Arabidopsis thaliana. This analysis revealed that COs are concentrated in regions of a few kilobases where their rates reach up to 50 times the genome average. The hotspots themselves tend to cluster in regions less than 8 kilobases in size with overlapping CO distribution. Non-crossover (NCO) events also occurred in the two hotspots but at very different levels  and their track lengths were quite small (a few hundred base pairs). We also showed that the ZMM protein MSH4 plays a role in CO formation and somewhat unexpectedly we also found that it is involved in the generation of NCOs but with a different level of effect. Finally, factors acting in cis and in trans appear to shape the rate and distribution of COs at meiotic recombination hotspots.The vast majority of meiotic recombination events (crossovers (COs) and non-crossovers (NCOs)) cluster in narrow hotspots surrounded by large regions devoid of recombinational activity. Here, using a new molecular approach in plants, called “pollen-typing”, we detected and characterized hundreds of CO and NCO molecules in two different hotspot regions in  Arabidopsis thaliana revealing several new features: (i) crossovers are concentrated in hotspots where their rate reaches up to 50 times the genome average; (ii) non-crossovers events,  also occur in hotspots but at very different levels; and (iii) in the absence of the recombination protein MSH4, the crossover rate is dramatically reduced (70 times less than the wild-type level) and the crossover distribution within a hotspot is also largely modified; unexpectedly, the non-crossover rate was also altered (15% of the wild-type level at a hotspot). Finally we showed that factors acting in cis and in trans may influence the level and distribution of crossovers at and between hotspots.During meiosis, genomes are reshuffled by recombination between homologous chromosomes. Reciprocal recombination events called crossovers are clustered in several kilobase-wide regions called hotspots, where their frequency is greatly enhanced compared to adjacent regions. Our understanding of hotspot organization is based on analyses performed in only a few species and rules differ between species. For the first time, hundreds of recombination events were analyzed in  Meiosis reduces the level of ploidy by half. To fulfill this goal, homologous chromosomes (homologs) are segregated at the first meiotic division. In most eukaryotes, accurate segregation is ensured by the formation of at least one reciprocal recombination event or crossover (CO) between the chromatids of homologs In all eukaryotes, CO distribution along chromosomes is not homogeneous. COs tend to be clustered in narrow regions (two to three kilobases wide) called hotspots where CO frequencies are greatly enhanced compared to large adjacent regions almost devoid of any recombinational activity Saccharomyces cerevisiae and Schizosacchamomyces pombe. As most of the proteins involved in the meiotic recombination process are evolutionary conserved, it is thought that their basic features are similar in all eukaryotes: meiotic recombination is initiated by DNA double-strand breaks (DSBs) formed early in meiotic prophase at the leptotene stage by the Spo11 protein The molecular organization of hotspots has been deciphered in the two yeasts per se are the major determinant of linkage disequilibrium (non-random association of genetic markers) breakdown. In addition, the gene conversion tracks contained in both COs and NCOs shape the haplotype landscape. Indeed, CO associated gene conversion events soften the boundaries between haplotype blocks while NCOs create holes within blocks S. cerevisiae with a very low CO to NCO ratio next to telomeres (excess of NCOs) and repression of both CO and NCOs close to centromeres Arabidopsis, to date little information is available. Using antibodies directed against the meiotic DSB repair protein DMC1, DSB sites have been estimated to be between 100 to 200 per meiosis in male meiosis of various accessions Arabidopsis as in other higher eukaryotes. Several recent studies have tried to tackle the question of the meiotic NCOs rate in A. thaliana. Genome-wide studies using Next Generation Sequencing (NGS) gave contradictory results with NCOs found to be either a rare meiotic event 6 tetrads, the others being associated with COs Most studies of meiotic recombination hotspots characterized COs whereas because NCOs are difficult to detect these have rarely been analyzed. COs Caenorabditis elegans has only interfering COs while all COs in S. pombe are interference-free. Both pathways appear to contribute equally in S. cerevisiae. However, in most higher eukaryotes, it seems that a vast majority of COs belong to the interfering pathway: 90 to 95% in mice and Humans, 85% in A. thalianaMeiotic hotspots are under the control of a series of genes that channel DSB repair toward different pathways. In most organisms, two CO pathways coexist. One is dependent on a group of proteins called ZMM A. thaliana. We set up a “pollen-typing” molecular approach  and “Landsberg erecta-4” (Ler), identified the regions 14a and 130× as good candidates for true CO hotspots er hybrids and precisely quantified by PCR . Taking into account the CO frequency estimated by our genetic map, the gDNA was then diluted to obtain less than one putative recombinant molecule per PCR reaction. Recombinant products were detected by two rounds of allelic PCRs (Arabidopsis genome but rather to a complex mixture of loci from different chromosomes (data not shown) in contrast to products obtained from pollen DNA (see below).A previous study of CO distribution over the entire lic PCRs . When anlic PCRs . Indeed,er hybrid plants (167 and 104 COs at 130× and 14a respectively). The CO rate in pollen gDNA was 0.55% : 0.29–0.95) at 14a, and 0.53% (CI:0.34–0.78) at 130×. The recombinant molecules were confirmed by sequencing and their exchange point mapped precisely. All CO molecules characterized contained a single transition between the parental haplotypes.We were able to amplify and characterize hundreds of recombinant CO molecules at both regions in gDNA extracted from the pollen of ColxLTwo distinct CO peaks were observed in the 14a region (subsequently referred to as 14a1 and 14a2), which both fit a Gaussian distribution . The widA. thaliana.At 130×, the CO rate was also null on both sides of the region and reached a maximum (close to the center) at 167 cM/Mb, which is 35 times the chromosomal average . The diser” and “Ler to Col” (i.e. ‘CtoL’ and ‘LtoC’) were observed: ‘LtoC’ exchanges were shifted to the left of ‘CtoL’ exchanges , while it was only barely significant  for the 14a2 hotspot, probably due to the lower CO number. These patterns are consistent with the hypothesis that the Ler allele has a stronger initiation activity than the Col allele at these hotspots We then looked at the distribution of exchange points in the recombinant molecules in each orientation at both loci . At the xchanges . A compaxchanges . At 14a1At meiotic recombination hotspots, DSBs are repaired as either COs or NCOs. In plants, very few meiotic NCOs have been characterized because of the difficulty in detecting molecular events unless they are linked to a phenotypic change. We characterized NCO events at both the 14a1 and 130× hotspots, with different molecular approaches adapted to the polymorphisms available at each hotspot .er leaves. Positives clones obtained with pollen DNA were sequenced . This result strengthens the hypothesis that initiation occurs preferentially on the Ler allele at the 14a1 hotspot (see above).We recovered unequal numbers of NCOs in both directions: two ‘LtoCtoL’ and six ‘CtoLtoC’ were detected at polymorphism #35 while one ‘LtoCtoL’ and six ‘CtoLtoC’ at polymorphism #37 . When aler specific primers on 96 samples, each containing 4,145 F1 pollen genomes or 4,800 F1 leaf genomes. Then, to specifically detect NCO molecules, allele-specific PCR was carried out at three different SNPs , while polymorphisms to the left were co-converted in only five tracts, but over a greater distance (up to 791 bp) ; similarWe also noticed that as observed for 14a1, an excess of ‘LtoCtoL’ (36) NCOs compared to ‘CtoLtoC’ (23) NCOs were detected at both #21 and #44. At #52, only ‘LtoCtoL’ NCOs were obtained. In the latter case, it was not possible to determine whether there was an absence of ‘CtoLtoC’ NCOs in our starting pool of almost 398000 genomes or if these were missed by pollen-typing. However, when the results were pooled for #21 and #44, the difference was not significant (‘LtoCtoL’: 0.009% (CI: 0.007–0.013); ‘CtoLtoC’: 0.006% (CI: 0.004–0.009)).A. thaliana, on the basis of chiasma counts in mutant backgrounds, it is assumed that 85% of COs belong to the interference dependent pathway (class I), while the remaining 15% are interference-free (class II) atmsh4 mutant background in which interfering COs are absent. Crosses were made between hemizygous Col and Ler lines containing a T-DNA insertion in the AtMSH4 gene . Meiosis appeared regular in both AtMSH4+/− Col and Ler parents and the F1s AtMSH4+/+ or AtMSH4+/−. Meiosis, however, was disturbed in the F1 Atmsh4−/− with a dramatic reduction in chiasma number as described in . We set up pools of Atmsh4−/− or AtMSH4+/− or AtMSH4+/+ F1 plants, extracted gDNA from their pollen and performed pollen-typing PCR to detect CO molecules. CO rates were not statistically different in AtMSH4+/+ and AtMSH4+/− at either 14a or 130× (data not shown). Thus pollen gDNA from AtMSH4+/+ and AtMSH4+/− was pooled and is referred to as “MSH4” in the following experiments. As expected, in the Atmsh4−/− pollen, when we conducted the experiment at the 14a locus, we detected a dramatic decrease (12 fold) in CO frequency compared to the “MSH4” CO rate  occurred in 14a1 (ratio 14a1/14a2: 1.6). At contrario in Atmsh4−/−, the proportion of COs between 14a1 and 14a2 was inversed  , and right (78 times lower) sections of the loci (In  CO rate . However CO rate . Surpris.7 10−5) ; Table 2.7 10−5) . We alsothe loci ; Table 1Atmsh4 mutant background and “MSH4” at both loci. At the SNP#35 in the center of 14a1, 41 NCOs were detected and confirmed among 11,896 colonies in pollen DNA extracted from Atmsh4−/− and 20 among 3,600 colonies for “MSH4” pollen DNA. Thus, at this marker the NCO rate was comparable in both genetic backgrounds . In “MSH4”, the NCO frequency (84 events/545844 genomes) was significantly higher at the same SNP 0.015% (0.012–0.019)   . TherefoArabidopsis accessions for which we could use the same allele-specific primers to perform pollen typing but which have different levels of polymorphisms within the DNA sequence at the 14a hotspot: Pyl-1 (8AV) and Ws-4 (530AV), . Between Col and the three accessions Ler, Pyl1 and Ws-4, there are 0.43%, 0.53% and 0.63% of polymorphisms distributed along the 5 kb of the 14a hotspot  but significantly less than in “MSH4” , whereas no significant variation was obtained at 14a (MSH4” but distant in ColxLer (see above)  but as mentioned above the hotspot sequences are identical in these two lines.Surprisingly, we observed that the CO and NCO rates and CO distribution at 130× differed significantly between the two ColxLd at 14a . We alsoe above) . MoreoveoC’ NCOs . Thus thA. thaliana chromosome 4. Both the rate and distribution of COs and the occurrence of NCOs across these regions confirm that they are indeed true meiotic recombination hotspots.Here, we characterized several meiotic recombination hotspots on HS22 hotspot in vitroAt 14a, CO distribution patterns fit with the existence of two independent hotspots located very close to each other. 14a1 has a very high peak rate of COs and is narrow  while 14a2 is broader  and peaks at less than half the rate of 14a1. At 130×, the CO landscape is more complex: the CO distribution is broad and does not conform to a single Gaussian curve. Instead, it is irregular with alternating peaks and valleys. In this region, COs may originate from a single initiation zone: the irregularities observed in the distribution pattern could be explained by the presence of several insertions/deletions - which are 20, 13, 12, 70, 10, 7, 11 and, 3 bp wide respectively - along the region . Such hea1bz) regions a1 the pattern is similar to that of 130× with COs distributed throughout a wide region (10 kb) with peaks and valleys but with no region devoid of COs between two peaks. At bz, there are at least three sections where hotspots have been detected in a 99 kb region but based on the data provided whether each section is itself a cluster of hotspots or a unique hotspot could not be determined NID2a-NID2b, MSTM1a-MSTM1bArabidopsis there are regions of tens of kb that are permissive for recombination and that within these regions, recombination hotspots arise at sites where particular sequence motifs and/or chromatin modifications target the activity of Spo11 Interestingly, in maize, there are two regions where the fine-scale distribution of COs has been characterized  Mu and the chromatin modification H3K4me3 was also reported in maize. As Mu insertion sites also correlate strongly with recombination sites, it was suggested that the local chromatin structure could play a key role in both mechanisms S. pombeC. elegans modulate meiotic DSB formation A. thaliana, the 3a and 14a hotspots but not 130× lie in a low nucleosome density region S. cerevisiae and Arabidopsis hotspots in promoter regions could be due to the resemblance of their genomic structures. Both are compact, have a high density of genes along chromosomes and small intergenic regions The center of all three of the hotspots described here lies close to gene promoters, which are active in MSH4” but even more strikingly, the hotspot almost disappeared in ColxPyl1. There were even less COs than in the Atmsh4−/− background. Both Ws and Pyl1 exhibit differences at the DNA sequence level compared to Col  but nevertheless they have evolved since they were isolated and it could be that some key mutations were selected that changed in “trans the local behavior of the region. Alternatively, the epigenetic status of the two F1s may have changed leading to differences in the activity of hotspots.Although the 8.8 kb sequence of the 14a locus in the two other F1s, ColxLArabidopsis thaliana. Three recent studies addressed the detection and rate of meiotic NCOs in A. thaliana. One recorded gene conversion events associated or not with COs at seven loci but only one NCO was detected at one locus A. thaliana, meiotic DSBs sites have been estimated at between 100 and 200 per meiotic cell based on the number of RAD51 or DMC1 foci (two DSB repair proteins) at mid-prophase We isolated and characterize dozens of meiotic NCOs at two loci for the first time in We also detected three NCO events with a discontinuous conversion pattern: two ‘CtoLtoCtoLtoC’ and one ‘LtoCtoLtoCtoL’ . In all S. cerevisiae, mammals and plants) most if not all NCO events arise through a “Synthesis Dependant Strand Annealing” (SDSA) mechanism whereas COs are formed by two distinct pathways, which generate either interfering COs (class I) or non-interfering COs (class II). MSH4 belongs to a group of highly conserved proteins, called ZMM, that are essential for the “class I” CO pathway S. cerevisiae  at the onset of zygotene, where it appears to play a role in the orderly progression of the pairing and synapsis processes Surprisingly, we also found that in the Arabidopsis thaliana. We have also established that COs and NCOs occur at very high rates at these hotspots, as observed in yeast and mammals. However, we have shown that the pattern of COs and NCOs differs from that described in other species. There is therefore a need for the analysis of more hotspots in a diverse range of species in order to understand the underlying mechanisms that control them.In conclusion, we have formally demonstrated that true meiotic recombination hotspots exist in the plant Arabidopsis thaliana accessions “Columbia-0” , “Landsberg erecta” , Pyla-1  and Wassilewskija-4  were obtained from the “Centre de Ressources Biologiques” at the “Institut Jean Pierre Bourgin”, Versailles, France. The MSH4 mutant lines SALK_136296 (Col-0) er) http://nasc.nott.ac.uk/). All plants were grown in the greenhouse under standard conditions.The AtMSH4+/+,+/− and −/− plants among the F1. The oligonucleotides used were: (i) N636296U CTTCTTGCAGGTTfGTGTTTG - N636296L GCCAGCTGTTTTTGTTGTC and N636296L - LbSalk2 TCCCGCTCAGAAGAACTC to genotype the wild type and the mutant allele respectively in Col (ii) GT14269U CCGTTCAAATGTTTGCCATAC - GT14269L TTTCACCTTCCTAACGGTGC and CSHLds5-4 TACGATAACGGTCGGTACGG - GT14269L to genotype the wild type and the mutant allele respectively in Ler.After crossing, we used PCR screening to select homozygous er and “MSH4” as described in Characterization of male meiosis by cytology was carried out as described in Genomic DNA from pollen was extracted as described in Genomic DNA was extracted from young leaves as described in Taq DNA polymerase and 0.1 U Pfu DNA polymerase. Whenever less than 100 pg/µl of genomic DNA was used, herring sperm DNA (Clontech) was added into the reactions (1 ng/µl). Primers (sequence and genomic coordinates) are listed in Quantification of gDNAs was performed as described in m;30 sec)))×30). After the second PCR, the proportion of negative wells among a set of aliquot reactions was approximated by e−m, where ‘m’ is the mean number of DNA molecules per well in the first reaction. Parental molecules were thus quantified using ASOs all specific to either Col or Ler DNA.Products amplified from the gDNA extracts were quantified in a series of dilutions through two rounds of PCR, using the nested allele-specific oligonucleotides (ASOs) listed in er allele on one side, and Ler or Col respectively on the other side  confirm the NCO event and (ii) map the recombinant point  between the BamHI and XbaI unique sites, using standard procedures. The ligation products were then used to transform DH10B E. coli strain by electroporation. Transformed cells were spread onto LB agar plates containing 100 µg/ml carbenicillin, 0.2 mM IPTG and 40 µg/m X-gal. Following blue/white screening, individual colonies were transferred to 200 µl of LB medium containing 100 µg/ml carbenicillin in 1 ml MASTERBLOCK microplates (Greiner Bio-One ref. 780215) and grown with gentle shaking at 37°C for 16 hours. Then, 100 µl of each cell culture was transferred to 96 well V-bottom microplates and spun down at 3200×g for 10 min. Cell pellets were resuspended in 100 µl of sterile water.For 14a1, we followed the procedure described . Twenty  primers . Then afBacterial clones were then genotyped using the “Chemicon Amplifluor SNPs Genotyping System”. For this purpose, oligonucleotides either specific to each parent at polymorphisms #33, #35 and #37, or non-specific primers, were designed using the Amplifluor AssayArchitect software .Genotyping was then performed as described in CLleft, COCLright, COLCleft, COLCright; (ii) these numbers were grouped in a contingency table for testing the association between left/right and CL/LC classification using the two-tailed Fisher's exact test.The differences between CO distribution in reciprocal orientations at a given hotspot were tested as follows: (i) CO breakpoints located on each side of the median position were grouped separately for the two reciprocal orientations (‘CtoL’ CL and ‘LtoC’ LC), thus providing four numbers COThe difference between ‘CtoLtoC’ and ‘LtoCtoL’ NCO rates at polymorphisms #35 and #37 in the 14a1 hotspot was tested using the one tailed Fisher's exact test.The parameters of the best fitting Gaussian distributions were calculated using an R script , which cEstimated CO frequencies and associated confidence intervals were analyzed as follows:C) was used as the template for all experiments, but at different dilutions. An iS series of experiments (indexed with i) was performed with the first primer pair, and an jS series (indexed with j) with the second primer pair. For the kth series, a total of kN PCR reactions was carried out using as template the initial DNA sample diluted at the rate kD. Let us note ky as the number of reactions that did not produce a product.Repeated PCR experiments were performed on highly diluted pollen DNA samples collected on a F1 plant obtained from a cross between two homozygous parents carrying different alleles at two marker loci. Two primer pairs were used for PCR amplification. The first pair was specific for molecules carrying the alleles of the first parent at both loci, and could thus amplify half of the non-recombinant molecules. The second primer pair was specific for molecules carrying the first parental allele at the first locus and the second parental allele at the second locus, and could then amplify half of the recombinant molecules. A PCR reaction was considered positive if the template contained at least one molecule corresponding to the primer pair used. The same initial pollen DNA sample  is: C and r are independent, the a posteriori probability of parameters C and r knowing the observations is: Cf and rf are the prior density functions of C and r respectively.Because r and C were taken uniformly distributed in  and r<10%, and that at least 10% positive reactions are expected with the least concentrated sample.The prior distributions for a posteriori distributions of r and C were numerically computed by a two-dimensional scan of the parameter space , the Col and the Ws-4 accession (purple triangle), the Col and the Pyl-1 accession (green dot). The numbers on the left refer to the percentage of polymorphisms between Col and the other accessions. B. CO distribution at 14 a in various F1s.Polymorphisms at 14a in various F1. A. Localization of polymorphisms between the Col and the L(PPT)Click here for additional data file.Figure S2er and “MSH4.” For both reciprocal orientations, cumulated (from left to right) relative CO rates were scored at successive polymorphic sites along the region. Blue curves: cumulated relative ‘CtoL’ CO rates; dark blue: “MSH4”; light blue: ColxLer. Red curves: cumulated relative ‘LtoC’ CO rates; dark red: “MSH4”; light red: ColxLer. Vertical lines: median positions of the hotspots; dotted: “MSH4”; filled: ColxLer.Cumulative CO rates at 14a in ColxL(PPT)Click here for additional data file.Figure S3Atmsh4−/−.. Position of SNPs genotyped are indicated by green filled circles (SNPs) or triangles (indel). The polymorphisms are indicated by filled black circles along the chromosome coordinate axes. Triangles: insertion or deletion above 7 nt. Thick red or blue horizontal lines: converted SNPs, Thick grey dotted horizontal lines: interval in which NCO tract ends are located. Blue: Col; Red: Ler. Yellow line: chimeric SNPs.NCO at 130× in “MSH4” and (PPT)Click here for additional data file.Figure S4Chiasma number per meiosis in “MSH4” and ColxLer F1s. The number of chiasma per male meiocytes was obtained as described in (PPT)Click here for additional data file.File S1R Script to determine the parameters of the best fitting Gaussian distribution of a CO distribution.(DOCX)Click here for additional data file.Table S1Sequences of primers.(DOC)Click here for additional data file.Table S2Primers for DNA quantification and CO detection.(DOC)Click here for additional data file.Table S3Primers for NCO detection at 130×.(DOC)Click here for additional data file.Table S4Primers for NCO detection at 14a1 hotspot.(DOC)Click here for additional data file.


In this review we will discuss recent findings concerning the biological role of the ACE2/Ang-(1–7)/Mas arm in the cardiovascular and pulmonary system. Also, we will highlight the initiatives to develop potential therapeutic strategies based on this axis.Angiotensin (Ang)-(1–7) is now recognized as a biologically active component of the renin-angiotensin system (RAS). The discovery of the angiotensin-converting enzyme homologue ACE2 revealed important metabolic pathways involved in the Ang-(1–7) synthesis. This enzyme can form Ang-(1–7) from Ang II or less efficiently through hydrolysis of Ang I to Ang-(1–9) with subsequent Ang-(1–7) formation. Additionally, it is well established that the G protein-coupled receptor Mas is a functional ligand site for Ang-(1–7). The axis formed by ACE2/Ang-(1–7)/Mas represents an endogenous counter regulatory pathway within the RAS whose actions are opposite to the vasoconstrictor/proliferative arm of the RAS constituted by ACE/Ang II/AT Importantly, these new components of the RAS, due to their counter regulatory actions, are candidates to serve as a concept to develop new cardiovascular and respiratory drugs.


The individual sensitivity for ones internal bodily signals (“interoceptive awareness”) has been shown to be of relevance for a broad range of cognitive and affective functions. Interoceptive awareness has been primarily assessed via measuring the sensitivity for ones cardiac signals (“cardiac awareness”) which can be non-invasively measured by heartbeat perception tasks. It is an open question whether cardiac awareness is related to the sensitivity for other bodily, visceral functions. This study investigated the relationship between cardiac awareness and the sensitivity for gastric functions in healthy female persons by using non-invasive methods. Heartbeat perception as a measure for cardiac awareness was assessed by a heartbeat tracking task and gastric sensitivity was assessed by a water load test. Gastric myoelectrical activity was measured by electrogastrography (EGG) and subjective feelings of fullness, valence, arousal and nausea were assessed. The results show that cardiac awareness was inversely correlated with ingested water volume and with normogastric activity after water load. However, persons with good and poor cardiac awareness did not differ in their subjective ratings of fullness, nausea and affective feelings after drinking. This suggests that good heartbeat perceivers ingested less water because they subjectively felt more intense signals of fullness during this lower amount of water intake compared to poor heartbeat perceivers who ingested more water until feeling the same signs of fullness. These findings demonstrate that cardiac awareness is related to greater sensitivity for gastric functions, suggesting that there is a general sensitivity for interoceptive processes across the gastric and cardiac modality. The perception and feedback of visceral signals is considered to be an essential variable in many theories of emotion Attempts to quantify the individual degree of accurately perceiving one’s internal bodily signals have predominantly focused on the individual sensitivity for cardiac signals (“cardiac awareness”) that has been assumed to represent an indicator of general “interoceptive awareness”. Investigating cardiac awareness is primarily based on the advantage that the sensitivity for cardiac signals can be easily and non-invasively measured by heartbeat perception tasks Although cardiac awareness can be reliably detected, the implicit assumption of cardiac awareness representing an indicator of “general” interoceptive awareness for bodily signals is still unresolved. It is not clear whether the individual sensitivity for cardiac activity is indeed related to the sensitivity for other bodily, visceral functions that would justify speaking of an individual general interoceptive awareness across different visceral bodily modalities.Anatomical studies demonstrated a class of afferent fibers that monitor the physiological state of all internal organs of the body According to this evidence and because there are quite stable inter-individual, trait-like differences in perceiving ones cardiac signals Studies investigating interoception across different visceral modalities in healthy persons under controlled conditions are very sparse and findings are ambivalent One bodily system that can be manipulated and measured using standardized methods and that shows spontaneous activity which can be perceived comparably to cardiac activity under controlled conditions is the gastrointestinal (GI) system. Although there has been longstanding interest in interoceptive processes in the GI tract Using invasive methods of assessing gastric sensitivity has several limitations which may potentially influence the relevant dependent functions. Using this method implies that participants should tolerate the procedure and are taught to swallow a stomach tube during preparatory sessions before the experiment Thus, the principal aim of the present study was to investigate the relationship between cardiac awareness as assessed by a standard heartbeat tracking task For this purpose we employed a standardized water load test (WLT) representing an easily performed, well tolerated and reliable method to induce gastric distension and postingestion gastric neuromuscular activity, in order to assess gastric sensation In healthy persons water loads have been demonstrated to stimulate normal slow-wave frequency of the stomach in humans which is 3 cpm (cycles per minute) On the basis of this evidence showing that the WLT is a sensitive marker for gastric sensitivity, and proposing that interoceptive awareness implies interoception across different bodily systems, we hypothesized that cardiac awareness should be accompanied by less water ingestion in the WLT until feeling first signs of fullness, signaling more sensitive perception of gastric function. In accordance with earlier findings Sixty-eight right-handed female students were recruited by notices posted on the campus of the University of Tuebingen. All interested women filled in screening questionnaires regarding sociodemographic and health information. Items included age, educational status, body weight and body height, current and former illnesses , medication use, and sporting activities. Questions on gastrointestinal symptoms also included low-level conditions such as heartburn, dyspepsia, bloating or irritable bowel syndrome (IBS).Only healthy female participants without any diseases, without use of medication and without substance abuse were selected for inclusion in the study. As it is known that regular exercise influences autonomic tone, especially the vagal component We aimed at avoiding sex differences and therefore investigated only a sample of women because it has been shown that men potentially demonstrate more accurate heartbeat perception than women M = 25.19; SD = 4.30) and a body mass index (BMI) of M = 21.35 (SD = 1.99). None of these final participants had received out- or inpatient treatment for somatic diseases, mental disorders or eating disorders and none suffered from cardiovascular or gastrointestinal diseases. There was no medication use and all participants had an equivalent educational background and were students. All participants gave written informed consent and all experiments were conducted in accordance with the declaration of Helsinki. Ethical approval was obtained from the Institutional Review Board of the University Clinic of Tuebingen.Based on these criteria nineteen students had to be excluded. The final sample comprised 49 healthy female students with an age range of 21 to 45 years  and electrogastrogram (EGG) recording and filled in the State Trait Anxiety Inventory (STAI) Cardiac awareness was assessed using a heartbeat tracking task A heartbeat perception score was calculated as the mean score across four heartbeat perception intervals according to the following transformation: ¼ Σ (1– (|recorded heartbeats – counted heartbeats|)/recorded heartbeats). The heartbeat perception score varies between 0 and 1. The maximum score of 1 indicates absolute accuracy of heartbeat perception.Furthermore, the sample was divided into good (good HP) and poor heartbeat perceivers (poor HP) according to the heartbeat perception score. Participants scoring above 0.85 were assigned to the good heartbeat perception group (N = 20), whereas the remainder formed the group of poor heartbeat perceivers (N = 29). The selected cut-off score of 0.85 was used in accordance with many previous studies WLT was performed by having subjects drink room temperature, non-carbonated water ad libitum over a 5-min period until reaching the point of individually perceived fullness After skin preparation, three standard Ag-AgCl electrodes  for EGG were positioned on the anterior abdominal surface. All electrode sites were cleaned with abrasive gel and wiped with a gauze pad prior to electrode placement. Electrogastrogram (EGG) data were assessed using the UFI 3991 x/3 GPP BioLog  and EGG data were collected at a sampling rate of 10 Hz. Respiration was used for cross referencing with the EGG in order to identify and eliminate data with respiration artifacts. EGG recordings were visually inspected for strong respiratory artifacts that could make the EGG data unusable. This occurs when the EGG picks up respiration instead of the electrical activity of the stomach. This can occur when the diaphragm is in close proximity to the stomach and the respiratory cycle moves the stomach in a way that the EGG records respiration instead of the electrical activity. Periods deemed to have strong respiratory artifacts were excluded from analysis. This was not necessary in any of the participants.The EGG signal was analyzed using a Fast-Fourier running spectral analysis ECG was recorded by a computer-based data acquisition system and the corresponding software . ECG was measured by means of lead II, Einthoven, with a sampling rate of 1000 Hz. Heart rate data were used for assessing the heartbeat perception score.Since former data suggested a positive association between cardiac awareness and anxiety Changes in EGG activity from 15 minutes baseline to reactivity during/after the WLT (5 minutes WLT procedure plus 30 minutes after WLT) were analyzed by means of repeated measures analyses of variance (ANOVAs) with the within-subjects factor “water ingestion” (activity during baseline vs. activity during WLT) for each EGG frequency band.Pearson correlations were calculated between the heartbeat perception score and the amount of water volume (ml) each participant ingested until feeling full in order to investigate the relationship between heartbeat perception sensitivity and the sensitivity for gastric functions. Additionally, Pearson correlations were analyzed between the heartbeat perception score and the percentages of total power for bradygastria (%bradygastria), normogastria (%3 cpm), and tachyarrhythmia (%tachyarrhythmia) frequency bands during baseline and during and after 30 minutes of WLT. Furthermore, bivariate Pearson correlations were calculated between ingested water volume (ml) and % 3 cpm activity during the WLT. In order to analyze if the association between heartbeat perception score and %3 cpm activity during the WLT was mediated by the ingested water volume (ml), ingested water volume (ml) was included as a predictor in linear multiple regression analyses with %3 cpm activity as the criterion and heartbeat perception score as a predictor.Correlations were also examined between the heartbeat perception score and subjective ratings of fullness, pleasant and unpleasant feelings and nausea as well as between the water load (ml) and recorded feelings of hunger and thirst at the beginning of the study.Differences between good and poor heartbeat perceivers regarding ingested water volume (ml) and subjective ratings of perceived fullness, nausea, valence and arousal during the WLT procedure were analyzed by means of ANOVAs with “group” (good vs. poor heartbeat perceivers) as between-subjects factor. Differences between both groups in baseline EGG activity and EGG reactivity scores to the WLT procedure in all frequency bands  were analyzed by using ANOVAs with “group” as between-subjects factor. Where appropriate (when the assumption of sphericity was not met), degrees of freedom were adjusted according to Greenhouse and Geisser Finally, Pearson correlations were analyzed between state and trait anxiety scores and heartbeat perception scores. Possible group differences in anxiety scores were analyzed by use of ANOVAs.F = 4.83, p = .03, 2η = .09. There were no significant changes in %bradygastria, F = 0.70, p = .45, 2 = η.01 and in %tachyarrhythmia, F = 0.90, p = .34, 2η = .02  was r = −.50, p = .001) , F = 16.59, p = .005, 2η = .23 (see There was a significant negative correlation between the heartbeat perception scores and the amount of ingested water volume (001) see . In acco .23 see .r = −.29, p = .04) showing that cardiac awareness was associated with less %3 cpm activity during the WLT procedure. There were no significant correlations between %3 cpm and heartbeat perception score during baseline  and between %bradygastria  or %tachygastria  during/after WLT or during baseline  and heartbeat perception scores.Additionally, there was a significant negative correlation between heartbeat perception scores and %3 cpm EGG activity during and 30 minutes after the WLT procedure  and that the heartbeat perception score significantly predicted ingested water volume (ml) . Additionally, ingested water volume (ml) significantly predicted %3 cpm activity .There was also a significant correlation between %3 cpm activity and ingested water volume (ml) in the WLT . In accordance with the results of the bivariate correlation analyses the results of the regression analyses demonstrated that the heartbeat perception score significantly predicted %3 cpm activity during the WLT  while the heartbeat perception score no longer significantly predicted %3 cpm activity .When entering ingested water volume (ml) in addition to the heartbeat perception score as predictors into the linear multiple regression analyses with %3 cpm activity as criterion the results demonstrated that ingested water volume (ml) continued to significantly predict %3 cpm  showed significantly less %3 cpm increase during WLT than poor heartbeat perceivers , F = 6.95, p = .01, 2η = .13) , F = .14, p = .71, 2η = .003; %bradygastria , F = .13, p = .73, 2η = .003; %tachygastria , F = .46, p = .50, 2η = .01.ANOVA analyses for group differences regarding EGG reactivity demonstrated that good heartbeat perceivers (.13) see . There wM = 3.17, SD = 3.85, poor HP: M = 3.36, SD = 2.82, F = ,002, p = .96, η2 = .001), arousal  = .67, p = .67, 2η = .004), pleasant feelings  = 1.52, p = .22, 2η = .03), unpleasant feelings  = 2.65, p = .21, 2η = .04) and nausea  = .87, p = .35, 2η = .02).ANOVAs demonstrated no significant differences between good and poor heartbeat perceivers in the subjective ratings of fullness  and heartbeat perception score as well as no significant correlation between heartbeat perception score and subjective ratings of fullness , arousal , pleasant feelings , unpleasant feelings  and nausea .Correlation analyses demonstrated that there was no significant association between state  or trait anxiety  and poor heartbeat perceivers  = .62, p = .43, 2η = .01; state anxiety: F = .08, p = .78, 2η = .001). Additionally, there was no significant correlation between the volume of ingested water in the WLT and state anxiety  or trait anxiety  as well as no significant correlation between ingested water (ml) and subjective feelings of hunger  and thirst  at the beginning of the study.There were no significant differences between good (trait anxiety: The present study investigated the relationship between cardiac awareness using a heartbeat tracking task and the sensitivity for gastric functions assessed by means of a water load test (WLT), in association with gastric myoelectrical activity as measured by non-invasive electrogastrogram (EGG) in healthy persons. The results demonstrate that there is a significant negative association between cardiac awareness and the amount of water ingested ad libitum that represents an indicator of subjectively felt fullness The results of the present study extend this earlier finding with respect to several relevant aspects. Whereas Whitehead and Drescher used a heartbeat discrimination task that is based on simultaneity paradigms, this study investigated cardiac awareness by means of a heartbeat tracking task. Thus, the findings of both studies confirm a positive association between cardiac awareness and the sensitivity for gastric signals that is independent of the applied heartbeat perception technique. An important aspect of the present study is that non-invasive methods of measuring gastric sensitivity and functions were used in order to assess interoceptive awareness across both, cardiac and gastric, organ systems. As has been stated before, the earlier study applied invasive methods to measure gastric perception which implies potential relevant limitations regarding a pre-selection of appropriate and compliant study participants for these assessments as well as a potential bias of sensitization for gastric signals by repetitively practicing and getting used to the swallowing of gastric tubes. Furthermore, the invasively applied distension stimuli are to a great degree artificial This is underscored by both, observed changes in gastric activity and subjective experience during water load. As expected, the EGG results demonstrated that ingestion and post-ingestion of drinking water in the whole group resulted in a significant increase of the percentage of normogastria (%3 cpm), but no significant changes in bradygastria and tachyarrhythmia. This represents a typical and normal gastric reaction after water load in healthy humans These findings suggest that persons with good cardiac awareness indeed ingested less water because they subjectively felt more intense signals of fullness during this lower amount of water intake compared to persons with poor cardiac awareness who ingested more water until feeling the same signs of fullness. This obviously highlights that persons with good cardiac awareness felt gastric signs of fullness more sensitively at a lower level of water intake and controlled water intake according to a more subtle recognition of visceral gastric signals compared to persons with poor cardiac awareness. This is in accordance with findings demonstrating that interoceptive awareness as measured by cardiac awareness is positively related to behavioral self-control according to a more sensitive perception of internal bodily signals such as cardiovascular changes and feelings of fatigue in a setting that allows persons to decide upon behavioral control of e.g. physical effort These results also speak in favor of the validity of the ingested amount of water during the WLT as an indicator for individual differences in gastric sensations of fullness or satiety and argues against the possibility that the amount of ingested water volume could potentially also reflect individual differences in further possibly intervening variables, such as personal preferences for or against water or different feelings of nausea that could have influenced the WLT procedure. Moreover, there was no relationship neither between anxiety measures and heartbeat perception nor anxiety indicators and ingested water volume underscoring that results were not confounded by anxiety.This interpretation is corroborated by the EGG data showing that, while there were no associations between cardiac awareness and EGG activity during baseline, there was a significant negative association between %3 cpm increase and heartbeat perception. Furthermore, good compared to poor heartbeat perceivers reacted with less %3 cpm increase during WLT. In conjunction with the findings outlined above showing that cardiac awareness is associated with a less amount of water ingestion according to a more sensitive perception of gastric signals of fullness, the results that cardiac awareness is associated with less %3 cpm reactivity are in line with expectations. As in healthy subjects the volume required to produce fullness in a drink test has been demonstrated to correspond to an increase of 3 cpm activity of the EGG In summary our results demonstrate that there is significant overlap or coherence of interoceptive awareness across different visceral modalities regarding cardiac and gastric signals, which both represent bodily cues that also show perceivable changes during situations of daily life .These findings are in line with the characterization of interoception representing the sense of the physiological condition of the entire body and of different bodily signals with the anterior insula as a relevant brain site for the integration of homeostatic states of different bodily tissues with motivational and emotional processes, supporting feeling states and giving rise to conscious visceral perception At this point, one crucial aspect has to be discussed which is of interest for future studies investigating the neural correlates of interoceptive awareness across different modalities. It has been proposed On the basis of this assumption, and in the context of results showing greater activity of the anterior insula cortex in persons with greater cardiac awareness Furthermore, recent results demonstrated that cardiac awareness is positively associated with benefits in selective and divided attention Finally, our findings are the first to demonstrate a congruency between the sensitivity for cardiac and gastric signals without using invasive methods that are limited by potentially influencing and manipulating sensitivity of different bodily modalities. “Interoceptive awareness” as assessed by heartbeat perception seems to represent a better ability to focus, to perceive and to process internal bodily information across visceral modalities, such as gastric signals, with cardiac and gastric signals both representing bodily cues that show perceivable activity changes during situations of everyday life. Future studies are encouraged, combining brain imaging and measures of interoceptive awareness, in order to elucidate the role of the anterior insula regarding somatotopic re-representations of different visceral signals lending support to interoception.


In response to the increased organ shortage, organs derived from donation after cardiac death (DCD) donors are becoming an acceptable option once again for clinical use in transplantation. However, transplant outcomes in cases where DCD organs are used are not as favorable as those from donation after brain death or living donors. Different methods of organ preservation are a key factor that may influence the outcomes of DCD kidney transplantation.We compared the transplant outcomes in patients receiving DCD kidneys preserved by machine perfusion (MP) or by static cold storage (CS) preservation by conducting a meta-analysis. The MEDLINE, EMBASE and Cochrane Library databases were searched. All studies reporting outcomes for MP versus CS preserved DCD kidneys were further considered for inclusion in this meta-analysis. Odds ratios and 95% confidence intervals (CI) were calculated to compare the pooled data between groups that were transplanted with kidneys that were preserved by MP or CS.Four prospective, randomized, controlled trials, involving 175 MP and 176 CS preserved DCD kidney transplant recipients, were included. MP preserved DCD kidney transplant recipients had a decreased incidence of delayed graft function (DGF) with an odd ration of 0.56  compared to CS. However, no significant differences were seen between the two technologies in incidence of primary non-function, one year graft survival, or one year patient survival.MP preservation of DCD kidneys is superior to CS in terms of reducing DGF rate post-transplant. However, primary non-function, one year graft survival, and one year patient survival were not affected by the use of MP or CS for preservation. In order to address the severe shortage of organs available for clinical transplantation, donation after cardiac death (DCD) donors have reemerged as an additional source of organs in many countries during the last decade Since the time that kidney transplantation was first implemented as a clinical standard of care, two preservation technologies have been developed to minimize ischemic injury to kidney allografts prior to transplantation, namely static cold storage (CS) and pulsatile machine perfusion (MP) Considering the current controversy, we conducted a meta-analysis of the available relevant prospective RCTs to better understand whether MP is able to gain better outcomes in DCD kidney transplantation when compared to CS. These data convey an important message for clinical transplant professionals to decide the best way to preserve DCD kidneys regarding the transplant outcomes.A search of the PubMed/Medline, Embase and Cochrane library databases was performed using the terms  AND (“DCD” OR “donation after cardiac death” OR “NHBD” OR “non-heart-beating donors”). The search was conducted in May 2012. Publications were limited to those written in English and to those reporting results from human subjects. Review articles were excluded after limit filtering. A manual search of the references of the relevant publications was also performed.Studies reporting outcomes of DCD kidney transplantation using MP preservation were included. Exclusion criteria were: (1) Overlapping cohort studies from the same institution (avoid duplication). (2) Studies lacking a control group (CS preservation). (3) Studies that included DBD donors. (4) Retrospective, non-randomized, or uncontrolled design studies. (5) Studies published greater than 20 years ago were excluded due to the significant technological changes that occurred in preservation techniques and other advances in transplantation surgery after that time Publications were reviewed and data were extracted by two independent investigators with disagreements being resolved through discussion and consensus. The primary outcome was the incidence of DGF, defined as the requirement of dialysis in the first week after transplantation. Secondary outcomes included primary non-function (PNF), one year graft survival, and one year patient survival rates.2 and 2I statistical tests. Heterogeneity was considered significant when P<0.05 or 2I>50%, and a random effect model was adopted. A random effect model is a kind of hierarchical linear model which assumes that the data set being analyzed consists of a hierarchy of different populations whose differences relate to that hierarchy. When P>0.05 for χ2 or 2I<50% for 2I statistical tests, indicating low statistical heterogeneity in both cases, a fixed effect model was used. A fixed effect model is a statistical model that represents the observed quantities in terms of explanatory variables that are treated as if the quantities were non-random. Publication bias was assessed by a funnel plot. A funnel plot is designed to check the existence of publication bias in systematic reviews and meta-analyses. The largest studies will be near the average while small studies will be spread on both sides of the average. Variation can indicate publication bias. All statistical analyses for the current study were performed with Review Manager .Pooled odds ratios (OR) were used to evaluate the event rates, and the results were reported with 95% confidence intervals (CI). A P value <0.05 was considered a significant difference in the values between the two groups. Heterogeneity through all the included studies was evaluated by χThe search algorithm and results based on the search strategies and selection criteria described above are outlined in 2 = 4.14, P = 0.25; I2 = 28%). Thus, a fixed effect model was used. Combined result of the 4 studies showed that the OR for DGF was 0.56 (95% CI = 0.36–0.86) for MP over CS (P = 0.008)  for MP and 66.5% (55.6%–84.6%) for CS kidneys. There was no significant heterogeneity among the 4 studies (χ= 0.008) .2 = 1.19, P = 0.75; I2 = 0%), thus a fixed effect model was adopted. The OR for developing PNF in transplant recipients receiving MP preserved kidneys was 1.30 (95% CI = 0.49–3.44) compared to those receiving CS preserved kidneys, which was not statistically significant (P = 0.60) (All four  = 0.60) .2 = 0.45, P = 0.80; I2 = 0%) Similarly, no significant heterogeneity was observed between the two studies that reported one year patient survival rates . Thus, a fixed effect model was used to pool the overall results for one year graft survival rate and one year patient survival rate. For MP preservation, the OR for one year graft survival was 0.64 (95% CI = 0.28–1.46), and there was no significant difference between the two preservation techniques (P = 0.29)  . For oneWe attempted to evaluate the length of stay (LOS), mean duration of dialysis for DGF period and serum creatinine levels at 3 months post-transplantation. However, only one study Because of the increasing number of patients added to the transplant waiting list and the lack of sufficient standard criteria donors (SCD) (brain death donors less than 50 years old) to meet this demand, DCD kidneys are now being accepted by many organ transplant centers all over the world as a potential organ source We found no difference in the incidence of PNF between the two preservation methods in the current analysis. This may be true or may be due to an inability to detect an effect of MP because of the relatively low incidence of PNF (∼5%) after DCD kidney transplant. One would expect that the reduction of DGF by machine perfusion might lead to an increase in graft and patient survivals since previous studies suggested that DGF was associated with reduced graft survival, which was observed in DBD kidneys. However, in our study, the one year graft and patient survival rates were similar between MP and CS preserved DCD kidneys. One US study Notably, MP needs additional logistic requirements and costs in comparison with conventional CS preservation before the kidneys are implanted. However, reduced requirement for dialysis after transplantation due to a lower rate of DGF and a shorter duration of DGF after transplantation could compensate for these additional logistic requirement costs by MP. No included study assessed the cost-effectiveness of MP and CS for DD kidneys in this analysis, but one multicenter RCT study There are some limitations in the present study. Firstly, clinical heterogeneity between studies might exist although we had strict enrollment criteria of references (only included prospective RCTs). One of the studies In conclusion, MP preservation of kidneys obtained from DCD donors can reduce DGF incidence after transplantation in comparison to conventional CS preservation. However, PNF incidence and one year graft and patient survivals were not different in patients using the two technologies. MP preservation is recommended for DCD kidneys to minimize the risk of DGF.PRISMA Checklist S1PRISMA 2009 Checklist.(DOC)Click here for additional data file.Flow Diagram S1PRISMA 2009 Flow Diagram.(DOC)Click here for additional data file.


Tokenization is an important component of language processing yet there is no widely accepted tokenization method for English texts, including biomedical texts. Other than rule based techniques, tokenization in the biomedical domain has been regarded as a classification task. Biomedical classifier-based tokenizers either split or join textual objects through classification to form tokens. The idiosyncratic nature of each biomedical tokenizer’s output complicates adoption and reuse. Furthermore, biomedical tokenizers generally lack guidance on how to apply an existing tokenizer to a new domain (subdomain). We identify and complete a novel tokenizer design pattern and suggest a systematic approach to tokenizer creation. We implement a tokenizer based on our design pattern that combines regular expressions and machine learning. Our machine learning approach differs from the previous split-join classification approaches. We evaluate our approach against three other tokenizers on the task of tokenizing biomedical text.Medpost and our adapted Viterbi tokenizer performed best with a 92.9% and 92.4% accuracy respectively.Our evaluation of our design pattern and guidelines supports our claim that the design pattern and guidelines are a viable approach to tokenizer construction (producing tokenizers matching leading custom-built tokenizers in a particular domain). Our evaluation also demonstrates that ambiguous tokenizations can be disambiguated through POS tagging. In doing so, POS tag sequences and training data have a significant impact on proper text tokenization. Natural language processing (NLP) is the computer processing of human language . It is aTokenization typically plays a role in processing text. Tokenization is broadly defined as the segmentation of text for subsequent processing. The definition’s breadth reflects the ambiguity and differences of tokenization strategies. Tokenization strategies can vary depending on language ,3, softwNoun to the token paper. Similarly, the CKY algorithm is a widely accepted solution for syntactic parsing , given  ε ℕ0 × sT and some s ε S′. A value of  indicates that the transducer could not be applied.Applying an implementation of the transduce function to the example string “10mg of” might result in: The token transducer formalization assumes that the token transducer operates from the input string’s beginning. An alternate formalization includes an index into the input string specifying the location on which to apply the transducer.To complete the tokenizer’s components, an algorithm is required that chooses the best path (tokenization) from the token lattice and one that constructs the token lattice from token transducer output. The token lattice’s best path is the most likely path through the token lattice given some language model. An algorithm exists for best path selection (e.g. adapted Viterbi ).To construct a token lattice, a lattice constructor applies every transducer to each character position in the input text. The result of applying a lattice constructor on “The patient’s 10mg tablet.” is seen in Figure Given:• Σ is a finite set of symbols called the alphabet.S is the set of all finite strings over Σ.• G :=  is a directed graph consisting of a finite set of vertices and a finite set of labelled edges, E ⊆ V × S × V.• G is constructed for some text s ε S as follows:The token lattice L := {i : i ε ℕ0, 0 ≤ i ≤ length(s)}.• Let s′ is a slice of s; s′ := s[i : length(s)] given an i ε L.• i ε Vv for i ε L. These vertices represent a position between characters in s.• s and corresponding token transducer output τ = ), a path of edges, , in the token lattice, G, is constructed where the first and last vertices of the path correspond to a position between characters, e0[0] = iv and me[2] = iv+l, and an edge is associated with a token by label(je) = j.t• For every slice of Of the three described software components, only the lattice constructor’s pseudocode is presented. This is due to token transducer code being specific to a token transducer’s objective and due to existing documentation of a best-path selection algorithm (e.g. ).Given our token lattice design pattern, a biomedical tokenizer can be created by:• Choosing a set of documented POS tags such as the Penn Treebank’s.• Choosing a best path selection algorithm. Implement the algorithm, if necessary.• Identifying the token transducers. Implement the transducers, if necessary.The proposed tokenizer design pattern does not provide a method for identifying token transducers. Token transducers will vary depending on the tokenizer’s input. For example, the token transducers required for English will likely differ from the token transducers required for Spanish. In this section, we propose a systematic approach to token transducer identification. The guidelines are as follows:• Select a set of documented POS tags such as the Penn Treebank’s.• Collect text segments (e.g. sentences) from the input texts that are representative of the input texts’ diversity. This may be via random sampling or another method.• For each text segment, identify its tokens.– Adhere to POS tag definitions– Insure that each token corresponds to at least one POS tag.P >t using the introduced notation .– Do not segment text when segmentation results in an unlikely POS-tag sequence such as segmenting “di-trans,poly-cis-Undecaprenyl-diphosphate” into (space separated) “di trans , poly cis Undecaprenyl diphosphate”. This can be captured as – Segment text when text ambiguously maps to multiple POS tags and segmenting establishes a single POS tag per token (e.g. “2.4kilograms” becomes “2.4” and “kilograms”)• Categorize the identified tokens into token classes .– Base classes on POS tag definitions, named entities , abbreviations and acronyms.– Minimize the number of classes and multi-class tokens.• Create a token transducer for each class of token.What follows is an example application of the token transducer guidelines using the Penn Treebank’s POS tag set, an author’s language model and the following sample descriptions:1. Entire upper dental arch (body structure)Segmentation: Entire upper dental arch ( body structure )2. Royal Navy - non-commissioned personnel (occupation)Segmentation: Royal Navy - non-commissioned personnel ( occupation )3. Primidone 50mg tabletSegmentation: Primidone 50 mg tablet4. Primary Sjogren’s syndrome with organ/system involvement (disorder)Segmentation: Primary Sjogren ’s syndrome with organ and system involvement ( disorder )5. Posterior cervical spinal cord injury, without spinal injury, C1-4Segmentation: Posterior cervical spinal cord injury , without spinal injury , C1 to 46. Precorrin-3B C17-methyltransferaseSegmentation: Precorrin-3B C17-methyltransferase7. Salmonella III arizonae 47:k:1,5,7Segmentation: Salmonella III arizonae 47:k:1,5,7Item 1 is an example of a simple segmentation.: whereas the second use, a hyphen in the token non-commissioned, is more difficult to assess. The hyphen could have been removed resulting in two tokens. Since hyphen removal might decrease POS tag sequence likelihood, non-commissioned was segmented as one token. For this limited example, either segmentation could be considered acceptable.Item 2 includes two uses of the symbol -. The first use is assigned the POS tag 50mg of Item 3 is segmented because segmenting establishes a single POS tag per token. The text would otherwise be a partial match to at least two POS category descriptions. For similar reasons, C1-4 of Item 5 is segmented into multiple tokens.The text ’s is split from Sjogren’s.The Penn Treebank specifies possessives as a separate POS category. Given this definition, the possessive 47:k:1,5,7, Precorrin-3B and C17-methyltransferase remain as one token, whereas organ/system and C1-4 are modified.Items 4, 5, 6 and 7 are segmented to maintain likely POS tag sequences. That is, Given these segmentations the resulting token transducers are:• Alphabetic • Possessive (’s)• Independents • Numeric (50)to and / for and)• Abbreviations • Substances We applied the design pattern and the token transducer identification guidelines in the creation of a tokenizer for biomedical concept descriptions and compared our tokenizer to three other tokenizer methods.Biomedical concept descriptions were extracted from SNOMED CT . SNOMED We randomly selected 2781 current SNOMED CT concept descriptions to create the ground truth (gold standard) tokenizations. An example concept description is “Posterior cervical spinal cord injury, without spinal injury, C1-4”. An author manually segmented each description by following our definitions and guidelines. He is a native English speaker. A second individual also segmented the concept descriptions after reading instructions and practicing on several examples. The instructions and examples can be found in Appendix . The second individual has a health sciences background but is not a native English speaker.separated units of meaning”. The segmentor was encouraged to develop their own segmentation strategy given that this strategy included the two rules provided in the instructions.The second segmentor was provided with open-ended segmenting instructions and five examples. The segmentor read the instructions and segmented the examples, after which the preferred segmentations were presented. This was sufficient for the segmentor to conclude that segmentation “The greatest effect of our segmentation definitions and guidelines was to expand closed-class words into their regular form. For example, plus and slash separated lists were converted to regular lists (e.g. “paracetamol + caffeine” became “paracetamol and caffeine”). Similarly, dashes representing the word “to” were replaced (e.g. “C1-4” becomes “C1 to 4”) and slashes representing the word “per” were replaced (e.g. “ml/g” becomes “ml per g”). Knowing that these abbreviated forms were generally absent in the training data, their expansion was to satisfy the requirement of likely POS tag sequences.Segmentation agreement is presented in Table There was weak preliminary agreement (CK 0.139) because descriptions ending with a parenthesized word such as “(finding)” were considered one segment by the second segmentor. She judged these parenthesized endings to have a single meaning and thus a single segmentation.  When the second segmentor encountered descriptions ending with several words within parentheses, she opted for segmentation consistency (not separating parentheses) rather than changing completed segmentations (changing single parenthesized words).An author segmented the parentheses and agreement was recalculated. This single change of separating parentheses from their adjoining words, for words located at the end of concept descriptions, resulted in a CK of 0.888. Further minor corrections to both segmentor’s results such as segmenting missed possessives resulted in a CK of 0.916. The author’s corrected segmentations were adopted for testing. These segmentations appear to be reasonable segmentations given a CK of 0.916 with another segmentor.We constructed a baseline whitespace-only tokenizer and selected tokenizers specifically designed for biomedical text from the list provided by He and Kayaalp . Specialjava -classpath nlpProject.jar gov/nih/nlm/nls/utils/Tokenize –inputType=freeText –tokens.Specialist is written in Java. Specialist considers a contiguous run of alpha-numeric characters bounded by white space as a token, as well as individual punctuation. Specialist over-segments and repairs the segmentation into meaningful tokens at a latter stage. For example, “2.4” is tokenized as (space delimited) “2 . 4” and corrected post-tokenization. Specialist was run using the following command: medpost -text.Medpost is written in C++ and uses 33 interdependent heuristics to tokenize biomedical text. It segments text for further processing which includes POS tagging. Medpost’s POS tag set is based on the Penn Treebank’s POS tag set. Medpost was run using the following command: P(state|state').We implemented the adapted Viterbi algorithm  to chooshttp://www.python.org) and use NLTK  [Our tokenization methods are written in Python (n 2.0b8) , a naturIn one case, we augmented the sample Penn Treebank corpus with %10 of the publicly available MedPost POS tagged corpus . The MedTo identify token transducers, we segmented concept descriptions by whitespace and constructed a set from these segmentations. Prior examination of the concept descriptions had shown that whitespace was rarely found within a token. We randomly selected 1900 items from the set of segmentations. These segmentations were separated into tokens by following our guidelines and using the Penn Treebank’s POS tags. Several segmentations were tokenized in context of their associated descriptions because the text segment contained insufficient information to perform tokenization (e.g. the “+” in “Paracetamol + caffeine”). Table The tokenizers were applied to our ground truth data (45.5 percent of the data contained ambiguous token boundaries). A segmentation identical to the ground truth’s was considered successful and any other tokenization was considered in error. Table Specialist performed poorly because it takes a different approach to tokenization, that of over-segment and repair. Specialist also removes symbols from the output tokens, such as brackets, resulting in poorer performance than the baseline whitespace-only tokenizer.MedPost’s most consistent error was leaving a quantity and its unit joined rather than segmenting them. For example, MedPost would leave “10mg” as a token whereas our approach was to segment “10mg” into “10” and “mg”.Our most accurate tokenizer’s most consistent error was separating decimal numbers. For example, our algorithm would separate “0.123” into “0 . 123” (space separated). One explanation could be that our training data contained an insufficient quantity of decimal numbers. Unless the HMM had been trained with the decimal number then the token was unknown to our HMM. Training an HMM using token features as well as the token itself would likely improve our most accurate tokenizer.The adapted Viterbi tokenizer, implemented using our proposed design pattern and our token transducer identification guidelines, performed as well or better than current biomedical text tokenizers. The results suggest that the design pattern and guidelines are a viable alternative to current biomedical tokenization methods.POS tag sequences and training data have a significant impact on proper text tokenization. The 0-order HMM disregards transition probabilities and consequently POS tag sequences, whereas the 1st-order HMM considers one previous state. Considering one previous state improves tokenization by approximately 15%. A further improvement of approximately 10% is achieved by training the HMM on data that has greater resemblance to the testing data. In other words, ambiguous tokenizations can be disambiguated through POS tagging.Dividing software into well defined components can increase software extensibility and reuse . Our desWe presented our tokenizer design pattern named the token lattice design pattern and associated token identification guidelines. We described the tokenizer’s input, output and components. The components are a token lattice and lattice constructor, a best lattice-path chooser and token transducers. Our evaluation of our design pattern and guidelines supports our claim that the design pattern and guidelines are a viable approach to tokenization. The token lattice design pattern is expected to apply to domains other than the biomedical domain.Our evaluation demonstrates that ambiguous tokenizations can be disambiguated through POS tagging. In doing so, POS tag sequences and training data have a significant impact on proper text tokenization. Our approach of tokenization through POS tagging differs from previous split-join classification approaches.Our tokenizer formalization suggests how various biomedical text processing components such as machine learning of named entities can interact cooperatively (as token transducers). Our formalization also demonstrates that machine learning algorithms are appropriate for choosing the best-lattice path from a  token lattice.Our research results support further investigation of machine learning on token lattices for selecting the best-lattice path. Future work includes applying the tokenizer pattern to other biomedical texts  and testing new best lattice-path chooser algorithms. Improvements to token transducers and the best lattice-path chooser are expected to further improve tokenization.The authors declare that they have no competing interestsNB is a PhD student at the University of Victoria. This work has been created as part of his PhD research. JWJ is a faculty member at the university and NB’s supervisor.You are asked to segment a sentence into its tokens (pieces). Here’s an example :A car, faster than lighting, was painted red.Acar,fasterthanlighting,waspaintedredWhen segmenting a sentence you are permitted to 1) separate and 2) delete pieces of the sentence. In the example above, spaces were deleted and punctuation was separated from its adjoining word.may choose to do the following:Tokens may have spaces (whitespace). Some people New York is a big city.New YorkisabigcityBelow are segmenting rules that you must follow, These rules apply to very few situations. For most cases, you will decide how to segment a sentence.• Consider the following as separate tokens (upper or lower case): ’ll ’re ’ve n’t ’s ’• Abbreviations of closed-class words must be expanded. Example: The sentence ”Jon/Roger are running.” would become ”Jon and Roger are running.” Here is a list of closed-class words: a about above across after against all along although among an and another any anybody anyone anything around as at because before behind below beneath beside between beyond both but by despite down during each either enough ever every everybody everyone everything except few for from he her hers herself him himself his how i if in inside into it its itself like many me mine myself near neither no nobody none nor of off on once one onto or ours ourselves out outside over past per several she since so some somebody someone sufficient than that the theirs them themselves these they this those though through throughout till to toward under underneath until up upon us we what whatever when where whether which whichever while who whoever whom whomever with within without yet you yours yourself yourselvesApply what you’ve just learned to these examples:Entire upper dental arch (body structure)Entireupperdentalarch(bodystructure)Royal Navy - non-commissioned personnel (occupation)RoyalNavy-non-commissionedpersonnel(occupation)Posterior cervical spinal cord injury, without spinal injury, C1-4Posteriorcervicalspinalcordinjury,withoutspinalinjury,c1to4Primidone 50mg tabletPrimidone50mgtabletPrecorrin-3B C17-methyltransferasePrecorrin-3BC17-methyltransferase


T) in the delivery room, causing lung inflammation and injury, but little is known about their effects on the brain. The aim of this study was to compare an initial 15 min of high VT resuscitation strategy to a less injurious resuscitation strategy on cerebral haemodynamics, inflammation and injury.Preterm infants can be inadvertently exposed to high tidal volumes (VT targeting 10–12 mL/kg for the first 15 min (n = 6) or 2) a protective resuscitation strategy (Prot VT), consisting of prophylactic surfactant, a 20 s sustained inflation and a lower initial VT . Both groups were subsequently ventilated with a VT 7 mL/kg. Blood gases, arterial pressures and carotid blood flows were recorded. Cerebral blood volume and oxygenation were assessed using near infrared spectroscopy. The brain was collected for biochemical and histologic assessment of inflammation, injury, vascular extravasation, hemorrhage and oxidative injury. Unventilated controls  were used for comparison.Preterm lambs at 126 d gestation were surgically instrumented prior to receiving resuscitation with either: 1) High VT lambs had worse oxygenation and required greater ventilatory support than Prot VT lambs. High VT resulted in cerebral haemodynamic instability during the initial 15 min, adverse cerebral tissue oxygenation index and cerebral vasoparalysis. While both resuscitation strategies increased lung and brain inflammation and oxidative stress, High VT resuscitation significantly amplified the effect (p = 0.014 and p<0.001). Vascular extravasation was evident in the brains of 60% of High VT lambs, but not in UVC or Prot VT lambs.High VT resulted in greater cerebral haemodynamic instability, increased brain inflammation, oxidative stress and vascular extravasation than a Prot VT strategy. The initiation of resuscitation targeting Prot VT may reduce the severity of brain injury in preterm neonates.High V Brain injury, particularly white matter injury and intracranial hemorrhage, is a major problem in very premature infants and the incidence and severity increases with decreasing gestational age and birth weight T) during the initial resuscitation in the delivery room T is poorly controlled and not routinely measured T, can induce an inflammatory response leading to chronic diseases of the lung such as bronchopulmonary dysplasia Recent clinical trials find that many preterm infants are inadvertently exposed to high tidal volumes  animal ethics committee at Monash University.via cesarean section. A skin incision was made in the fetal neck for placement of ultrasonic flow transducers  around the left and right carotid arteries. Carotid blood flow correlates highly with cerebral blood flow At 126±2 d (mean ± SD), the ewe and fetus were anesthetized  and the fetal head and neck were exposed T group; n = 6) or to protect the lung from injury . These were: 1) High VT, targeting a volume of 12–15 mL/kg within the first postnatal breaths using a neonatal positive pressure ventilator , which was maintained for 15 minutes to mirror the time taken for clinical neonatal resuscitation and transfer to NICU T, consisting of prophylactic surfactant  followed by a 20 second sustained inflation delivered by a Neopuff  using a peak inspiratory pressure (PIP) of 35 cmH2O. This second package of care was chosen to minimize pulmonary and systemic inflammation and improve the cardiopulmonary circulatory transition at birth T group and after 15 min of ventilation in High VT group, all lambs were ventilated in volume guarantee mode  for the remainder of the experiment. The high VT group did not receive surfactant. The total ventilation time for both groups was 90 minutes. In all lambs, ventilation was conducted using warmed, humidified air with the fraction of inspired oxygen initially set at 0.4 but adjusted to maintain arterial oxygen saturation (SaO2) between 88–95%. Well-being of the newborn lamb was monitored by regular blood gas analysis  and pre-ductal transcutaneous oxyhemoglobin saturation.Lambs were randomly assigned to receive either of two strategies that, based on previous studies, would be expected to either cause lung injury (High VLeft ventricular output and the proportion of left-to-right to right-to-left shunting through the ductus arteriosus was detected using pulsed Doppler ultrasound as described previously dyn), adjusted for weight, was calculated as VT/kg/(PIP - PEEP). Ventilatory efficiency index (VEI) was determined as 3800/(ΔP ·f ·PaCO2) where 3800 is a constant for the production of carbon dioxide (mL·mmHg·kg−1·min−1), ΔP = PIP-PEEP and f is the respiratory frequency 2) iO2 · mean airway pressure)/PaO2 · ΔCBV = (ΔHBT · MWhemoglobin·10−6)/(tHb · 10−2 · CLVHR · Dt · 10) where MWhemoglobin  =  molecular weight of hemoglobin  = 64,500, tHb  =  concentration of hemoglobin in large vessels in g · 100 mL−1, CLVHR  =  cerebral to large vessel hematocrit ratio  = 0.69, and Dt  =  brain tissue density in g·mL−1 = 1.05.Dynamic compliance -1β, IL-6 and IL-8 mRNA expression using quantitative RT-PCR 2 . H&E and albumin analysis was conducted using six random high-powered fields per section, 2 sections per lamb, from the deep periventricular or subcortical white matter . For H&E sections, each field was designated a qualitative score for the assessment of brain injury, where 0 =  none, 1 =  mild (<50% of the field of view occupied by brain damage), 2 =  severe (>50% of the field of view occupied by brain damage). Albumin extravasation was determined by the presence or absence of albumin within the tissue surrounding blood vessels; only the incidence was noted.The brain was removed and sectioned as described previously Data are presented as mean ± SEM. Serial data were compared between groups using two-way ANOVA with repeated measures . The first 15 minutes was analysed separated as above. Posthoc comparisons were performed using the Holm-Sidak method. ANOVA was used to compare biochemical and histologic indices of injury. Statistical significance was accepted for p<0.05.2: 67.0±5.8 mmHg: PaO2: 28.8±5.1 mmHg; SaO2: 60.3±8.7%). Lamb body weights were not different between groups .Umbilical arterial blood gas and acid/base status at birth were normal for all lambs and not different between groups  while in the Prot VT group it was 7.0±0.2  in High VT lambs from 30 min .Peak inspiratory pressure was higher in the High Vp<0.001; , while rPaO2) was higher at 10 and 15 min in High VT lambs but variability precluded significance  and was not different between groups (p = 0.393). Calculated cerebral oxygen delivery was not different between groups at any of the blood-gas time points (p = 0.840). Body temperature (p = 0.784), blood lactate (p = 0.893) or blood glucose (p = 0.435) were not different between groups (data not shown).The partial pressure of arterial oxygen ; the majority of the variability was observed within the first 15 min. Maximum CaBF (p<0.001) and the amplitude of CaBF (p = 0.002) was significantly higher within the first 15 min in High VT lambs compared to Prot VT. Mean arterial pressure was significantly higher in High VT lambs between 10–15 min and at 60 min compared to Prot VT lambs , while the high VT group remained unchanged.NIRS data was successfully obtained from 4 Prot Von Index  was not p<0.001; . The ProT lambs compared to Prot VT lambs and UVC . There were no differences observed in the periventricular white matter between the groups (p = 0.271). The number of 4HNE-positive cells, indicative of oxidative injury, was significantly higher in High VT lambs within the periventricular white matter compared to UVC (p<0.001) and showed a strong trend to be greater than Prot VT lambs (p = 0.054). There was no difference observed in the subcortical white matter  blood oxygenation levels, suggesting that the normal postnatal adaptation of the cerebral circulation has been altered in these lambs. Furthermore, as these lambs also had lower PaCO2 levels during the 1st 15 min, we would expect this to enhance the cerebrovasoconstriction caused by increased oxygenation, providing further evidence that the cerebral vascular response was abnormal in High VT lambs. It is possible that this reflects injury to the cerebral vessels and likely contributed to the vascular leakage observed in the High VT group, particularly as relative cerebral hyperemia and cerebral vasoparalysis are known features in neonatal brain injury In the Prot VOur study highlights the ability to detect some aspects of brain injury, including inflammation, oxidative stress and microscopic hemorrhage in brains within 90 minutes after the onset of resuscitation/ventilation at birth. This has not been previously reported, with most commentaries indicating that it takes 24 to 48 hours before brain pathologies can be detected. In the clinical setting, most cerebral ultrasounds are performed more than 24 hours after birth, with MRI studies conducted much later, and only if the baby can be moved. The study protocol allowed us to determine the effects of the initial resuscitation strategy, without the confounding influences of prolonged maintenance of respiratory and life support and other postnatal influences including nutrition and sepsis.T resuscitation impacts the immature brain, we compared these lambs with lambs resuscitated with a strategy we considered to be less injurious (Prot VT) and to minimally interfere with the cardiovascular transition at birth. The Prot VT group received prophylactic surfactant, a 20 second sustained inflation followed by volume guarantee ventilation at 7 mL/kg, representing the normal spontaneous tidal volume of preterm lambs T group displayed normal postnatal adaptive changes within the cerebral circulation and had reduced brain inflammation compared to the more injurious group. This suggests that improved resuscitation procedures, including appropriate lung recruitment and minimizing lung injury, may reduce the risk and severity of brain injury.To assess how High VT for 15 min initiates a pulmonary inflammatory response T resuscitation increased biomarkers of inflammation within the lung and brain compared to the Prot VT group. Up-regulation of systemic pro-inflammatory cytokines  are known to compromise integrity of the blood brain barrier and neuro-vascular circulation T lambs, which was not present in unventilated control or Prot VT lambs, confirming a strong association between increased systemic cytokines levels and blood brain barrier integrity.This study confirms that resuscitation with high VT lambs had elevated cellular lipid peroxidation within the periventricular white matter, potentially resulting in increased white matter injury within this group. The findings of increased brain inflammation and oxidative stress resulting from resuscitation, and particularly high VT resuscitation, indicates that the immediate resuscitation period in the delivery room is likely a critical window for the development of white matter injury.Oxidative stress is another notable mechanism implicated in white matter injury, such as periventricular leukomalacia (PVL) T resuscitation strategy further exacerbated cerebral hemodynamic instability, brain inflammation and injury. This study highlights the critical role that the initial respiratory support has on the development of brain inflammation and injury, and the requirement for better monitoring of delivered tidal volumes to preterm infants in the delivery room. It also demonstrates that brain injury can be seen within 90 minutes of the onset of an injurious stimulus, and the severity of brain injury can be reduced by protective resuscitation strategies in the delivery room.In summary, this study has shown that resuscitation increases brain inflammation and oxidative stress, but an initial high V