Variation within individual genomes ranges from single nucleotide polymorphisms (SNPs) to kilobase, and even megabase, sized structural variants (SVs), such as deletions, insertions, inversions, and more complex rearrangements. Although much is known about the extent of SVs in humans and mice, species in which they exert significant effects on phenotypes, very little is known about the extent of SVs in the 2.5-times smaller and less repetitive genome of the chicken.We identified hundreds of shared and divergent SVs in four commercial chicken lines relative to the reference chicken genome. The majority of SVs were found in intronic and intergenic regions, and we also found SVs in the coding regions. To identify the SVs, we combined high-throughput short read paired-end sequencing of genomic reduced representation libraries (RRLs) of pooled samples from 25 individuals and computational mapping of DNA sequences from a reference genome.We provide a first glimpse of the high abundance of small structural genomic variations in the chicken. Extrapolating our results, we estimate that there are thousands of rearrangements in the chicken genome, the majority of which are located in non-coding regions. We observed that structural variation contributes to genetic differentiation among current domesticated chicken breeds and the Red Jungle Fowl. We expect that, because of their high abundance, SVs might explain phenotypic differences and play a role in the evolution of the chicken genome. Finally, our study exemplifies an efficient and cost-effective approach for identifying structural variation in sequenced genomes. Structural variation within the genome, including insertions, duplications, deletions, and inversions of up to multiple kilobase pairs, have recently been described in a variety of species, including humans -3, mice The contribution of structural variants (SVs) to complex phenotypes has been measured by association analyses of variance in gene expression levels (traits) and the presence of SVs. SNPs and SVs have been shown to account for 83.6% and 17.7%, respectively, of the total detected genetic variation in gene expression, with only a limited overlap . The effhttp://projects.tcag.ca/variation/). The resolution of array-CGH, though extremely costly, can be improved by using high-resolution whole-genome tiling arrays. Most of these SVs have been identified by methods that do not resolve SV end points at the base pair level. In addition, methods like array-CGH are based on a reference genome that currently does not encompass all SVs within the population and, thus, is limited in scope. Genomic regions that are the result of deletions not present in the reference genome are not captured by the array and not analyzed for SVs.Until recently, comparative genomic hybridization (array-CGH) was the most commonly used method for detecting SVs . Fosmid Next generation sequencing (NGS) technology was recently shown to be a powerful alternative to array-CGH for identifying genomic structural variation ,7,19. UsHere, we used PEM on reduced representation libraries (RRLs) of pooled chicken DNA samples. In the chicken genome, only 43 (larger) SVs have been described thus far . These SAluI RRLs for a white egg layer line, brown egg layer line, and two different broiler lines. For the white and brown egg layer lines, the 150-200 bp AluI fragments were used for creating the RRL; for the two broiler lines, 125-200 bp AluI fragments were used. From the brown and white egg layer RRLs, we obtained 31.61 million and 29.70 million raw reads, respectively, and from broiler 1 and broiler 2 we obtained a total of 34.8 million and 32.4 million raw reads, respectively. Reads were filtered for the presence of the restriction enzyme tag and trimmed to 32 bases. We required a phred quality score [To identify genomic rearrangements in the chicken genome, we applied massively parallel sequencing using the Illumina Genome Analyzer platform to sequence both ends of the genomic DNA fragments derived from the RRLs. We used pooled samples from 25 individuals to construct ty score  of at lein silico AluI digest of the chicken genome build WASHUC2, which resulted in 583,826 fragments of 150-200 bp, whereas 947,538 fragments of 125-200 bp were obtained. We calculated RRL sequence coverage based on the paired-end reads that passed our sequence quality filters. Coverage of the RRLs ranged from 11-13X in broiler lines to 18-20X in the layer lines, indicating that we analyzed, on average, 22-40% of the haplotypes of the 25 individuals used for constructing the RRL  predicted SVs Table . For theThe results suggest that the presence of concordantly mapping reads partly overlapping the predicted SV region did not correlate with the quality of SV prediction, whereas reference errors in the predicted SV region correlated negatively. Furthermore, the results indicate that putative SVs predicted by a single or a few discordantly mapping read pairs that mapped a slightly different distance than expected were false positives, whereas the majority of putative SVs with greatly deviating mapping distances were confirmed as being true SVs. With this limited number of observations, we formulated a simple but fitting rule to determine SV clusters with a high likelihood of representing a genomic rearrangement from false positives.We hypothesize that the size range of targeted DNA fragments isolated from the gel might contain a very small fraction of fragments outside the established size thresholds Figure . This laGenotyping results suggested that the presence or absence of SVs in a particular breed is fairly well predicted by the sequencing data. Therefore, we further analyzed 186 rearrangements (deletions) validated by our rule for breed specificity. We also analyzed breed specificity for 280 putative deletions that resulted from applying a less stringent read mapping quality constraint, which was also applied in previous SV detection studies ,25. The The majority of detected SVs were small Figure ; approxiin silico transcript to which it was mapped. The majority of intronic deletions resulted in a loss of a variety of known repetitive motifs  deletions and insertions with comparable or shorter length than the standard deviation of paired-end insert sizes requires de novo assemblies, because such SVs cannot reliably be identified by mapping approaches. Moreover, reference-based approaches, included mapping approaches, are biased to the completeness of the reference and, thus, ignore variants in regions that are missing from the reference genome due to structural variation. Finally, de novo assembly has the advantage of resolving SVs to a single base pair level, and inserted sequences can be obtained [Currently, sequence-based genome-wide surveys of SVs involve the preparation of whole genome fragment libraries in combination with paired-end sequencing. Such approaches require relatively large investments, particularly if multiple individuals from multiple breeds have to be screened. This study demonstrated the potential of massive parallel paired-end sequencing of RRLs constructed from the pooled DNA of multiple individuals. SVs were predicted based on the read pair information from the paired-end sequenced small insert RRL, which was purposely created for SNP detection. The small RRL size allowed for PCR-based confirmation and characterization of the SV at the base pair level of acquired deletions and small insertions with minimal sequencing efforts. Revealing inversion and translocation breakpoints is much more laborious due to the limited information RRL approaches provide. We showed that read pair analysis of a paired-end sequenced RRL is already sufficient for obtaining a first glimpse of SVs in a particular sequenced species. This RRL based strategy put constraints on the quality of the reference genome because assembly errors will result in false positive SV predictions in reference based detection approaches. Uncertainty about the quality of assembly of some of the smaller micro-chromosomes together with computational limits at the time of this study were the reasons why we did not analyze the whole genome for SVs. An enhanced assembly of the chicken reference genome and the increasing computational power allow for improvement in the detection of SVs using our approach. Furthermore the use of multiple RRLs including large and small fragments pools, that are separately tagged and paired-end sequenced together in bulk, will considerably improve SV detection at small increase of cost. More demanding is PEM of a randomly sheared and size-selected whole genome library providing a more complete catalog of rearrangements characterized between a sample and a reference ,19. An eobtained .We used a NGS approach to identify genomic rearrangements within four commercial chicken breeds by comparing their genomes to the sequenced chicken genome (Red Jungle Fowl). We excluded several classes of sequence reads from further analysis, including reads that did not show the restriction enzyme tag and those that showed more than one mismatch in the alignment. The first constraint was applied to eliminate false positive insertion predictions due to a breakdown of the RRL resulting in shorter spans of paired-end reads, whereas the second constraint was applied to reduce the number of false predictions due to sequencing errors. However, we realize that by taking these measures we also discard many read pairs because of true nucleotide variation, which occur in one of every 200 bp in the chicken . The incAluI sites may have caused one or both SV alleles to not be sampled and are, thus, not predicted to be present in that breed, consequently affecting our sequencing-based estimation of SV distribution across breeds. Conversely, our PCR-based genotyping approach with pooled samples was not affected by sampling limitation or AluI SNPs and revealed the presence of SVs in a breed even at allele frequencies of 0.1 (data not shown).Theoretically, our approach for identifying SVs allows the prediction of SVs and insight into how a predicted SV is distributed across breeds. We showed that the observed distribution of SVs is a good predictor for the actual distribution of the SV in breeds. Even with limited sampling, predicted SV distributions correlated with the PCR-based genotyping results of pooled samples Table . In geneBecause of the difference in the predicted presence of a SV in a breed and the genotyping results, we realize that the 186 SVs with which we estimated breed specificity might not be fully representative. The use of different RRL sizes (150-200 bp in layers and 125-200 in broilers) is reflected in a 1.5-2-fold difference in the SVs detected in broilers and layers. The fairly large percentage of SVs shared in broilers can be interpreted as being due to the effects of selection during line development by commercial companies and is consistent with the results of recent SNP genotyping , but it The reduction in the percentage of the genome covered by sequencing a RRL instead of randomly sampling the whole genome placed high constraints on the detection of SVs. The actual amount of SVs is likely much higher because we only sampled those that are flanked by restriction sites, and such that the intermediate sequence length of the variant was in the size range of the RRL. Large insertions were not expected to be detected because our RRL approach only allows for the detection of up to about 170 bp, the size between the maximum RRL fragment size (~200 bp) minus the mapping size of two completely overlapping reads (32 bp)Although the larger SVs are most likely under-represented in our data due to the constraints of the applied detection method, we can conclude that the majority of SVs in the chicken genome are smaller than 1 kb Figure . This fiWe provided a first glimpse of the abundance and genomic locations of structural variation in the chicken genome by identifying 188, mostly small, rearrangements, some of which were in coding regions, though a majority was located in non-coding regions. Based on the present data, we expect to find thousands of small (<1 kb) and hundreds of larger rearrangements in the whole chicken genome, encompassing more nucleotides than SNPs, and that are putatively involved in phenotypic variation. We observed that structural variation has contributed to genetic differentiation among current domesticated chicken breeds and the Red Jungle Fowl. Finally, we showed that little sequencing effort on a reduced representation of a genome is sufficient for the detection and base pair level annotation of a variety of SVs in a sequenced genome.Individual DNA samples were pooled according to breed and the genome complexity reduced by isolating a fraction of a complete genome digest. The isolated reduced representation library (RRL) was paired-end sequenced using Illumina genome Analyzer technology. The paired-end reads were aligned to the reference chicken genome WASHUC2 build and SVs are identified as significant differences between the mapping distances identified by the paired-end reads and the size range used for constructing the RRLs. Deletions relative to the reference genome were identified by paired ends spanning a genomic region in the reference genome longer than the size in the RRL, whereas insertions were identified by paired ends spanning a shorter genomic region in the reference sequence than expected based on the RRL. Inversion breakpoints were detected by paired ends that mapped in a different relative orientation compared to the reference genome.0 individuals from brown and white egg layer lines and two broiler lines consisting of 13 males and 12 females (Broiler 1) and 25 males (Broiler 2) using a Puregene DNA isolation kit .Genomic DNA was extracted from 30 μl of blood from 25 unrelated FAluI in a total volume of 240 μl. The selection of the restriction enzyme was based on the 10-fold reduction of genome complexity in the optimum size range (100-200 bp) of the sequencing technology platform . The digested DNA sample was fractionated on a 10% precast polyacrylamide gel (Biorad) at 100 V for 3 h and stained with ethidium bromide. The size fractions were sliced out of the gel and the DNA was mechanically sheared and and eluted over night in 300 μl recovery buffer  and precipitated with isopropanol. The DNA was washed with ethanol and re-suspended in DNA hydration solution .The RRLs were prepared by digesting 25 μg of pooled DNA using 1,000 units of the restriction enzyme We prepared the Genome Analyzer paired-end flow cell according to the manufacturer's protocol.Five picomole aliquots of the RRLs were processed using the Illumina Cluster Generation Station  following the manufacturer's recommendations. The Illumina GAII Genome Analyzer  was programmed to produce a theoretical fixed read length of 36 bp.Images from the instrument were processed using the manufacturer's software to generate FASTQ sequence files. Paired reads that had both the RRL restriction tag and a per base phred  quality score of at least 20 were selected using custom Perl scripts and aligned to the chicken genome (WASHUC2) using the MAQ  algorithAlignment results were analysed according to the MAQ  documentFor each SV cluster, we recorded the number of reads spanning the rearrangement, regardless of whether a normally mapping pair was observed or whether a sequence gap in the WASHUC2 build was present within the genomic range in which the deletion was predicted. SV clusters were prioritized for validation as follows: (i) an alternative mapping quality score of at least 60, (ii) both reads of a discordantly mapping pair mapped within a single predicted Ensembl exon or gene , and  were sequenced and mapped to the reference to ensure that it originated from the expected genomic position.Confirmed SVs were defined as those for which PCR reactions resulted in a distinct band in the expected size range in at least the breed for which the rearrangement was predicted and with no matching band in the UCD001 reference animal. The PCR results had to be supported by unambiguous sequencing data mapping confirming the rearrangement.http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi) under accession no. SRA026771.The data from this paper have been submitted to the NCBI Short Read Archive (The SVs identified in this study that have not been confirmed and annotated at the base pair level are available upon request, awaiting a central repository of structural variation in genomes.HHDK designed and developed the SV prediction method and wrote the manuscript. BWD and RPMAC prepared the samples and performed the initial validation and genotyping analysis. AV and RO selected the animals to be sequenced and collected the samples. MAMG and RPMAC coordinated and supervised experiment implementation and assisted in the preparation of the manuscript. All authors read and approved the final manuscript.


Cardiovascular diseases are complex pathologies that include alterations of various cell functions at the levels of intact tissue, single cells and subcellular signalling compartments. Conventional techniques to study these processes are extremely divergent and rely on a combination of individual methods, which usually provide spatially and temporally limited information on single parameters of interest. This review describes scanning ion conductance microscopy (SICM) as a novel versatile technique capable of simultaneously reporting various structural and functional parameters at nanometre resolution in living cardiovascular cells at the level of the whole tissue, single cells and at the subcellular level, to investigate the mechanisms of cardiovascular disease. SICM is a multimodal imaging technology that allows concurrent and dynamic analysis of membrane morphology and various functional parameters  in intact living cardiovascular cells and tissues with nanometre resolution at different levels of organization . Using this technique, we showed that at the tissue level, cell orientation in the inner and outer aortic arch distinguishes atheroprone and atheroprotected regions. At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio. We also demonstrated the capability of SICM to measure the entire cell volume as an index of cellular hypertrophy. This method can be further combined with fluorescence to simultaneously measure cardiomyocyte contraction and intracellular calcium transients or to map subcellular localization of membrane receptors coupled to cyclic adenosine monophosphate production. The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents. In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies. Heart tissue is highly organized in a three-dimensional manner at the levels of the intact tissue (macroscopic level), single cells (microscopic level) and at the nanoscale level of subcellular compartments. Classically, a broad range of conventional techniques has been employed to study these individual levels of organization, while a universal approach integrating this multi-dimensional information has been lacking. For example, biochemical and molecular biological techniques provide insights into various cellular functions but require the destruction of the sample , which does not allow continuous dynamic measurements. On the other hand, more physiological measurements are possible at the single-cell level but such techniques rarely reach the nanoscale level of the cell organization.in vitro models assessed by multiple extracellular electrodes . U. U2 muscmyocytes b,c, whil6.A hierarchical level of organization within living cardiovascular tissues requires the application of various methods to study the structure and function. High-resolution multi-parametric techniques and investigation of cell function at various levels of tissue organization (from macroscopic to nanoscopic) is one of the major current interests in cardiovascular biology. The aim is to correlate physiological function of the tissue with cellular and subcellular processes. However, contemporary technologies diverge and they are difficult to use together on the same biological substrate. SICM represents a versatile universal platform for all these studies. Using various modification of the SICM technique, one can investigate such diverse processes as arrhythmias, HF, atherosclerosis, hypertrophy, valvular heart disease and mechanical dysfunction of the heart. Studies using SICM may facilitate efforts to uncover the mechanisms of various cardiovascular diseases and to identify potential novel therapeutic strategies.


We report a case of Wegener's granulomatosis clinically mistaken for carcinoma in a 21-year-old girl presenting with an ulcerated mass of the nasopharynx associated with enlarged laterocervical nodes. The lesion was clinically suspected as malignant on the basis of clinical and radiological findings . However, multiple biopsies were not conclusive for malignancy showing histological change suggestive of Wegener's granulomatosis. A serum determination of cANCA supported the diagnosis of Wegener's granulomatosis. Clinical findings and image studies suggested an erroneous diagnosis of malignancy whereas a definitive diagnosis of Wegener's granulomatosis was achieved only after repeated biopsies thus leading to a correct therapeutic approach. The Wegener granulomatosis must be added to the list of the differential diagnoses of the masses of the nasopharynx associated with or without enlarged laterocervical nodes. Wegener's granulomatosis (WG) is a systemic, autoimmune disease histologically characterised by a necrotizing granulomatous inflammation with vasculitis. It belongs to the group of antineutrophil cytoplasmic antibodies- (ANCA-) associated vasculitis which are responsible for the inflammation of WG. The diagnosis of WG is typically based on the recognition of the clinical picture and the detection of ANCA in the serum, especially cANCA anti-PR3 , 2.The sinonasal tract is the most frequently affected site within the head and neck, and this site may be the only affected organ by WG . HoweverAs far as histology is concerned, the presence of necrotizing granulomas inflammation and vasculitis is the hallmark of WG. However, these pathognomonic features may be missed by the pathologists either in the early stage of the disease or when facing with small fragments of tissue. We herein present a case of sinonasal WG clinically and radiologically mistaken for an undifferentiated carcinoma of the nasopharynx.A 21-year-old girl was admitted to our hospital because of a 3-month history of otitis. A clinical evaluation, which included a nasal fibroscopic exam, revealed the presence of a vegetative and ulcerated mass of the rhinopharynx involving the left Eustachian tube, suggestive of malignancy .max] = 13.2) . The whole body scan was performed 50 min after intravenous injection of 208 MBq (5.6 mCi) of 18F-FDG revealing a focal area of FDG-6-phosphate accumulation in rhinopharinx involving left parapharyngeal space  .Even the PET finding supported the clinical diagnosis of malignancy. A biopsy was performed. Histology was negative for carcinoma and lymphoma on the basis of morphological and immunohistochemical findings .Because of the clinical and radiological suspicions of malignancy, an additional biopsy was performed. Histological examination revealed morphological immunohistochemical findings identical to the previous ones leading to a descriptive diagnosis of “no evidence of malignancy.” An aspecific medical therapy was started. Since the clinical symptoms were not responsive to the therapy, a third biopsy was performed showing the presence of a granulomatous inflammation with scattered giant cells and vasculitis suggestive of Wegener's disease .On the basis of these histological findings, a serum determination of ANCA was performed, showing strong (+ + +) cANCA positivity and thus supporting the histological diagnosis of WG. Clinical evaluation ruled out the pulmonary and renal involvement of the disease. The patients started atherapy with Rituximab  and suddenly improved the clinical symptoms.WG is an autoimmune disease histologically characterised by a necrotizing granulomatous inflammation associated with vasculitis of small and medium vessels. WG belongs to the group of antineutrophil cytoplasmic antibodies- (ANCA-) associated vasculitis which are responsible for the inflammation of WG. The determination of serum ANCA can assist in the diagnosis of WG, but ANCA positivity is not always conclusive for diagnosis, and the negativity for ANCAs is not sufficient to reject the diagnosis , 2.Although lungs and kidneys are typically involved in the course of WG, many other organs, including the upper respiratory tract, may be affected , 5. HoweIn the present case, the suspicions of malignancy was highly supported either by the clinical presentation or by image analysis . Carcinomas of the sinonasal tract are highly aggressive neoplasm arising from the nasal cavity and paranasal sinus. Although sinonasal carcinoma is rare, it must be suspected when facing with an ulcerated mucosa of this tract even in young patients . ClinicaMoreover, even the image analysis together with the PET study oriented and supported the clinical diagnosis of malignancy. Preliminary studies have reported promising results when malignant diseases in the head and neck were assessed with combined PET/CT since 18F-FDG PET has been reported sensitive for the diagnosis and staging of several types of malignancy , 10. HowAlthough clinically suggestive of malignancy, a definite diagnosis relies on the histological confirmation of the disease. In the present case, two consecutive biopsies were negative for malignancy. It should be underlined that a clinical diagnosis different from “malignancy” should be suspected when facing with consecutive histological diagnosis of “no evidence of disease” on bioptic samples. WG should be one of the differential diagnoses of 18F-FDG PET/CT positive scans in nonmalignant conditions, in conjunction with appropriate clinical background. False positive results are possible with 18FDG-PET alone because an infectious or inflammatory process producing uptake might be present, and because physiologic uptake into structures such as tonsils, salivary glands, and muscles might occur , 15. The recognition of WG in its early phase is of paramount importance since that if left untreated the disease runs an accelerated clinical course. Our case underlines that sinonasal involvement of WG may clinically overlap sinonasal carcinoma and should be included in the list of differential diagnoses of the masses of this region. On the other hand, pathologists have to consider that WG may not be histologically evident in small bioptic samples and that minimal changes including focal necrosis or granulomatous inflammation could be suggestive of WG. In conclusion, including the WG in the list of the differential diagnoses of sinonasal masses may have as a practical consequence to test serum ANCA in order to confirm or support the diagnosis of WG. This could have a more cost effectiveness effect in the diagnosis and treatment of an unusual but underdiagnosed condition of the sinonasal tract.


Trypanosoma vivax, one of the leading parasites responsible for Animal African Trypanosomosis (Nagana), is generally cyclically transmitted by Glossina spp. but in areas devoid of the tsetse flies in Africa or in Latin American countries is mechanically transmitted across vertebrate hosts by other haematophagous insects, including tabanids. We followed on from our recent studies on the maintenance of this parasite in vivo and in vitro, and its genetic manipulation, by constructing a West African IL1392 T. vivax strain that stably expresses firefly luciferase and is fully virulent for immunocompetent mice. We report here on a study where murine infection with this strain was monitored in vivo using a non-invasive method. Study findings fully support the use of this strain in the assessment of parasite dynamics in vivo since a strong correlation was found between whole body light emission measured over the course of the infection and parasitemia determined microscopically. In addition, parasitemia and survival rates were very similar for mice infected by the intraperitoneal and sub-cutaneous routes, except for a longer prepatent period following sub-cutaneous inoculation with the parasite. Our results clearly show that when administered by the subcutaneous route, the parasite is retained few days in the skin close to the inoculation site where it multiplies before passing into the bloodstream. Ex vivo bioluminescence analyses of organs isolated from infected mice corroborated our previous histopathological observations with parasite infiltration into spleen, liver and lungs. Finally, our study reinforces previous observations on the presence of the parasite in the central nervous system and consequently the brain commitment in the very late phases of the experimental infection. Trypanosoma vivax for decades, but the recent development of murine infection models and axenic cultures has enabled the genetic manipulation of this parasite and has opened the door to a more in-depth understanding of its biology and the infectious process that leads to animal trypanosomosis. We report herein the characterization of a luciferase-expressing strain that can be used to follow parasite dynamics in vivo in real time using a non-invasive method. Regardless of the inoculation parasite route and some minor differences concerning the length of the prepatent period of infection following the subcutaneous injection of the parasites, we highlight the general commitment of the organs triggered by the infection and particularly the presence of the parasite in the brain at late phases of disease. The study presented herein provides new insights into the interaction between T. vivax and its mammalian host and assesses new tools for in vivo drug screening.Very little work has been performed on  Trypanosoma vivax, T. congolense and T. b. brucei. T. vivax accounts for up to half of all AAT prevalence in West Africa where it is considered to be the major pathogen that together with T. congolense causes 3 million cattle deaths annually T. vivax but also T. equiperdum and T. evansi trigger different pathologies  and are species that have spread to South America. Globalization and livestock trade between countries, coupled with the lack of rapid diagnostic tools and the transport of infected animals to non-endemic areas have a huge impact on agriculture and, consequently, on the economy of breeding and endurance.Animal African trypanosomosis (AAT) is a major protozoan disease due to trypanosomes. The disease which is endemic in Africa is mainly caused by T. vivax compared to other animal trypanosomes (i.e. T. brucei spp and T. congolense) is its ability to be transmitted not only by Glossina spp. (tsetse) flies but also by other biting flies of the Tabanidae and Muscidae families that can mechanically transmit the parasite among mammalian hosts Glossina spp. are the only vectors in which T. vivax is able to multiply and pursue its differentiation into metacyclic forms. In contrast, T. vivax is unable to grow or multiply in other insects that can only mechanically transmit the parasite. Regardless of the natural type of transmission , T. vivax is inoculated in the subcutaneous tissue and the infective forms join the bloodstream via the lymphatic system. After one or more parasitemia peaks, the animals generally show neurological disorders in late phases of infection and perish T. vivax show a range of tissue damage and the diversity of the pathognomonic signs and the severity of the disease frequently correlate with the degree to which the host shows resistance (“tolerance”) or susceptibility to the parasite. Few studies have been conducted to compare the infective process following a bite by tsetse or tabanids, or experimental infections by intraperitoneal or subcutaneous inoculation routes One of the specificities of T. vivax infection and pathology in the field, we recently developed murine models that deliver sustained and reproducible infections which successfully mimic the parasitological, histological and pathological features of the infection and closely resemble those observed in cattle trypanosomosis T. vivax, its tissue tropism, the temporal course of its invasion and the crucial question of the permeabilization of the blood brain barrier is still incomplete.In efforts to overcome the problems encountered when studying T. vivax genetic manipulation in vitro and in vivo characterization of the T. vivax luciferase strain and the validation of real-time biophotonic detection systems employed to study the propagation of this parasite in vivo. We determined method limits of detection and linearity ranges to better correlate mouse parasitemia with luminescence measured in vivo, and analyzed the course of the infection and parasite tissue distribution over time. Finally, we compared infection dynamics and organ commitment after subcutaneous and intraperitoneal inoculations with the parasite. Our results confirmed the usefulness of real-time biophotonic analysis in the study and monitoring of the T. vivax infectious process in vivo. Irrespective of the causes that have conducted each mouse to perish during early phases of infection, such as anemia, hyperparasitemia or organ failure, our data provide important evidence that at long-term trypanosomes attain the central nervous system of all the animals which have showed a extended survival just some days before death.In order to address some of these questions and supplement the conventional anatomic pathology examinations conducted during studies of the infectious process, we took full advantage of the latest advances made in Comité Régional d'Ethique pour l'Expérimentation Animale (CREEA), Ile de France. Animal housing conditions and the procedures used in the work described herein were approved by the “Direction des Transports et de la Protection du Public, Sous-Direction de la Protection Sanitaire et de l'Environnement, Police Sanitaire des Animaux” under number B 75-15-28, in accordance with the Ethics Charter of animal experimentation that includes appropriate procedures to minimize pain and animal suffering. PM is authorized to perform experiments on vertebrate animals  and is responsible for all the experiments conducted personally or under her supervision as governed by the laws and regulations relating to the protection of animals.All mice were housed in our animal care facility in compliance with European animal welfare regulations. Institut Pasteur is a member of Committee #1 of the Trypanosoma (Dutonella) vivax IL 1392 was originally derived from the Zaria Y486 Nigerian isolate T. vivax (102 parasites/mouse). Parasitemia was determined as previously described 3 LB960; Thoiry, France) and light emission was measured for 0.1 s. Luminescence was expressed in Relative Light Units (RLU).A luciferase assay kit  was used to monitor luciferase expression. Serial dilutions of parasite suspensions were washed in PBS and pellets were suspended in 150 µl of cell lysis buffer. The lysates were then transferred into white, 96-well microplates . Light emission was initiated by adding the luciferin-containing reagent, in accordance with manufacturer instructions. The plates were immediately transferred to the luminometer , the luciferase substrate, at a dose of 150 mg/kg before any bioluminescence measurements were made. They were anaesthetized in a 2.5% isoflurane atmosphere  for 5 minutes and kept in the imaging chamber for analysis. Emitted photons were acquired for 1 minute by a charge couple device (CCD) camera  set in high resolution (medium binning) mode. The analysis was then performed after defining a region of interest (ROI). The same ROI was used for all animals and all time points. Total photons emitted from the image of each mouse were quantified using Living Image software , and results were expressed as number of photons/sec/ROI.T. vivax strain that stably expresses firefly luciferase (TvLrDNA-luc) have been described elsewhere T. vivax. Luciferase expression levels in non infective epimastigote axenic forms (EPI) and in infective bloodstream trypomastigote (BSF) forms of TvLrDNA-luc were compared by measuring the luciferase activity of equivalent numbers of parasites purified from axenic cultures or mouse blood, respectively. Serial dilutions of EPI and BSF were washed, lysed and the extract supernatants assayed in parallel for in vitro luciferase activity by measuring relative light emission (RLU) initiated by adding luciferin substrate. TvLrDNA-luc extract supernatants. Since no bioluminescence was detected in the WT EPI and BSF parasites included in the assays (<50 RLU), these results indicate that this light emission is specific to bioluminescent (luciferase-expressing) parasites.The methods used to engineer this in vivo do not result in any loss of the construction carrying the luciferase and the resistance marker genes, TvLrDNA-luc parasite strain was maintained in vivo without drug pressure for 3, 7 or 15 sequential passages and compared for light emission. in vivo. The results obtained confirmed that the differences observed between EPI and BSF were not due to any in vivo loss of the construction. These differences therefore may reflect dissimilar stabilities of the enzyme at different temperatures or, like for other trypanosomatids T.vivax was shown to be stable over at least 15 consecutive passages, corresponding to 15 weeks.Limits of detection were about 300 EPI and 8000 BSF using the bioluminescence assay. EPI clearly gave 7 to 10 fold the luciferase activity of purified BSF. To check that BSF serial passages T. vivax-infected mouse model previously studied in the laboratory, was followed by using sensitive, non-invasive optical imaging to track TvLrDNA-luc parasite strain in live mice. The bioluminescent signal obtained in vivo with TvLrDNA-luc parasites was validated by considering criteria such as background spontaneous signals obtained from whole-body images of mice infected with WT parasites and optimal time period between substrate administration to live animals and image capture. Firstly, a group of mice were infected with WT parasites, injected with D-Luciferin and subsequently exposed to photon detection under the IVIS Lumina Imaging System (IVIS) to determine background emissions across the entire body. This resulted in 106 ph./s being considered as the background level for further in vivo experiments using the TvLrDNA-luc parasite strain (not shown). Secondly, another group of mice were infected with TvLrDNA-luc parasite strain and analyzed once the infection had resulted in moderate parasitemia (106 parasites/mL). Total body light emission was determined under the IVIS at time points 1, 3, 5, 10, 15 and 20 minutes after D-Luciferin injection. As can be seen in in vivo measurements were then made 10 minutes after the D-Luciferin injection. Lastly, we checked whether or not the light emitted correlated with the T. vivax infectious process in vivo. To do this, we determined whether or not the parasitemia observed microscopically correlated with the bioluminescence measured over the whole animal. Parasites were counted under a light microscope in five microliters of blood harvested individually from the tail vein of mice infected with 102TvLrDNA-luc parasites, and parasitemia was expressed as number of parasites per mL of blood. Mice were immediately injected with D-Luciferin and submitted to whole-body imaging. An extensive and increasing light emission was observed during the course of the infection, as shown in 8 parasites/mL. The exhaustive plotting of bioluminescence versus parasitemia depicted in TvLrDNA-luc bioluminescent strain and the baseline imaging parameters necessary to analyze and monitor T. vivax infection and disease progression in vivo.Parasite dissemination and disease progression in a T. vivax multiplication is confined to the vascular compartment, we compared the parasite dissemination after infection by two different routes. The conventional intraperitoneal (IP) route commonly used for mouse experimental infections was compared to the subcutaneous (SC) inoculation route that closely resembles the natural infections, cyclic or mechanically conveyed by the insects. The course of the resulting infection together with parasitemia and survival rates were therefore studied in mice infected subcutaneously or intraperitoneally with 102TvLrDNA-luc BSF parasites. As can be seen in 4 parasites per ml of blood). As shown in T. congolense, as reported previously In order to gain a clearer insight into disease progression, and in particular determine whether 2TvLrDNA-luc by the SC route and monitored bioluminescence every day during the prepatent period. Light emissions were seen to increase between days 8 and 9 post-infection, suggesting that the parasite load was increasing but remained below the limit of detection of the microscopy visualization technique (<104/mL). By the time parasitemia had become detectable (day 9–10), the mice showed a bright spot on the right lateral flank close to the injection site , as determined microscopically. 5–106 parasites/mL), photons were detected along the entire body, with hotspots corresponding to spleen, lungs and liver. Attempts were made to accurately define the dynamics of parasite dissemination by segmenting the images obtained for each animal into several areas corresponding to whole body (R), head (R1), thoracic region (R2), abdomen (R3), inguinal (R4) and testis areas (R5) (T. vivax is confined to the vascular compartment.eas (R5) . The bioeas (R5)  for someT. vivax at key time points in the infectious process by ex vivo examination of the main organs affected after injection of the TvLrDNA-luc strain. Groups of 3 mice per time point were sacrificed and the light emitted by spleen, lungs, liver and brain was promptly recorded in the presence of excess of D-Luciferin. As can be seen in 7±6×106 ph./s, not shown) is recorded for the mouse hearts to attain more than 1000 times the background levels for the organ by day 28 of infection.In a further set of experiments we compared the distribution of T. vivax in vivo also allowed the validation of previous data which showed that the parasite may cross the blood brain vessels and lodge into the brain parenchyma Use of bioluminescent T. vivax is one of the leading parasites responsible for AAT, or Nagana, that still ranks among the most neglected diseases. One of its main particularities that can explain its capacity to emerge in areas free from tsetse flies is its ability to also be transmitted mechanically by a broad spectra of haematophagous insects T. vivax infection using mice infected with the ILRAD1392 reference strain T. vivaxTvLrDNA-luc). Then, in detailed studies using this bioluminescent parasite, we ascertained that it has the same virulence in immunocompetent mouse models and behaves in the same manner as WT parasites. We established that these TvLrDNA-luc mutant parasites can be used successfully to i) monitor in vivo the infectious processes triggered by T. vivax and ii) evaluate organ infiltration by these parasites in vivo and ex-vivo. To the best of our knowledge, the study reported herein is the first to use a systematic imaging method to study the experimental model of T. vivax infection in vivo and the first to demonstrate the presence of the parasite in the brain by simple bioluminescent signal.in vivoPlasmodium berghei, Leishmania major, Toxoplasma gondii, Trypanosoma cruzi and T. bruceiin vivo without animal sacrifice. The technique is widely used for instance to screen new active compounds with the intention of discovering novel chemotherapies TvLrDNA-luc strain to follow the dynamics of T. vivax infection in vivo from the start to the end of the infectious process. We compared the results obtained with two different routes of infection, i.e. the conventional experimental intraperitoneal (IP) route and subcutaneous inoculation that mimics natural transmission of the parasite by the vector. The only difference observed between these routes was that the subcutaneous injection resulted in a more prolonged prepatent period and in parasite multiplication in situ for some days before it reached the bloodstream. These observations are fully consistent with data previously obtained mainly with T. congolense and T. evansi where it was shown that the inoculation of metacyclics into the animal's hypodermis either by insect bite or syringe resulted in the development of a local inflammatory reaction, called inoculation chancre T. congolense bloodstream forms, at least in low numbers, are unable to induce a metacyclics-like inflammatory reaction The use of bioluminescent parasites is a strategy of choice for investigating and following infectious processes T. vivax are fully consistent with our previous reports 2 concentration 2 in this organ. Our previous report T. vivax and T.b. bruceiAlthough it is generally recognized that African trypanosomes migrate from the skin into the blood via the lymph system, our results did not demonstrate whether or not parasite multiplication in this compartment contributes to the infection spreading or to the orientation of the immune response. However, the light emissions we measured clearly demonstrated that growing parasites were accumulating close to the inoculation site. The results we obtained with bioluminescent T. vivax both in the cerebrospinal fluid and the nervous tissue parenchyma of goats T.b. brucei infection in rat and mouse models, the parasite actively migrate out of the cerebral blood vessels, cross the endothelial basement membrane, the perivascular space and the parenchymal membrane to invade the brain parenchyma, with no signs of plasma protein leakage into the brain Our observations together with earlier histopathological studies TvLrDNA-luc strain for detailed in vivo studies of the infectious process triggered by T. vivax. In particular, our data showed that the TvLrDNA-luc strain is highly appropriate to ascertain the evolution of the infection and the mechanisms involved in the progression of the disease. A more in-depth comprehension of the strategies set in place by the parasite to persist inside the host could open up perspectives for the development of a new therapeutic strategy against AAT. Our data also validate the use of bioluminescent T. vivax in high throughput drug screening strategies.Altogether, the results reported herein strongly support use of the


Mobile mapping systems have been widely applied for acquiring spatial information in applications such as spatial information systems and 3D city models. Nowadays the most common technologies used for positioning and orientation of a mobile mapping system include a Global Positioning System (GPS) as the major positioning sensor and an Inertial Navigation System (INS) as the major orientation sensor. In the classical approach, the limitations of the Kalman Filter (KF) method and the overall price of multi-sensor systems have limited the popularization of most land-based mobile mapping applications. Although intelligent sensor positioning and orientation schemes consisting of Multi-layer Feed-forward Neural Networks (MFNNs), one of the most famous Artificial Neural Networks (ANNs), and KF/smoothers, have been proposed in order to enhance the performance of low cost Micro Electro Mechanical System (MEMS) INS/GPS integrated systems, the automation of the MFNN applied has not proven as easy as initially expected. Therefore, this study not only addresses the problems of insufficient automation in the conventional methodology that has been applied in MFNN-KF/smoother algorithms for INS/GPS integrated systems proposed in previous studies, but also exploits and analyzes the idea of developing alternative intelligent sensor positioning and orientation schemes that integrate various sensors in more automatic ways. The proposed schemes are implemented using one of the most famous constructive neural networks—the Cascade Correlation Neural Network (CCNNs)—to overcome the limitations of conventional techniques based on KF/smoother algorithms as well as previously developed MFNN-smoother schemes. The CCNNs applied also have the advantage of a more flexible topology compared to MFNNs. Based on the experimental data utilized the preliminary results presented in this article illustrate the effectiveness of the proposed schemes compared to smoother algorithms as well as the MFNN-smoother schemes. StartSince the early nineties, advances in satellite and inertial technology made it possible to think about mobile mapping in a new way. Instead of using ground control points as references for orienting the images in space, the trajectory and orientation of the imager platform can now be determined directly . CamerasAn INS is a self-contained navigation technique in which measurements provided by accelerometers and gyroscopes are used to track the position and orientation of an object relative to a known starting point, orientation and velocity . In addii.e., the variance and the correlation time) of each sensor system has to be accurately known [In the classical approach, the KF is applied in real-time applications to fuse different data from various sensors while optimal smoothing is applied in the post-mission mode. The basic idea of using the KF in INS/GPS integration is to fuse independent and redundant sources of navigation information with a reference navigation solution to obtain an optimal estimate of navigation states, such as position, velocity and orientation. However, the limitations of the KF have been reported by several researchers –15. The ly known –15. Furtly known . As indily known , the EKFly known ,19. Onlyly known , second These limitations, in turn, may result in sub-optimal performance or even filter divergence if the assumption of local linearity is violated –20. EachIn addition to these limitations, the problem of poor observation of inertial error states becomes the most critical issue, especially when integrating a low cost MEMS IMU with GPS ,21. PoorOptimal smoothing algorithms, also known as smoothers, have been applied for the purpose of accurate positioning and orientation parameter determination through post-processing for most of surveying and mobile mapping applications with integrated sensors ,14. In c2.As mentioned in the previous section, the accuracies of the KF solutions sometimes cannot fulfill the requirements of mobile mapping applications. An integrated system has to predict state parameters such as position, velocity and orientation using KF when GPS signal blockages appear . But in In order to achieve high accuracy for positioning and orientation determination in mobile mapping applications, the data is processed in post-mission mode with an optimal smoothing algorithm. Most of the commercial mobile mapping systems use an optimal smoothing algorithm to provide accurate information on position and orientation for direct geo-referencing ,22. HoweAnother effective way to improve the accuracies of low cost MEMS INS/GPS integrated solutions is through the improvement of sensor fusion algorithms. Compared to the hardware perspective mentioned above, the software perspective can be considered as a cost effective solution to develop a low cost sensor fusion solution for certain mobile mapping applications. a posteriori. The updated KF engine is triggered at every GPS measurement using the difference between GPS and INS solutions as input. Hence, the KF generates an updated estimate for reducing the INS errors using measurement update equations. Whenever GPS measurements are not available, the KF works in the time prediction mode to estimate the error state vector. The optimal smoothing is performed after the filtering stage and thus it relies on the previously filtered solutions. Consequently, an accurate filtering procedure is required for accurate smoothing process [i.e., 0 and N) are fixed. Compared to other fixed-interval smoothers, the Rauch-Tung-Striebel backward smoother has the advantage of being the simplest to implement [i.e., at epoch N), see [The process of the KF is divided into two groups, those for prediction and updating ,13. The  process . A fixedmplement . It cons N), see  for detaThe smoothed estimates at any epoch k are computed as a linear combination of the filtered estimate at that epoch and the smoothed estimate at the heading epoch k + 1. Thus, these smoothed estimates can be considered as updating the forward filtered solution to obtain improved estimates. The computation of the smoothed estimates at each epoch requires the storage of the KF predicted and updated (filtered) estimates and their corresponding covariances at each epoch ,14. This3.Three approaches concerning the development of alternative multi-sensor integration algorithms to improve the ability of analysis and prediction of complicated kinematic and nonlinear models to reduce the magnitude of accumulated positional and orientation errors during frequent GPS outages in land applications have been identified . The firet al. [The advantage of the sampling based approach is that the computation of derivatives is not applied . Particlet al.  indicateet al. . Particlet al. .et al. [Bergman  applied et al.  proposedet al. . Moreoveet al. .Aggarwal  proposeda priori density is assumed to be the sum of several normal distributions. However the first order Taylor series approximation is applied for updating each distribution similarly to the EKF. Therefore, it suffers the impact of high nonlinearity similarly to the classic EKF. Kubu and Wang [According to Kubo and Wang , the Gauand Wang  proposedand Wang  for detaet al. [To overcome the limitations of EKF instabilities and Jacobian evaluations, Julier and Uhlmann  proposedet al.  applied et al. , the diset al.  also comGenerally speaking, the results shown in –25 illuset al. [i.e., IMU stand-alone mode). These results are well beyond the performance characteristics expected from the IMU used in the NovAtel BDS GPS/IMU system.The artificial intelligence approach distinguishes itself from other types of estimation approaches by using inexplicit models, known as black box, to approximate the nonlinear relationships between system dynamics and measurements . Chiang et al.  proposedet al.  is trainet al. [Chiang et al.  introducet al.  have proet al. [Wang et al.  presenteSharaf and Noureldin  introducet al. [El-Sheimy et al.  developeAlthough artificial intelligence approaches are easier to design and implement, there are also limitations to these types of approaches . Most ofet al. [In addition, if the dynamics experienced by the vehicle exceeds the ranges of training data significantly, the performance of these approaches tends to deteriorate accordingly. Therefore, a frequent re-training procedure is required to guarantee the performance of these approaches. Chiang et al.  proposedet al.  for realet al. . In addiThe navigation parameters provided by these artificial intelligent models are limited to positional parameters only because they rely on GPS solutions to provide training target and the use of a GPS receiver is unable to provide any reliable orientation parameters. Therefore, all of these artificial intelligent models are implemented to provide 2-D or 3-D positional parameters and bridge the gaps during GPS outages for land vehicular navigation applications –29,38. Het al. [et al. [The hybrid approach is implemented by combing conventional EKF or smoother based approaches with artificial intelligent models. These artificial intelligent models are applied to model the error behaviors of conventional EKF or smoother and compensate for the errors of the positional and orientation parameters estimated by KF and Smoother during GPS outages. Goodall et al.  proposed [et al.  proposed [et al. ,31. The  [et al. ,31. The Generally speaking, a MFNN with an optimal topology is expected to provide the best approximation accuracy to the unknown model using the most appropriate number of hidden neurons and hidden layers ,40. TherTo avoid these limitations, several methods have been proposed in the last two decades to construct a neural network successively and automatically during the learning process. These methods are often recognized as constructive networks. The common principle is to start from a small network and then add hidden neurons and hidden layers as needed during the learning procedure using special algorithms. In other words, the networks are able to decide the appropriate topology based on the task given without human intervention. An overview of current constructive algorithms can be found in . Among tConsequently, the objectives of this article were to: (1) develop a CCNN-smoother scheme for precise sensor positioning and orientation, (2) verify the performance of the proposed scheme using a low cost MEMS INS/GPS integrated system and (3) compare the performance with previously developed MFNN-smoother scheme in terms of the topology applied and estimated accuracy during GPS outages.4.ϕ(•) for limiting the amplitude of the neuron output and the final output [ax and the weight links connecting input and hidden layers are given as a weight matrix, ϕ(h) and ϕ(o), respectively.ANN methodologies have been motivated by the recognition that the human brain works in an entirely different way from a conventional digital computer . The siml output ,40. FiguaZ and y, are given below:(h)s and (o)s represent the outputs of the adders in hidden and output neurons, respectively.Therefore, the outputs of hidden and output layers, y should relate to input vector, ax. The process of obtaining these weights from these desired examples is called supervised learning and it is basically a conventional estimation or approximation process [ process . That is process ,40.A simplest way to understand how to adjust weight is to use standard backpropagation learning algorithm whose error function E is given below :(3)E=12η is learning rate parameterThen the weight update formation is given as :(4)Δw=−The primary goal of developing ANN-aided schemes is to improve the positioning and orientation accuracies during frequent GPS outages, which are crucial for land mobile mapping applications. As shown in Therefore, the inputs applied in this study include positional and orientation states estimated by the EKF and smoother along with time information while the outputs include the errors of those estimated states during GPS outages. In other words, the proposed topology realizes the nonlinear mapping relationships between system dynamics, the length of GPS outage and the error behaviors of the EKF and smoother during GPS outages. Through using proper training strategy with sufficient training data, the proposed scheme is able to generalize the nonlinear mapping relationships between system dynamics, the length of GPS outage and the error behaviors of the EKF and smoother during outages.i.e., position or orientation errors) behaves under the influences of dynamics of the platform, the error characteristics of the inertial sensors being used and the length of GPS outage applied. As mentioned previously, the residual error compensation scheme developed involves a set of complicated non-linear function approximations to adapt to the influence of the variations of vehicle dynamics and sensor errors on KF and smoother during GPS outages [In this study, one of the constructive ANNs, the CCNN, is implemented to learn and compensate for the residual errors of the KF and smoother, respectively, to improve the accuracies of the positional and orientation parameters. The proposed scheme is capable of learning how the state vector . According to , an epocn neuron .According to , the useBased on the training data applied in this study, the topologies of the proposed schemes are shown in i.e., they have more hidden layers). Based on the results presented in As indicated in 5.The EKF applied in this study has 21 states, which are given as follows:ab,1×3 and gb,1×3) and scale factors  can be estimated and feed back to the INS mechanization to correct these raw measurements provided by an IMU. The scope of this study is to improve the accuracies of positional and orientation parameters during GPS outages, only the components concerning these parameters are shown in As shown in The errors of positional and orientation parameters estimated by the KF and smoother during GPS outages are used as the desired outputs or target values during the training process of various proposed ANN architectures, including MFNNs and CCNNs. The positional and orientation parameters estimated by the KF and smoother along with the time information in each scenario are used as the inputs of the proposed architectures. The goal of the proposed schemes is to compensate for the errors of the positional and orientation parameters estimated by the KF and smoother during GPS outages ,31. A suAn ANN with optimal topology is expected to provide the best approximation accuracy for the unknown model using the most appropriate number of hidden neurons and hidden layers ,40. The After being well trained, the proposed ANN compensation schemes are added to a loosely coupled INS/GPS integration architecture (closed loop), as shown in 6.Three field tests were used to evaluate the performance of the proposed schemes. The tests were conducted in land vehicle environments using different integrated systems consisting of one tactical grade IMU, Litton LN200 (1 deg/hr), a low cost MEMS IMU, BEI MotionPak II and two NovATel OEM-4 receivers. In this study, those IMUs were applied to collect inertial measurements in the field and then these along with carrier phase DGPS solutions were fed into software that has an inertial navigation algorithm and EKF to estimate inertial states optimally. The integrated system with LN200 IMU was used as the reference. The measurements and navigation solutions provided by the integrated system with MotionPak II were used to verify the performance of proposed schemes. The GPS measurements were processed using GrafNavTM software (Waypoint Consulting Inc.) in carrier phase DGPS to achieve ten centimeter level accuracy. The reference trajectories were generated by the integrated system with LN 200 IMU. They were determined using 21-state EKF and smoother implemented in the Aided Inertial Navigation Software (AINS) from the Department of Geomatics Engineering at of the University of Calgary. These parameters of EKF and the smoother applied in this study were well tuned so that they can represent the best achievable navigation accuracy for tactical grade IMUs.Ten GPS outages, marked with circles and each lasting 30 seconds, were simulated using the measurements collected in the third field test, as indicated in In addition, a total sixty four GPS outages, each has 30 seconds in length, were simulated randomly in four scenarios using the measurements collected in the first and second field tests using the INS/GPS integrated with the MotionPak II (MEMS) IMU. Both field test data sets are applied to verify the performance of the proposed schemes.6.1.As show in Similarly, the 99-Percentile of errors of CCNN-KF/smoother and MFNN-KF/smoother based schemes are reduced by 99% on average compared to KF/smother solutions. However, the performance of the proposed schemes still needs to be verified using other independent data sets and this is presented in the next section.6.2.Since the objective of this study was to verify the performance of proposed CCNN-smoother and compare to previously developed MFNN-smoother and commercially available smoother based schemes, those results concerning CCNN-KF, MFNN-KF, and commercially available KF are not provided to condense the discussions of this study. To verify the performance of well-trained ANN-smoother schemes, four sets of testing scenario are created. Each scenario consists of eight simulated GPS outages (30 seconds each) which are randomly selected from test trajectory 1 and trajectory 2, respectively. Therefore, the proposed algorithms are validated with 16 randomly selected GPS outages in each scenario. As shown in Since it is decided through empirical trials during the training stage, the number of hidden layers and neurons are determined by the designer. Insufficient or excess hidden layers and hidden also lead to the impact of overtraining or over-fitting, although this can be mitigated by reducing the number of training epoch, increasing the number of training goal or adjusting the number of hidden neurons. However, the ratios of improvement are not as good as with the CCNN-smoother scheme. The number of training epochs for MFNN applied in this study is reduced from 500 to 200. As shown in As shown in Generally speaking, the majority of positional and orientation errors produced by proposed CCNN-smoother and previously developed MFNN-smoother schemes concentrate around 0, which means that they are able to compensate various systematic errors automatically and increase the stability. On the other hand, the distributions of positional and orientation errors produced by CCNN-smoother and MFNN-smoother schemes are comparable.On the other hand, the performances of proposed CCNN-smoother and previously developed MFNN-smoother schemes are similar in terms of RMS errors, medians and 99-Percentile of positional and orientation errors. The results presented in this study indicate that the proposed CCNN-smoother scheme is able to achieve comparable performance when undertaking the prediction task with fewer hidden neurons and less training efforts. In addition, based on the characteristics of the INS/GPS integration applications, the scheme applied is able to reflect the impact of new information and so catch the latest dynamic and sensor error variations. In a neural network, this can occur with a continuous learning process to adjust the weights and appropriate variation of topology if needed. Consequently, self-growing or construct networks such as CCNNs are better than fixed topology networks such as MFNNs.The error behaviors of KF and smoother during GPS outage are coupled with vehicle dynamics, inertial sensor errors, the length of GPS outage and the quality of the system and measurement model applied. However, these error behaviors are too complicated to describe through proper mathematical models. The general idea of ANNs is to build up the nonlinear mapping relationship between inputs and outputs and learns for examples and generalizes for applications. Therefore, the proposed topology realizes the nonlinear mapping relationships between system dynamics, the length of GPS outage and the error behaviors of the EKF and smoother during GPS outages. Through using proper training strategy with sufficient training data, the proposed scheme is able to generalize the nonlinear mapping relationships between system dynamics, the length of GPS outage and the error behaviors of the EKF and smoother during outagesTherefore, the proposed CCNN-smoother scheme significantly improves the accuracies of all these positional and orientation parameters estimated by smoother applied in most of the commercial packages for mobile mapping applications for a low cost MEMS integrated system based on the field test data applied. Among these parameters compensated by proposed CCNN-smoother scheme, the improvement of the orientation parameters is more significant than that of positional ones. However, more training data sets are required in future studies to generalize the contributions of this study.In addition, the use of CCNNs for developing an alternative INS/GPS fusion scheme has several advantages over MFNNs. First, the best topology can be decided automatically based on the complexity of the applications, and there is no need to perform extensive empirical trials to determine the size and depth of the network. 7.This study developed a CCNN embedded sensor fusion algorithm to improve the accuracy of positional and orientation parameters during GPS outages using a novel procedure that combines CCNN architecture and the smoother applied in most of the software packages for mobile mapping applications for post-mission processing.The preliminary results presented in this study indicate that the proposed CCNN-smoother scheme outperforms commercially available smoother schemes using a low cost MEMS INS/GPS integrated system in terms of RMS errors, medians and 99-Percentile of orientation errors by 80% in average based on the field test data applied in this study. Similarly, it outperforms smoothers in terms of RMS errors, medians and 99-Percentile of positional errors by 60% in average. Among these parameters compensated by proposed CCNN-smoother scheme, the improvement of orientation parameters is more significant than that of positional ones.On the other hand, the performances of proposed CCNN-smoother and previously developed MFNN-smoother schemes are comparable in terms of RMS errors, medians and 99-Percentile of positional and orientation errors. The results presented in this study indicate that the proposed CCNN-smoother scheme is able to achieve comparable performance when undertaking the prediction task with a simpler and flexible topology decided automatically and with less training efforts. However, more training data sets are required to generalize the contributions and validate the applicability of propose algorithms for real life applications in future studies.


The solid variant of aneurysmal bone cyst is rare, and only 13 cases involving the spine have been reported to date, including seven in the thoracic vertebrae. The diagnosis is difficult to secure radiographically before biopsy or surgery.An 18-year-old Hispanic man presented to our facility with a one-year history of left chest pain without any significant neurological deficits. An MRI scan demonstrated a 6 cm diameter enhancing multi-cystic mass centered at the T6 vertebral body with involvement of the left proximal sixth rib and extension into the pleural cavity; the spinal cord was severely compressed with evidence of abnormal T2 signal changes. Our patient was taken to the operating room for a total spondylectomy of T6 with resection of the left sixth rib from a single-stage posterior-only approach. The vertebral column was reconstructed in a 360° manner with an expandable titanium cage and pedicle screw fixation. Histologically, the resected specimen showed predominant solid fibroblastic proliferation, with minor foci of reactive osteoid formation, an area of osteoclastic-like giant cells, and cyst-like areas filled with erythrocytes and focal hemorrhage, consistent with a predominantly solid variant of aneurysmal bone cyst. At 16 months after surgery, our patient remains neurologically intact with resolution of his chest and back pain.Because of its rarity, location, and radical treatment approach, we considered this case worthy of reporting. The solid variant of aneurysmal bone cyst is difficult to diagnose radiologically before biopsy or surgery, and we hope to remind other physicians that it should be included in the differential diagnosis of any lytic expansile destructive lesion of the spine. Histologically, aneurysmal bone cyst is typically characterized by cavernous channels surrounded by a spindle cell stroma with osteoclast-like giant cells and osteoid production . There iet al.  in 1983;et al.  occurrinWe report a case of the solid variant of aneurysmal bone cyst occurring in the T6 vertebra with extensive involvement of the left sixth rib and pleural cavity in an 18-year-old Hispanic man. We review the 13 prior cases that have been reported in the literature and discuss the unique features of these unusual tumor-like lesions of the vertebral column.An 18-year-old, previously healthy Hispanic man presented to our institution with a one-year history of left paraspinal tenderness and radiation into the left chest. Our patient denied weakness or numbness of the legs and bowel or bladder incontinence. He had no difficulties with ambulation or balance.On physical examination, tenderness could be elicited on palpation of the spinous processes of the mid-thoracic spine. No motor or sensory deficits were observed. There were no signs of myelopathy. A rectal examination showed good volitional rectal tone and no perineal anesthesia. The post-void residual volume of urine was negligible.A computed tomography (CT) scan of the thoracic spine Figure  demonstrConsideration was given to pre-operative spinal angiography and possible embolization of large arterial feeders to the mass. However, the risk of spinal cord infarction with embolization was deemed to be too high by the experienced interventional radiologists at our institution, and subsequently, this was not performed.in situ with no further attempts at correction of the kyphosis. Morselized bone graft from the osteotomized laminae and cancellous morselized allograft were used as graft material.Our patient was taken to the operating room where a single stage posterior-only approach for total T6 spondylectomy with left sixth rib removal and circumferential reconstruction of vertebral column was planned Figure . Spinal Post-operatively, our patient was neurologically intact. However, he did develop a pleural effusion on the left side on the first post-operative day that necessitated chest tube placement. The effusion was likely from irritation of the pleura and post-operative oozing of fluid/blood from the operative bed directly into the pleural cavity. There was no hematoma, and there were no signs of infection. After the chest tube was removed two days later, our patient progressed quickly in his recovery from surgery and was subsequently discharged home. Imaging revealed gross total resection of the mass lesion, adequate screw placement and moderate kyphosis of 34°  remains to be seen in longer-term follow-up, and is the subject of future studies.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.GA was responsible for the concept and design of the manuscript and for writing and editing of the manuscript. KR aided in the illustration of the manuscript. AA analyzed and interpreted the pathological data for our patient. WEW aided in the editing of the manuscript. DJC aided in the editing of the manuscript. TGL aided in the editing of the manuscript. AJ was responsible for the concept and design of the manuscript and for writing and/or editing the manuscript. All authors read and approved the final manuscript.


In the Netherlands a successful MRSA Search and Destroy policy is applied in healthcare institutes. We determined the effect of an adjustment in the MRSA Search and Destroy policy for patients in the outpatient clinic on the MRSA transmission to health care workers (HCW).In June 2008 an adjustment in the policy for outpatients was introduced in a large teaching hospital. Following this adjustment MRSA positive patients and patients at risk could be seen and treated applying general precautions, without additional protective measures. Also, disinfection of the room after the patient had left was abandoned. To monitor the effect of this policy on the transmission of MRSA all physicians and health care workers of the outpatient clinic were screened for MRSA carriage repeatedly.Spa type t011. As far as we could verify, no patients with this Spa-type had been cared for by the health care worker.Before the introduction of the adjusted policy all physicians and HCW of the outpatient clinic were screened . None of them was found to be MRSA positive. After introduction of the policy in June 2008 the screening was repeated in October 2008  and April 2009 . In April 2009 one health care worker was MRSA positive resulting in a mean prevalence of 0.09%. This is lower than the known prevalence in HCW. The health care worker was colonized with the livestock-related The adjusted MRSA policy did not lead to detectable transmission of MRSA to HCW and was associated with less disturbances in the work flow. Staphylococcus aureus (MRSA) is one of the leading pathogens causing infections in patients admitted to healthcare institutes all over the world. To prevent transmission of MSRA in healthcare institutes, a stringent 'Search and Destroy (S&D) policy’ is applied in the Netherlands and Scandinavian Countries .,19.11,19Our study shows that universal precautions in the outpatient department prevent widespread transmission of MRSA to HCW. The absence of MRSA positive HCW contributes to the final goal of the S&D policy: prevention of MRSA in patients. The adjusted policy is safe for HCW, leads to better logistics on the outpatient clinic and it is a more patient friendly approach.Staphylococcus aureus; MRSA: Methicillin-resistant Staphylococcus aureus; PPE: Personal protective equipment; S&D: Search and destroy.HCW: Health care workers; LA-MRSA: Livestock-associated methicillin-resistant The authors declare that they have no competing interests.MvR designed and coordinated the study, gathered en interpreted data, and drafted the manuscript. JK coordinated the study and participated in interpretation of the data. He also helped to draft the manuscript. Both authors read and approved the final manuscript.S. aureus.MvR and JK coordinate and give advice about the implementation of infection control guidelines in the hospital. Furthermore, they conduct multiple studies for methicillin-resistant


The surgical management of renal cell carcinoma with invasion of the renal vein or inferior vena cava is associated with significant rates of perioperative morbidity and mortality. In this report we propose a surgical checklist aimed at reducing adverse events associated with the resection of these tumors.This review describes the development of an evidence- and experience-based surgical checklist aimed at improving the perioperative safety of patients undergoing radical nephrectomy and tumor thrombectomy.Reducing the risk of complications during the surgical management of renal tumors with venous invasion begins with appropriate pre-operative imaging aimed at defining the cranial extent of the tumor thrombus, thus facilitating accurate preoperative planning. Other key elements of the checklist are aimed at ensuring clear and precise pre-, intra- and postoperative communication between members of the multidisciplinary-care team.A standardized surgical checklist may help to increase the perioperative safety of patients undergoing radical nephrectomy and tumor thrombectomy. Future validation studies are required to determine the clinical feasibility and post-implementation safety profile of this new checklist. Renal cell carcinoma (RCC) is known to invade the renal vein or inferior vena cava (IVC) in 4 to 10% of cases -4. The hBorrowing from lessons learned in the aviation industry, in 2008 the World Health Organization (WHO) unveiled a universal surgical safety checklist aimed at minimizing perioperative morbidity and mortality . Only onIn the field of urology, surgical checklists have recently been described for the performance of transurethral resection of bladder tumor as well as partial nephrectomy ,9. HereiA safety checklist for the surgical management of RCC with tumor thrombus was developed based on a review of the English-language literature and our own operative experience Table . More spSurgery is indicated in patients without evidence of metastatic disease who are otherwise fit for surgery. In the absence of metastatic disease, those undergoing surgery have 5-year survival rates approaching 70% . AdditioA complete history and physical examination should be performed for all patients. Special attention should be paid to the patient’s past medical history and performance status, as many patients will require referral to a cardiologist or internist for medical optimization prior to surgery. Further, the surgeon should document the presence or absence of lower extremity edema, which is an important factor in determining whether a patient can tolerate a surgical interruption of the IVC. The absence of lower extremity edema suggests the development of venous collaterals.Other components of the preoperative workup include routine lab work , chest x-ray and possibly a nutritional assessment. Working in collaboration with a medicine specialist, additional studies may be required to evaluate a patient’s cardiac and respiratory status. This may include a stress test and/or an echocardiogram. In addition, cessation of preoperative anticoagulation and the need for bridging therapy should also be addressed.The most important information needed for operative planning is the level of tumor extension. Adverse event rates have been shown to increase with higher levels of the tumor thrombus . PreoperThe presence of a coexisting bland thrombus increases the possibility that the patient will need to undergo an IVC resection, or less preferably placement of an IVC filter . MagnetiPrior to the day of surgery, the patient should be evaluated by the anesthesia team. This should include an assessment of the patient’s airway, prior anesthesia history, and medical comorbidities. In addition, the surgeon should begin communication with other surgical specialists who may be required to aid in the procedure. Commonly this includes a vascular and/or cardiothoracic surgeon. For complex cases, it is often necessary to hold a multidisciplinary meeting several days in advance of the procedure.On the day of surgery, operative cases that may require the assistance of ancillary care teams should not start until the appropriate teams have been notified or reminded of their possible need. Patients postoperatively going to the intensive care unit should be presented to the ICU team the morning of surgery. Finally, any specific surgical items or equipment should be requested at this point to allow sufficient time for them to be acquired prior to the beginning of the surgery.Since serious complications can arise even with the management of level I tumor thrombi, all patients should have an arterial, peripheral, and central line placed and also have blood products readily available. For level I and level II tumor thrombi, our group has had a median estimated blood loss of 275 mL with 75% of patients requiring at least 1 transfused unit of blood . HoweverP= 0.003) and inpatient complications from 11.0% to 7% (P< 0.001) in first 30 days after surgery [Prior to incision, a “time out” should be performed in accordance with the recommendations of the WHO . Importa surgery .Depending on the level of the tumor thrombus, the surgical management can be more or less complex. Our group has previously described the important steps involved in the surgical procedures for the management of different levels of tumor thrombi -21. We bVVBP can be used in the treatment of level III and IV tumor thrombi to avoid complications associated with decreased venous return to the heart. However, VVBP introduces its own set of risks including the possibility for lymphocele, infection, vessel injury due to vascular access, and air embolism ,23 . VVBFor tumor thrombi extending above the diaphragm, the traditional approach has been the use of a sternotomy and CPBP with DHCA ,26. CPBPIntraoperative TEE is indicated for all tumors which extend to at least the level of the major hepatic veins. In these cases, TEE is essential as it provides real-time information regarding the proximal extent of the tumor which may change with manipulation of the IVC or arterial clamping.Resection of the vena caval wall should be avoided when the tumor is free floating and therefore can be easily extirpated following a simple cavotomy. However, it may be necessary to resect the IVC in cases of adherent or invasive tumors so as to ensure complete local resection. Of note, one report showed no 5-year survivors in the setting of incomplete local resection . In a reConcomitant bland thombus is present in 15-20% of cases with level II-IV tumors . While sPrior to closure of the surgical incision, the surgical team should ensure that the instrument, sharp, and towel counts are correct. Any surgical specimens being sent for pathological analysis should be appropriately marked and identified. A brief operative note should be completed prior to patient transport to ensure accurate communication to teams in the post-operative recovery area or ICU. At the time of patient handoff, the surgeon should speak directly with the receiving team to ensure continuity of care.Radical nephrectomy for RCC with venous invasion is associated with high rates of perioperative morbidity and mortality. The proposed surgical checklist aims to improve the perioperative safety for patients undergoing this procedure. Future validation studies are required to determine the clinical feasibility and post-implementation safety profile of the proposed checklist.CPBP: Cardiopulmonary bypass; DHCA: Deep hypothermic circulatory arrest; ICU: Intensive care unit; IVC: Inferior vena cava; MRI: Magnetic resonance imaging; RCC: Renal cell carcinoma; TEE: Transesophageal echocardiography; VVBP: Venovenous bypass; WHO: World health organization.The authors declare that they have no competing interests.GC and RA conceived the manuscript. SJ and MAG drafted the manuscript. All authors participated in the critical revision of the manuscript. All authors read and approved the final manuscript.