However, it will need to be determined by rigorous at pre-clinical and clinic testing. We hope this review will be beneficial for current research in the area of immunotherapy or radio-immunotherapy.Rituximab has been revolutionized and validated CD20 targeting monoclonal antibody. Although, it is widely used for lymphoma therapy and many patients have been benefited. However significant numbers of patients are refractory or developed resistance to current therapies due to low level of CD20 expression and/or availability on cells surface. Thus development of novel anti-CD20 mAbs with great cell killing ability and enhance CD20 levels on cell surface can potentially exploit lymphoma therapy. In this scenario, we are summarizing the recently developed mAbs against CD20 and compounds that have ability to induce CD20 expression at significant level. We also are providing information regarding combination strategy for use of radiation and anti-CD20 mAbs  Cancer remain is a global concern and great challenge to medical management. It has emerged as the second leading cause of death globally after cardiovascular diseases. The International Agency for Research on Cancer (IARC) recently estimated that 8.2 million deaths worldwide were due to cancer with 14.1 million new cases per year being reported worldwide [It is a type of blood cancer that occurs when lymphocytes begin behaving abnormally. Lymphocytes are white blood cells that protect the body from infection and disease. Abnormal lymphocytes may divide faster than normal cells or they may live longer than they are supposed to. Lymphoma may develop in many parts of the body such as the lymph nodes, spleen, bone marrow, blood or other organs of the human body.Hodgkin lymphoma (HL): There are 6 types of HL an uncommon form of lymphoma that involves the Reed-Sternberg cells.Non-Hodgkin lymphoma (NHL): There are more than 61 types of NHL some of which are more common than others. In other words any lymphoma that does not involve Reed-Sternberg cells is classified as non-Hodgkin lymphoma.There are two main types of lymphomas:◦B-cell lymphomas: B-cell lymphomas make up most (about 85%) of non-Hodgkin lymphomas in the United States (http://www.cancer.org/cancer/non-hodgkinlymphoma).◦T-cell lymphomas: T-cell lymphomas make up less than 15% of non-Hodgkin lymphomas in the United States. There are many types of T-cell lymphoma but they are all fairly rare (http://www.cancer.org/cancer/non-hodgkinlymphoma).Classification of non-Hodgkin lymphoma (NHL) can be quite confusing (even for doctors) because there are so many types and several different organs are involved. The most recent WHO classification is based on microscopic observations, the chromosome features of the lymphoma cells and the presence of certain proteins on the surface of the cellsLow grade (indolent): These tend to grow very slowlyHigh grade (aggressive): These tend to grow more quicklyDoctors put non-Hodgkin lymphomas into two groups depending on how quickly they are likely to grow and spread .Low graCurrently different treatment modalities are used for treatment of cancer for instance surgery, radiation therapy, chemotherapy, and immunotherapy (targeted immunotherapy). Traditionally radiation therapy (RT) plays an important role in the management of NHL. RT alone may be used as curative treatment for stages I and II in patients with indolent NHL. For the more extensive and aggressive conditions RT is used in combination with chemotherapeutic substances. While indolent and aggressive NHLs are responsive to RT and chemotherapy 50%–70% of patients are relapsed ,4. Most The concept of targeted immunotherapy was known almost a century before. Paul Ehrlich 1854–1915) the founder of immunology discovered a ‘magic bullet’ on the surface of an infected cell which able to selectively deliver a toxin to the bacterium inside the cell while sparing other tissues. This led to a discovery of therapy for syphilis in the pre-penicillin era for which Ehrlich received a Nobel Prize in 1908 . The con54–1915 tThe clinical success of CD20-targeted immunotherapy is limited expression of CD20 molecules. It is specifically expressed on tumor cells; it is not expressed in hematopoietic stem cell and differentiated B-cells. Therefore the B-cell hematopoiesis and other cell lineages are not in danger. CD20 is a non-glycosylated transmembrane phosphoprotein with four transmembrane domains. It has been a superb biomarker for immunotherapies targeting B-cell derived diseases defined by the monoclonal antibody tositumomab –13. The Learning about the limitations of rituximab and other monoclonal antibodies lead to the development of new treatments strategies, appropriate modifications in the Fc region of mAbs or development of novel anti-CD20 mAbs as well as screening and identification of small molecules which have ability to increases levels of CD20 on surface of human tumor cells. The increases in CD20 levels on cell surface and developing these novel mAbs may be increase more CD20 and antibody associations, increases their binding affinity, reducing immunogenicity and improving ADCC, CDC and PCD. In an effort to increase their cytotoxic activity mAbs have also been conjugated to radioisotopes, chemo-toxins and made various modifications in Fc region. The purpose of this article is to update the scientific readers those are working in the area of recent advances in the biotechnology for the development of novel anti-CD20 mAbs and identification of CD20 modulators for the improvement of immunotherapeutic responses against lymphoproliferative disorders.The development of anti-CD20 mAbs against lymphoma diseases were started from concept of magic bullets in 1879. The main induction of monoclonal antibodies technology or generation was initiated after the Kohler and Milstein. His scientific work directed to a great expectation that mAbs would provide effective targeted therapy for cancer. Although the CD20 specific antibody B1 (renamed tositumomab) was first discovered in 1981. However rituximab became the first mAb approved by the U.S. Food and Drug Administration (FDA) for use in relapsed and indolent lymphoma . RituximWhile successes, limitations and elucidations of the mechanism of action of rituximab have increased our understanding or knowledge and helped in our goal to improve the efficacy and decreasing the associated adverse effects as well as providing effective therapies for those patients who have developed resistance to rituximab. The interest in development of anti-CD20 mAbs continues to provide a major focus for scientific and clinical investigators alike and it seems to be highly probable that this research interest will continue to grow as the new generation of anti-CD20 mAbs have developed and tested clinically. Currently, there are several new generation anti-CD20 mAbs have been engineered and/or modified to improve antitumor activity and Fc binding affinity and provide advantages over rituximab that are currently undergoing clinical investigation. They may be grouped in two categories: second or third generation anti-CD20 mAb.Second generation mAbs designed as humanized or fully human with unmodified Fc domain, the purpose of reducing immunogenicity compared to chimeric mAb rituximab. Second generation mAbs include Ocrelizumab, veltuzumab and ofatumumab. Ocrelizumab and veltuzumab are humanized while ofatumumab is fully human antibody. Ocrelizumab  is a humanized type I anti-CD20 IgG1 mAb. It has differences in several amino acid positions within the CDRs variable regions of the light and heavy chain as compared to rituximab. Thus it demonstrated superior binding affinity for the low-affinity variants of the FcγRIIIa receptor (CD16). Moreover it showed higher ADCC and lower CDC activity as compared to rituximab toward lymphoid malignancies. Currently, this mAb has been evaluated through a phase I/II study in patients with relapsed/ refractory follicular lymphoma (FL) after rituximab failed therapy and showed superior efficacy and safety ,33. VeltLikewise, the third generation humanized mAbs are modified mAbs in the Fc region. The Fc domain was engineered with the glycol or protein. The main goal of this modification is to improving the therapeutic efficacy in all patients; particularly patients in which expression with low affinity version of the Fc receptor are found on their tumor cells.Third generation anti-CD20 mAbs include AME133v, Pro131921 (v114), GA101, R603/EMAB-6 and TRU-015. They are ongoing in early phases of clinical development. AME-133v  is an Fc protein engineered humanized type I IgG1 mAb which currently being evaluated in a Phase I/II dose escalation study in patients with relapsed/refractory follicular B-cell NHL . In vitrA number of CD20 mAbs are now used in clinical practice or are in different stages of development . Most ofIn clinical applications, the efficacy of anti-CD20 mAbs seems to be decline after a period of months of treatments due to therapeutic resistance. Actually the explanation for this therapeutic resistance is not clear. The possible mechanisms of this resistance of B-cell NHLs against anti-CD20 mAbs therapy may be include three patterns: (I) Protection of the tumor cells from mAbs mediated depletion of B-cell lymphoma by ADCC, CDC and apoptotic stimulation (II) Inadequate binding of mAbs to the CD20 molecule and (III) Low levels of CD20 antigens on cells surface or reduce CD20 surface levels on cells.Although, some investigators provide information that decreased levels of CD20 expression and/ or harbor low levels of CD20 on surface of malignant B-cells may be one of the major contributing factors for antibody response ,104. HowThe bryostatin-1 induced increases in CD20 expression were found at the transcriptional level. The effects of bryostatin-1 on CD20 expression in NHL derived cells was apparently mediated through the MAPK/ERK signal transduction pathway and involved protein kinase C . An incrIn conclusion over the last 10 years rituximab is used against the treatment of all common B-cell malignancies. Based on this success, limitations and elucidation of the mechanism various novel anti-CD20 mAbs has been developed to improved clinical outcomes with outstanding performance in ADCC, CDC and PCD and reduced immunogenicity. Although, the mechanisms of action of each anti-CD20 mAbs has been well studied in preclinical settings. However, the variability seen in clinical responses of these mAbs may be depend on level of CD20 expression, levels of circulating soluble CD20, presence of effector cells, CD20 binding epitope and kinetics, binding with Fc receptors, tissue distribution and tumor burden. Singh et al. recently published a report provide information that sub-lethal dose of radiation can induced CD20 surface levels on cells determined efficacy of both type I (rituximab) and type II (tositumomab) anti-CD20 mAbs in vitro. However, more preclinical and clinical investigations need to be confirmed. Therefore, the ability to selectively control CD20 expression and appropriate modifications in Fc domain of mAbs may be great importance in enhancing therapeutic values and in optimizing anti-CD20 immunotherapy and radio-immunotherapy. The modulation in CD20 expression may provide more binding sites for anti-CD20 mAbs and may play a major role in therapeutic response. Based on this information and previous data we suggested that use of external beam radiotherapy (in a site selective manner) just prior to immunotherapy may be beneficial for tumor clearance and maximum clinical outcomes.


Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting millions of individuals worldwide, and a major risk factor for disabling cerebral embolic stroke. Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant inherited disorder, characterized by vascular abnormalities with high-bleeding tendency and therefore intolerance for oral anticoagulation. We report a case of percutaneous closure of the left atrial appendage, which might be a good alternative strategy instead of chronic oral anticoagulation to protect patients with high-risk AF and HHT from cerebral embolic strokes. Atrial fibrillation (AF) is the most common cardiac arrhythmia and a major risk factor for cerebral embolic stroke. Although oral anticoagulation (OAC) is highly effective in stroke prevention, a substantial number of patients is unable to sustain chronic OAC because of high-bleeding risks. Among these are patients with hereditary hemorrhagic telangiectasia (HHT), who frequently encounter inconvenient epistaxis, gastrointestinal bleedings, or life-threatening bleedings from cerebral or pulmonary arteriovenous malformations. Since most thromboembolic complications in patients with AF arise from the left atrial appendage (LAA), a percutaneous transcatheter LAA closure device was recently developed. This is the first paper about the feasibility of percutaneous LAA closure in patients with high-risk AF and HHT, which can be a promising strategy to protect these patients against embolic stroke, while avoiding the need for long-term OAC.2-score of four indicated a high risk of recurrent stroke (8.5%/year) [We present a 79-year-old man who had been previously diagnosed with HHT, based on the clinical Curaçao criteria ; his fam5%/year) . BecauseThis is the first paper about the feasibility of a percutaneous LAA closure device in patients with high-risk AF and HHT. Percutaneous closure of the LAA might be a new strategy to protect this population against cerebral embolic stroke without the need for chronic OAC and subsequent high-bleeding risks. HHT-related gene mutations result in abnormal angiogenesis and fragile vessels with increased bleeding tendency . RecurreBecause of this inconvenient and sometimes life-threatening bleeding tendency, the decision to start OAC in patients with high-risk AF and HHT should be based on a careful individual risk assessment of both thromboembolic and hemorrhagic complications. Previous studies demonstrated that the LAA is the source of thrombi in more than 90% of patients with nonvalvular AF , which l Percutaneous closure of the LAA might provide an alternative strategy to chronic anticoagulation therapy for stroke prevention in patients with high-risk AF and HHT.


Pathway analysis of these genes revealed networks related to hematopoietic stem cell activity or function. qPCR was used to confirm the differential expression of a subset of these genes, focusing on genes that may represent novel AhR targets due to the presence of a putative AhR binding site in their upstream regulatory region. We verified differential expression of PDGF-D, Smo, Wdfy1, Zbtb37 and Zfp382. Pathway analysis of this subset of genes revealed overlap between cellular functions of the novel AhR targets and AhR itself. Lentiviral-mediated knockdown of AhR in lineage-negative hematopoietic cells was sufficient to induce changes in all five of the candidate AhR targets identified. Taken together, these data suggest a role for AhR in HSC functional regulation, and identify novel HSC AhR target genes that may contribute to the phenotypes observed in AhR-KO mice.Dysregulation of hematopoietic stem cell (HSC) signaling can contribute to the development of diseases of the blood system. Lack of aryl hydrocarbon receptor (AhR) has been associated with alterations in gene expression related to HSC function and the subsequent development of a myeloproliferative disorder in aging female mice. We sorted the most primitive population of HSCs with the highest stem cell potential  from 18-month-old AhR-null-allele (AhR-KO) and WT mice and analyzed gene expression using microarray to determine alterations in gene expression and cell signaling networks in HSCs that could potentially contribute to the aging phenotype of AhR-KO mice. Comparisons with previous array data from 8-week old mice indicated that aging alone is sufficient to alter gene expression. In addition, a significant number of gene expression differences were observed in aged LT-HSCs that are dependent on both aging  Hematopoietic stem cells (HSCs) are the source for continuous lifelong replacement of differentiated cells in peripheral blood and the immune system. Loss of mature hematopoietic cells due to normal attrition, infection, radiation/chemotherapy, stress or environmental exposures can stimulate HSCs to divide and differentiate in order to sustain peripheral blood and immune cell populations. However, like all adult stem cells, HSCs are incapable of sustaining an indefinite number of divisions, and prolonged or excessive proliferation can lead to premature exhaustion. There are significant age-related changes in HSCs, and HSC senescence and/or exhaustion may contribute to disease processes that occur at greater frequency with age –3. Howevex vivo exposure to AhR antagonists promotes the expansion of human HSCs/progenitors [Ahr gene is down regulated during proliferation and self-renewal of HSCs, and is expressed during quiescence [The aryl hydrocarbon receptor (AhR) is a ligand activated transcription factor that was originally identified as a mediator of the toxicity associated with a variety of persistent environmental pollutants such as dioxins (e.g. TCDD) and certain polychlorinated biphenyls (PCBs) . Howevergenitors . Similargenitors . Exposurgenitors . Aging Agenitors . Togetheiescence .A recent analysis of mouse-HSC-specific gene expression and the proximal promoters of 322 HSC-enriched genes identified the AhR response element (AHRE) as one of four motifs for the binding of transcription factors  that control genes critical for HSC function . HoweverAll mice were treated in accordance with approved protocols for both handling and experimental procedures at the University of Rochester Medical Center which are set by the University Committee on Animal Resources (UCAR). All lentiviral experiments were carried out in accordance with policies established and approved by the Institutional Biosafety Committee at the University of Rochester. All experiments described in this work were specifically approved by UCAR under protocol number 93-188R. All efforts were taken to minimize animal suffering.Ahrtm1Bra/J), [Female AhR-KO mice (B6.129-Bra/J), , were obPeripheral blood was collected from the retroorbital plexus vein using capillary tube in BD Microtainer tubes with EDTA . Hematological profiles of these mice were analyzed using a HESKA Hematology Analyzer .Femur and tibia BM cells from female mice were harvested as previously described . Cells f-ΔΔCT method. CFX Manager Software  was used for analysis of qPCR data. Differences were considered significant when relative mRNA expression was 1.5 fold higher or lower than WT control with a p-value of less than 0.05. Genes found to be altered according to these criteria were analyzed using Ingenuity Pathway Analysis to determine altered physiological networks and functions, as well as the relationship of those functions and genes to canonical AhR signaling and reported functions from the literature.Quantitative mRNA expression was performed using a custom 96-well PrimePCR Array  containing primer assays for a subset of genes found to be altered in the microarray experiments and containing putative AhR transcription factor binding sites. LT-HSCs (SLAM+) were sorted as described above and total RNA was extracted using an RNeasy Mini Kit (Qiagen). cDNA was prepared as described above and 10 ng of cDNA was used in qPCR reactions via a Bio-Rad CFX96 Real Time PCR instrument. Expression of mRNA for each gene was normalized using the expression of HPRT and GAPDH. Values from AhR-KO mice and WT mice were compared according to the 2Lineage negative cells were prepared as described above and placed into Stemspan media  containing 10ng/mL mSCF. Cells were cultured for 24 hrs. The media was aspirated and replaced with DMEM containing lentiviral particles encoding AhR shRNA (TRC0000218025) from The RNAi Consortium at the Broad Institute (Harvard and Massachusetts Institute of Technology). Cells were then allowed to incubate for 24 hours. Fresh viral media was added, and cells were cultured for an additional 24 hours. Successful transduction was confirmed by visualizing GFP expression on a Zoe Fluorescent Cell Imager . RNA and cDNA preparation were performed as described above, and samples were analyzed by qPCR at the University of Rochester Medical Center Functional Genomics Core facility.Hepa1c1c7 cells were cultured in DMEM and transduced as described previously, using virus encoding multiple shRNA sequences. At the end of transduction, culture media was removed, cells washed twice with PBS, and lysed . Lysates were stored at −80°C. Lysate proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) (8.4% acrylamide resolving gel) and transferred to polyvinylidene fluoride (PVDF) membrane which was then blocked using 5% nonfat milk in wash buffer . Antibodies used were AhR  and β-actin . The appropriate horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson Immunoresearch . Proteins were visualized by chemiluminescence using LumiGlo reagents .Unless otherwise indicated, results were analyzed and plotted using Graphpad Prism . When appropriate, a two-tailed Student’s t-test was used to analyze statistical significance. A p-value of less than 0.05 was considered to be statistically significant. For validation using qPCR Arrays, a p-value of less than or equal to 0.01 was considered to be statistically significant. A right-tailed Fisher exact test was used to calculate a p-value to determine the probability that each biological function or disease assigned to the data set was by chance alone.Our lab previously reported that young AhR-KO mice display a hematopoietic phenotype that includes elevated numbers of white blood cells, alterations in splenic cell populations and increases in the number of phenotypically defined HSC and hematopoietic progenitor populations . StrikinIn order to determine underlying alterations in gene expression and signaling mechanisms in LT-HSCs that could be contributing to the myeloproliferation and decrease in HSC engraftment capability reported in AhR-KO mice, we performed genomic analysis on a population of cells greatly enriched for LT-HSCs. We chose to use 18-month-old mice, as this age represents a time point when maximum levels of myeloproliferation have yet to be achieved ; and a tIn order to ascribe possible biological functions to the network of gene expression changes observed in LT-HSC (Comparing HSCs from old AhR-KO mice vs. HSCs from old WT mice), data from our microarray were uploaded to Ingenuity Pathway Analysis (IPA). The top biological networks and functions altered in IPA  in HSCs and increased age. This 203 gene set included 141 genes up-regulated in aged AhR-KO mice, and 62 genes down-regulated in aged AhR-KO mice . A subset of these genes were determined to be differentially expressed by qPCR . We overlaid pathways generated for Cancer, Cell Proliferation, Hematopoiesis and Cell Migration, and AhR  Figs. ThIn order to test the hypothesis that AhR is a direct regulator of PDGF-D, Smo, Wdfy1, Zbtb37 or Zfp382, we utilized a lentiviral vector to deliver AhR shRNA to primary Lin- murine bone marrow cells. All novel target genes examined displayed differential expression in shRNA transduced cells relative to an empty vector control , which iThe data presented here suggest a role for the AhR in regulating gene expression in LT-HSCs during aging. Importantly, we identified several novel target genes that AhR may directly regulate during aging of the hematopoietic system. We previously reported that lack of AhR produces a myeloproliferative phenotype in aging mice . The preThese data are consistent with the proliferative phenotype observed in AhR-KO mice, and further support the hypothesis that AhR regulates the expression of genes involved in HSC proliferation and function. If lack of AhR promotes a proliferative phenotype in HSCs, this could lead to enhanced loss of hematopoietic capacity and/or lineage skewing at earlier ages than would be expected. Abnormal proliferation of HSCs can lead to premature exhaustion of the hematopoietic system –39, or aand lack of AhR) indicated that they may be involved in cell functions such as Cancer, Cell Growth and Proliferation, and Hematological System Development and Function. A subset of the latter genes was selected based on the presence of a putative AhRE for validation by qPCR. The genes differentially expressed included PDGF-D, Smo, Wdfy1, Zbtb37 and Zfp382. While the observed relative expression changes in these genes in vivo and in vitro following knockdown of AhR were not exactly the same, the direction of the changes were consistent. This is consistent with the hypothesis that AhR does regulate these genes, but also suggests that additional mechanisms may influence the expression values observed in vivo. Wdfy1, Zbtb37 and Zfp382 are largely undescribed in hematopoiesis and as such represent novel candidate target genes for AhR regulation of HSC function. Additional work is needed to define the exact role of these genes in HSC biology and their relationship to AhR.In order to identify altered genes that are affected by both lack of AhR expression AND age, we utilized a reductive strategy to exclude genes that are altered with age alone in either AhR-KO mice or WT mice. Pathway analysis of the remaining genes  is a member of the sonic hedgehog signaling (Shh) pathway, and both stromal and HSC intrinsic Shh signaling has been implicated as an important regulator of hematopoiesis –44. It hIn order to determine functional overlap with AhR, Pathway Analysis was performed using Ingenuity. Our IPA network builds which included our 5 novel AhR targets  as well as described AhR functions, revealed overlap between biological functions such as number of hematopoietic progenitor cells, number of leukocytes, and self-renewal of hematopoietic progenitors  Figs. ThS1 ARRIVE ChecklistThis work has been described in accordance with the ARRIVE guidelines .(PDF)Click here for additional data file.S1 FigLin- cells from WT or AhR-KO mice were isolated and stained for CD34 (Red B), cKit (Green A), Sca1 (Violet H), CD48 (Blue B) and CD150 (Red C). Events were acquired on a BD LSR11 18-color Flow Cytometer. Events were gated using geometric gating to exclude doublets. A gate was applied to CD34- events. CD34-LSK+ cells were then gated on CD48-CD150+ to identify LT-HSCs (LSK-SLAM+).(TIF)Click here for additional data file.S2 FigThe subset of genes whose differential expression was dependent on age and lack of AhR expression, and subsequently validated by qPCR , was analyzed to determine possible functional overlap with the AhR in the context of biological functions and properties of cancer.(TIF)Click here for additional data file.S3 FigThe subset of genes whose differential expression was dependent on age and lack of AhR expression, and subsequently validated by qPCR , was analyzed to determine possible functional overlap with the AhR in the context of cell proliferation, which is an important aspect of the regulation of hematopoietic stem cell function and maintenance.(TIF)Click here for additional data file.S4 FigThe subset of genes whose differential expression was dependent on age and lack of AhR expression, and subsequently validated by qPCR , was analyzed to determine possible functional overlap with the AhR in the context of cell migration.(TIF)Click here for additional data file.S5 FigLin- bone marrow cells were transduced as described, and the percent of GFP positive cells was determined. (A) Representative fluorescent profiles for a single untransduced and transduced sample. (B) Summary data for transduction efficiency (n = 3). (C) Level of reduction of AhR mRNA in lin- cells (n = 2) (D) extent of reduction of AhR protein in hepatoma cells using various shRNA clones. TRC0000218025 was determined to yield >90% reduction. AhR levels were normalized to the beta-actin band for each sample loaded.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 TableThe gene set analyzed was previously found to be altered in aging HSCs and reported to the Broad Institute Molecular Signatures Database. AhR-KO mice display alterations in this gene set relative to WT controls.(TIF)Click here for additional data file.


Ultra-sensitive laboratory assays based on the Polymerase Chain Reaction (PCR) are playing an increasingly important role in HIV cure-related research. This article reviews the different assays available and how they have evolved. There is a great need for their standardization and for the establishment of reference reagents and testing algorithms to evaluate potential HIV cure-related treatments. The HIV capsid surrounds two copies of genomic ribonucleic acid (RNA). Replication proceeds with reverse transcription of genomic RNA into a deoxyribonucleic acid (DNA) intermediate. This DNA intermediate is integrated into the host genome where it is referred to as proviral DNA. Unintegrated DNA is present in cells in linear and circular forms. It has a short half-life and disintegrates soon after it is formed .HIV replicates in an error-prone manner that generates a mutation virtually every time the virus replicates. This ongoing mutation allows the emergence of different variants in the host, such as drug-resistant strains or immunological escape mutants. HIV is categorized into HIV type 1 and HIV type 2. HIV-1 is further divided into groups; the major (M) group, the more divergent outlier group (O) group; the non-M, non-O group (N) and the P group. Most HIV infections occur within group M, which is differentiated into subtypes A, B, C, AE, AG, H, J and K. All subtypes and most Circulating Recombinant Forms (CRF's) are found in sub-Saharan Africa. Subtype B is the predominant strain in the US, Europe, Canada and Australia but the prevalence of non-B subtypes in these countries is increasing [The most sensitive FDA approved HIV Nucleic Acid Test (NAT) on the market today is the Abbott Real Time HIV-1 Assay. It has an analytical sensitivity of approximately 25 copies/ml for the 1 ml application. It is approved for the detection of HIV RNA in plasma samples [Resting memory CD4+ T cells have the ability to harbor latent HIV infection and have been established as an HIV reservoir. The gold standard assay for measuring the frequency of resting memory CD4+ T cells containing latent but replication-competent virus is a viral outgrowth assay that involves harvesting large volumes of blood from an infected patient, sort purifying resting memory CD4+ T cells and activating limiting dilutions of the cells in culture with phytohemagglutinin (PHA). The cells are co-cultured with CD4+ T lymphoblasts from an HIV-negative donor to amplify any virus released from the cells. A p24 Enzyme-Linked Immunosorbent Assay (ELISA) is used to measure infectious units per million cells (IUPM) after two to three weeks of culture. The assay is expensive and labor intensive. It requires large volumes of blood, highly skilled staff and specialized laboratory equipment. The assay has a wide coefficient of variation and cannot be performed with tissue biopsies. The assay may not perform well for eradication approaches that produce only small (1 log) reductions in the size of the latent reservoir .There are an increasing number of ultra-sensitive laboratory developed PCR-based assays in use that are capable of detecting lower concentrations of HIV RNA and are capable of detecting HIV DNA. The main advantage of most of the PCR-based assays is that they can be performed on small volumes of fresh and frozen samples including blood and tissue. They are relatively faster and simpler to perform when compared to the gold standard assay. These assays are playing an increasing role in HIV cure-related research. A system needs to be devised for their evaluation and standardization.Currently, most Taqman PCR assays designed to quantify HIV-1 DNA are optimized for Subtype B and may not be suitable for non-B subtypes. HIV-1 molecular assays do not detect HIV-2. There is a lack of sero-conversion panels for non-B HIV-1 and HIV-2 infections .The most recent ultra-sensitive NATs reported in the literature are addressing this problem by basing oligonucleotide sequences on the Long Terminal Repeat (LTR) region of the HIV genome where sequence conservation across subtypes is at its greatest. Examples include a whole blood leukocyte assay – pbs-rtPCR - that is reported to have 100% sensitivity for 2 input copies of DNA even in the presence of high amounts of genomic DNA (1μg) . AnotherThere are currently two general approaches to HIV cure-related research. One approach involves strategies such as very early initiation of HAART and the use of agents that reverse latent infection and thus reduce the latent reservoir. The second approach involves the use of agents that re-activate the latent reservoir and then target and reduce the reactivated virus. The successful outcomes of these potential treatments are classified into “sterilization cures” or “functional cures”.A sterilization cure occurs when HIV-1 DNA and RNA becomes undetectable in samples from the treated patient when analyzed using ultra-sensitive assays and the patient remains free from disease over a prolonged period without antiretroviral treatment. The first reported case of a sterilization cure from HIV was in 2009 ,8. A GerA functional cure occurs when low grade HIV-1 DNA and/or RNA is detectable in the treated patient but the patient remains free of HIV-associated disease over a prolonged period of time. Elite controllers of HIV infection are considered prototype cases of a functional cure against HIV .The Ultra-sensitive viral load assay or SCA was one of the first ultra-sensitive PCR-based viral load assays developed against HIV that is successfully able to detect down to 1 copy of HIV-1 RNA per ml of plasma -11. It hAssays that measure total (unintegrated and integrated) HIV DNA have been in clinical use for a number of years. Standardized International quality controls are available for them. They quantify replication competent and incompetent virus. They are currently the most feasible tool available for large-scale clinical trials and cohort studies ,15-20.Despite a general correlation between total DNA and integrated proviral DNA, there are studies that have shown discordance between the measurements. Some researchers argue that specifically measuring integrated DNA is required as it is a better prognostic marker for treatment failure and success .Measuring proviral DNA in sort purified resting CD4+ T cells or cells derived from other potential HIV reservoirs is considered a marker for latency. Original assays used linker ligation , inverseThese assays can be labor intensive. Multiple methods are used in different studies and the reproducibility across multiple laboratories is unknown ,25,31-33Detection of low grade viral activation will be important for strategies that rely on the reactivation and targeting of previously latent / recently reactivated infected cells -36. VariA transient increase in 2-LTR circles in peripheral lymphocytes is interpreted as an indication of ongoing viral replication ,37-39 anThese assays measure HIV transcription in productively and latently infected cells. No extracellular HIV RNA is expected in latently infected cells if viral replication has been blocked by HAART or another means. There is a block in nuclear export of multiply spliced RNA and inefficient production of unspliced RNA in these cells. These assays could be used to quantify any increase in transcription following HIV latency reactivation treatment strategies. There are currently very few published studies using these techniques and reproducibility across multiple laboratories is therefore unknown. Assaying for HIV RNA in latent cells may, however, serve as a better marker for reactivation as compared to 2-LTR circles ,42-50.Droplet digital PCR (ddPCR) is a nucleic acid detection method that uses the same primers and probes as traditional Taqman RT-PCR. The aqueous reaction mixture is emulsified into thermostable oil and micro-partitioned into picoliter droplets that can contain a single copy or less of target DNA. Following PCR amplification, enumeration of both fluorescing and non-fluorescing droplets allows absolute quantitation of target sequences without relying on the use of standard curves. This greatly reduces the amount of assay “background noise” and allows for greater accuracy and precision. Using 96-well plates, 2 million PCR reactions can be performed simultaneously ,52. EmerThis technology provides a viable alternative to traditional RT-PCR and should be optimized and validated further for use in HIV cure-related research.Both endpoint and kinetic PCR protocols have been applied to these assays. Generally endpoint PCR is more sensitive but kinetic or quantitative / real-time PCR provides more accurate quantification. Kinetic PCR can control for false positive signals better than endpoint PCR .PCR-based assays and protocols are allowing for greater and greater sensitivity. Assays can now detect down to 1 input copy of HIV DNA or RNA with greater and greater confidence. The main limitation in sensitivity now lies in sample processing and the number of cell equivalents that can be incorporated into each test, i.e., the number of copies of DNA that can be detected per million cells.Chun et al. observed a patient with undetectable HIV DNA both in blood and in tissue, but who nonetheless experienced viremia rebound after ART cessation . This shSample collecting and handling can greatly affect Nucleic Acid Test Results. It is imperative that specimens be handled, stored and processed correctly and that standardized protocols be devised.Specimen concentration and enrichment protocols will need to be utilized to ensure maximum assay sensitivity.Researchers need to arrive at a consensus on all the possible reservoir sites within patients and standardize the protocols for sampling and processing cells from these sites.Specialized staff training and competency assessment will be required when implementing sample collection and ultrasensitive viral load testing.There will be a need for dedicated laboratory equipment with pre and post-amplification areas, reagents and equipment to minimize specimen carry-over and cross-contamination.False-positive results and specificity vary depending on the assay used. Low positive results will need to be repeated and assays may need to be used in combination to improve specificity.A universal reagent program that provides standardized reagents and panels should be devised.A standardized validation protocol needs to be devised for current and future ultra-sensitive laboratory developed assays that are to be used to evaluate potential treatment strategies.The HIV strains circulating in the population will need to be considered when selecting the assaysA standardized protocol for re-testing after a certain number of months or years needs to be devised that incorporates the gold standard assay whenever appropriate and possible.Clear definitions of what will be considered a “functional” versus a “sterilization” cure should be devised with wording that takes into account the assay limitations.As in clinical diagnosis and monitoring of HIV, different settings may require different assay protocols and algorithms.Some of the considerations that will need to be addressed are as follows:


The contemporary use of interictal scalp electroencephalography (EEG) in the context of focal epilepsy workup relies on the visual identification of interictal epileptiform discharges. The high-specificity performance of this marker comes, however, at a cost of only moderate sensitivity. Zero-crossing interval analysis is an alternative to Fourier analysis for the assessment of the rhythmic component of EEG signals. We applied this method to standard EEG recordings of 78 patients divided into 4 subgroups: temporal lobe epilepsy (TLE), frontal lobe epilepsy (FLE), psychogenic nonepileptic seizures (PNES) and nonepileptic patients with headache. Interval-analysis based markers were capable of effectively discriminating patients with epilepsy from those in control subgroups (AUC~0.8) with diagnostic sensitivity potentially exceeding that of visual analysis. The identified putative epilepsy-specific markers were sensitive to the properties of the alpha rhythm and displayed weak or non-significant dependences on the number of antiepileptic drugs (AEDs) taken by the patients. Significant AED-related effects were concentrated in the theta interval range and an associated marker allowed for identification of patients on AED polytherapy (AUC~0.9). Interval analysis may thus, in perspective, increase the diagnostic yield of interictal scalp EEG. Our findings point to the possible existence of alpha rhythm abnormalities in patients with epilepsy. Despite decades of experience, contemporary clinical EEG analysis remains largely focused on the identification of interictal epileptiform discharges (IEDs) - a fundamental high-specificity biomarker of epilepsyet al.10Since the advent of electroencephalography, changes in the alpha rhythm have been associated with epilepsy6et al.17EEG interval analysis was originally introduced as a computationally inexpensive alternative to Fourier spectral analysisWe retrospectively studied EEG recordings from 78 patients  who were hospitalized in the Departament of Neurology and Epileptology, Medical Centre for Postgraduate Education, Warsaw, Poland during the years 2009–2013. All data analysed were collected as part of routine clinical workup. The protocol of the study was approved by the Medical University of Gdańsk Independent Bioethics Commission for Research and all analysis was performed in accordance with the approved guidelines. The presented data do not contain any identifying information relating to the patients. All data analysis was performed in MATLAB .The patients were selected due to the definiteness of their diagnoses. Within the studied group 51 of the subjects were diagnosed with epilepsy (19 with TLE and 32 with FLE). A proportion of these patients were hospitalized because of the resistance to antiepileptic drugs but available documentation did not include complete medication history in some of the remaining subjects. The duration of epilepsy (time since the occurrence of their first seizure) was known in 44 subjects  and, since patients with epilepsy were not seizure free during the time preceding hospitalization, it was taken as an indirect measure of the degree of drug resistance. The control group comprised two subgroups: 13 patients with PNES and 14 patients who were admitted to hospital because of headache (of whom none were diagnosed with epilepsy or PNES). In all cases of PNES diagnosis was confirmed by video-EEG monitoring. Additional information on individual patients in the studied group is provided in 2 test) and patients given more AEDs were more likely to be diagnosed with epilepsy . AEDs were, however, also taken by the majority of the PNES patients (85%) and this control subgroup did not differ from the epilepsy group in this respect .N = 60 (77%) of the patients were receiving AEDs at the time of examination . The heterogeneity of drugs taken by the patients did not allow to distinguish single-AED effects. The potential impact of AEDs on the patients’ EEG was therefore assumed to be proportional to the number of different AEDs taken by a subject  regardless of individual drug dosing. Benzodiazepines with mainly sedative-hypnotic properties were also included in nAED due to their considerable influence on background EEG activity. Diagnosis and nAED were significantly correlated - i.e. patients with the diagnosis of epilepsy were more likely to be on AED therapy  and photic stimulation (Fs) using the same recording system for all patients . The average duration of recording was 20.2 minutes, range 19.5–22.1 minutes). Recording electrodes were placed according to the international 10–20 system and reference electrodes were placed on the patients’ nape, at the level of vertebra prominens. The signal was passed through an analogue 0.5Hz high-pass filter and digitized at 250 Hz. Data from the standard 19 scalp channels in referential montage were then analysed mathematically. The review summaries of EEG recordings were converted to a 4-level scale of pathology (the “EEG-score”): (0) no abnormalities, (1) normal EEG variants, (2) non epilepsy-specific abnormalities (including background slowing) and (3) epilepsy-specific abnormalities (IEDs). No studied recording contained any overt ictal electrographic activity.The signals from individual channels were first digitally band-passed using zero-phase shift FIR filters. The filter functions were constructed using the “fir2” Matlab function. The passband frequencies were fixed at theta-alpha [4–13 Hz] for initial statistical analysis and treated as a free parameters (in 1 Hz increments) in classifier fitting for cross-validation (see below). The frequency response of a typical filter is shown in Recording segments were defined according to the EEG recording protocol . Sub-segments of the post-Hv segment were also analysed (see Results). Eyes-open and eyes-closed conditions were not considered separately as this did not substantially influence the results (data not shown). Interval histograms obtained from different EEG channels were analysed as sums of symmetric channels  which allowed to reveal spatial effects regardless of the lateralization of epileptic foci. Pooling of all segments and channels together is referred to as “whole recording” analysis throughout the following text.Zero (i.e. isoelectric line) crossings of the pre-processed EEG signal were identified by linear interpolation . The distributions of the relative counts of time intervals between subsequent zero-crossings  were estimated by histogramming . This was followed by pooling of raw histograms over channels and/or segments (see above) and histogram normalization.The family of filters used for prefiltering was kept fixed. Hence the algorithm transforming the EEG signal to its interval spectrum had 4 free parameters: the lower passband (1), the upper passband (2) together with the choice of recording segments (3) and channels (4) to be included in the pooled interval histogram. In case of whole recording analysis only the passbands (1 and 2) needed to be specified. The power spectral densities of (prefiltered) signals were additionally estimated using Welch’s method (4s-window).min) which quantify the level of disorder associated with a probability distribution. The entropy functions are defined as follows:We assessed three types of interval spectrum-derived markers: (1) the relative counts of fixed-length intervals , (2) the standard descriptive statistical parameters of the interval spectrum  and (3) selected entropy measures of the interval spectrum , pThe distributions of analysed measures within patient subgroups did not pass normality and homoscedasticity tests. Therefore, in order to avoid violation of ANOVA assumptions, we tested the data using nonparametric one-way Kruskal-Wallis ANOVA separately for the effects of diagnosis (4 groups) and nAED (4 groups). Multiple statistical hypothesis testing has been accounted for by using Bonferroni’s correction. Post-hoc multiple comparisons were performed following Tukey’s procedure. In order to focus on robust candidate markers and reduce the number of statistical tests performed, we analysed only histogram bins for which the raw interval count was >100 for all patients.Discriminatory power of identified putative markers was assessed in the framework of receiver-operating-characteristic (ROC) analysis. Epilepsy (FLE and TLE) and control (headache and PNES) subgroups were joined to enable binary classification. The area under the ROC curve (AUC) was used as a measure of classifier performance. As a summary statistic AUC is equivalent to the probability that the score attributed to a randomly chosen case is higher than that attributed to a randomly chosen control. When calculated “naively” for the whole patient group AUC is, however, prone to optimistic bias due to overfitting23As our primary interest was to study the alpha rhythm and its possible slowing, initial statistical analysis was performed for signals band-passed in the theta-alpha [4–13 Hz] band. Markers significantly influenced by either the patients’ diagnosis or nAED were further evaluated using ROC analysis with cross-validation (see the following section).The shapes of averaged interval spectra (obtained from whole recordings) for different patient subgroups are shown in Simultaneous one-way ANOVA  showed that significant effects lie in two disjoint portions of the interval spectrum . Within S and Hmin respectively) with no significant effects of nAED  are presented in Interestingly, despite the fact that the signals were initially band-passed in the theta-alpha band, a small number of beta and gamma range intervals were detected – particularly in patients with epilepsy  the “alpha score” - a classifier based on epilepsy-related reduction of interval spectrum magnitude in the alpha range and  two “entropy scores” - basing on increased entropy of the interval spectrum in patients with epilepsy . Discrimination of AED polytherapy from monotherapy patients was assessed for (4) a classifier based on increased interval spectrum magnitude in the theta range - the “theta score”. Discriminative power (AUC* and AUC** for one- and two-step optimization respectively) was estimated by cross validation (see Methods) and the results are summarized in The alpha-score classifier was optimized by minimizing AUC over the alpha interval range  and this classifier benefited from relatively narrow-band pre-filtering [7–13 Hz]. As for initial statistical analysis (see previous section) no significant dependence of either of entropy scores on nAED was found (P = 0.0770 and P = 0.3696 for Shannon- and min-entropy scores respectively).The theta-score classifier was optimized by maximizing AUC over the theta interval range  with AUC* = 0.8057 and 0.7829 respectively . The results are summarized in The potential gain obtained from analysing specific channel/segment combinations instead of whole recordings is expressed by mean AUC ratios . For alpha- and Shannon entropy-scores AUC ratios were consistently greatest in temporal-occipital leads and towards the end of the recording  and did not display any particular temporal pattern see  and 2b3.We next analysed how the performance of alpha- and Shannon entropy-score based classifiers depended on the length of the analysed recording segment. Instead of analysing the whole T6 + T5 & post-Hv segment, a random subsegment of fixed duration was chosen independently for each patient and “naive” AUC values for alpha- and entropy-scores were computed. This procedure was repeated 100 times and the results (mean AUC ± s.d. as a function of subsegment length) were normalized by AUC obtained for the whole T6 + T5 & post-Hv segment. As shown in The comparison of ROC curves  shown in Our results demonstrate the possibility to distinguish patients with epilepsy from non-epileptic controls by interval analysis of interictal EEG recordings. Classification in the studied patient group was possible at relatively high sensitivity and specificity (AUC~0.8). This level of performance is comparable to published ANN-based methods10Alpha-score and Shannon entropy-score based classifiers performed best for signals obtained from temporal and occipital leads  and for recording segments following hyperventilation. Effective classification may be possible with only ~1 minute long recording segments. As a practical consequence, the lack of requirement for a complete set of EEG leads and for prolonged recordings may potentially lead to an applicability of the method e.g. in the emergency department or outpatient setting.Decreases in alpha activity in patients with epilepsy as compared with controls were previously reported but were not notable in a subgroup of untreated patientsEven though a proportion of patients with epilepsy were refractory to AED treatment and often on AED polytherapy, the inclusion of PNES patients  in the control group allowed for statistical analysis which revealed weak and non-significant AED dependence of alpha- and Shannon entropy-scores respectively. AED-related effects were concentrated in the theta interval range and allowed for identification of AED polytherapy patients in the studied group. Differing spatial patterns of optimal epilepsy vs control and theta-score based AED polytherapy vs monotherapy classification further support the distinctness of epilepsy and AED-related phenomena. The association of theta band effects with AEDs has been reported in previous studies9When interval and Fourier spectral analysis were applied analogously to the same data, consistent trends were seen in the alpha and theta bands. Interval analysis, however, outperformed Fourier analysis in the alpha range and effective classification is likely not to be possible using simple Fourier spectrum based classifiers. We hypothesize that this may, at least in part, result from different sensitivity of both methods to episodic low-frequency EEG artefacts . In case of interval analysis, large deflections of the signal give rise to few unusually long intervals which are likely to fall outside the region of interest identified in our study . Contrastingly, time-localized waveforms typically make a broadband contribution to Fourier power spectra and may contaminate the bands of interest. The transformation of the signal to its interval spectrum could thus naturally “filter out” recording segments contaminated with considerable low-frequency artefacts. High-frequency artefacts were, on the other hand, mostly removed by pre-filtering (see Methods). An in-depth comparison of Fourier and interval-based EEG analysis requires futher study.The preservation of discriminative power of interval spectrum markers and the EEG score when analysis was restricted to patients with normal and normal variant EEGs suggests that epilepsy-specific EEG abnormalities captured by the interval spectrum may not be IED-related. As in the case of episodic low-frequency artefacts it may be conjectured that interval analysis is in general not sensitive to IEDs as they are not likely to produce sufficient interval counts to influence the overall statistic. It must be stressed that our findings should be considered preliminary and require validation in a larger and homogenous patient populations. Nevertheless, it seems that the information extracted from EEG recordings by interval and visual analysis may be to some extent complementary.Some electroencephalographers make note of the qualitative features of background EEG rhythms. “Sharpened” and “irregular” alpha rhythm morphologies are often found in recordings from patients with epilepsy even in the absence of overt IEDs. While clinical reasoning based on such features is controversial and widely regarded as overinterpretationWe demonstrate that zero-crossing interval analysis of interictal EEG recordings may be used for the distinction of patients with focal epilepsy from non-epileptic controls including patients with PNES. With the development of this method it may become possible to augment the predictive value of the standard EEG for epilepsy work-up. The pathophysiological interpretation of our findings points to the possible existence of diffuse alpha rhythm abnormalities in patients with epilepsy.How to cite this article: Pyrzowski, J. et al. Interval analysis of interictal EEG: pathology of the alpha rhythm in focal epilepsy. Sci. Rep.5, 16230; doi: 10.1038/srep16230 (2015).


The GTEx Consortium reported that hierarchical clustering of RNA profiles from 25 unique tissue types among 1641 individuals accurately distinguished the tissue types, but a multidimensional scaling failed to generate a 2D projection of the data that separates tissue-subtypes. In this study we show that a projection by t-Distributed Stochastic Neighbor Embedding is in line with the cluster analysis which allows a more detailed examination and visualization of human tissue relationships. The use of dimensionality reduction methods such as multidimensional scaling (MDS) or principal component analysis (PCA) are popular techniques for data representation in low dimensions. As an eWhen mapping high-dimensional genomic data to lower dimensions, it is important that distances between similar samples are preserved . This allows separation of similar samples in the low dimensional representation, and hence discovery of relationships between nearby samples by visual inspection. We re-evaluated the projection of tissue types using the GTEx transcriptome data, consistNormalization of the data is performed as described previously, 2: geneWe utilized the Barnes-Hut t-SNE algorithThe tissue gene expression profiles in the 2D t-SNE map are clustered using DBSCAN. ClusterP≤0.0001   . However≤0.0001) . A compaTo investigate the value of the t-SNE mapping further, we evaluated the results of different clustering algorithms  followed by DBSCAN cluster analysis to determine the optimal number of clusters. Each square depicts the -log10(P) enrichment of the cluster label with the tissue label, and shown when (TIF)Click here for additional data file.S2 FigCluster associations with the tissue label are determined based on 20 different gene filtering cut-offs, i.e., , and are subsequently summarized. Per cut-off we determined the clustering, and each detected cluster is subsequently associated with the tissue labels (as demonstrated in (TIF)Click here for additional data file.S3 FigFour cluster algorithms are compared in the original data space and in the low dimensional space. Panel A-C depicts the Davies Bouldin index (DBindex) score for the clustering of brain samples in original data space using HC, Kmeans and Mixture of Gaussians respectively. DBSCAN was not able to fit the data in the original data space. Panel C-F depicts the DBindex score for the clustering of brain samples in the low dimensional space using HC, Kmeans, Mixture of Gaussians, and DBSCAN respectively. The red vertical line depicts the optimum number of clusters under the restriction that at least 5 and at most 15 clusters can exists. Panel G-J depicts the brain samples in the low dimensional space and the corresponding cluster labels for HC, Kmeans, Mixture of Gaussians and DBSCAN respectively.(TIF)Click here for additional data file.S1 TableFour cluster algorithms are compared in the original data space  and in the low dimensional space (by first employing a t-SNE to two dimensions). Enrichment is computed for the detected cluster labels and the brain regions.(TIFF)Click here for additional data file.S2 TableColumns depict the tissue type. Rows depict the cluster number. Values within the cells depict the number of samples that do not map with a matching tissue label.(TIFF)Click here for additional data file.


Multi-robot task allocation determines the task sequence and distribution for a group of robots in multi-robot systems, which is one of constrained combinatorial optimization problems and more complex in case of cooperative tasks because they introduce additional spatial and temporal constraints. To solve multi-robot task allocation problems with cooperative tasks efficiently, a subpopulation-based genetic algorithm, a crossover-free genetic algorithm employing mutation operators and elitism selection in each subpopulation, is developed in this paper. Moreover, the impact of mutation operators  is analyzed when solving several industrial plant inspection problems. The experimental results show that: (1) the proposed genetic algorithm can obtain better solutions than the tested binary tournament genetic algorithm with partially mapped crossover; (2) inversion mutation performs better than other tested mutation operators when solving problems without cooperative tasks, and the swap-inversion combination performs better than other tested mutation operators/combinations when solving problems with cooperative tasks. As it is difficult to produce all desired effects with a single mutation operator, using multiple mutation operators (including both inversion and swap) is suggested when solving similar combinatorial optimization problems.The online version of this article (doi:10.1186/s40064-016-3027-2) contains supplementary material, which is available to authorized users. Multi-robot task allocation (MRTA) determines the task distribution and schedule for a group of robots in multi-robot systems  the development of the subpopulation-based genetic algorithm, this algorithm employs mutation operators and elitism selection in each subpopulation, and can solve the multi-robot tasks allocation problems for industrial plant inspection efficiently; and (2) the recommendation of mutation operators for solving multi-robot task allocation problems and similar optimization problems, i.e., using multiple mutation operators (including both inversion and swap) is suggested.To find good solutions efficiently for multi-robot task allocation problems, many approaches have been developed, such as genetic algorithms  Mitchell  is a cenSelection, crossover, and mutation operators maintain the population diversity  All robots are identical and start at the same time. (2) All robots start from their home bases that are predefined, and end at their home bases after finishing their assigned tasks. Between performing any two tasks, robots do not move back to their home bases. (3) Each single-robot task is assigned to one robot, while each cooperative task is assigned to two robots. Based on the described characteristics and assumptions of the multi-robot task allocation for inspection problems, the time of each robot finishing its assigned tasks includes three parts: the traveling time, the inspection time of each task, and the waiting time occurring when performing cooperative tasks. In this work, the traveling time is calculated using a modified A* algorithm Liu  that corThe objective of multi-robot task allocation problems is usually to minimize the total mission cost due to energy consumption, completion time, and/or traveled distance. Shorter duration of inspecting a plant permits a higher frequency of repetition of the inspections, and also may reduce costs of the inspection. Therefore, the objective of multi-robot task allocation problems in this paper is to find the optimal solution J of each admissible solution Formally, given a set of k-th robot assigned d tasks is denoted as For task allocation problems with only single-robot tasks, instead of mentclass2pt{minimFor task allocation problems with cooperative tasks, each single-robot task is denoted as a subtask mentclass2pt{minimEach cooperative task is carried out by two different robots.Two subtasks of each cooperative task are started at the same time.When two robots are scheduled to carry out a cooperative task, all tasks that are assigned to both robots before this cooperative task have to be executable and finished.In addition, the following three executability constraints (EC) must also be satisfied to ensure that the task allocation A subpopulation-based genetic algorithm is developed to solve the multi-robot task allocation problems with cooperative tasks in this section. At the beginning of this section, the solution representation is introduced. After that, the implementation of the proposed genetic algorithm is illustrated. At the end, the subpopulation-based and standard genetic algorithms  The genotype of a solution represents the task sequence of each robot and consists of two parts:chromosome is a string of genes and represents the sequence of all A gene-apportion is a set of A The genotypes represent the initial allocation that each task is assigned to one robot and robot coalitions of cooperative tasks have not yet built. Genetic operators are applied to the genotypes of solutions not phenotypes of them. Both the chromosome and gene-apportion of each genotype in the initial population is generated at random. For example, Fig. mentclass2pt{minimDecoding (Phenotype) The phenotypes represent complete feasible task allocations including the distribution and the sequence of all subtasks. The completion time of each solution candidate is calculated according to its phenotype not genotype. In a genotype, each cooperative task is only assigned to one robot, thus the other robot is required to carry out this task simultaneously. A genotype is decoded as a phenotype via two steps:For single-robot tasks, each gene is directly decoded as its corresponding task; see Fig. One subtask of Subtask (S2.1) Calculate the arriving time (S2.2) Sort (S2.3) For the first cooperative task of (S2.4) Repeat (S2.2)–(S2.3) until all cooperative tasks are decoded. For instance, the decoding outcome of the step “S1” is shown in Fig. For cooperative tasks, two subtasks of each cooperative task should be decoded. The main idea of the following decoding is to minimize the waiting time. According to the genotype, it is obvious that each cooperative task is already assigned to a robot As illustrated above, this decoding can satisfy all executability constraints, i.e., all decoded phenotypes are feasible for execution.Using this representation, each individual (solution candidate) includes a genotype and a phenotype. In the proposed genetic algorithm, the chromosomes of the genotypes are mutated for generating offspring; phenotypes are used to calculate the completion time.The developed genetic algorithm in this paper is based on subpopulations. The main idea of this algorithm is that selection and genetic operators are applied separately in each subpopulation. Elites selection is based on the fitness of individuals in a subpopulation not in the whole population, thus more elites including both global elites and local elites will be kept in the new generation. These elites avoid losing the best found solution and local optimal solutions. The pseudo code of our proposed subpopulation-based genetic algorithm is presented in Algorithm 1. Parameters Parameters of the genetic algorithm are set at the beginning, such as population size  is suggested when solving similar combinatorial optimization problems.For mutation combinations, it is difficult to analyze which is the only operator that guides the evolutionary search, because: (1) an operator cannot guarantee to produce better offspring than parents; and (2) the best individual in The problem complexity significantly increases if cooperative tasks are involved because they introduce additional spatial and temporal constraints. To solve the multi-robot task allocation problems without/with cooperative tasks for industrial plant inspection, a subpopulation-based genetic algorithm is developed. The proposed subpopulation-based genetic algorithm using just inversion mutation and selection obtains better solutions than the tested binary tournament genetic algorithm with partially mapped crossover (PMX) and inversion mutation. This provides the possibility of crossover-free genetic algorithms. Succeeding, the impact of four mutation operators and four mutation operator combinations in the subpopulation-based genetic algorithm is analyzed to find suitable mutation operators for multi-robot task allocation problems. The results indicate that inversion mutation performs well when solving problems without cooperative tasks, and the swap-inversion combination performs well when solving problems with cooperative tasks. As it is difficult to produce all desired effects with a single mutation operator, using multiple mutation operators (including both inversion and swap) is suggested when solving similar combinatorial optimization problems.


Inflammatory Bowel Diseases (IBD) are disorders of the gastrointestinal tract characterized by recurrent inflammation that requires lifelong treatments. Probiotic microorganisms appear as an alternative for these patients; however, probiotic characteristics are strain dependent and each probiotic needs to be tested to understand the underlining mechanisms involved in their beneficial properties. Genetic modification of lactic acid bacteria (LAB) was also described as a tool for new IBD treatments. The first part of this review shows different genetically modified LAB (GM-LAB) described for IBD treatment since 2000. Then, the two principally studied strategies are discussed (i) GM-LAB producing antioxidant enzymes and (ii) GM-LAB producing the anti-inflammatory cytokine IL-10. Different delivery systems, including protein delivery and DNA delivery, will also be discussed. Studies show the efficacy of GM-LAB (using different expression systems) for the prevention and treatment of IBD, highlighting the importance of the bacterial strain selection (with anti-inflammatory innate properties) as a promising alternative. These microorganisms could be used in the near future for the development of therapeutic products with anti-inflammatory properties that can improve the quality of life of IBD patients. Inflammatory Bowel Diseases (IBD) describe a group of disorders of the gastrointestinal tract characterized by recurrent inflammation, with periods of relapse and remission, and epithelial injury. Ulcerative colitis (UC) and Crohn's disease (CD) are the two most frequent forms of IBD, clinically characterized by different intestinal location, nature, and the histological features of the inflammatory lesions as well as their association with specific deregulation of the host's immune response. The exact etiology of these pathologies is still unknown; however, it was described that in IBD patients there existed aberrant features of the interaction between intestinal microorganisms and gut immune and epithelial cells, which is manifested as chronic intestinal inflammation .IBD require lifelong treatments, and although they are not generally associated with increased mortality, they can cause significant morbidity. Probiotic microorganisms have appeared as an alternative for IBD patients and their efficiency has been analyzed in experimental animal models and also in clinical trials , 3.This paper will describe some of the mechanisms by which probiotic microorganisms can exert specific benefits against IBD, followed by a revision about the potential use of genetically modify lactic acid bacteria in the prevention and treatment of these recurrent diseases.Lactic acid bacteria are the most common microorganisms used as probiotics. Because of specific documented beneficial effects, certain strains of LAB have been designated as probiotics, which have been defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” . Among tIn this sense, it has been shown that LAB and other probiotic microorganisms can counteract inflammatory processes in the gastrointestinal tract (GIT) through different mechanisms. Lactobacillus (L.) reuteri could be used to prevent colitis in IL-10−/− mice by increasing the number of lactobacilli in the GIT [ L. salivarius UCC118 reduced the prevalence of colon cancer and inflammatory activity in the mucosa of IL-10−/− mice by modifying the intestinal microbiota in these animals: Clostridium (C.) perfringens, coliforms, and enterococci counts also decreased significantly in the group treated with the probiotic [ Lactobacillus have been reported [ Lactococcus (Lc.) lactis, inhibited a wide range of genetically distinct C. difficile strains isolated from healthy subjects and also from patients with IBD [One of these is the modulation of the intestinal microbiota related to probiotic administration. It has been shown that the GIT . In a plrobiotic . Anotherreported . The inh L. salivarius Ls 33 required the presence of NOD2 receptors to exert its protective effect, which was also related to the local production of IL-10 [ L. plantarum Lp91 caused a significant decrease in the levels of TNFα (tumor necrosis factor α) and COX-2 (cyclooxygenase-2) in a mouse model of colitis, and this effect was related to significant increases of IL-10 expression [ in vitro and ex vivo studies, that milk fermented with L. paracasei L74 CBA inhibited proinflammatory cytokines without affecting the levels of anti-inflammatory cytokines and that this anti-inflammatory activity depended on metabolic products released during the fermentation process [ Streptococcus (S.) salivarius inhibited the activation of the NF-κB in vitro by demonstrating anti-inflammatory properties in vivo [Numerous studies have shown that certain strains of LAB may modulate the host's immune response by regulating the production of cytokines which are involved in regulation, activation, growth, and differentiation of immune cells. One of the ways by which probiotics may exert immunomodulatory activities is by stimulating the production of IL-10 (an anti-inflammatory cytokine). However, not all probiotic strains act in the same way. The anti-inflammatory effects such as the stimulation of IL-10 producing cells are strain-dependent characteristics, and their effectiveness is also dependent on the concentrations used and the method of administration. Thus, the anti-inflammatory effect associated with the administration of a potential probiotic yoghurt to mice was studied in models of acute and chronic intestinal inflammation induced by trinitrobenzene sulfonic acid (TNBS). Animals receiving yoghurt continuously improved immune response with increases in IL-10 and decreases in IL-17 (a proinflammatory cytokine) in the GIT of mice , 10. In of IL-10 . By studpression . It was  process . A new s in vivo . Lactobacilli, Bifidobacterium, and Streptococcus species) normalized the colonic physiological function and integrity of the mucosal barrier in IL-10−/− mice [ L. plantarum DSM 9843 and L. reuteri R2LC also improved barrier function in a model of enterocolitis induced by methotrexate in rats [ muc genes and mucus secretion [The improvement of the intestinal barrier function is another mechanism by which probiotic bacteria can benefit the host. The exact mechanism by which probiotics enhance the barrier function and the intestinal mucus is unclear; however, it may be related to alterations in mucus secretion or changes in intercellular interactions of the mucosa and cell stability by modulating the phosphorylation of cytoskeletal proteins and tight junctions , 16. Ora−/− mice . L. plan in rats . Some precretion . L. rhamnosus CNCM I-3690, selected for its antioxidant properties in vitro, showed anti-inflammatory activity in a model of colitis in vivo [Probiotic can also act by reducing oxidative stress, which is characterized by an uncontrolled increase in the concentration of reactive oxidative species (ROS) in the GIT. Thus, another suggested mechanism to prevent inflammation by LAB administration is through the expression of enzymes that are be able to decrease the concentrations of ROS or affect their formation. The levels of these enzymes are often depleted in patients with IBD . Probiot in vivo .It is important to consider that probiotic characteristics are strain dependent and each probiotic should be tested to know if it has specific beneficial effects, and to describe the mechanism/s involved in their health-promoting properties. In addition, not many mechanisms are usually associated with one individual strain. So, genetic modification of LAB has been also described as a tool to development new treatments for IBD, using microorganisms with GRAS  status. L. lactis is a LAB used in various processes in the food industry and is has been characterized because it does not survive in the digestive tract of animals and humans and thus has the potential to be used without the possibility of survival through the gastrointestinal tract [ L. lactis is normally used as a LAB model because (i) its genome has been completely sequenced, (ii) it is easy to manipulate genetically, and (iii) many genetic tools have already been developed for this species. Based on the identification and isolation of plasmids from native strains of L. lactis and other LAB, several cloning vectors have been developed. Using molecular biology techniques, these vectors have been engineered to become important tools for cloning genes of interest, and their products can be controlled with constitutive or inducible promoters.It is theoretically possible, using genetic engineering techniques, to obtain LAB strains that possess a variety of beneficial properties.al tract . L. lactThe development of efficient systems to express genes and suitable protein secretion systems for use in LAB can permit these microorganisms to be used for the production and secretion of a number of heterologous proteins . So, as The production of antioxidant enzymes and the production of the anti-inflammatory cytokine IL-10 are two of the most studied anti-inflammatory strategies using GM-LAB and both will be revised in the following sections along with a few examples of other anti-inflammatory compounds that have successfully been produced by GM-LAB. L. plantarum and L. lactis that are able to produce and release SOD exhibited anti-inflammatory effects in a TNBS induced colitis model [ L. gasseri strain producing SOD with an associated reduction of the severity of colitis in IL-10 deficient mice [ L. casei BL23 producing SOD was able to significantly reduce the damage induced by TNBS in mice as shown by increased survival, decreased weight loss, lower microbial translocation to liver, and more importantly a decrease in the damage of the large intestines [As mentioned previously, in patients with IBD, oxidative stress occurs as the result of an abnormal and recurrent inflammation associated with increased concentrations of ROS. Because few microorganisms produce antioxidant enzymes in the concentrations required to exert biological effects, genetic engineering strategies have been used to obtain more efficient antioxidant producing LAB. Spyropoulos et al. have shown the potential uses of such strains in the treatment of IBD using a variety of animal models . LAB havis model . Anotherent mice . L. casetestines . These rtestines . L. lactis lacks catalase , the heme catalase gene katE from B. subtilis was added to this industrially important organism resulting in a GM strain able to produce catalase that provided active catalase activities [ L. lactis producing catalase was able to prevent the development of colon tumors in mice using a chemical induced model [ L. casei BL23 was modified to produce heme-independent catalase and this in turn decreased the intestinal inflammatory damage in a TNBS induced mouse model [ L. casei BL23 and the derived GM strain producing catalase reduced inflammation degrees in the colon and cecum of mice, using a DSS induced model [Becausetivities . It was ed model . In anotse model . This reed model .S. thermophilus CRL807 is a strain that was present in the starter mix, together with 12 other LAB, used to prepare yoghurt with anti-inflammatory and anticancer effects [ in vitro and in vivo [ S. thermophilus CRL 807 [ L. lactis, it was observed that the unmodified strain exerted anti-inflammatory effects in a TNBS-induced colitis model in mice. It was also observed that both genetically modified S. thermophilus CRL 807:CAT and S. thermophilus CRL807:SOD (used as suspension or in fermented milks) decreased the severity of inflammation, and these beneficial changes were increased compared to those observed in mice that received the wild type strain. The mixture of both GM-streptococci were also evaluated and shown to exert more anti-inflammatory properties than when each strain was given individually. These results prove that the use of LAB strains that are able to modulate the immune response (innate capacity of S. thermophilus CRL 807) and also express antioxidant enzymes show a combined effect and may be a useful strategy in the development of new therapeutics for patients suffering from IBD. effects , 32. The in vivo . So, the CRL 807 . Unlike Interleukin-10 (IL-10), as explained in the introduction, is one of the major anti-inflammatory cytokines involved in maintaining the homeostasis of the intestinal immune response. It is recognized for its ability to regulate inflammatory responses through the suppression of the proinflammatory cytokine cascades , and thi L. lactis secreting IL-10 prevented the onset of colitis in IL-10−/− mice [ L. lactis for intestinal delivery of human IL-10 cytokine [ L. lactis was replaced by the human IL-10 gene, making this strain unable to grow in the absence of thymidine or thymine. This strain did not contain any antibiotic resistance marker and since thymidine is auxotrophic, the strain could not be spread to the environment making it one of the safest built GM-LAB so far. This containment system was evaluated in patients with CD and was shown not to produce any adverse side effects, and these GM-LAB could only be recovered in feces with the addition of thymidine [The first evidence of GM-LAB as therapeutic vehicle for IL-10 was published in 2000, when it was shown that a strain of−/− mice  and redu−/− mice . An impocytokine . In thathymidine . Howeverhymidine –49.L. lactis subsp. lactis NCDO2118 pXYL:IL-10 is a LAB that produced IL-10 using an expression system that has a food-grade inducer in the xylose-inducible expression system (XIES) [L. lactis subsp. lactis NCDO2118) in a DSS induced colitis model in mice [ L. lactis NCDO2118 pXYLCYT:IL-10, strain capable to produce and maintain IL-10 in the bacterial cytoplasm, exerted an anti-inflammatory effect in an acute TNBS induced model of IBD in mice, which was more pronounced than the ones observed with milk fermented by the wild type strain [ L. lactis. In this system milk acts as a matrix to protect the bacteria and the cytokine during the passage through the GIT. This new approach could lead to the development of new therapeutic fermented products , appropriate for a specific population that suffers gastrointestinal disorders.m (XIES) . Anti-in in mice . It was e strain . This efL. lactis subsp. cremoris MG1363 pGroESL:IL-10 produces IL-10 using the protein delivery expression system SICE (Stress-Inducible Controlled Expression) and is based on a stress inducible promoter (pGroESL) that allows the production of the heterologous protein in situ  [ L. lactis capable of delivering the IL-10 protein using the SICE system has the advantage that it does not require an inductor because the adverse conditions of the GIT can by themselves induce this system and all the IL-10 can be locally produced by this LAB in the intestine. Recent studies showed that this strain exerted a protective effect against inflammation in a model of low-grade colon inflammation [, colon) . L. lactammation . L. lactis subsp. cremoris MG1363 pValac:IL-10 is a GM-LAB used for the delivery of IL-10 cDNA. This system is based on a new vector for DNA delivery using lactococci called pValac (vaccination using lactic acid bacteria) that delivers the DNA to the intestinal cells and gives these cells the capacity to produce IL-10 directly at the site of inflammation [ L. lactis MG1363 engineered to express fibronectin binding protein A (FnBPA) was used as a vehicle to deliver the cDNA for IL-10 using the plasmid pValac::il-10. L. lactis MG1363 FnBPA + pValac:IL-10 exerted a significant anti-inflammatory effect in a TNBS induced acute model of colitis in mice maintaining high ratios of anti-/proinflammatory cytokines in the intestinal fluids and tissues [ L. lactis that expresses FnBPA under the control of the nisin inducible expression system [ L. lactis subsp. cremoris MG1363 pValac:IL-10 was also able to provide the IL-10 cDNA to the host's cells and exerted an in-inflammatory effect in a DSS induced colitis model in mice [Even though protein delivery by GM-LAB showed promising results, DNA delivery by GM-LAB was also evaluated for the production of IL-10 locally by the host's intestinal cells.ammation . It was  tissues . The impn system ; however in mice  showing  L. lactis pValac:IL-10 (noninvasive strain) exerted similar anti-inflammatory effects than L. lactis pGroESL:IL-10, when they were administered to the mice during the remission period [The effectiveness of both protein and DNA delivery systems was also recently compared using a TNBS induced chronic inflammation model. It was demonstrated thatn period . Even th L. lactis was engineered to secrete monovalent and bivalent murine (m)TNF-neutralizing nanobodies as therapeutic proteins. It was shown that the oral administration of nanobody-secreting L. lactis resulted in local delivery of anti-mTNF nanobodies at the colon that significantly reduced inflammation in DSS-induce chronic colitis in mice [Since TNF is one of the most important proinflammatory cytokines in immune regulated inflammatory processes, the objective of certain groups has been not only to reduce the expression of this cytokine but also to prevent it from being active. in mice . L. lactis together with a signal sequence which allowed for this cytokine to be secreted. This IL-27 secreting strain was able to reduce colitis, induced via transfer of CD4(+)CD45RB(hi) T cells into Rag(−/−) mice, by increasing the production of IL-10 [Interleukin-27 (IL-27) plays a role in the regulation of T helper (Th) cell differentiation inducing Th1 differentiation and suppressing immune responses. For this reason, it has been proposed that IL-27 could be useful in therapy of diseases mediated by inflammatory cytokines . The 2 gof IL-10 .β1 (TGF-β) is an inhibitory cytokine recognized as a key regulator of immunological homeostasis and inflammatory responses and was successfully expressed by L. lactis and when administered to DSS-induced mice, this GM-LAB decreased colon damage scores [Transforming Growth Factor-e scores . L. lactis led to active delivery of TFF at the mucosa of the colon and, in contrast to administration of purified TFF, proved to be very effective in prevention and healing of acute DSS-induced colitis [ L. lactis secreting human TFF1 which provided a safe and efficient therapeutic tool for treating oral mucositis [Besides immune regulators, other compounds have also been shown to possess anti-inflammatory potential. The Trefoil Factor (TFF) family of peptides, TFF1 (formerly pS2), TFF2 , and TFF3 , is involved in the protection of the gastrointestinal tract since they play an essential role in epithelial restitution and repair of mucosal damage . Intraga colitis . These sucositis . L. lactis and were shown to be very effective anti-inflammatory molecules in a DSS-induced mouse model of colitis [It was previously shown that colonic tissues of patients with IBD have increased proteolytic activity and that the use of protease inhibitor Elafin was able to prevent intestinal inflammation in mouse models of colitis . For thi colitis . L. lactis strain 15-LOX-1 was effective in the prevention of the intestinal damage associated with inflammatory bowel disease in a TNBS murine model [The enzyme 15-lipoxygenase-1 (15-LOX-1) is another molecule that has been proposed as a potential candidate for the resolution of IBD because of its potent anti-inflammatory action. It was shown that the administration of milk fermented by ane model .Although there is no scientific evidence to support the notion that GM organisms are dangerous for human consumption, it is necessary to show that it is safe to use genetically modified probiotics designed to extend the range of applications covered by natural probiotics.Consumption of GM microorganisms by human is still a highly controversial issue due to the public perception that genetic manipulation is not “natural.” Scientists need through well-designed studies to report the results for the general population to inform consumers of the benefits that these techniques can confer with minimal risk to the health and the environment, such was the case of the IL-10 producing LAB that was shown to be safe in human clinical trials .The revision presented here not only showed the potential associated with probiotic microorganisms to be used in patients suffering diseases associated with gastrointestinal inflammation, but also showed the potential use of GM-LAB as new therapies for these patients. It is important to restate the genetic modification of lactic acid bacteria with innate anti-inflammatory properties to produce anti-inflammatory compounds (such as antioxidant enzymes or anti-inflammatory cytokines). It is a promissory tool to obtain new more effective strains with potential applications for IBD patients. These powerful strains should be given as an adjunct treatment to current protocols for IBD patients, and because of the beneficial properties, these could actually improve the quality of life of these patients and aid in preventing the unbalance of beneficial/pathogenic microbiota present in the GIT.


N>4000) representative for the population in Germany. Standardized norm data for men and women in different age groups are provided through percentile ranks and T-values.Standardized norm data for three scales of a 19-item self-report measure on environmental spatial strategies are provided. This self-report measure comprises egocentric spatial strategies, an allocentric mental map strategy and knowledge of cardinal directions as three separate scales, “Validation of a 3-factor structure of spatial strategies and relations to possession and usage of navigational aids”  [3]. The data are based on a large sample ( Specifications TableValue of the data•Standardized norm data based on a large, representative sample helps assessing measured individual differences in self-reported environmental abilities•Standardized norm data based on a large, representative sample helps assessing measured self-reported environmental abilities in particular samples •Separation of standardized norm data by sex and different age groups helps to assess individual differences with respect to relevant reference groups and age cohorts.1N>4000). Standardized norm data for men and women and for different age groups from <30 years to 50–80 years are separately provided – see T-values . Data were collected through the GESIS panel http://www.gesis.org/en/services/data-collection/gesis-panel/gesis-panel-data-usage/).Standardized norm data for each of the three scales of a 19-item self-report measure on environmental spatial strategies are provided based on a representative sample  and about 35% of the panelists participated offline (mail questionnaires are sent to those participants who are not able or do not want to participate online). Every interviewed person was incentivized for the recruitment interview. Likewise, participants are incentivized for the data collection waves. The present data collection was integrated in the GESIS panel data collection wave "ba" .Data were collected between February and April 2014 from 2223 female and 2057 male participants. The web-based online survey was answered by 2790 participants. Another 824 participants answered an offline, paper-based questionnaire with the same items. For another 666 participants, information about the online/offline format was not provided.2.2missing completely at random (MCAR) using the non-parametric test of the MissMech-R-package In 243 cases, missing values occurred in some individual items of the 19-item German Questionnaire on Spatial Strategies Calculating norm data for female and male participants and different age groups required to separate the data with respect to sex and age group. For these analyses, another twelve participants were excluded because of missing age data. The MCAR analysis showed no systemic changes by deleting these cases from the original data set. Accordingly, 4025 participants  were included in the analyses.2.3Participants answered the 19 items of the Questionnaire on Spatial Strategies The first two factors of the self-report measure FRS reflect the distinction between an egocentric (Factor I) and an allocentric (Factor II) spatial reference frame. Corresponding to its core meaning, the item indicating self-reported sense of direction loaded on the egocentric factor, but not on the allocentric factor. The egocentric strategies involved both directional strategies and route-based strategies. In addition, knowledge about cardinal directions was found to be a separate factor (Factor III). The factor structure was confirmed using confirmatory factor analyses http://www.gesis.org/en/services/data-collection/gesis-panel/gesis-panel-data-usage/). Each survey wave has about twenty minutes interviewing time and consists of two parts. About fifteen minutes of survey time are reserved for submitted studies (such as the present study). The second part of each survey wave (about five minutes of interviewing time) is reserved for longitudinal core study topics developed by GESIS.Participants also answered questions included in further studies that were carried out within the same data collection wave. GESIS provides reports and study descriptions on these studies  and age groups. The first T-values were computed based on means and standard deviations conserving the interval scale information. The second T-values were computed according to McCall T-value distribution .For each of the three scales of the FRS, the percentile rank and two T-value according to McCall The standardized norm tables for the three scales are provided in T-value is 50  strategy scale  strategy scale means that 47% (T-value is needed. For statements about how many people have a certain raw value or whether a certain person lies in a specific population area, the T-value according to McCall To compute group statistics, the non-corrected


To reduce data processing and postal costs, and to potentially enhance data quality in a large long-term mail-based randomised trial, the ASCEND study team developed a bespoke online web-based version of the standard 6-monthly paper follow-up questionnaire. The form incorporates verification checks and study staff were trained to provide IT support for participants. The key IT challenges included ensuring screen format compatibility with different computer devices and software compatibility with multiple browsers and versions, as well as ensuring data security which was enhanced by using a two factor user authentication scheme. The aim was to assess the impact of providing the option to study participants in a small pilot study.In November 2014, 200 randomly selected participants [aged 46 -89 years] were mailed their paper questionnaire with a flyer giving details of the new web option. Of the 200 participants, 188 [94%] responded with 29 [15% of total responders] completing their questionnaire on-line [aged 46-84 years]. For web forms the data quality was significantly better with reduced data processing time compared with the equivalent paper form. Users provided positive feedback with 8 IT-related queries. A subsequent mailing to the same group of 200 resulted in 39 web responders, suggesting that the uptake may increase by continuing to advertise the option at the time of each mailing coupled with an awareness campaign via the study newsletter.These early results provide encouraging evidence for adopting a web-based approach for similar studies where email addresses can be captured at screening.