Jean-Martin Charcot has first described multiple sclerosis (MS) as a disease of the central nervous system (CNS) over a century ago. MS remains incurable today, and treatment options are limited to disease modifying drugs. Over the years, significant advances in understanding disease pathology have been made in autoimmune and neurodegenerative components. Despite the fact that brain is the most lipid rich organ in human body, the importance of lipid metabolism has not been extensively studied in this disorder. In MS, the CNS is under attack by a person’s own immune system. Autoantigens and autoantibodies are known to cause devastation of myelin through up regulation of T-cells and cytokines, which penetrate through the blood–brain barrier to cause inflammation and myelin destruction. The anti-inflammatory role of high-density lipoproteins (HDLs) has been implicated in a plethora of biological processes: vasodilation, immunity to infection, oxidation, inflammation, and apoptosis. However, it is not known what role HDL plays in neurological function and myelin repair in MS. Understanding of lipid metabolism in the CNS and in the periphery might unveil new therapeutic targets and explain the partial success of some existing MS therapies. Lipoproteins contain triacylglycerols, cholesterol esters, cholesterol and phospholipids. They exist in two main forms: high- and low-density lipoproteins (LDLs). These hydrophobic aggregates covalently bind to apoproteins to form apolipoproteins. As major components of lipoproteins, apoproteins determine their structure, metabolism, receptor interaction and function. The primary apolipoprotein of high-density lipoprotein (HDL) is apolipoprotein A-I or ApoA-I, while major component of LDL is ApoB.In mammals peripheral cholesterol is produced in the liver and the small intestine. Cholesterol biosynthesis involves as many as 30 enzymatic steps. Cells produce cholesterol from acetyl-CoA, which is reduced to mevalonate by 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). Through a series of reactions, mevalonate is converted to squalene and lanosterol and then to 7-dehydrocholesterol, 7-dehydrocholesterol reductase produces a cholesterol molecule. Cholesterol can be hydroxylated to 24-, 25-, and 27-hydroxycholesterol (27-OHC) by the CYP46 hydroxylase. Healthy cholesterol homeostasis is necessary for membrane formation, function of steroid hormones, vitamin D, and adequate brain function. Cholesterol is also an important myelin component and a precursor of oxysterols, steroid hormones and bile acids.Excess cholesterol is removed from tissues through a process known as reverse cholesterol transport, during which cholesterol from the periphery is returned to the liver for biliary excretion. In plasma a membrane associated ATP-binding cassette transporter A1 (ABCA1) promotes efflux of free cholesterol (FC), sphingomyelin (SM), glycerophosphocholine (PC) to ApoA-I to form nascent HDL nHDL; . This prIn the central nervous system (CNS), lipid rafts are present in neurons and glia and are believed to play a role in neuronal signaling, cell adhesion and axonal guidance . CholestLipoproteins, apoproteins, and apolipoproteins can be found in circulation in free or nascent form, but lipid rafts are always associated with cellular membranes. It is unclear whether apolipoproteins require any scaffolding support of the raft for their function, but it is certain that lipid rafts need the presence of cholesterol, lipids, and proteins.The exact role of lipid rafts in the pathogenesis of multiple sclerosis (MS) and other diseases remains to be discovered. Different brain regions have diverse cholesterol requirements and enrichment in membrane rafts . In addiSphingolipids are metabolized to generate ceramide and sphingosine. Sphingosine can be phosphorylated by sphingosine kinase to generate S1P  are expressed in the CNS and are involved in regulation of lipid metabolism, control of inflammation, and cholesterol transport . PPARs fApolipoproteins play a major role in cholesterol recycling process—reverse cholesterol transport within the CNS and in the periphery. Production of cholesterol in the brain peaks during myelogenesis, when glial cells and neurons produce it. Mature neurons seems to loose cholesterol-producing capacity and rely instead on cholesterol delivering lipoproteins to maintain ongoing needs. Several studies indicate a role for apolipoproteins in cholesterol transfer and lipid metabolism in the CNS . Among sApoA-I is not synthetized in the brain, however it has the ability to penetrate the BBB and solicit anti-inflammatory and neuroprotective effects in the brain Figure .In addition to participating in the CNS reverse cholesterol transport, ApoA-I blocks macrophage interactions with T-cells. This results in reduction of the Th1 and Th17 associated cytokines  is a transcription factor expressed in the liver, macrophages, and neurons. It is noteworthy that 24S-OH is a ligand for LXRs, which activates expression of ABCA1, ApoA-I, and ApoE. Mice lacking LXRs had impaired cholesterol removal . Murine ApoA-I is the most abundant component of HDL . HDL-assApoA-I has been described as a putative clinical biomarker for interferon beta (INFβ) treatment . Study sWe discovered that advanced MS patients have lower plasma ApoA-I levels in comparison to patents with stable relapsing remitting disease and healthy age-matched controls . PatientAnother group assessed serum lipid profiles of 492 MS patients for associations with disability and MRI outcomes . The autGiven the fact that ApoA-I is expressed at high levels in spinal fluid, and perturbations in lipid metabolism negatively affect myelin, factors that control ApoA-I production and turnover should receive special consideration . Levels A pilot study of 30 MS patients demonstrated a significant decrease in the number and volume of contrast enhancing lesions with 80 mg of simvastatin treatment . A largeLow ApoA-I presence in progressive patients’ plasma as we discovered in our study  could exApolipoproteins are important players in cholesterol homeostasis. ApoA-I acting as a major HDL component is involved in both HDL biosynthesis and transport. In CNS diseases with compromised BBB, healthy cholesterol turnover becomes extremely important for neuronal homeostasis and regeneration. In as much as the S1P receptor agonist fingolimod, Apo-I’s function is not completely understood in MS. However, data suggests a positive neuroprotective effect of this apolipoprotein on the immune and the CNSs. Reduction in ApoA-I levels has not been shown to cause MS. However for progressive patients, maintaining normal levels of ApoA-I might result in better neuronal health. Agents designed to improve ApoA-I production should be considered for therapeutic purposes. For example, PPAR agonists are one class of medications that regulate ApoA-I and subsequently HDL synthesis. Therefore, studies aimed at compounds responsible for ApoA-I expression during periods of inflammation could provide important information about the mechanisms of HDL regulation and its role in MS pathogenesis.Dr. Gardner became an employee of Novartis after initial submission of this manuscript. 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.


Pinus taeda, genome.Conventional short read sequences derived from haploid DNA were extended into long super-reads enabling assembly of the massive 22 Gbp loblolly pine, http://genomebiology.com/2014/15/3/R59See related research  Arabidopsis thaliana, a homozygous inbred diploid, which had been selected specifically for its relatively small size (119 Mb assembly), limited repetitive sequence content, and homozygosity. However, not all species in the plant kingdom have simplistic genomes like Arabidopsis. Indeed, for the vascular land plants, the mean C value for angiosperms is 5.79 pg , while that for gymnosperms is 18.08 pg . These large genome sizes are attributable to amplification of repetitive sequences, whole or segmental genome duplication, and/or polyploidy - all of which pose a technical challenge in genome assembly with current short-read assembly approaches. In addition, many species are self-incompatible and/or outcrossing in nature, which can result in a high degree of heterozygosity that further confounds genome assembly. An article in this issue of Genome Biology overcomes many of these technical challenges to achieve a near complete assembly of the 22 Gbp loblolly pine genome [The first plant genome sequenced was that of Fragaria vesca[To reduce the complexity of plant genomes, researchers have been able to capitalize on genetics and/or reproduction to simplify the sequencing and assembly of a reference genome sequence Figure . For exaria vesca with a gGenome Biology by Neale et al.[Pinus taeda) genome. In conifer seeds such as those of the loblolly pine, the haploid maternally derived megagametophye surrounds the developing embryo, providing it with nutrients, and is analogous to the endosperm in maize kernels. Due to the relatively large size of the megagametophyte, sufficient DNA for 11 paired-end libraries was isolated from a single, haploid loblolly pine megagametophyte to bypass the heterozygosity present in diploid tissues, such as needles. Using a novel computational approach described below, the authors complemented these haploid-derived libraries with 48 mate-paired and 9 fosmid ditag libraries constructed from ample DNA isolated from diploid needles, and constructed an assembly that spans 23.2 Gbp (20.1 Gbp non-gapped sequence), with an impressive N50 scaffold size of 66.9 Kbp. A total of 50,172 genes were annotated in the assembly and the quality of their assembly was evident in the detection of 201 of the 248 core CEGMA [Picea abies and Picea sitchensis, and restricted to loblolly pine [The article in this issue of le et al., and twole et al.,6, is anThe first plant genomes were sequenced using Sanger sequencing and initially utilized bacterial artificial chromosome (BAC)-by-BAC approaches, which later evolved into WGS. Assembly of the BACs and genomes was achieved using the overlap layout consensus (OLC) approach where all-versus-all pairwise read overlaps are identified, the read layout is calculated, and a contig consensus sequence is generated. The OLC assembly approach works well for high-quality long reads generated through Sanger sequencing. When ultra high-throughput next generation sequencing-by-ligation (SBL) and sequencing-by-synthesis (SBS) technologies emerged, a burst of innovation in genome assembly software occurred. The shorter read lengths, higher error rates and increased sequencing depth were incompatible with OLC assemblers due to very high memory usage and long assembly run times. A number of assemblers were developed based on the de Bruijn graph, an approach initially used for short reads generated by sequencing-by-hybridization and resurrected to assemble SBL and SBS reads efficiently. In the last few years, numerous improvements in short-read assembly have been made, including error correction of reads, trimming and filtering of low quality reads, merging of overlapping paired-end reads, scaffolding of contigs using large insert mate-pairs or RNA-sequencing reads, incorporating long reads, and gap filling using paired-end reads.For large and complex genomes, the computational requirements of de Bruijn graph-based short-read assemblers can exceed what is available, even on large memory multi-core servers. Approaches such as parallel assemblers that distribute the memory and computational requirements across a cluster of interconnected nodes are one way to address this issue. Other options include assemblers optimized for low memory usage or assemblers that run in a specialized cloud-based distributed computing environment. However, more innovation in genome assembly and/or reduction in genome complexity will be needed to access large, complex genomes using current short-read sequences. One such strategy is that developed for the loblolly pine genome, which introduces a novel approach for assembling large complex genomes ,5. The cet al. to sequence and assemble the loblolly pine genome provides a method of generating a high-quality draft assembly of a complex 22 Gbp plant genome using current and cost-efficient sequencing platforms.With regard to biology, genome sequencing technology improvements that began in the late 1990s have continued through the present day, igniting research across a wide range of biological disciplines and creating entirely new fields of research, including genome biology and personal genomic medicine. None of this would have been possible without parallel improvements in algorithms, computer-processing capabilities, and applied computing solutions. Current long-read sequencing technologies such as Pacific Biosciences SMRT Sequencing  are compBAC: Bacterial artificial chromosome; OLC: Overlap layout consensus; SBL: Sequencing-by-ligation; SBS: Sequencing-by-synthesis; WGS: Whole genome shotgun sequencing.The authors declare that they have no competing interests.


Datacenters consolidate diverse applications to improve utilization. However when multiple applications are colocated on such platforms, contention for shared resources like networks-on-chip (NoCs) can degrade the performance of latency-critical online services (high-priority applications). Recently proposed bufferless NoCs  have the advantages of requiring less area and power, but they pose challenges in quality-of-service (QoS) support, which usually relies on buffer-based virtual channels (VCs). We propose QBLESS, a QoS-aware bufferless NoC scheme for datacenters. QBLESS consists of two components: a routing mechanism (QBLESS-R) that can substantially reduce flit deflection for high-priority applications and a congestion-control mechanism (QBLESS-CC) that guarantees performance for high-priority applications and improves overall system throughput. We use trace-driven simulation to model a 64-core system, finding that, when compared to BLESS, a previous state-of-the-art bufferless NoC design, QBLESS, improves performance of high-priority applications by an average of 33.2% and reduces network-hops by an average of 42.8%. Web service companies such as Google, Yahoo, Amazon, and Microsoft deploy datacenters with hundreds to thousands of machines to host millions of users, all of whom may be running large, data-intensive applications . LatencyNetworks-on-chip are important shared resources in the manycore devices that will likely be used to build future datacenters. The NoCs in such chips are responsible for conveying operands between cores, accessing main memory, managing coherence, and performing I/O –4.Traditional NoCs use router buffers to reduce the number of dropped or deflected (misrouted) packets. These buffers, however, improve effective bandwidth at the expense of design complexity, chip area, and power consumption , 5. FurtIn contrast, bufferless NoCs eliminate on-chip router buffers so that when a flit arrives, a router must immediately select an appropriate output port to forward it. Although previous studies show that bufferless NoCs can reduce router area by 60% and save power consumption by 40% , the difTo address the problem, we propose QBLESS, a QoS-aware bufferless NoC scheme targeting datacenters. Instead of using the prevalent VC-based QoS mechanisms, QBLESS tags flits with priority bits and leverages this information in its deflection routing and congestion-control mechanisms. The flits of latency-critical applications are assigned a high priority, making them privileged with respect to routing in the QBLESS NoC.N hops, where N is a system parameter influenced by factors such as application memory access characteristics and network size  servers. CMPs rely on efficient networks-on-chip to synchronize cores and to coordinate access to shared memory and I/O resources. Here we present background specific to datacenter NoCs and briefly survey the most relevant prior work.In datacenters using CMPs with tens of cores, more and more workloads are deployed on a single server, and thus they must share resources. Kambadur et al.  point ouModern datacenters sacrifice server utilization to guarantee the QoS of online services by separating them from batch workloads. Previous efforts to increase utilization while keeping a high level of QoS have colocated the two incompatible kinds of workloads on the same node to eliminate interference. Tang et al. explore the impact of the shared memory subsystem (including the last level cache (LLC) and front side bus (FSB)) on Google datacenter applications . They prPrevious work on guaranteeing datacenter QoS mainly focuses on the on-chip and off-chip memory subsystems. However, just as the security level is defined by the weakest component, QoS is dictated by the least robust participant: this means that all shared resources must be QoS-aware if any are to meet service-level agreements (SLAs). Improving NoC QoS technology for interactive datacenter applications is thus one promising direction for achieving higher throughput and greater energy-efficiency.Dally and Towles  show thaMany buffer-based QoS approaches have thus been proposed for NoCs. For example, MANGO  guaranteSome recent work focuses on alternative designs that are tradeoff power consumption, die area, and performance. One promising direction is bufferless routing , which tBufferless routing yields significant network power savings with minimal performance loss when the network load is low-to-medium. In such bufferless NoCs, router area is reduced by 40–75% and power consumption is reduced by 20–40% , 18. HowTo bridge the performance gap between the buffered and bufferless NoCs at high network load, one possible approach is to directly improve the efficiency of bufferless deflection routing. Previous work , 19–21 uAnother approach is to make a hybrid network that can adaptively switch between the higher-capacity buffered mode and lower-cost bufferless mode. Jafri et al.  propose Previous proposals are effective in improving throughput and fairness of bufferless NoCs but they are not suited for datacenter environments with mixed workloads where the performance of latency-critical applications might be substantially degraded. In this work, we investigate both congestion control and deflection routing, finding that the latter is more effective in guaranteeing QoS in bufferless NoCs.Since almost all QoS-support techniques are based on buffer-based VCs, implementing QoS-support in bufferless NoCs remains an open problem.NoCs are shared by many cores; a QoS-oblivious bufferless NoC may substantially degrade performance for latency-critical applications, even if overall throughput is high. To investigate this, we simulate a 64-core system and measure the impact of NoC contention. We designate h264ref from SPEC CPU2006  to be a On the one hand, bufferless NoCs have the advantages of small area and low power. On the other hand, SLA-level QoS-support is essential for improving datacenter utilization through resource sharing. These factors motivate us to investigate how to design and implement QoS on bufferless NoCs.We propose QBLESS, a QoS-aware bufferless NoC design, for datacenter environments. To integrate these mechanisms into NoCs, we need to add a set of registers and to modify some components in the routers. Previous routing algorithms usually adopt age-based arbitration to determine which flit to deflect, regardless of the priority of the data flits. Therefore, in this case, because the age of the low-priority flit is one hop larger than that of the high-priority flit, the high-priority flit is deflected to router number 4, as shown in To achieve this, a QBLESS router must perform two tasks: ranking flits to select an appropriate flit candidate (flit-ranking) and prioritizing available output ports to select an appropriate one (port-prioritizing).Previous routing algorithms usually adopt age-based arbitration, for example, BLESS using an oldest-first (OF) ranking policy that performs best in most scenarios in terms of latency, deflection-rate, and energy-efficiency . However privilege-age when injected into the NoC, which means that the age of the high-priority flit is a certain number of hops ahead of low-priority flits. As shown in  privilege-age is stored in a register and is programmable via software.In QBLESS, a high-priority flit obtains a privilege-age is critical to effectiveness of the QBLESS routing. The value should be large enough to allow high-priority applications to always beat low-priority ones. For low-priority applications, privilege-age should be small enough to avoid livelock.Determining the value of the privilege-age is determined by the network parameters , the individual application characteristics , and the network confliction. In practice, privilege-age is an empirical parameter that reflects QBLESS's ability to guarantee high-priority applications. Since it is programmable, we can dynamically adjust its value according to NoC performance. We evaluate the impact of privilege-age in The value ofWhen a flit arrives at the router, the router first tries to assign it to its preferred port. If the preferred port is occupied, the router assigns it to a deflecting port. The routing algorithm must guarantee that low-priority flits are not deflected indefinitely.Starvation occurs due to congestion when a router exhausts all its ports and cannot inject new flits into the NoC. For bufferless NoCs, congestion increases the deflection-rate, and deflections exacerbate congestion. Thus, congestion control is important for both throughput and latency.Throttling a specific application is an effective approach for mitigating starvation, but it degrades the performance of the victim application. Previous studies , 19–21 tIn each epoch, a global controller collects the IPF and starvation rate of each router. If any router's starvation rate exceeds a threshold, the network is deemed to be congested. Note that each router has its own threshold:Equation  defines Generally, high-priority applications are not targeted for throttling. Low-priority applications whose IPFs are lower than the average value are selected as throttling candidates.Lower IPF indicates less NoC sensitivity, and thus applications with lower IPF can be throttled more than others. In particular, we adopt the algorithm of BLESS-T  and add Each flit has a  priority tag. For example, one bit can be used to indicate two priority levels. To make the QBLESS scheme easy to understand, we use just one bit to present our design. In practice, priority levels can be extended . priority tags are obtained from application QoS registers in the CPU cores. Specifically, we leverage a QoS framework that adds priority information to each process control block (PCB), and we add a corresponding QoS-register to each core  by using two priority bits. We perform experiments to evaluate QBLESS in a 100-core system. As shown in The major source of hardware overhead of QBLESS is the modification of router architecture, which is required to measure the starvation rate at each node and to throttle injection. As shown in We propose QBLESS, a hardware programmable approach for reducing in-network contention in bufferless NoCs. QBLESS adaptively selects the routed output port and throttling rate of low-priority applications to ensure the QoS of high-priority latency-critical corunners. We examine both application level and network level performance in 8 × 8 and 10 × 10 networks and show significant QoS improvements for latency-critical applications on a variety of real workloads.


Delirium is associated with substantial morbidity and mortality rates in elderly hospitalised patients, and a growing problem due to increase in life expectancy. Implementation of standardised non-pharmacological delirium prevention strategies is challenging and adherence remains low. Pharmacological delirium prevention with haloperidol, currently the drug of choice for delirium, seems promising. However, the generalisability of randomised controlled trial results is questionable since studies have only been performed in selected postoperative hip-surgery and intensive care unit patient populations. We therefore present the design of the multicenter, randomised, double-blind, placebo-controlled clinical trial on early pharmacological intervention to prevent delirium: haloperidol prophylaxis in older emergency department patients (The HARPOON study).In six Dutch hospitals, at-risk patients aged 70 years or older acutely admitted through the emergency department for general medicine and surgical specialties are randomised (n = 390) for treatment with prophylactic haloperidol 1 mg or placebo twice daily for a maximum of seven consecutive days. Primary outcome measure is the incidence of in-hospital delirium within seven days of start of the study intervention, diagnosed with the Confusion Assessment Method, and the Diagnostic and Statistical Manual of Mental Disorders, fourth edition criteria for delirium. Secondary outcome measures include delirium severity and duration assessed with the Delirium Rating Scale Revised 98; number of delirium-free days; adverse events; hospital length-of-stay; all-cause mortality; new institutionalisation;  Activities of Daily Living assessed with the Katz Index of ADL, and Lawton IADL scale; cognitive function assessed with the Six-item Cognitive Impairment Test, and the Dutch short form Informant Questionnaire on Cognitive Decline in the Elderly. Patients will be contacted by telephone three and six months post-discharge to collect data on cognitive- and physical function, home residency, all-cause hospital admissions, and all-cause mortality.The HARPOON study will provide relevant information on the efficacy and safety of prophylactic haloperidol treatment for in-hospital delirium and its effects on relevant clinical outcomes in elderly at-risk medical and surgical patients.NCT01530308; Dutch Clinical Trial Registry:NTR3207EudraCT Number: 201100476215; ClinicalTrials.gov Identifier: Delirium is a serious and frequently occurring complication of (acute) illness in older hospitalised patients with prevalence rates of more than 30%. DeliriuIn older patients, development of in-hospital delirium is associated with a range of negative patient outcomes such as increased hospital length-of-stay, long-term cognitive- and functional decline, new institutionalisation, and increased mortality-6, thereThis paper describes the design of the multicenter, randomised, double-blind, placebo-controlled clinical trial to evaluate the efficacy and safety of early prophylactic treatment with low dose haloperidol on in-hospital delirium, and post-discharge outcomes in acutely admitted older medical and surgical patients.To study the differences in in-hospital seven-day delirium incidence after acute admission for both general medicine and surgical specialties in older at-risk patients treated early with prophylactic haloperidol or placebo.Severity and duration of delirium; number of delirium-free days; hospital length-of-stay; adverse events; all-cause mortality in-hospital and within six months post-discharge; cognitive and physical function at baseline, three, and six months post-discharge; new institutionalisation post-discharge; number of hospital admissions within six months post-discharge.The HARPOON study is a multicenter, investigator initiated, stratified randomised, double-blind, placebo-controlled clinical trial with a six-month follow-up period. The study design is demonstrated in Figure 1. Malnutrition, including stimulation and measurement of nutritional intake by nutrition assistants, and dietary consultation;2. Falls and mobility impairments, including adequate footwear, stimulation of physical activity in or out of bed, and if needed, (daily) occupational and physical therapy; and3. Delirium.With respect to delirium, the VMS program based standardised hospital care covers identification and modulation of precipitating delirium risk factors such as medication reviews and orientation and noise-reduction measures, creating an optimal background regimen for conduction of this trial in all participating hospitals. Patients who positively confirm one or more of the VMS delirium risk questions (VMS ≥1) are considered at-risk for developing delirium during hospital admission. Patients with VMS 0 on first evaluation are not eligible for this study. Eligible patients will be asked for informed consent. In case of inability to write, patients must provide verbal consent in the presence of an independent witness (preferably patients’ proxy) who may then sign the informed consent form. The study medication, haloperidol 1 mg or identical placebo tablets, will be given orally to participants twice daily at 12 pm and 8 pm for a maximum of seven consecutive days (14 treatment dosages) on top of standardised care based on the implemented VMS program ‘Vulnerable elderly’. During the seven-day intervention period nursing staff will screen participants for delirium symptoms. Additionally, a trained observer (physician or geriatric nurse) will daily visit participants to detect the presence of delirium, register potential adverse events based on 24-hour nursing observations, clinical judgement, and electrocardiogram (ECG) monitoring, and to check for adherence to standardised adequate environmental manipulations for delirium such as orientation measures. For patients who have not developed delirium within seven days of initiation of the study intervention, daily assessment stops. Patients who develop delirium are evaluated daily by the observer to record delirium severity and duration until symptoms diminish. Study drug administration is terminated early if a participant develops delirium, is transferred to a non-participating care unit such as the ICU, refuses further participation in the trial, the QTc interval is more than 500 milliseconds (ms), is discharged from the hospital, or dies within seven-day of initiation of the study intervention. In case of established delirium or if the assigned study intervention is thought to have a direct influence on the patient’s treatment, the treating physician will request for unblinding and may further decide on treatment. For unblinding of treatment assignment, the treating physician is able to contact the VUmc consultant pharmacist through a central telephone number 24 hours a day, seven days a week.This study is conducted according to the Declaration of Helsinki. This trial is approved by the accredited Medical Ethical Committee of VU University Medical Center (VUmc) Amsterdam, the Netherlands (reference number: 2012.177). The executive boards of Isala Hospital Zwolle, Spaarne Hospital Hoofddorp, Rijnland Hospital Leiderdorp, Jeroen Bosch Hospital’s Hertogenbosch, and Medical Centre Alkmaar (MCA), all located in the Netherlands, have provided local feasibility approval.This study is primarily conducted at VUmc, a 733-bed teaching hospital located in Amsterdam, the Netherlands. Secondary study locations include the Isala Hospital Zwolle (976 beds), Spaarne Hospital Hoofddorp (455 beds), Rijnland Hospital Leiderdorp (420 beds), Jeroen Bosch Hospital ’s Hertogenbosch (730 beds), and MCA Alkmaar (485 beds), the Netherlands.Patients aged 70 years and over are eligible when acutely admitted to the hospital through the ED for general medicine or surgical specialties, are able to speak and understand Dutch or English language, are at-risk for delirium on admission (VMS ≥1), are available for inclusion within 24 hours after admission, and are able to provide informed consent. Patients are considered competent to provide informed consent if – after having spoken with the patient – the observer and/or the treating physician agree on the ability of the patient to understand and/or recall relevant study information, including the purpose of the study, its experimental nature, potential benefits and risks, the right to withdraw, and confidentiality of collected data, and to make a voluntary decision to participate. If their cognitive competence is in question, the patient will be regarded as ineligible.Exclusion criteria for participation are delirium on admission according to the DSM-IV criteria; VMS 0; patients not able to take study medication according to protocol (including nil per os); specific heart conditions on admission including QTc interval of more than 500 ms, recent myocardial infarction, decompensated heart failure, second- or third-degree AV block, (history of) ventricular arrhythmias or Torsade de Pointes (TdP), uncorrected hypokalemia with serum potassium level 3.0 milliequivalents per liter (mEq/L) or less, clinical significant bradycardia; concomitant use of pharmacodynamically interacting medication; use of antipsychotic or dopaminergic drugs; use of QT prolonging drugs in the presence of prolonged QTc interval  on baseline ECG; Parkinson’s Disease; Vascular or Lewy Body Dementia; Hypokinetic Movement Disorder; (history of) Neuroleptic Malignant Syndrome; Central Anticholinergic Syndrome; substance abuse and dependence according to the DSM-IV criteria; (history of) epilepsy; concomitant participation in another clinical trial with the exception of observational studies; not competent to provide informed consent.Two well-known variables associated with increased risk of delirium are age 80 years or over and surgical treatment/anaesthesia. To balance population representatives in both treatment groups, four strata are constructed with separate randomisation schedules: (1) age 70 to 80 years, planned surgery on admission; (2) age 70 to 80 years, no planned surgery on admission; (3) age 80 years or over, planned surgery on admission; (4) age 80 years or over, no planned surgery on admission. Each stratum consists of 20 pre-randomised individual study numbers, 10 assigned to haloperidol and 10 to placebo treatment, prepared by the Central Pharmacy for The Hague Hospitals . Participants, observers, physicians, nurses, other care givers, at the investigative sites are all blinded to the assigned intervention. The VUmc clinical pharmacy holds the randomisation list.AHZ is responsible for preparing, labelling, packaging and distributing the study medication according to Good Manufacturing Procedure (GMP) guidelines. Both the haloperidol (as a verum) and placebo tablets are identical in appearance, have fixed concentrations of 1 mg per tablet, and are packaged in identically labelled medicine carton boxes. Study medication prescription is standardised in every study centre’s automated prescription system. On prescription by the local investigator(s), a carton box containing study medication  can be collected. To ensure study medication access 24 hours a day, seven days a week, medicine boxes are securely stored in the ED or on the wards rather than in the local clinical pharmacy.th revision (ICD-10)[Data will be mainly collected by two observers, a physician (EJMS) and geriatric nurse (AV) trained according to the Good Clinical Practice (GCP) principles. Participants’ demographic data, medical history, (prescription) medication history on admission (including the use of sleep medication and antidepressants), presence of sleep-wake cycle disturbances, visual and/or hearing impairment, substance use, and vital parameters will be documented. Medical history and (prescription) medications will be coded according to the World Health Organization’s International Statistical Classification of Diseases and Related Health Problems 10(ICD-10), and the(ICD-10). Severit(ICD-10),19. Phys(ICD-10),21 and t(ICD-10). The Six(ICD-10)-25. Prim(ICD-10). The Con(ICD-10). Nurses (ICD-10),29. Once(ICD-10). An ECG Three and six months after discharge, patients are contacted by telephone by one of the observers to perform the Katz ADL, the Lawton IADL scale, and the 6-CIT, and to obtain data on home residency, hospital admissions, (recurrent) delirium, and all-cause mortality.This study will be monitored by two members – the manager and an independent clinical research assistant – of the Clinical Research Unit, Department of Internal Medicine, VUmc Amsterdam, the Netherlands. During the monitoring visits at respectively the beginning, half-way and end of the study, the consistency of data collection, documentation and the presence and completeness of the Investigator Site File are checked for compliance with GCP guidelines. Findings are systematically recorded and signed by the monitors.Because this study is a pharmacological intervention study recruiting (vulnerable) older patients, we appointed a Data Safety Monitoring Board (DSMB). The primary mandate of the DSMB is to monitor patient safety during the trial. The DSMB for this study consists of four independent members: two emeritus professors of internal medicine, a professor of clinical epidemiology, and a consultant physician in geriatric medicine. The DSMB reviews unblinded results and convenes prior to the beginning of the study, and thereafter, every six months from the first randomisation date. On request, the DSMB may obtain unblinded study results from the hospital pharmacy. When a study participant dies, the DSMB evaluates the event and mortality rates in both study arms. After 125 patients have completed the entire study period including follow-up in each study arm, the DSMB will perform a safety interim analysis with respect to all-cause mortality.The primary study objective is to evaluate the effect of prophylactic haloperidol compared to placebo for the incidence of in-hospital delirium within seven days of start of the study intervention. Based on previous studies, we expect the incidence of delirium in our target patient population currently to be 20%. Aiming for an absolute reduction of 10% in the haloperidol intervention group, with the significance level set at 5%, inclusion of n = 195 patients per study arm  will give us a power of 80% to detect differences with a two-sided alpha of 0.05.t test. The Mann–Whitney U test is used for comparison of non-normal distributed data such as secondary outcome measures concerning efficacy of prophylactic haloperidol treatment. Log rank test will be used to establish the time to (first) delirium diagnosis (number of delirium-free days), which will be compared between groups. Adverse events in both groups will be globally described. Kaplan-Meier estimates will be used to display survival probability within 6 months after hospital discharge for patients with and without diagnosed in-hospital delirium.For the data analysis IBM SPSS Statistics 20 will be used. Clinical trial data will be primarily registered in paper case report forms and later transcribed into the electronic data capture system of OpenClinica, open source clinical trail software. The primary analysis is aimed at evaluation of the primary outcome of this study: the incidence of in-hospital delirium according to the DSM-IV criteria in older at-risk patients aged 70 years or over who are acutely admitted through the ED for a general medicine or surgical specialty within seven days of initiation of the study intervention. Data from all randomised patients will be analysed according to the Intention-to-treat principal. The primary outcome delirium incidence is considered a binary outcome, present or absent. Patients who for example leave the hospital  within seven days of start of the study intervention without experiencing delirium will be regarded negative on the primary outcome. Patients who do not complete the intervention will be monitored during their in-hospital stay for occurrence of delirium. The number of patients diagnosed with delirium at least once in the seven days following initiation of the study intervention will be calculated for both the haloperidol and placebo group, and subsequently this number is divided by the total amount of patients assigned to the matching group, reflecting in-hospital delirium incidence ratio within seven days of initiation of the study intervention. Pearson’s chi-squared test will be used to compare this number between the two groups. Continuous normally distributed data including patient demographic data will be tested with Student’s This stratified randomised, placebo-controlled, double-blind clinical trial studies the efficacy and safety of haloperidol prophylaxis for prevention of delirium in older at-risk patients aged 70 years or over who are acutely admitted to the hospital through the ED for general medicine or surgical specialties, because generalisability of existing study results from well-designed RCTs in this field are limited to postoperative (mainly elective) hip-surgery and ICU In general, in-hospital delirium occurs within three or four days after admission or surgery,31, but In addition to extrapyramidal side effects, more rare side effects as ventricular arrhythmia’s and TdP are a great concern for physicians initiating haloperidol treatment in a patient. To date, there are no consensus practice guidelines on QT(c) monitoring for the prevention of TdP in the hospital setting, and recommendations are primarily based on clinical experience-38. DuriEligibility criteria include that a subject has the cognitive capacity to understand the scope of the study and is able to provide informed consent independently. Based on the sample size calculation, a large numbers of subjects must be screened. Therefore, a brief and valid screening instrument was selected to assess cognitive function (6-CIT),25 on adst 2015.In conclusion, the HARPOON study is a well-designed double-blind, placebo-controlled RCT that we believe will provide relevant information on the efficacy and safety of early administration of low-dose haloperidol for preventing delirium in at-risk elderly patients in the hospital setting, and its effects on post discharge outcomes. The predicted study completion is May 1th revision; ICU: Intensive care unit; IQCODE-N: Informant Questionnaire on Cognitive Decline in the Elderly; IADL: The Lawton Instrumental Activities of Daily Living scale; MCA: Medical Centre Alkmaar; mEq/L: Milliequivalents per liter; mg: Milligrams; ml: Millilitres; ms: Milliseconds; QTc: QT interval corrected for heart rate; TdP: Torsade de Pointes; VMS: Dutch Hospital Patient Safety Program (in Dutch: Veiligheidsprogramma); VUmc: VU University Medical Center.6-CIT: Six-item Cognitive Impairment Test; ADL: The Katz Index of Independence of Activities of Daily Living; AHZ: Central Pharmacy for The Hague Hospitals ; ATC: Anatomical Therapeutic Chemical (Classification System); CAM: Confusion Assessment Method; DOS: Delirium Observation Screening ; DRS-R-98: Delirium Rating Scale Revised 98; DSMB: Data Safety Monitoring Board; DSM-IV: Diagnostic and Statistical Manual of Mental Disorders, Fourth edition; ECG: Electrocardiogram; ED: Emergency department; EudraCT: European drug regulatory affairs clinical trials; GCP: Good Clinical Practice; GMP: Good Manufacturing Procedure; ICD-10: International Statistical Classification of Diseases and Related Health Problems 10The authors declare that they have no competing interests.EJMS, OJV, PMB, and PWN conceived of the study and drafted the study protocol. EJMS, OJV, AV, KG, PMB, AMK, SHAD, SA, AS, EK, AML, RJM, AMS, LB, DP, MHHK, and PWBN participate in the design and coordination of the study. EJMS, AV, and KG support the data collection. PWBN has overall responsibility for the trial. EJMS, OJV, PMV, and PWBN performed the statistical analysis for the study protocol and the manuscript, and will primarily be responsible for interpretation of the study results and primary data analysis. EJMS drafted and revised the manuscript. OJV, PMV, PWBN critically revised the manuscript for important intellectual content. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2318/14/96/prepub


Among other effects, post-translational modifications (PTMs) have been shown to exert their function via the modulation of protein-protein interactions. For twelve different main PTM-types and associated subtypes and across 9 diverse species, we investigated whether particular PTM-types are associated with proteins with specific and possibly “strategic” placements in the network of all protein interactions by determining informative network-theoretic properties. Proteins undergoing a PTM were observed to engage in more interactions and positioned in more central locations than non-PTM proteins. Among the twelve considered PTM-types, phosphorylated proteins were identified most consistently as being situated in central network locations and with the broadest interaction spectrum to proteins carrying other PTM-types, while glycosylated proteins are preferentially located at the network periphery. For the human interactome, proteins undergoing sumoylation or proteolytic cleavage were found with the most characteristic network properties. PTM-type-specific protein interaction network (PIN) properties can be rationalized with regard to the function of the respective PTM-carrying proteins. For example, glycosylation sites were found enriched in proteins with plasma membrane localizations and transporter or receptor activity, which generally have fewer interacting partners. The involvement in disease processes of human proteins undergoing PTMs was also found associated with characteristic PIN properties. By integrating global protein interaction networks and specific PTMs, our study offers a novel approach to unraveling the role of PTMs in cellular processes. The function of proteins is frequently modulated by chemical modifications introduced after translation from RNA. These post-translational modifications (PTMs) have been shown to also influence the interaction between proteins carrying them. We tested whether specific PTM-types characterized by attaching different chemical groups are associated with proteins with characteristic and possibly strategic positions within the network of all protein interactions in cellular systems. Based on network-theoretic analyses of PTMs in the context of protein interaction networks of nine selected species, we indeed observed distinctive properties of twelve PTM-types tested. Phosphorylation was found associated with proteins in central locations with the broadest interaction scope, while glycosylation was more prominent in proteins at the periphery of the web of all protein interactions. The involvement in disease processes of human proteins undergoing PTMs was also found associated with characteristic protein interaction network properties. Our study highlights common and specific roles of the various PTM types in the orchestration of molecular interactions in cells. While t UniProt . With te UniProt . Several UniProt , dbPTM [ UniProt , PTMCura UniProt , PTMcode UniProt  and many UniProt –14 offerMycoplasma pneumoniae [Many studies on PTMs have focused on specific types and their relevance for protein function with phosphorylation representing the most actively researched PTM-type –19. Moreeumoniae . Furthereumoniae . A more eumoniae . Carboxyeumoniae .In addition to PTMs, protein function is also regulated and mediated by non-covalent protein-protein interactions –33. As mArabidopsis thaliana [Therefore, it appears plausible that proteins carrying a particular PTM-type may possess specific interaction characteristics. Indeed, for the important and intensively investigated PTM-type phosphorylation, it was found that in yeast, phospho-proteins engage in many more protein-protein interaction than proteins without phosphorylation sites , found sthaliana . Thus, pAs the different PTMs modify proteins in specific ways, it appears furthermore likely that their consequences on protein-protein interactions may be different as well. This hypothesis formed the starting point for the present study. Specifically, we asked whether different PTM-types are associated with characteristic protein-protein network properties, such as interaction degree, clustering coefficient, and closeness centrality, for those proteins carrying them. We selected these three properties as they each reflect on potential functional role such as scope of impact (degree), diversity of responses (clustering coefficient), and placement within a possible signaling cascade . Furthermore, we investigated whether those characteristics are conserved across different species, and if the particular functions the different PTMs fulfil may become apparent when inspected from the viewpoint of protein-protein interactions.Homo sapiens, Mus musculus, Rattus norvegicus, Bos taurus), an invertebrate (Caenorhabditis elegans), the insect Drosophila melanogaster, fungi , and the plant Arabidopsis thaliana  or N-linked (arginine and asparagine residues) attachments of simple or complex carbohydrates  to proteins. Upon Hydroxylation, hydroxyl groups are attached to proline residues. Methylation refers to transfer of methyl groups to arginine or lysine residues. Nitrosylation leads to the incorporation of nitric oxide into the thiol group of cysteine residues. Protein phosphorylation is associated with attaching phosphate groups to serine, threonine, or tyrosine protein residues. Both sumoylation and ubiquitination are characterized by the attachment of small proteins to target proteins modifying their function or stability or, in the case of ubiquitination, tagging them for degradation. While the previous 10 PTM-types are characterized by the attachment of chemical moieties to proteins, disulfide bond and proteolytic cleavage lead to posttranslational modifications via changing the chemical bond structure within a given protein and in the process removing atoms from it, either by forming a covalent bond  or by breaking peptide bonds (proteolytic cleavage removing HOH). As the latter two met our established count-criteria and do indeed modify the protein, we retained them for the initial analyses. However, as data were available for human only, and to confine the analyses to moiety-addition-type PTMs, both PTM-types were left out in the subsequent analyses of network properties. For several PTM-types, several subtypes exist, e.g. Ser/Thr/Tyr phosphorylation. Generally, we considered PTM-types based on the added moiety, but also repeated selected analyzes with a further subsetting of the datasets based on the receiving group on the protein.To base our analyses on high-confidence PTM-instances, we restricted our analyses to PTM-sites that have been identified experimentally leaving out all annotated PTMs that are based on computational predictions alone. PTM-site information was collected from 11 different database resources and consolidated into a single set via sequence position information. Currently available datasets proved sufficiently large to conduct statistical analyses on the role of PTMs in the context of protein interaction networks for the following twelve PTM-types: acetylation, amidation, carboxylation, disulfide bond, glycosylation, hydroxylation, methylation, nitrosylation, phosphorylation, proteolytic cleavage, sumoylation, and ubiquitination associated with a set of nine diverse eukaryotic species covering several lineages and kingdoms: mammals  across the nine selected species were first investigated for PTM-type specific biological process involvement based on GO-term enrichment statistics as well as probed for significant co-occurrence patterns of different PTM-types on the same protein. Subsequently, we investigated whether protein sets associated with particular types of PTMs exhibit characteristic protein interaction network (PIN) properties. For the latter, we computed three basic and commonly used network properties, the degree, the clustering coefficient, and the closeness centrality, associated with proteins belonging to different PTM-specific protein sets when mapped onto the species-specific PIN. Finally, we tested whether pairs of interacting proteins exhibit preferences with regard to PTM-types they carry.Frequently, proteins are modified not only by a single PTM event, but by several and of different PTM-types. Thus, if we wish to understand the role of individual PTM-types in the context of protein-protein interactions, we first need to understand their co-occurrence on the same protein as well as their functional profile as it seems plausible that PTM-types associated with similar functional involvement will also exhibit similar characteristics with regard to their protein interactions.The functional significance of specific PTM-types and the respective proteins carrying them has been amply investigated , 36, 37.The co-occurrence patterns of different PTM-types on the same protein are critical confounding factors for the analysis of the individual PTM-types in the context of protein interactions. Evidently, frequent PTM-types will have a greater chance of co-occurring with other PTM-types on the same protein. Indeed, as judged by the Jaccard-distance, protein sets in human associated with phosphorylation, acetylation, and ubiquitination—the three PTM-types with the most observed instances —exhibit In conclusion, the overlap of different PTM-types on the same proteins is extensive and greater than expected by chance. Even though suggested by the separate clustering of PTM-types based on their functional and location annotations , with redegree quantifies the average number of connections a protein engages in. Thus, it reflects on how many interaction partners may be affected by a PTM of a given protein. The clustering coefficient allows estimating whether the proteins connected to a central reference protein are in turn connected amongst themselves. High clustering coefficients would indicate a closely knit network of local interactions, whereas low clustering coefficients would suggest that separate molecular processes with little communication between them are modulated, when a central protein undergoes a PTM. Finally, the closeness centrality allows assessing how centrally a particular protein resides relative to the overall network. Proteins with high closeness centrality are situated in central network positions such that they may serve central information relay functions. By contrast, low closeness centrality corresponds to peripheral locations as typical of initial receptor molecules.  of the nine selected species . By compHomo sapiens. Overall, all PTM-types exhibit a tendency to have higher degrees, lower clustering coefficients, and higher closeness centralities than protein sets not carrying the respective PTM-type, which includes a set of human proteins  currently not known to undergo any of the 12 PTM-types considered here. The latter set (no PTM), was observed with lower degree, higher clustering coefficient, and lower closeness centrality than proteins undergoing a PTM  showing slightly increased values for STRING-based PINs and decreased values in the remaining species. However, clearly more table cells are colored blue signifying lowered values than for the other PTM types further supported by negative fold-change values for IntAct PINs. Thus, overall, glycosylation appears to generally be associated with no significantly changed, or slightly lowered closeness centrality relative to control protein sets.Despite these data limitations, a similar overall picture emerges as observed in human. All examined PTM-types appear to be associated with proteins with increased degree and closeness centrality, but decreased clustering coefficient relative to protein sets not carrying the respective PTM-type. Glycosylation is a notable exception and seems associated with slightly decreased, rather than increased, degree centrality, while exhibiting similarly decreased clustering coefficients across all species as the other PTM-types. With regard to closeness centrality, the results for glycosylation are mixed with a few species  and their combinatorial interplay has attracted considerable attention in recent years –27. In tGeneralized across all PTM-types and species investigated here, PTM-carrying proteins appear engage in more physical contacts, with a reduced clustering coefficient among those proteins they are interacting with, and elevated closeness centrality than their respective protein sets devoid of the particular PTM-type  or that,Focusing on human PIN and PTM data, sumoylation and proteolytic cleavage stand out as being associated with the largest relative increase of degree and closeness centrality relative to reference sets. Proteolytic cleavage has been associated with activation processes and protein targeting events  and constitute a “dramatic” modification as the relative change of molecular composition of a protein can be significant. Furthermore, transporting proteins to different compartments will inevitably influence the possible interaction scope. The significance of sumoylation in a range of regulatory processes has been increasingly recognized . Our resPhosphorylation, the PTM-type with the largest data support, was identified as the PTM-type with the consistently central and with the largest potential influence scope . PhosphoBy contrast, glycosylation was found associated with proteins of low degree, low clustering coefficient, and low closeness centrality . In partAs shown in By including nine species from different kingdoms and lineages, we aimed to extract both general and species/lineage-specific trends. However, currently available datasets proved comprehensive enough for a few species only . In the case of phosphorylation, sufficient data were available across all nine species and provided a consistent result of increased degree and closeness centrality and a decreased clustering coefficient .The increased likelihood of a functional association of proteins with high interaction degree and their involvement in human disease has been reported before , 53. In Evidently, this study hinges on the completeness and accuracy of the available PTM and PIN data as well. Any bias towards a specific detection of particular protein classes and their associated PTM may further skew our results. By imposing a high significance cutoff for the PIN-data (confidence score > 0.9), and furthermore exploiting two data sources (STRING and IntAct), we believe to have taken proper precautionary steps even though some discrepancies were detected . Howeverr = 0.164) was detected suggesting that more heavily phosphorylated proteins occupy more central positions in the network of protein-protein interactions.A possible selection bias may also come from preferentially profiling those proteins for PTMs that possess “interesting” properties such has high degree. However, as PTMs are increasingly identified in massive, “shotgun” style omics studies, such selection bias may not be that critical. Rather, abundance may be a concern then. However, for phosphorylation it was reported that protein abundance is not correlated with network properties , 35. FurIn conclusion, proteins carrying different types of PTMs differ from average non-PTM-proteins and differ between each other with regard to their protein interaction characteristics. Thus, their location within the web of physical protein-protein interactions is not only non-random, but very likely indicates their specific functional roles in the orchestration of molecular processes mediated by the physical interactions between proteins.Post-translational modifications. Post-translational modifications annotated as “experimentally verified” and the associated proteins were extracted from UniProt [average(number of PTM sites)+3*sd(number of PTM sites)) were discarded as well. Twelve PTM types met those criteria in at least one species including 10 PTM-types in which a chemical moiety is attached: acetylation, amidation, carboxylation, glycosylation, hydroxylation, methylation, nitrosylation, phosphorylation, sumoylation, ubiquitination, and two PTM-types that modify the protein via forming or breaking bonds within the protein: disulfide bond and proteolytic cleavage. Nine species met those criteria for at least one PTM-type including representatives from the animal , plant , and fungal  kingdom, respectively . Then, protein sets were filtered further and referred to as “one-PTM-type-only” . For staProtein-protein interaction networks. For the nine species selected based on available PTM information, high confidence (confidence score>=0.9) protein-protein interactions (PINs) were extracted from STRING (version 9.05) [on 9.05) . In ordeon 9.05) , which sNetwork properties. The PINs associated with the selected species correspond to undirected networks. The degree of a node n is the number of edges linked to n [clustering coefficient [n is defined as. Cn = 2en/(kn(kn - 1)), where kn is the number of neighbors of n and. en is the number of connected pairs between all neighbors of n. It reflects the connectivity of adjacent nodes. The closeness centrality Cc(n) of a node n is defined as the reciprocal of the sum of shortest path length originating from n to all other nodes m [n and m. It ranges between 0 and 1. It corresponds to the inverse of the number of steps needed to traverse from all other nodes in the network to a selected node. The R package “igraph” (http://cran.r-project.org/web/packages/igraph/index.html) was used to compute the above mentioned network properties.ked to n . The clufficient  of a nod nodes m : Cc(n)=1p-values were corrected for multiple testing considering as the total number of tests all tests across species, all PTM-types for each network property. In all cases of multiple testing correction, the FDR method was used [For each PTM-type and across the selected species, the three selected network properties  were computed for all proteins carrying the respective PTM-type and compared to those proteins not carrying this particular PTM-type. Significant differences between the respective two distributions were detected based on a Mann–Whitney test. The was used . SignifiP-value was set to 0.01.For all species selected in this study, the available genome gene ontology (GO) process, function and cellular compartment annotations were extracted from UniProtKB-GOA  as the rA,SB)|/|Union|, where the set of proteins associated with A and B are denoted as SA and SB. Fisher’s exact test was used to test the significance of co-existence.For each pair of PTMs (A and B) in one species, The Jaccard index for the co-existence of A and B is defined as |Intersection as of May, 2014. In order to test the overlap between human diseases proteins with proteins associated with high (top 25%) or low (bottom 25%) network property values, Fisher’s exact test was used for all PTM-types. The p-values were corrected for multiple testing (FDR) across all PTM-types and network properties.Human disease proteins were downloaded from OMIM  threshold indicating significance was set to 0.01.The top five GO-terms were included that were found significantly enriched for each PTM-type. Each element in the heat map  represents the grey-scale-encoded p-value, in which a particular combination of PTM-type and GO-term was found significantly enriched. To he combined whole UniProtKB-GOA for all the selected species was used as the background set, Fisher’s exact test with FDR correction was used for the enrichment analysis, and the (TIF)Click here for additional data file.S2 Figp-value 0.05, **: p-value 0.01) according to a Mann-Whitney test.The red (blue) asterisks on the top of violin plot represents the corresponding non-PTM group has a significantly higher (lower) median value compared to the non-PTM group (*: (TIF)Click here for additional data file.S3 Figp<0.05 (p<0.01) by Mann-Whitney test with FDR correction, the values in red or blue text represent significantly higher or lower network properties which are inconsistent with the background color.The species are ordered according to their phylogenetic relationships as shown on the left. For every PTM-type, the log-2 of fold difference value for the degree/clustering coefficient/closeness centrality value relative to the respective value associated with proteins not carrying this particular PTM-type are given for PINs based on STRING and IntAct, respectively. Color scale indicates increased (red) or decreased (blue) values in the PTM-set relative to the non-PTM-set with symmetric color intervals  Bold-font (underlined) fold-changes indicate significant fold-changes at (TIF)Click here for additional data file.S4 Figp<0.05 (p<0.01) by Mann-Whitney test with FDR correction, the values in red or blue text represent significantly higher or lower network properties, which are inconsistent with the background color based on mean (not median) values.Protein sets were selected to contain one PTM-type only (one-PTM-type-only dataset). The species are ordered according to their phylogenetic relationships as shown on the left. For every PTM-type, the log-2 of fold difference value for the degree/clustering coefficient/closeness centrality value relative to the respective value associated with proteins not carrying this particular PTM-type are given for PINs based on STRING and IntAct, respectively. Color scale indicates increased (red) or decreased (blue) values in the PTM-set relative to the non-PTM-set with symmetric color intervals  Bold-font (underlined) fold-changes indicate significant fold-changes at (TIF)Click here for additional data file.S5 FigIncreased frequencies of protein-protein interactions  carrying the respective PTM-types relative to expectation. Line width is proportional to the number of species, which exhibit significant interactions of PTM-types carried by interacting proteins. The contingency table for the Fisher exact test contained the respective counts for number of proteins associated with a particular PTM-pair versus all alternative pairings and whether they have been reported to interact or not with applied FDR-corrected p-value threshold of <0.01.(TIF)Click here for additional data file.S6 Figr,  were: degree r = 0.056 (1.46E-06), clustering coefficient: r = -0.068 (6.27E-08), closeness centrality: r = 0.164 (0.00).The red line connects the mean network property values of proteins associated with different numbers of PTMs. Associated Pearson linear correlation coefficients, (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Central sensitization is modulated by the endogenous opioid system and plays a major role in the development and maintenance of pain. Recent animal studies performed following resolution of inflammatory pain showed reinstatement of tactile hypersensitivity induced by administration of a mu-opioid-antagonist, suggesting latent sensitization is mediated by endogenous opioids. In a recent crossover study in healthy volunteers, following resolution of a first-degree burn, 4 out of 12 volunteers developed large secondary areas of hyperalgesia areas after a naloxone infusion, while no volunteer developed significant secondary hyperalgesia after the placebo infusion. In order to consistently demonstrate latent sensitization in humans, a pain model inducing deep tissue inflammation, as used in animal studies, might be necessary. The aim of the present study is to examine whether a high-dose target-controlled naloxone infusion can reinstate pain and hyperalgesia following recovery from open groin hernia repair and thus consistently demonstrate opioid-mediated latent sensitization in humans.Patients submitted to unilateral, primary, open groin hernia repair will be included in this randomized, placebo-controlled, double-blind, crossover study. The experimental days take place 6–8 weeks after surgery, time-points at which patients are expected to be almost pain- free. Prior to administration of naloxone or placebo, the primary outcome  and the secondary outcomes  will be assessed. These assessments will be repeated at each step of the target-controlled infusion of placebo or naloxone at estimated median (95 % CI) plasma concentrations of 344 ng/ml , 1059 ng/ml  and 3196 ng/ml .We aim to demonstrate opioid-mediated latent sensitization in a post-surgical setting, using pain as a clinical relevant variable. Impairment of the protective endogenous opioid system may play an important role in the transition from acute to chronic pain. In order to sufficiently block the endogenous opioid system, a high-dose target-controlled naloxone-infusion is used, in accordance with recent findings in animal studies.EUDRACT: 2015-000793-36 (Registration date: 16 February 2015)NCT01992146 (Registration date: 12 December 2014)Clinicaltrials.gov:  In the translational study, 2 mg/kg of naloxone was administered to 3 pilot volunteers and 12 study volunteers. Six volunteers reported mild transient adverse effects, including tiredness, epigastralgia, frontal headache and photophobia. Furthermore, naloxone did not induce any overt changes in electrocardiography, blood pressure, heart and respiratory rate and oxygen saturation during the experimental days . AdditioIn regard to the risk of development of a sustained pain state after the naloxone administration in patients after GHR, we consider it highly unlikely. Naloxone is a short-acting reversible antagonist of opioid receptors and does not affect the production of endogenous opioids. Moreover, a prolonged reversal of endogenous opioids is unlikely due to the pharmacokinetic properties of naloxone. In adults, the distribution half-life of naloxone is 40 to 70 seconds , and theThe experimental days are taking place 6–8 weeks after surgery, time-points at which patients are nearly pain-free, albeit before the surgically induced tissue injury has fully resolved. Our primary outcome is a summated measure of reinstatement of pain , since pain is the most relevant outcome in the clinical setting. For the sample size calculation we opted to use a minimal relevant difference of 3 NRS-units, which is relatively low, considering our use of a summated measure (SPI) of 3 assessments . Baseline SPI is estimated to be 8 (mean per assessment = 2.7) NRS-units: a significant increase would then be ≥ 11 (mean per assessment ≥ 3.7) NRS-units: i.e., ≥ 38 %.Additionally, secondary outcomes , which are of exploratory nature, will provide information on sensory properties of the surgical site and of the mirror-site in the contralateral groin, and how they are modulated by naloxone.n = 4) cautious interpretation is required [Since secondary hyperalgesia is a measure of central sensitization, it is of interest to differentiate between subjects developing large secondary areas of hyperalgesia (high-sensitizers) and subjects developing small areas (low-sensitizers). Past research has shown that the size of the secondary area of hyperalgesia is reproducible within subjects and can thus be regarded as a phenotype . It is urequired . This isrequired , known trequired , 42. In In conclusion, by administrating a high-dose TCI of naloxone, the present study aims to show dose-dependent reinstatement of pain and hyperalgesia following recovery from a unilateral, primary, open GHR procedure. This would provide further evidence of opioid- mediated latent sensitization in humans. We consider that impairment of the protective endogenous opioid system may play a relevant role in the development of persistent post-surgical  or post-Inclusion of the first participants is expected in fall 2015.


Physicians are considered to be the most informed consumers in the use of medical services since they have more information about diseases or medical technology. However, although plenty of researchers have suggested that different medical seeking behavior exists among physicians, very few empirical studies have been conducted to investigate differences in medical utilization between physicians and the general population.We explored differences in the utilization of healthcare services between physicians and the general population using a population-based dataset.A cross-sectional study.Data for this study were sourced from the Taiwan Longitudinal Health Insurance Database 2000. We included 1426 physicians and 1426 sex- and age-matched comparison subjects.We used Wilcoxon-Mann-Whitney tests to explore differences in variables of healthcare resource utilization between physicians and comparison subjects. We further used Kruskal-Wallis tests to examine differences in variables of healthcare resource utilization between physician practice location and comparison subjects.p<0.001) and significantly lower outpatient costs  than comparison subjects. Furthermore, physicians had lower total health service costs than comparison subjects . This indicates that the mean total health service costs in the year 2010 was 1.66-fold greater for comparison subjects than for physicians. We also found that there were significant differences in the mean number of outpatient services (p<0.001), outpatient costs (p = 0.001), inpatients costs (p = 0.018), and total costs (p = 0.001) among office-based physicians, hospital-based physicians, and comparison subjects. Specifically, Scheffe contrast tests showed that office-based physicians had significantly more outpatient visits  and significantly higher outpatient costs  than hospital-based physicians.We found that physicians had significantly fewer outpatient visits (13.2 vs. 15.7, Physicians had less healthcare utilization than comparison subjects. Furthermore, hospital-based physicians had higher inpatient costs and less outpatient services and costs than office-based physicians. Physicians are considered to be the most informed consumers in the use of medical services since they have more information about diseases or medical technology. Prior studies reported that female doctors had a higher prevalence of use of hormone replacement therapy than other women ,2. AnothChristie & Ingstad indicated that when physicians are ill, they often have difficulty acting as patients . Many stTherefore, in this study, we explored differences in the utilization of healthcare services between physicians and the general population using a population-based dataset. In addition, this study further compared differences in the utilization of healthcare services between hospital-based physicians and office-based physicians.n = 23.72 million) under the NHI program by the Taiwan National Health Research Institute. The LHID2000, which was open to the researchers in Taiwan, was available from the National Health Insurance Institute (http://nhird.nhri.org.tw/date_01.html). Hundred studies have been published in international peer-reviewed journals utilizing data from the NHI program [Data on the health service utilization of sampled subjects used in this study were retrieved from the Longitudinal Health Insurance Database (LHID2000). The LHID2000 includes sociodemographic information and data on medical claims for 1 million enrollees since initiation of the Taiwanese National Health Insurance (NHI) program in 1995. These 1 million enrollees were randomly selected from all enrollees listed in the 2000 Registry of Beneficiaries  were excluded from the study. We also excluded those who died in the years 2010 and 2011 (n = 3) in order to assure equal follow-up periods for all selected physicians. Ultimately, we were left with 1426 physicians who met the study criteria. Of these physicians, 420 (29.5%) and 1006 (70.5%) were office-based and hospital-based physicians, respectively.This cross-sectional study consisted of a study group and a comparison group. To select the study group, we first identified 1508 physicians who were practicing medicine in January 1~December 31, 2010. In order to better reflect the actual scenario of physicians practicing in Taiwan, physicians aged over 80 years and under 25 years  from the remaining enrollees matched with study subjects by sex and age group  with the SAS SURVEYSELECT Procedure program . As a result, this study included 2852 study subjects including 1426 practicing physicians and 1426 matched comparison subjects.The following key variables of healthcare resource utilization were selected: numbers of outpatient visits, inpatient days, and costs of outpatient and inpatient treatment in the year 2010. Taiwan’s NHI is characterized by universal coverage, a single-payer payment system with the government as the sole insurer, comprehensive benefits, access to any medical institution of a patient’s choice, and a wide variety of providers well distributed throughout the country. Therefore, the costs of healthcare services include the costs of diagnoses, medications, treatments, laboratory tests, diagnostic imaging, and surgery.p values of ≤0.05.The SAS statistical package  was used for the statistical analyses in the dataset of this study (S1 File). Descriptive statistical analyses, including the frequency, percentage, mean, and standard deviation (SD), were performed on all of the identified variables of healthcare resource utilization. We used Wilcoxon-Mann-Whitney tests to explore differences in variables of healthcare resource utilization between physicians and comparison subjects. We further used Kruskal-Wallis tests to examine differences in variables of healthcare resource utilization between physician practice location and comparison subjects. Differences were considered significant for two-sided p = 0.711). After matching for gender and age group, there were significant differences in monthly incomes (p<0.001) between physicians and comparison subjects. Physicians were more likely to have a monthly income of ≥US$830 than were comparison subjects.p<0.001) and significantly lower outpatient costs  than comparison subjects. Furthermore, physicians had fewer inpatient days  and lower inpatients costs  compared to comparison subjects. Correspondingly, physicians had lower total health service costs than comparison subjects . This indicates that the mean total health service costs in the year 2010 was1.66-fold greater for comparison subjects than physicians.p<0.001), outpatient costs (p = 0.001), inpatients costs (p = 0.018), and total costs (p = 0.001) among office-based physicians, hospital-based physicians, and comparison subjects. Specifically, Scheffe contrast tests showed that office-based physicians had significantly more outpatient visits  and significantly higher outpatient costs  than hospital-based physicians. In other words, the mean number of outpatient visits was about 1.8-fold higher for office-based physicians than hospital-based physicians. However, differences in inpatient costs and total costs between office-based physicians and hospital-based physicians did not reach a significant level according to Scheffe contrasts tests.The use and costs of all healthcare services among office-based physicians, hospital-based physicians, and comparison subjects are presented in Based on our knowledge, this is the first population-based study to compare utilization of healthcare services between physicians and the general population. We found that physicians consistently had lower outpatient and inpatient services and costs compared to the general population.Our findings are in line with previous observations that physicians may perform self-diagnosis and treatment instead of seeking medical assistance from other health professionals –13. In aFurthermore, we found that office-based physicians incurred higher outpatient services and costs than hospital-based physicians. Prior studies indicated that office-based physicians work independently and tend to treat themselves when they have minor health problems –8. ThereFurthermore, the healthcare system in Taiwan is characterized as a closed-staff system, under which almost all physicians who practice in hospitals are employees of those hospitals, and physicians who practice in clinics do not have privileges at hospitals. Hospital-based physicians are compensated with highly variable combinations of salaries, bonuses, volume-driven fees, and teaching or research supplements. Therefore, their earnings do not fully exhibit a proportional relationship to their clinical efforts. However, incomes of office-based physicians are mainly based on patient volumes, so they have a strong financial incentive to provide more outpatient services including self-treatment in order to reach target incomes –18. ThisThis study also found that compared to office-based physicians, hospital-based physicians had higher inpatient costs. One study found that hospital-based physicians had a slightly higher risk of hospitalization compared to community practitioners in Taiwan . In contA key strength of our study is the use of a population-based dataset to investigate differences in the use of medical utilization among hospital-based physicians, office-based physicians, and the general population in Taiwan. This feature afforded sufficient statistical power and an adequate sample size to detect differences in inpatient and outpatient services among the three groups after adjusting for confounders.Nevertheless, there are several limitations of this study. First, the LHID2000 used in this study provides no information on working characteristics, lifestyles, health habits, or the disease severity of subjects. Second, most subjects included in our study were of Chinese ethnicity, so the ability to generalize the results to other ethnic groups is uncertain. Finally, this study only employed 1 year of data of healthcare utilization, and this might not fully represent long-term treatment-seeking behavior of the sampled subjects.Despite these limitations, our population-based study found that physicians had less healthcare utilization than comparison subjects. Furthermore, hospital-based physicians had higher inpatient costs and less outpatient services and costs than office-based physicians. Further large-scale long-period epidemiological studies are suggested to explore differences in the utilization of healthcare services between physicians and the general population in other regions and countries.


Metagenomics is limited in its ability to link distinct microbial populations to genetic potential due to a current lack of representative isolate genome sequences. Reference-independent approaches, which exploit for example inherent genomic signatures for the clustering of metagenomic fragments (binning), offer the prospect to resolve and reconstruct population-level genomic complements without the need for prior knowledge.We present VizBin, a Java™-based application which offers efficient and intuitive reference-independent visualization of metagenomic datasets from single samples for subsequent human-in-the-loop inspection and binning. The method is based on nonlinear dimension reduction of genomic signatures and exploits the superior pattern recognition capabilities of the human eye-brain system for cluster identification and delineation. We demonstrate the general applicability of VizBin for the analysis of metagenomic sequence data by presenting results from two cellulolytic microbial communities and one human-borne microbial consortium. The superior performance of our application compared to other analogous metagenomic visualization and binning methods is also presented.de novo for the visualization and subsequent binning of metagenomic datasets from single samples, and it can be used for the post hoc inspection and refinement of automatically generated bins. Due to its computational efficiency, it can be run on common desktop machines and enables the analysis of complex metagenomic datasets in a matter of minutes. The software implementation is available at https://claczny.github.io/VizBinunder the BSD License (four-clause) and runs under Microsoft Windows™, Apple Mac OS X™ (10.7 to 10.10), and Linux.VizBin can be applied The online version of this article (doi:10.1186/s40168-014-0066-1) contains supplementary material, which is available to authorized users. Mixed microbial communities are ubiquitous and play fundamental roles in the Earth’s biogeochemical cycles, as well as in human health. Shotgun sequencing of extracted DNA from microbial consortia allows for culture-independent analysis of their composition and/or genetic potential. So far, metagenomics has been applied to a panoply of microbial communities of differing complexities -6.In metagenomic analyses, the characterization of constituent populations is typically carried out using reference-based approaches whereby sequence fragments, e.g., filtered sequence reads or reconstructed genomic fragments (contigs), are aligned to previously characterized isolate genomes ,8. Howevkmers are counted over sequence fragments and counts are normalized to represent frequency distributions [k. So far, the exploration of the signature space has been hampered by the comparably high dimensionality of genomic signatures: for k=5, the vectors are embedded in a 1,024-dimensional space. To reduce the dimensionality of the data, approaches based on self-organizing maps (SOMs) have been used for the visualization and delineation of population-specific sequence clusters, e.g., emergent SOMs (ESOMs) [Reference-independent deconvolution of metagenomic datasets from single samples generally relies on the use of data-inherent characteristics, e.g., oligonucleotide composition , to grouibutions  resultin (ESOMs) ,13.https://claczny.github.io/VizBin).We have recently demonstrated  that nonhttps://github.com/claczny/VizBin. The only runtime requirements of VizBin are a working Java™ installation and the Java™ Standard Edition Runtime Environment . This work implements parallelization into the original C source code for BH-SNE (http://homepage.tudelft.nl/19j49/t-SNE.html) by integrating the OpenMP®; Application Programming Interface (API). VizBin incorporates this parallelized version of BH-SNE for the computation of the two-dimensional embedding which is then visualized by the GUI. A copy of the VizBin software can be downloaded from http://claczny.github.io/VizBin.VizBin is a graphical user interface (GUI)-based desktop application for Microsoft Windows™, Apple Mac OS X™ (10.7 to 10.10), and Linux. The GUI is written in Java™ and makes use of different Java™ libraries as described in https://github.com/claczny/VizBin/wiki).A key feature of VizBin is that it allows the visualization and subsequent binning of metagenomic fragments, e.g., contigs or long reads, for users without any bioinformatic background and exploits the superior pattern recognition capabilities of the human eye-brain system for cluster identification and delineation. The VizBin application presents the user with a simple dialogue to specify the input FASTA-file containing the metagenomic fragments of interest. The user can then choose a minimum input sequence length before the calculation of the genomic signatures. By default, the program will only consider fragments of at least 1,000 nt in length, but lower thresholds can also be defined. However, depending on the dataset, clusters are expected to overlap when the lengths approach 600 nt . Due to In this section, we compare the performance of our approach to a commonly used method for visualizing metagenomic data, i.e., ESOM-based visualization, as well as to a state-of-the-art fully automated binning method . The pede novo reference-independent visualization and subsequent human-augmented binning of metagenomic data. Despite a general accordance between VizBin and MaxBin, clusters are visible that share the same color but clearly are separate in the VizBin plot  which was recently used to asses the performance of the MaxBin approach . Individt Figure .Figure MaxBin  is a staWe used VizBin to inspect the original MaxBin-based bins of two cellulolytic microbial communities (37A and 37B)  and one Using the example of the 37B dataset, bins 37B.out.024 and 37B.out.026 exhibit each two pronounced and wellseparated subclusters . While ribed in , VizBin Project name: VizBinProject home page:https://claczny.github.io/VizBinOperating system(s): Platform independentProgramming language: Java™ version 7 or greaterOther requirements: Java™ Standard Edition Runtime Environment (JRE); local installation of BLAS/LAPACK for maximum performance (detailed information is provided in the project’s wiki)License: BSD License (4-clause). Detailed licensing information is available at https://github.com/claczny/VizBin.Restrictions: NoneAdditional file 1VizBin supplementary materials.Additional file 2ZIP archive containing VizBin visualization screenshots of the individual bins for the three datasets  originally reported in .rted in .


Introduction: Two principal findings of cardiovascular disease in end-stage renal disease patients undergoing regular hemodialysis are left ventricular hypertrophy (LVH) and arterial disease due to rapidly progressive atherosclerotic vascular disease that can be characterized by an enlargement and hypertrophy of arteries .Objectives: In this study, we sought to evaluate the relationship between left ventricular hypertrophy with intima-media complex thickening in end-stage renal disease patients undergoing regular hemodialysis.Patients and Methods: Sixty-one patients with end-stage renal disease (ESRD) who were undergoing regular and maintenance hemodialysis treatment  were studied. The subjects consisted of 50 non-diabetic hemodialysis patients  and 11 diabetic hemodialysis patients . For all the subjects, echocardiography and carotid intima-media thickness measuring by B-mode ultrasonography were performed. Results: In this study, there was a positive correlation between stages of LVH with duration of hemodialysis treatment, stages of hypertension (HTN), and with carotid-IMT. A positive correlation was also seen between stages of LVH and presence of chest pain, and more thickening of the intima-media complex was seen in the diabetic group. Diabetes mellitus was associated with the presence of chest pain, as was positive correlation between stages of HTN with IMT, and a reverse correlation was observed between IMT with the percent of cardiac ejection fraction.Conclusion: Prevalence of thickening in intima-media complex is more evident in hemodialysis subjects with LVH. When there is LVH, IMT is similar in severity to the LVH. Prevalence of thickening in intima-media complex is more evident in hemodialysis subjects with left ventricular hypertrophy. When there is left ventricular hypertrophy, intima-media complex thickening is similar in severity to the left ventricular hypertrophy.Cardiovascular disease is the principal cause of morbidity and mortality in hemodialysis (HD) patients -2. The pWe studied a group of hemodialysis patients consisting of diabetics and non-diabetics to find an association between IMT with LVH and IMT with severity of hypertension and chest pain.This cross-sectional study was done on 61 unselected patients ESRD, undergoing regular HD. Patient exclusion criteria were cigarette smoking, body mass index (BMI) more than 25, anti-lipid drug use, recent MI and vascular diseases as well as pericarditis and pericardial effusion in echocardiography. For stratification of hypertensive patients, according to the sixth and seventh report of the Joint National Committee on Prevention, Detection, Evaluation and Treatment of High Blood Pressure, we stratified hypertensive patients from stage one to three  . Stages Carotid sonography was done by a single sonologist unaware of history or lab data of patients with a Honda-HS-2000 Sonograph and 7.5 MHz linear probe to measure IMT ,8. The pThe research followed the tenets of the Declaration of Helsinki; written informed consent was obtained; and the research was approved by ethical committee of Shahrekord University of Medical Sciences.For statistical analysis, descriptive data are expressed as Mean±SD. Comparison between groups was performed using chi-squared test, Mann-Whitney U, as well as Kruskal-Wallis and Fisher’s exact test. For correlations, we used Spearman’s rho, Phi and Cramer’s V and also Eta tests. In addition, partial correlation test (with adjustment for age) was used. All statistical analysis was performed using SPSS version 11.00. Statistical significance was inferred at a p< 0.05.The total number of patients was 61 , which consisted of 50 non-diabetic hemodialysis patients  and 11 diabetic hemodialysis patients . et al. in a study on 62 patients on maintenance hemodialysis observed 65% prevalence of LV hypertrophy and showed that age, body mass index and duration of HTN were associated with LV hypertrophy and asymmetric septal hypertrophy  have been completely observed by the author.None.


Motivation: The repetitive nature of plant disease resistance genes encoding for nucleotide-binding leucine-rich repeat (NLR) proteins hampers their prediction with standard gene annotation software. Motif alignment and search tool (MAST) has previously been reported as a tool to support annotation of NLR-encoding genes. However, the decision if a motif combination represents an NLR protein was entirely manual.Results: The NLR-parser pipeline is designed to use the MAST output from six-frame translated amino acid sequences and filters for predefined biologically curated motif compositions. Input reads can be derived from, for example, raw long-read sequencing data or contigs and scaffolds coming from plant genome projects. The output is a tab-separated file with information on start and frame of the first NLR specific motif, whether the identified sequence is a TNL or CNL, potentially full or fragmented. In addition, the output of the NB-ARC domain sequence can directly be used for phylogenetic analyses. In comparison to other prediction software, the highly complex NB-ARC domain is described in detail using several individual motifs.Availability and implementation: The NLR-parser tool can be downloaded from Git-Hub (github.com/steuernb/NLR-Parser). It requires a valid Java installation as well as MAST as part of the MEME Suite. The tool is run from the command line.Contact:burkhard.steuernagel@jic.ac.uk; fjupe@salk.eduSupplementary information:Supplementary data are available at Bioinformatics online. R) genes from wild relatives (reviewed in (R genes encodes nucleotide-binding domain leucine-rich repeat proteins (NLRs or NB-LRRs). These are key receptors that recognize secreted pathogen effector molecules or their effect in the plant. On recognition, these proteins commonly lead to a hypersensitive response in the form of local cell death to prevent further spread of pathogens relying on living tissue . We use 20 previously biologically characterized motifs  for NLR gene products using the here presented MAST pipeline. In total, we identified 266 from within 35 386 Arabidopsis proteins as partial or complete NLRs. The original TAIR protein annotation provides 219 sequences with one of the following annotation terms: ‘Toll-Interleukin-Resistance (TIR) domain’, ‘NB-ARC’ or ‘NBS-LRR’ and 212 of these were also identified with our MAST pipeline. Blastp analyses of the seven remaining proteins identified two false-negatives with an NB-ARC and LRR domains, but five that had neither an NB-ARC nor an LRR domain and thus can be excluded. Detailed analysis shows that the two false-negatives correspond to the ancient and small group of NLRs with similarity to ADR1 . Therefore, we report a 100% specificity for the NLR-Parser.In a proof-of-concept study, we screened the available set of  to ADR1 . Here, tng HMMER  and founBrachypodium distachyon. The NLR-parser pipeline identified 586 partial or complete NLRs. All 190 proteins that the NLR-parser annotated as complete NLRs have previously been annotated as resistance genes (http://phytozome.jgi.doe.gov/). The general quality of the Brachypodium annotation, relying on similarity to Arabidopsis and rice does not allow a precise estimation of sensitivity and selectivity. However, there is a good consistency between annotation, found PFAM domains and NLR-Parser. Eight genes with NB-ARC domain and LRR have not been found by the NLR-Parser, including an ADR1-like. Conversely, the NLR-Parser annotated 47 proteins as complete NLRs while HMMER only detected the NB-ARC domain, not any LRR (Supplementary Table 2).We further tested the MEME motifs in our NLR-parser for their functionality in monocotyledonous plant genomes and screened the publicly available set of annotated genes from A. thaliana and B. distachyon, we were able to show the functionality of the 20 well-characterized MEME motifs in monocotyledonous and dicotyledonous plants. The output of this tool is directly usable for downstream applications including phylogenetic analyses, or visualization on the corresponding reference sequence. The tab delimited output format is publishable as a Supplementary Table.Due to the biological importance and relevance for breeding, the identification and annotation of NLR-type disease resistance genes has high priority in all plant genome sequencing projects. These annotations, however, rely heavily on gene-prediction software. In the past, we were able to show that up to 50% of the total NLR complement was either wrongly predicted or completely missing. Our MAST Parser tool provides high precision identification of NLR gene sequences from every input format that is available from genome sequencing projects including contigs, scaffolds, pseudomolecules or chromosomes. In two experiments with the model plants The MAST Parser pipeline that we present here will streamline NLR identification efforts within genome sequencing projects in monocotyledonous and dicotyledonous plants.