken from Tirosh et al. S. cerevisiae and S. bayanus were taken from Bullard et al. S. cerevisiae were taken from Holstege et al. D. melanogaster and D. simulans were taken from Ranz et al. D. melanogaster and D. sechellia were taken from McManus et al. Yeast genome sequences and gene coordinate were downloaded from the P-value The conservation of sequence motifs in promoters of closely related yeast species was analyzed and the loss of TF-binding sites was predicted by Doniger et al. in vivo and in vitro nucleosome occupancy data in S. cerevisiae were taken from Kaplan et al. in vivo and in vitro nucleosome occupancy in coding region for each gene, respectively. Genome-wide RNA polymerase II occupancy (RNA polymerase II subunit Rpo21) data in S. cerevisiae were taken from Venters et al. S. cerevisiae. We added the histone modification data from Pokholok et al. Genome-wide We performed the global alignment on gene sequences between orthologous genes. We used the rate of nonsynonymous substitutions (Ka) normalized by the rate of synonymous substitutions (Ks) as a measure of gene sequence evolutionary rate.S. cerevisiae and S. paradoxus, we calculated their absolute value of difference in CAI values, and defined the resulting value as its CAI divergence. We compared genes with significant cis-effects to gene expression divergence with the other genes in CAI divergence.We used the codon adaptation index (CAI) to indicate codon bias. We calculated CAI for each gene as a previous method We used 35 types of conformational and thermodynamic DNA di- or trinucleotide structural scales, which were mainly collected by two references For a DNA region, the sequence is divided into overlapping di- or trinucleotide sequences. Structural profiles from DNA sequences are calculated for each structural scale  as follows: The corresponding parametric value for each di- or trinucleotide was assigned to the first nucleotide of the di- or trinucleotide. In this way, the nucleotide sequence is converted into a sequence of numbers . For hydroxyl radical cleavage intensity data, structural profiles are calculated as the reference where the data was published Partial correlation can measure the degree of association between two variables with the effect of controlling variables removed. The DNA structural evolution rates in microarray probes which were used to measure gene expression divergence are calculated as follows. For each probe, we profiled the values of each specific structural scale versus its sequence positions, and called this graph its structural profile of this structural scale. For each pair of orthologous genes, we calculated the Euclidean distance between structural profiles of their two probes, and used the resulting values normalized by the length of the probe as a measure of evolution rate of DNA structure. For orthologous genes with more than one pair of probes, we calculated the Euclidean distance normalized by the length of the probe for each pair of probes, and used the average resulting distance value as a measure of DNA structural evolution rate. In this way, there were 35 measures of structural evolutionary rate in probe regions for each pair of orthologous genes.To evaluate the microarray probe bias on the measurement of chromatin remodeler occupancy, we calculated for each coding region the average structural value of each structural scale across its coding regions after excluding the sequences of its microarray probe. The resulting DNA structure values are still significantly correlated with chromatin remodeler occupancy (data not shown).For each probe in microarray that were used to measure histone modification level, we calculated the average structural value of each structural scale across its sequence positions. We found that histone modification levels are weakly correlated with the DNA structures in probe regions  Box plot of average values that correspond to levels of cis-effects on gene expression divergence are shown for genes with loss of TF-binding sites and genes without loss of TF-binding sites. (B) Box plot of average values that correspond to absolute values of pair-wise difference in levels of cis-effects to gene expression divergence are shown for divergent gene pairs and the other gene pairs. Values were normalized using the function (JPG)Click here for additional data file.Figure S2zscore in Matlab, such that their means are zero and standard deviations are one.The relationship between changes in primary nucleotide sequences of 3′ UTR and gene expression divergence. Box plot of average values that correspond to levels of cis-effects on gene expression divergence are shown for genes whose 3′ UTR cis-regulatory sequences are less conserved (divergent) and genes with conserved 3′ UTR cis-regulatory sequences. Values were normalized using the function (JPG)Click here for additional data file.Figure S3zscore in Matlab, such that their means are zero and standard deviations are one. We performed the global alignment on orthologous gene sequences between S. cerevisiae and S. paradoxus, and used the rate of nonsynonymous substitutions (Ka) normalized by the rate of synonymous substitutions (Ks) as a measure of gene sequence evolutionary rate.The relationship between changes in primary nucleotide sequences of coding regions and gene expression divergence. Box plot of average values that correspond to gene sequence evolutionary rates are shown for genes with significant cis-effects to gene expression divergence and the other genes. Values were normalized using the function (JPG)Click here for additional data file.Figure S4S. cerevisiae and S. paradoxus, we calculated gene sequence evolutionary rates and DNA structural evolution rate for each of the 35 DNA structural scales. The Pearson correlation coefficient between sequence evolutionary rates and structural evolution rates is shown for each of the 35 structural scales.The correlation of DNA structural evolution rates with primary nucleotide sequence evolution rate. For each pair of orthologous genes between (JPG)Click here for additional data file.Figure S5The number of structural scales in which each of the genes with significant cis-effects to gene expression divergence shows significantly high evolution rates ((JPG)Click here for additional data file.Figure S6Partial correlation of DNA structural evolution rate with cis-driven gene expression divergence is shown for each of the 35 DNA structural scales when controlling primary nucleotide sequence evolution rates. Each bar represents the resulting partial correlation coefficients.(JPG)Click here for additional data file.Figure S7S.cerevisiae and S. paradoxus with DNA structural evolution in microarray probe regions. We compared the difference in evolution rates of 35 DNA structural scales in microarray probe regions between genes with significant cis-effects to gene expression divergence and the other genes. P-values were calculated through Mann-Whitney U-test, and are shown for the 35 DNA structural scales. Red (green) indicates high (low) P-values that evaluate the difference.The relationship of cis-driven gene expression divergence between (JPG)Click here for additional data file.Figure S8S. cerevisiae and S. paradoxus with DNA structural evolution when restricting analysis to genes whose probe sequences have low structural evolution rates (the 50% percentile). For each of the 35 DNA structural scales, we excluded genes whose probe sequences have high structural evolution rates (the 50% percentile), and compared the difference in DNA structural evolution rate between genes with significant cis-effects to gene expression divergence and the other genes in their coding regions. P-values were calculated through Mann-Whitney U-test, and are shown for the 35 DNA structural scales. Red (green) indicates high (low) P-values that evaluate the difference.The relationship of cis-driven gene expression divergence between (JPG)Click here for additional data file.Figure S9S. cerevisiae and S. bayanus with DNA structural evolution. We compared the difference in evolution rates of 35 DNA structural scales between genes with significant cis-effects to gene expression divergence and the other genes in their coding regions. P-values were calculated through Mann-Whitney U-test, and are shown for the 35 DNA structural scales. Red (green) indicates high (low) P-values that evaluate the difference.The relationship of cis-driven gene expression divergence between (JPG)Click here for additional data file.Figure S10Drosophila species with DNA structural evolution. We compared the difference in evolution rates of 35 DNA structural scales between genes with significant cis-effects to gene expression divergence and the other genes in their coding regions. Comparison was also performed after normalizing DNA structural evolution rates by gene sequence evolution rates. P-values were calculated through Mann-Whitney U-test, and are shown for the 35 DNA structural scales. Red (green) indicates high (low) P-values that evaluate the difference.The relationship of cis-driven gene expression divergence in (JPG)Click here for additional data file.Figure S11The correlation of DNA structural levels in coding regions with transcription rates and RNA polymerase II occupancy in coding regions. Each bar represents the resulting Pearson correlation coefficients for the 14 DNA structural scales. 12 out of the 14 scales show significant correlation with these two features ((JPG)Click here for additional data file.Table S1List of dinucleotide/trinucleotide DNA structural scales and their corresponding parameters.(XLS)Click here for additional data file.Table S2P-value from Mann-Whitney U-test, which was performed between genes with significant cis-effects to gene expression divergence and the other genes, to represent the degree of contribution of this type of structural evolution to gene expression divergence.The correlation in the contribution of structural evolution to gene expression divergence among Drosophila and yeast species. For each type of structural evolution, we used the (XLS)Click here for additional data file.Table S3The correlation of DNA structural levels with histone modification levels in coding regions. The Pearson correlation coefficients are shown.(XLS)Click here for additional data file.Table S4P-values for Gene Ontology terms were derived using ‘GO term finder’ at the Saccharomyces Genome Database.The gene clusters which are clustered based on their DNA structural levels in coding regions. For each of the 14 structural scales, all genes are clustered into 5 groups based on their corresponding DNA structural levels, respectively. Biological processes and functions that the each gene cluster belongs to are shown. (XLS)Click here for additional data file.


Cytokines play a central role in the pathogenesis of many diseases, including HIV infection. However, the role of the cytokine network in early HIV infection is only now starting to be elucidated. A number of studies conducted in recent years have indicated that cytokines of the acute/early stages of HIV and SIV infection can impact viral set-point months later, and this is of critical importance since viral set-point during chronic HIV infection affects virus transmission and disease progression. This raises the question whether modulating the cytokine environment during acute/early HIV infection can be a target for novel approaches to develop a vaccine and therapeutics. In this review we focus on the kinetics and function of cytokines during acute HIV and SIV infection and how these may impact viral set-point. We also discuss unresolved questions that are essential for our understanding of the role of acute infection cytokines in HIV infection and that, if answered, may suggest novel therapeutic and vaccine strategies to control the worldwide HIV pandemic. Early events during acute HIV infection may determine progression and pathogenesis of infection, as the immunological milieu of the initial antigen encounter appears critical in dictating the long-term equilibrium between the host and the pathogen The complexity of the role of the cytokine milieu in acute HIV and SIV infection has only partially been addressed. The first reports examining cytokines in acute HIV infection were conducted in patients with symptomatic acute infection . Cytokine mRNA declined in parallel with the decrease of viral replication after day 14, suggesting that the virus directly or indirectly drives much of the cytokine production A more precise timing of cytokine kinetics, however, can be done in SIV-infected non-human primates. Several such studies have been conducted of very early SIV infection in non-human primates to analyze the cytokine production during the first weeks of infection and compare differences between non-pathogenic and pathogenic infections Comparison of cytokine gene expression profiles and kinetics in the non-pathogenic SIV infection of natural hosts (African green monkeys and sooty mangabeys) with pathogenic SIV infection of non-natural hosts (rhesus macaques and pigtailed macaques) can be used to link cytokine production with pathogenesis. IFNα, TGFβ, and IL-10 mRNA and protein expression is increased earlier in non-pathogenic SIV infection compared to pathogenic SIV infection The source and location of cytokine production during acute HIV/SIV infection is only partially understood. Cytokines in the very early phase of HIV and SIV infection are most likely produced locally, whereas after virus dissemination, cytokines are produced by more generalized and distant sites such as genital draining and distal LNs and gut-associated lymphoid tissue . However, at the same time, these cytokines can also increase the target cell pool for HIV/SIV by recruiting and activating CD4+ T cells, the primary targets for HIV/SIV infection. Immunosuppressive cytokines can dampen anti-viral immunity but also reduce inflammation and decrease the target cell pool of activated CD4+ T cells. Most cytokines exert pleiotropic and sometimes contrasting effects on the immune response and viral replication as suggested by a number of studies. Examples of these are pro-inflammatory cytokines such as TNFα that are produced during acute HIV/SIV infection A central question regarding cytokines during acute HIV/SIV infection is whether these cytokines are beneficial or detrimental for the host. Cytokines can upregulate or accelerate the anti-viral immune response, and this could contribute to viral control Recent studies have suggested that cytokines during acute HIV/SIV infection are important determinants of the viral set-point of chronic infection An association of acute infection cytokines with viral set-point was recently suggested in HIV infection. Higher plasma IL-7 and IL-15 levels during acute/early HIV infection appear detrimental and correlate with higher viral set-points, while conversely, plasma IL-12 and IFNγ levels appear beneficial and associate with lower viral set-points One prediction from the above human studies would be that addition of cytokines during acute SIV infection would alter viral set-point months later. Such studies have been conducted and remarkably agree with these predictions based on plasma level correlations in HIV infection. Treatment of acute SIV infection with simian IL-15 resulted in a dramatic 1,000-fold increase in viral set-point That cytokines present early during acute infection can affect viral set-point is further supported by a study testing whether the induction of antiviral cytokines at the vaginal mucosa would prevent SIV infection. The basic premise for this study was that Toll-like receptor (TLR) 7 and 9 agonists applied intravaginally would stimulate antiviral cytokines and induce an antiviral state in the vaginal mucosa. As expected, these agonists upregulated mRNA and protein for several cytokines in vaginal secretions, including IFNα, TNFα, IL-6, IP-10, and IFNγ The potential mechanisms by which acute HIV/SIV infection cytokines affect viral set-point are suggested by the above studies. Although the precise mechanism by which IL-15 is affecting viral set-point is not known, effects on host immunity and/or infection of target cells are suspected, as IL-15 decreases anti-SIV antibody responses but enhances the homeostatic proliferation of predominantly memory CD4+ T cells during acute infection, and both correlate with increased viral set-point From the data discussed above, it is clear that without a better understanding of the complex interactions between cytokines and the host during acute HIV/SIV infection, the accurate prediction of the effect of cytokine interventions will be difficult . A numbeSome of these questions can now be answered, as assays to measure multiple cytokines in non-human primates are being developed and validated. To investigate the cytokine profile in plasma and tissues, the cell type source and the production location will require a concerted effort of multiple laboratories specializing in non-human primate studies. Importantly, understanding the role and function of individual cytokines in determining viral set-point will require the inhibition or addition of individual cytokines during acute SIV infection. These types of experiments require significant resources for the production and validation of cytokines and blocking reagents. Finally, both extensive immunological and virological studies need to be conducted in the setting of cytokine administration or inhibition during acute SIV infection to determine the mechanism by which viral set-points are altered.Leishmania and Mycobacterium tuberculosis are formulated in oil-in-water emulsions they primarily induce Th2 responses, whereas the addition of TLR4 agonists such as monophosphoryl lipid A or glycopyranosyl lipid Findings from studies above can help identify cytokines that can be targeted to alter HIV viral set-point, transmission, and disease progression. This could lead to cytokines or cytokine inhibitors being used as pharmacological agents in acute/early infection with the goal of altering the course of infection and lowering viral set-points. Cytokine hierarchies have been exploited therapeutically in patients with rheumatoid arthritis (RA) and inflammatory bowel disease (IBD) where blocking TNFα, a key driver of the inflammatory cytokine network in RA and IBD, has a major impact on inflammation We have reviewed above the evidence that viral set-point can be manipulated by cytokine interventions during acute HIV/SIV infection. This raises the question whether we can target cytokines during acute HIV infection to alter viral set-point through immunotherapeutics and potentially vaccines. Treating with cytokines or blocking cytokines is clearly feasible but will depend on the window of opportunity after initial HIV exposure, during which such treatments are still effective at lowering viral set-point. Non-sterilizing vaccine strategies that alter the cytokines produced during acute infection will be challenging, but the reward of a vaccine that reduces viral set-point below transmission levels would be tremendous. Further understanding of the cytokine network and the potential cytokine hierarchies during acute HIV/SIV infection may uncover critical cytokines that control viral set-point. This may not only provide fundamental insight into the poorly understood mechanisms that control viral set-point but could instruct the development of novel immunotherapeutics or vaccine adjuvants against HIV.


Kaposi sarcoma-associated herpesvirus (KSHV) is a human γ-herpesvirus associated with several human malignancies. The replication and transcription activator (RTA) is necessary and sufficient for the switch from KSHV latency to lytic replication. Toll-interleukin-1 receptor (TIR) domain-containing adaptor-inducing β-interferon ) is a signaling adaptor molecule that is critically involved in the Toll-like receptor 3 (TLR-3) and TLR-4 signaling pathways for type I interferon (IFN) production, a key component of innate immunity against microbial infection. Previously we have identified that RTA blocks TLR3 signaling activation by degrading cellular TRIF, and this RTA-mediated degradation is at least partially mediated through the ubiquitin-proteasome pathway. In this report, we have identified a new mechanism that innate immunity regulates KSHV replication. We find that TRIF increases the expression of KSHV RTA. The enhancement of RTA expression and the degradation of TRIF are two independent pathways. TRIF specifically enhances the translation efficiency of RTA mRNA. Because RTA may not directly interact with TRIF, the functional interactions between TRIF and RTA may be indirect through unknown mediators.Taken together, these data suggest that KSHV employs a novel mechanism to block the innate immunity by degrading TRIF protein, and at the same time, use the innate immune system to boost viral replication by increasing the expression of KSHV RTA. This regulatory circuit may be an important part of the KSHV-host interactions for the initial infections. This work may contribute to our understandings on how KSHV interacts with the host immune system for its survival in vivo.


Implicit learning may be shown by improvements in motor performance, which occur unconsciously with practice and are typically restricted to the task that was practiced. The purpose of this study was to examine behaviorally relevant brain activation associated with change in motor behavior during sequence-specific motor learning of a perceptuomotor continuous tracking (CT) task in middle-aged adults. To gain further insight into the neural structures associated with change in motor behavior, overall improvement in tracking  was decomposed into two components—temporal precision and spatial accuracy. We hypothesized that individual differences in CT task performance would be evident in unique networks of brain activation that supported overall tracking behavior as well-temporal and spatial tracking accuracy. A group of middle-aged healthy individuals performed the CT task, which contains repeated and random segments for seven days. Functional magnetic resonance imaging (fMRI) data was collected on the first and seventh day while the participants performed the task. Subjects did not gain explicit awareness of the sequence. To assess behaviorally-relevant changes in the blood oxygenation level-dependent (BOLD) response associated with individual sequence-specific tracking performance, separate statistical images were created for each participant and weighted by the difference score between repeated and random performance for days 1 and 7. Given the similarity of performance for random and repeated sequences during early practice, there were no unique networks evident at day 1. On Day 7 the resultant group statistical fMRI image demonstrated a positive correlation between RMSE difference score and bilateral cerebellar activation (lobule VI). In addition, individuals who showed greater sequence-specific temporal precision demonstrated increased activation in the precentral gyrus, middle occipital gyrus, and putamen of the right hemisphere and the thalamus, cuneus, and cerebellum of the left hemisphere. Activation of this neural network further confirms its involvement in timing of movements as it has been previously associated with task performance when individuals are instructed to emphasize speed over accuracy. In the present study, behavioral performance was associated with neural correlates of individual variation in motor learning that characterized the ability to implicitly learn a sequence-specific CT task. Implicit learning supporting the performance of complex motor sequences can be achieved in the absence of conscious knowledge of what was learned or that any learning took place  studies of implicit learning is differences in experimental designs. For example, SRT task practice of shorter repeated sequences (less than 12 elements) can be acquired explicitly . We created sequence-specific weighted images to evaluate the correlations between increased brain activation during repeated sequence performance and the behavioral change score at a delayed retention test. We hypothesize that positive difference scores from repeated to random performance at retention will be associated with a distinct behavioral relevant fMRI activation changes in middle-aged adults following the completion of long-term practice of a CT task.SD = 8.5 years; six females). None presented with any evidence of dementia   were not studied. Additional exclusion criteria were adopted for fMRI scanning .A target circle outlined in white was visible on a black background as it moved up and down on a computer screen for a total of 20 s. Participants used their non-dominant arm to track the vertical path of the target with wrist movements that controlled a non-ferrous joystick  analysis, which reflects the overall movement error of tracking, the random patterns were ranked for each participant and then the ranking averaged across all subjects. This measure clearly identified segments that subjects consistently performed well or poorly. The average velocity for each random segment was calculated. There was a strong pattern showing segments with the lowest RMSE rankings (ranked as “easy”) also had low average velocity. The average velocity for the repeated cycle was calculated and compared with the values of the random segments. The value of the repeated segment average velocity was well above that of the “easy” random segments. Based on this analysis, an average velocity minimum was determined and segments with an average velocity lower than this value were eliminated from consideration. Both of these screening methods were employed as post processing. The random segments were analyzed; both for appropriate range of motion and average velocity, and segments not meeting these criterions were eliminated from further analysis.The order of presentation of random and repeated segments were counterbalanced such that there was always an equal chance of performing either type of sequence first. The trajectories of the target and participants' movements did not leave a trail on the screen and thus, participants could not visualize the entire target pattern . Participants' initial behavioral performance and blood oxygenation level-dependent (BOLD) response at baseline was assessed on Day 1. A separate retention test on Day 7 following the 5 days of practice was used to assess motor sequence learning and associated BOLD response. During these 2 days of fMRI acquisition, participant performed four separate runs of CT task performance that were comprised of 2 blocks presented in a block design (40 s rest/150 s stimulation/40 s rest/150 s stimulation/40 s rest) such that each block of stimulation consisted of either random or repeated sequences with the order of presentation within a scan counterbalanced across scans. During both behavioral practice and fMRI sessions the beginning and end of segments were not marked in any way, one segment simply ran into the next. The visual display was back projected  onto an opaque screen located above the participants' head and visible via a reflecting mirror located in the radio frequency head coil. Before the first fMRI session all participants were familiarized with the motor task. Prior to functional imaging, high-resolution 3D T1 images were collected for anatomic localization and co-registration .Functional and anatomical imaging was performed at the UBC MRI Research Centre on a Philips Achieva 3.0 T whole body MRI scanner  using a sensitivity encoding head coil (SENSE). Participants lay supine in the scanner with foam padding around their head to limit motion. The scanner was equipped with a three-axis local gradient radio frequency coil to collect whole brain fMRI . Functional imaging data were collected as axial echo-planar images, using a single-shot, blipped gradient-echo echo-planar pulse sequence . Four, 7 min runs of functional data were collected (210 images). Prior to functional imaging, high-resolution 3D T1 images were collected for anatomic localization and co-registration . Total scan time was ~45 min.Motor performance was evaluated across practice and retention in two ways. First we considered changes in RMSE, which is the average difference between the target pattern and participant movements and reflects overall tracking errors in the kinematic patternR2) reflect the spatial accuracy of tracking performance and the position of highest correlation was taken as the time lag as compared to target motion. Time lag of tracking represents the temporal distance from the target. The temporal offset was converted to milliseconds to characterize the temporal precision of tracking. Time lag scores that shifted from negative numbers to approach 0  indicate improved motor performance. Finally, correcting for the contribution of time lag in the overall RMSE scores allowed us to determine spatial tracking error . Only continuously overlapping sections of target and position data were correlated to avoid sample size biasing from different temporal shifts across trials and participants. Correlation coefficients  of the repeated than of the random sequence at the delayed retention test ; individual time-course BOLD signal data were fit to the design matrix. In this fMRI block design, the model included the boxcar regressor of each condition of the design matrix  convolved with a hemodynamic response function (HDF). In addition, six predictors of no interest were included in the model to account for translational and rotational motion in the x, y, and z planes. Functional data were spatially smoothed using a 4 mm full-width-half-maximum Gaussian kernel. Anatomic images were registered in Talairach space and then co-registered with functional images , temporal precision, and spatial accuracy were created  = 0.279, p > 0.05]. Outliers were deemed and removed if the changes in tracking were greater than two standards deviations of the group mean.Changes in tracking  were analyzed across motor sequence practice sessions (Day 2–6) and in the MRI scanner (Day1 and 7). To evaluate the performance related changes in sequence specific learning separate repeated measures for Day (2–6) and Day (1–7) by Sequence  ANOVAs were conducted for overall movement error, temporal precision, and spatial accuracy of tracking change scores. Mauchly's test indicated that the assumption of sphericity had not been violated . However, a main effect of day demonstrated that practice benefitted tracking performance for both types of sequences   = 6.01; p = 0.001; η2 = 0.40; δ = 0.97] was observed  = 2.836; p = 0.04; η2 = 0.706; δ = 0.262]  (2–6)  Figure . When ded Figure . Perform] Figure . There wParticipants failed to gain explicit knowledge as shown by their poor ability to correctly recognize the repeated sequences. Explicit recognition was at chance; only 55% of sequences were correctly identified.There was no overlap between behaviorally correlated brain activation from Day 1 to retention, demonstrating the existence of a sequence-specific motor network. The correlational analysis for the positive differences scores at the retention test produced overall movement error (RMSE) and temporal precision sequence-specific weighted statistical images. Spatial accuracy did not show any positive correlations associated with increase brain activation at the retention test. The positive difference score in overall tracking performance was evident in three out of the 10 participants Table . DecompoThis study confirms past work showing that a broad network of brain activity supports sequence-specific learning of perceptuomotor tasks. Sequence-specific motor learning, as measured by the difference score in overall tracking error (RMSE) between repeated and random sequences at retention, was associated with increased activity in large bilateral clusters within the cerebellum (lobule VI). However, as this positive difference in behavior was only evident in three of the 10 subjects. Sequence-specific motor improvements were largely the result of improved  time lag of tracking (eight of the 10 subjects), denoted following the decomposition of overall movement error into the temporal and spatial components. We observed a relationship between improvements in temporal precision during sequence-specific motor learning and activation within a specific bihemispheric network of the brain—the precentral gyrus, middle occipital gyrus, putamen, thalamus, cuneus, and cerebellum. Limited literature exists on the long-term neural changes associated with improvements in the components motor learning in middle-aged adults, thus, the whole brain correlational analysis employed here provides a more comprehensive portrait of cortical reorganization associated with implicit motor sequence learning.The sequence-specific weighted correlational analysis identified activation within both hemispheres of the cerebellum associated with sequence-specific motor learning as measured by changes in RMSE from repeated to random sequence performance. Activation associated with superior repeated motor learning was observed in bilateral lobule VI of the cerebellum. Past work suggested that during motor sequence learning, activation within areas of the cerebellum is dependent on the phase of learning (Doyon et al., Practice-related changes were tracked across the five practice days outside the scanner. Consistent with previous findings, older adults showed repeated sequence-specific improvements during practice of the CT task (Howard and Howard, The effect of practice is related to changes in the movement structure of the motor sequence as learner's transition into the motor processing phase of learning (Park and Shea, The premotor regions with the thalamus and striatum form the cortico-striato-thalamo-cortical circuits (Middleton and Strick, Feedforward tracts project from the cortex via pontine nuclei, decussate and terminate within the lobules of the cerebellum, forming the cerebo-ponto-cerebellar pathway (Salmi et al., During 5-days of practice middle-aged adults showed overall improvement in movement error of the CT task and demonstrated sequence-specific motor learning as shown by superior repeated sequence performance. The importance of a subject-level analysis was fundamental in dissociating behaviorally-related changes of fMRI activation associated positive differences scores between repeated and random sequence performance at retention. The decomposition of overall tracking accuracy into the spatial and temporal components showed a behavior-related difference between repeated and random sequence performance. Sequence-specific motor learning was greatly reliant upon improvements in temporal precision and accompanied by activation of a specific neural network—premotor, thalamus, putamen, and cerebellum. Sequence-specific motor learning required participants to use top-down control as demonstrated by activation of the premotor area and the striatum. Activation of the premotor area shows participants were preparing for the upcoming learned movement pattern, whereas striatum activation supports the increased speed of movement. Thus, the observed brain areas associated with behavioral changes at retention provide insight into the neural correlates associated with different learning strategies during a CT task in middle-aged adults.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fungal pathogens cause devastating losses in economically important cereal crops by utilising pathogen proteins to infect host plants. Secreted pathogen proteins are referred to as effectors and have thus far been identified by selecting small, cysteine-rich peptides from the secretome despite increasing evidence that not all effectors share these attributes.Stagonospora nodorum and Fusarium oxysporum as well as the N-terminal Y/F/WxC-motif from the barley powdery mildew pathogen. Application to the cereal pathogen Fusarium graminearum reveals a secreted phosphorylcholine phosphatase that is characteristic of hemibiotrophic and necrotrophic cereal pathogens and shares an ancient selection process with bacterial plant pathogens. Three F. graminearum protein clusters are found with an enriched secretion signal. One of these putative effector clusters contains proteins that share a [SG]-P-C-[KR]-P sequence motif in the N-terminal and show features not commonly associated with fungal effectors. This motif is conserved in secreted pathogenic Fusarium proteins and a prime candidate for functional testing.We take advantage of the availability of sequenced fungal genomes and present an unbiased method for finding putative pathogen proteins and secreted effectors in a query genome via comparative hidden Markov model analyses followed by unsupervised protein clustering. Our method returns experimentally validated fungal effectors in Our pipeline has successfully uncovered conservation patterns, putative effectors and motifs of fungal pathogens that would have been overlooked by existing approaches that identify effectors as small, secreted, cysteine-rich peptides. It can be applied to any pathogenic proteome data, such as microbial pathogen data of plants and other organisms. Fungal pathogens represent one of the most important restraints of food production worldwide and can also threaten natural plant populations ,2. Any oPathogens utilise a wide range of virulence strategies to bring about disease. These include the production of small secreted proteins (or effectors) that interfere with host functions, the production of small secondary metabolites that may have effector-like function or be toxic to host cells and the production of reactive oxygen species. Pathogens also possess mechanisms to defend themselves against the host response to infection such as proteins to mask other molecules that may otherwise be recognised by the host, enzymes to metabolise toxic plant derived compounds and enzymes to detoxify reactive oxygen species. However, our understanding of the virulence arsenal of most pathogens is fragmentary. This is in part because most virulence strategies possessed by a single pathogen contribute in a quantitative manner to the outcome of an interaction, making their detection difficult. Proteinaceous effectors that act as avirulence determinants in specific host genotypes are an exception to a quantitative contribution to an interaction, but the virulence function of these in compatible hosts are also poorly defined.De novo prediction of pathogen virulence mechanisms in fungi based on common sequence features has to date been difficult. In contrast, proteinaceous effectors of bacteria and oomycete pathogens can be predicted from conserved targeting signals in the N-terminal regions with comparative ease. Using whole-genome sequence and deduced protein-coding gene sequences of these organisms, proteins with certain conserved amino-acid motifs in their N-terminal region have been observed to be strongly associated with predicted secreted proteins. In bacteria, this is the signal that targets proteins to the type III secretion system which directly delivers proteins to the host cytoplasm -P-C-[KR]-P motif and that functional testing is necessary to reveal its role in the host-pathogen interaction.Cluster C8 contains 110 proteins and has sequence-derived features that are distinct from Cluster C7. A striking difference to the other clusters is the enriched threonine content and molecular weight as well as the depleted leucine content and protein charge Table . We ran Ma et al., howeverl.-C-x-T-Y-x-S-T-Y-L.Click here for fileTable listing the properties of the predicted pathogen-associated proteins in B. graminis.Click here for fileContain information about the predicted Y/F/WxC-motif proteins B. graminis.Click here for fileContain information about the predicted Y/F/WxC-motif proteins B. graminis.Click here for file