Difficulty with turning is a major contributor to mobility disability and falls in people with movement disorders, such as Parkinson's disease (PD). Turning often results in freezing and/or falling in patients with PD. However, asking a patient to execute a turn in the clinic often does not reveal their impairments. Continuous monitoring of turning with wearable sensors during spontaneous daily activities may help clinicians and patients determine who is at risk of falls and could benefit from preventative interventions. In this study, we show that continuous monitoring of natural turning with wearable sensors during daily activities inside and outside the home is feasible for people with PD and elderly people. We developed an algorithm to detect and characterize turns during gait, using wearable inertial sensors. First, we validate the turning algorithm in the laboratory against a Motion Analysis system and against a video analysis of 21 PD patients and 19 control (CT) subjects wearing an inertial sensor on the pelvis. Compared to Motion Analysis and video, the algorithm maintained a sensitivity of 0.90 and 0.76 and a specificity of 0.75 and 0.65, respectively. Second, we apply the turning algorithm to data collected in the home from 12 PD and 18 CT subjects. The algorithm successfully detects turn characteristics, and the results show that, compared to controls, PD subjects tend to take shorter turns with smaller turn angles and more steps. Furthermore, PD subjects show more variability in all turn metrics throughout the day and the week. Difficulty turning during gait is a major contributor to mobility disability, falls and reduced quality of life in older people and people with movement disorders. Studies have found that measurements of turning discriminate elderly fallers from non-fallers . Falls dCurrent clinical measures to assess mobility in PD and other movement disorders are coarse and insensitive. The most common objective method of tracking movement is based on optical Motion Analysis systems. However, these systems are costly, time consuming and can only be used in large laboratories. The need to overcome these limitations has led to an upsurge in research on the use of wearable inertial sensors for the study of human movement . SensorsGait research has focused primarily on straight-ahead walking ,23, and The assessment of gait and balance in the clinic does not adequately reflect mobility function during daily life, and little is known about turning characteristics in the home or community settings. Neurologists currently rely upon patient interviews and unreliable diaries for medical decision-making regarding fluctuations in motor function. As PD advances, patients experience frequent fluctuations in their motor function. Movement disorder specialists spend much of their effort on reducing these fluctuations by adjusting medication schedules and dosages, changing medications and referring to deep brain stimulation surgery. A brief medical examination of PD patients misses these diurnal fluctuations.et al. used activity monitors  to quantify changes in ambulatory activity following deep brain stimulation in advanced PD over a seven-day period. They found a significant increase in the length and variability of walking bouts, but the total number of steps per day did not change [Clinicians and patients would benefit from a system they can easily use to measure daily mobility and assess its fluctuations throughout the day, evaluate their risk of falling and measure the effects of treatment and exercise. However, no current system actually characterizes the quality of gait or turning or mobility fluctuations across days and weeks, because of the lack of sophisticated analysis and adequate technology. A few earlier studies to measure movement for long periods of time utilized activity monitors (Actigraphs) ,31. Theyt change . Human mIn this study, we use wearable inertial sensors to detect and analyze prescribed and spontaneous turns during gait in the laboratory and home. In addition to turning onset, the turn detection algorithm estimates other turn metrics, including duration, peak and mean velocity, number of steps to complete a turn and body jerk during a turn. We demonstrate the validity of our inertial algorithm in both the laboratory and home environment. In the laboratory, the sensitivity and specificity of the inertial algorithm is assessed using a Motion Analysis system and video data from a waist-mounted video camera aimed at the feet. We also evaluate the performance of the inertial algorithm during seven days of continuous data collected in subjects' homes. To the best of our knowledge, our study is the first to characterize spontaneous walking and turning in the home for an extended period of one week.2.In order to develop and validate the accuracy and reliability of the turn detection algorithm, we collected two sets of data. The first set was collected in the Balance Disorders Laboratory at the Oregon Health and Science University (OHSU). A second set of continuous monitoring data was collected in subjects' homes throughout a period of seven days. The following section describes the subjects, data collection protocol, and the algorithm for detecting turns and corresponding metrics.2.1.We examined 21 PD subjects  version III 24.5 ± 7.5) and 19 control subjects (67 ± 9 years) wearing an Opal inertial sensor  on the lumbar spine, as shown in 2.2.q in quaternion form and can be used directly to transform body frame sensor measurements into the inertial frame. We transform the angular velocity measurements from the body frame, bω, to the inertial frame, iω, using the following quaternion multiplication:iω to extract the z component, Angular rotational rate of the pelvis, measured by the gyroscope about the vertical axis, is an ideal signal to detect turns. The direction of gravity, measured by the accelerometer during a stationary period, can be used to project the gyroscope measurements on to the vertical axis throughout the trial, as described in . In our It is difficult for humans to make more than a very slight turn with a duration <0.5 s or to complete an extremely slow turn with a duration >10 s during gait. Therefore, only turns with durations between 0.5 and 10 s and turn angles over 45° were considered. We combine any turns in the same direction separated by a brief pause <50 ms. Relative turn angles are obtained by integrating Algorithm 1 Turn detection algorithm. 1:iωRotate angular velocity to Earth frame,  2:ωziExtract vertical component,  3:ωzi with cf = 1.5 HzLow pass filter  4:|ωzi|≥15°/sFind maxima of absolute value,  5:for each maximum do 6: find 5°/ s crossings preceding and following maximum 7: turn duration = final zero crossing - initial zero crossing 8:end for 9:for each turn do 10:if (intra-turn duration < 0.05 s) and (previous turn is same direction) then  11:  combine with previous turn 12:end if  13:end for 14:for each remaining turn do 15:if (turn duration > 10 s) or (turn duration < 0.5 s) then  16:  eliminate turn 17:end if  18:zθ = Σzω × sT turn angle  19:if turn angle zθ < 45° then  20:  eliminate turn 21:end if  22:end for2.3.We enrolled 12 subjects with PD  and 18 older control subjects (86 ± 7.5 years) in the home for seven days. On the morning of the first day, a study coordinator met to wear them and charge them at the end of each day. Three Opal sensors were worn: one on the pelvis at the lumbar vertebral level and one on each foot. The Opal's on-board data storage can hold 720 h worth of data. Opal sensors have sufficient battery life to continuously record data over 16 h throughout the day. Participants wore the Opal sensors all day for the rest of the seven days, recharging them each night. We expanded the algorithm to capture walking and turning events during spontaneous activities using the data collected in the home. The algorithm identifies periods of walking activities during normal daily activities in the community and calculates the hourly frequency of turning, the duration of each turn, the number of steps needed to complete a turn, the peak and average rotational turning rate and jerk, as well as the variability of these measures throughout the day and week.tω, of the lumbar sensor:tω exceeded a threshold of 15 °/ s for 10 s or longer. Consecutive bouts that were less than 10 s apart were merged together and considered one bout.Periods of walking (bouts) were detected from the total rotational rate, The number and duration of each step during detected walking bouts and turns were calculated using the rotational rate data collected from the Opal sensors attached to the subject's feet. For each step, the midpoint of the foot swinging motion (midswing) was first detected by the peak of the pitch angular velocity. Initial and terminal contact of the foot with the ground, marked by the zero-crossing of the pitch angular rate around the midswing, were used to detect each step and its duration.σ, to the mean, μ.We us the coefficient of variation (CV) to analyze the variability of the turn metrics throughout the day and week among the PD and control subjects. The CV summarizes the amount of variation as the ratio of the standard deviation, 3.First, we present the results related to the validation of the inertial sensor algorithms based on optical and video data collected in the laboratory. Then, we compare turning and characteristics between the subjects with and without PD obtained from the continuous inertial data collected in the home.3.1.The video data was reviewed by two independent raters who annotated the beginning and end time of each turn based on the rotation of the feet over the ground. The time indices of turns detected by the two video raters were upsampled from 30 to 128 Hz to match the sampling rate of the inertial data. Similarly, all turn metrics from Motion Analysis were resampled at 128 Hz. Rather than compare the inertial algorithm to video and Motion Analysis based on turn-by-turn detection accuracy, we validate the performance and agreement of the systems based on sample-by-sample accuracy. Although this might degrade the performance results, the timing accuracy of turn detection is important for characterizing turn duration. Therefore, a true positive turn sample was declared when detected by the inertial algorithm and the other gold-standard system. A false negative was declared when a turn sample was detected by the gold-standard system, but not detached by the inertial algorithm.A true negative was declared when no turn sample was detected by either the inertial algorithm or the gold-standard. A false positive was declared when a turn sample was detached by the inertial algorithm, but not by the gold-standard. The specificity of the inertial algorithm is 0.75, 0.69 and 0.60 with Motion Analysis and the two video raters.Motion Analysis has a better specificity than the inertial algorithm compared to the video raters; see Despite the good agreement between the inertial algorithm and the other gold-standard systems, some disagreement could be attributed to the different definition of a turn used with each system. For example, the video raters were instructed to annotate the time of the heel strike prior to and following the end of each turn. The heel strikes were chosen because the onset and end of each turn starting at the subject's waist is often ambiguous from the video recording. This, however, differs from the way that humans tend to turn by first rotating the pelvis around the vertical axis before turning their feet. This is consistent with the turn definition used for the inertial algorithm, which is based on the angular velocity of the lumbar. The optical marker-based algorithm is qualitatively similar, in that it first estimates a continuously varying yaw angle and then detects turns based on the turn angle. This might explain the better agreement between the Motion Analysis and inertial detected turns.The inertial sensor-based algorithm can be used to detect turns during walking at least as well as the Motion Analysis system. The number of detected turns for the inertial algorithm averaged 10.9 ± 1.6 across all subjects and trials. Similarly, the algorithm based on optical markers detected an average of 10.4 ± 3.4 turns. Subjects were inconsistent with one particular turn, when they were asked to pick up a light basket and turn. Some subjects avoided the turn altogether by picking up the basket without any turning at the waist. In this close pattern with the rapid series of turns, some subjects blended two turns together as they transitioned fluidly from one to the next rather than following the line precisely. Both of these factors contribute to variability in the number of detected turns, especially in the fast trials.Analysis of the pelvic rotation shows that PD subjects had a slower velocity compared to controls while turning at their self-determined slow, preferred and faster gait speeds. Even when matched for the same gait speed, patients with PD show impaired turning because of the significant challenge to their dynamic balance. However, turn duration was not significantly different between PD and control subjects.It has been shown that PD patients appear to walk better when they are examined in an outpatient clinic or in a research laboratory than caregivers report about their daily lives . This is3.2.Using the inertial algorithm validated with the laboratory data, we analyze the continuous data collected in the home. We expanded the algorithm to capture walking and turning events during spontaneous activities. The algorithm identifies periods of walking activities inside and outside the home and calculates the number of turns and corresponding turn metrics during each hour of the day. p-value for a t-test at a 5% level of significance to determine if PD and CT bout and turn metrics significantly differ from each other. Active rate is defined as the percentage of time when the subject is walking or turning compared to the full time of monitoring during the day. Bout duration and active rate are statistically significantly different between PD and control subjects. Active rate and number of bouts per hour show the largest variation among all subjects, with a clear trend of subjects being more active during the first three hours of the day. Activity rate declines for an average of three hours, then increases again during the 7–8th h. PD and control subjects generally complied with wearing the inertial sensors as instructed for the entire week, for an average of ten hours per day.p-value for the t-test.Subjects exhibit a great variability of the number of turns throughout the day, with an average of 65.2 and 67.3 turns/h for control and PD subjects, respectively. PD subjects tend to complete shorter turns with smaller turn angles, compared to the control group. Data also shows that PD subjects complete a turn with a larger number of steps. Consistent with the results obtained from the laboratory data, PD subjects show more variability than controls in all turn metrics throughout the day and the week.Compared to turn duration during the prescribed tasks in the laboratory (1.4 s), both control and PD subjects completed a turn with a longer average duration of 2.2 s and 2.0 s, respectively. Similarly, analysis of the pelvic rotational rate during turning shows that subjects significantly decreased their rotational rate in the home, compared to in the laboratory. In a previous study, it was demonstrated that even an identical instrumented test of mobility conducted in the home environment resulted in slower turns and walking than when conducted in the laboratory .4.In this study, we developed a wearable inertial system for continuous monitoring to detect and distinguish turning characteristics in healthy subjects and individuals with Parkinson's disease (PD). Assessment of the system in the laboratory showed that the inertial system can be used to detect turns with a comparable accuracy to that of the gold-standard Motion Analysis. We demonstrated that we could use the inertial system to measure locomotor activities and to characterize turns in the home throughout the day and week, using three wearable sensors. The algorithm successfully quantifies turns, and results show significant differences between turn characteristics of PD and control subjects. Compared to controls, PD subjects tend to take shorter turns with smaller turn angles and more steps. Furthermore, PD subjects show more variability in all turn metrics throughout the day and the week. Precise measures of turning mobility will improve intervention for balance disorders and fall prevention in the elderly and in patients with neurological diseases.


Exon recognition occurs through base-pairing interactions between two guide sequences on the ribozyme domain dI known as EBS1 and EBS2 and two stretches of sequence known as IBS1 and IBS2 on the 5' exon, whereas the 3' exon is recognized through interaction with the sequence immediately upstream from EBS1 [(δ-δ' interaction (subgroup IIA)] or with a nucleotide [(EBS3-IBS3 interaction (subgroup IIB and IIC))] located in the coordination-loop of dI. The δ nucleotide is involved in base pairing with another intron residue (δ') in subgroup IIB introns and this interaction facilitates base pairing between the 5' exon and the intron.Group II intron splicing proceeds through two sequential transesterification reactions in which the 5' and 3'-exons are joined together and the lariat intron is released. The intron-encoded protein (IEP) assists the splicing of the intron in vivo. We found that the EBS1-IBS1 interaction was required and sufficient for RmInt1 excision. In addition, we provide evidence for the occurrence of canonical δ-δ' pairing and its importance for the intron excision in vivo.In this study, we investigated nucleotide requirements in the distal 5'- and 3' exon regions, EBS-IBS interactions and δ-δ' pairing for excision of the group IIB intron RmInt1 in vivo of the RmInt1 intron is a favored process, with very few constraints for sequence recognition in both the 5' and 3'-exons. Our results contribute to understand how group II introns spread in nature, and might facilitate the use of RmInt1 in gene targeting.The excision  By contrast, most IIB and IIC introns display no statistical evidence of pairing of this δ position with the 3' exon. These group II ribozymes recognize the first nucleotide in the 3' exon, referred to as IBS3, rather than δ', by canonical base-pairing with an intron nucleotide, denoted EBS3 rather than δ, located in the "coordination loop" of dI [Group II introns are ribozymes and mobile retroelements consisting of a highly structured RNA organized into six distinct domains (dI to dVI). They have a multifunctional intron-encoded protein (IEP) ORF in dIV, encoding a reverse transcriptase-maturase that is required tructure . Group Itructure . In the tructure . In the p" of dI . The δ pSinorhizobium meliloti, a group IIB intron, is a mobile intron that self-splices in vitro in the absence of the IEP [in vivo and observed a low efficiency; only 0.07% of exons were joined [The RmInt1 intron of  the IEP ,6. Recene joined , but is in vivo. We found that exon recognition by the intron is subject to few constraints, the most critical of which is the EBS1-IBS1 interaction and the δ-δ' pairing. Our findings might have practical implications for the development of RmInt1 as a highly efficient gene targeting vector and support the extraordinary ability of RmInt1 for its dispersal in nature.In this study, we analyzed the nucleotide requirements in the distal 5' and 3' exon regions, EBS-IBS interactions and δ-δ' pairing for excision of the IIB intron RmInt1 in vitro trans-splicing depends on exon-intron binding sequences, the EBS1-IBS1 and EBS2-IBS2 pairings, whereas normal cis-splicing requires only the EBS1-IBS1 pairings [in vivo, by introducing single and multiple nucleotide substitutions into the IBS1 and IBS2 elements , primer extension or cRT-PCR (data not shown). Moreover, we showed previously that the lariat form accumulates in the splicing process and that this product is free of lariat-3' exon reaction intermediates Likewise, the RmInt1 splicing process was inefficient, shown by a low detection of ligated exons, more likely to be susceptible to cell degradation [The excision reaction was characterized by primer extension; with a primer P complementary to a sequence located 80-97 nt from the 5' end of the intron. In these assays, the excised intron RNA was detected as a 97 nt extension product Figure . In addiThe IBS2 stretch encompasses 5 nt (positions -13 to -9) in the 5' exon of RmInt1 that form Watson-Crick pairs with the EBS2 intron RNA sequence . The extension of the linker nucleotide between IBS2 and IBS1 by the addition of one nucleotide  led to a moderate decrease in excision efficiency (~87% of wild-type levels). Thus, in contrast to the critical positions of the EBS-IBS interactions in the DNA Target [By contrast, more heterogeneous results were obtained for nucleotide substitutions within the IBS1 element. The IBS1 stretch encompasses 7 nt, positions -7 to -1 with respect for the intron insertion site, that base-pair with the EBS1 sequence in the intron RNA  that rendered excision undetectable, and simultaneous changes of the -4, -3 and -2 nucleotides , which strongly reduced intron excision (~13% of wild-type levels). These results identified the IBS1 element in the 5' exon as the most critical for the intron excision reaction. A combined mutant (M2.3/-5G) lacking the canonical IBS2 sequence plus the substitution of the U-5 nucleotides by a G residue in IBS1 rendered intron excision barely detectable (lane 9), which reveals the additional contribution of the IBS2-EBS2 pairing to the efficiency of the intron excision reaction.(iv)[in vivo.In class IIB introns, position +1 at the 3' exon, known as IBS3 (C+1 residue in the RmInt1 3' exon), base-pairs with the EBS3 element in the intron RNA (position 329G), which is located in the "coordination loop" at dI and displaced only one nucleotide from the 3' branch of helix ID(iv). Interes(iv), resultein vivo is a highly favored process, with IBS1-EBS1 pairing the most critical step. As the intron lariat released from the 3'exon remains associated with the IEP, forming a ribonucleoprotein particle complex (RNP) that mediates intron mobility, we investigated whether the observed decrease in excision was correlated with reduced intron mobility. We carried out a homing assay with intron donor and recipient plasmids, in which intron mobility was assessed by DNA hybridization. Once RmInt1 was excised in vivo from the exon-mutant donor constructs, the RNPs produced were wild-type. Following introduction of either the wild-type intron (pKG2.5) or mutant constructs into a S. meliloti RMO17 strain harboring the target-recipient plasmid pJB0.6as, a broad correspondence was observed between the excision level detected by primer extension and homing efficiency  and reverse (intron retrohoming) splicing reactions. Our results contribute to understand how group II introns spread in nature, and might facilitate the use of RmInt1 in gene targeting.We show here that the intron-exon interaction IBS1-EBS1 and δ-δ' interaction in the domain dI of the rybozime, play an important role in the RmInt1 excision in vivo is subject to few constraints, the most critical of which is the EBS1-IBS1 interaction. Thus, the spread of group II introns into bacterial genomes from ectopic sites to homing sites may occur efficiently, increasing their ability to spread in nature.Group II introns can move site-specifically to homologous intronless genes, in a process known as retrohoming or, at much lower frequencies, to novel ectopic sites, in a process known as retrotransposition. It is believed that the latter process has led to the wide dispersal of mobile group II introns among bacterial species and also may have been used to invade eukaryotic genomes, where mobile group II intron are thought to have evolved into spliceosomal introns and non-long-terminal-repeat retrotransposons . Intron Gene targeting technology based on group II introns requires the modification of IBS2, IBS1 and IBS3, to render these sites complementary to the retargeted EBS2, EBS1 and EBS3 sequences for efficient RNA splicing ,14. We hS. meliloti RMO17, lacking the RmInt1 intron, was grown at 28°C on TY or defined minimal medium [E. coli DH5α was used for cloning and the maintenance of recombinant plasmids; it was cultured at 37°C in Luria-Bertani medium [S. meliloti and 50 μg/ml for E. coli; ampicillin, 200 μg/ml and tetracycline, 10 μg/ml.Rhizobial strain l medium . E. coliAltered Sites II in vitro Mutagenesis System , using pAL2.5 as a template. This plasmid was generated by inserting a 2.5 kb SphI fragment containing the entire RmInt1 sequence and flanking exons (-174/+466) from pGEmex2.5 [BamHI/SpeI fragment and inserted into pKG0 (pKG2.5 and mutant derivatives), which constitutively expresses RmInt1 and its derivatives under control of the promoter of the Km resistance gene [RmInt1 donor derivatives containing point or accumulative substitutions in the flanking regions of intron insertion sites and ribozyme domain dI, including the δ and δ' sequences, were generated with the GEmex2.5  into thence gene .600 nm of 0.6 to 0.9).RNPs were isolated essentially as previously described ,20. RNPsThese experiments were carried out on RNP preparations, since the lariat intron seems to be less susceptible to degradation than ligated exons, due to its association with the IEP forming ribonucleoprotein particles (RNPs). Primer extension reactions were essentially carried out as previously described . SamplesS.meliloti specific primers Sino 452 (5'-CGTTGTTCGGAATTACTGG) and Sino 952 (5'-TGTCTCCGATCCAGC). Primer specificity was determined by melting-curve analysis and gel electrophoresis , with the final volume made up to 23 μl with DEPC water. The PCR cycling conditions were as follows: hot start, with heating at 94°C for 3 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. Controls without a template were also included. Two independent RNA preparations from each strain were tested. Relative quantification was carried out for splicing, based on the 2-ΔΔCT method [-ΔΔCT where ΔΔCT = ΔCT (DV* or +1A) - ΔCT (WT). ΔCT (DV* or +1A) = CT (DV or +1A mRNA amplified by 5-6 primers) - CT (DV or +1A mRNA amplified by S. meliloti specific 16S primers Sino 452 and Sino952). ΔCT (WT) = CT (WT mRNA amplified by 5-6 primers) - CT (WT mRNA amplified by S. meliloti specific 16S rRNA primers Sino 452 and Sino952). The result is expressed as a percentage related to WT construction. The error is calculated applying the formula: 2-ΔΔCT · ln2 · ΣSE.For the quantification of ligated RmInt1 flanking exons, a 283 bp fragment was amplified with 3'RTSp (5'-ATTCAACGGGAAACGTCTTG) and +9 (5'-AGCGGCGCCATGTTCGTCTT) primers. To normalize the loading amount of the starting material, a 500 bp fragment of the 16S ribosomal subunit RNA was amplified with T method . BrieflyS. meliloti RmInt1-less strain RMO17. We studied homing between compatible intron donor (pKG2.5 and mutant derivatives) and target-site recipient plasmids (pJB0.6as (-174/+466) or pJB-20/+5) as previously described [E. coli DH5α to rhizobial strains by tri-parental mating, using pRK2013 as a helper plasmid [Rm2011-2 element .Click here for file


PaSSU intron in the rRNA small subunit gene of Phialophora americana isolate Wang 1046 is capable of in vitro splicing using a standard group I intron pathway, thus qualifying it as a functional ribozyme.Group I introns  are common in the nuclear ribosomal RNA genes of fungi. While most range in length from more than 200 to nearly 1800 nucleotides (nt) in length, several small putative (or degenerate) group I introns have been described that are between 56 and 81 nt. Although small, previously we demonstrated that the Phialophora isolates, a fungal genus that ranges from saprobic to pathogenic on plants and animals. All contain putative pairing regions P1, P7, and P10, as well as a pairing region formed between the middle of the intron and part of the 3' exon. The other pairing regions common in the core of standard group I introns are absent. However, parts of the 3' exon may aid in the stabilization of these small introns. Although the eight putative group I introns were from at least three species of Phialophora, phylogenetic analysis indicated that the eight are monophyletic. They are also monophyletic with the small introns of two lichen-forming fungi, Porpidia crustulata and Arthonia lapidicola.Here, we describe eight short putative group I introns, ranging in length from 63 to 75 nt, in the rRNA small subunit genes of Phialophora have common features that may represent group I introns at their minima. They appear to have a single origin as indicated by their monophyly in phylogenetic analyses.The small putative group I introns in  A few hof fungi -5. This There are no known advantageous effects of group I introns to host organisms . They haCenococcum geophilum [Phialophora americana Wang 1046 [Phialophora species (anamorphic dematiaceous fungi) in order to study the structure and evolution of these introns after their establishment in the rRNA genes. We show that the small introns differ in sequence, but retain similar structural features.In previous studies we reported the presence of self-splicing group I introns in the rRNA small subunit (SSU) genes of eophilum , as wellang 1046 ,14. ThisPhialophora isolates  were obtained from Dr. CJK Wang, State University of New York, College of Environmental Science and Forestry, Syracuse, New York. They had been isolated from plant and human sources (Table DNA from mycelia was extracted using a CTAB (cetyltrimethylammonium bromide) preparation method -19. The http://www.ebi.ac.uk/Tools/clustalw2/index.html. The alignments were examined, and manual adjustments were performed. Phylogenetic analyses were performed with PAUP  using MThe intron sequences were subjected to secondary structure analysis using Mfold . BecausPaSSU) from P. americana Wang 1046 was demonstrated to splice in vivo [in vitro, and phylogenetically falls within the IC1 subgroup [PaSSU, PvSSU, or PeSSU) located in the SSU rRNA gene at position 1516 (relative to E. coli) that can form similar secondary structures  and Arthonia lapidicola (63 nt intron) also fall within the same clade with all of the Phialophora small putative introns, suggesting that this intron may be widespread among certain groups of fungi and might have been generated by a single mutational event.Previously, the small intron  varied to a greater extent in sequence, which is typical of group I ribozymes. All contained a G·U pair at the 5' splice site within the P1 pairing region, which consisted of part of the 5' exon, and the first 20-30 nucleotides of the intron . Each of the other small introns also had a U that is similar to the one in isolate Wang 1046 at the 3' end of the intron , located in the rRNA SSU gene of P. americana  as the PaSSU group I intron in isolate Wang 1046  of the exon was included, splicing occurred in vitro [Secondary structure conservation is evident in these small introns. Pairing regions P1, P7, and P10, as well as another pairing region between the middle of the introns and the 3' exons (termed PM) all were maintained in each of the introns Figure . Furtherin vitro . TherefoPaSSU intron in isolate Wang 1046, and therefore they may be pseudogenes. Vertical transmissions, mutations and losses are common in the evolution of group I introns in fungi [Phialophora, all appear to have originated from a single unequal crossover event that occurred in a common ancestor [Ribosomal RNA genes are arranged in tandem arrays of 60-200 copies in fungi. Recombination and gene conversion act to homogenize the individual repeats. However, mutations can lead to a heterogeneous combination of sequences. In one case, an intron-less allele coexisted with intron-containing versions. Sequence results demonstrated that there often were one or more mutant versions of the introns in each of the isolates (not shown). While it is unknown whether the mutant introns are functional, some differed from the functional in fungi . The smaancestor . This isPhialophora species. Certainly, a protein encoded from these small putative introns is implausible, and in general, open reading frames are absent from introns in the 1506-1521 location of the rRNA SSU genes in fungi. However, the different location for the Porpidia crustulata small degenerate intron (at position 516) suggests that it might have inserted into the rRNA locus through horizontal transfer or transposition.Many studies have shown evidence for stable maintenance of group I introns over long periods of time ,26,27, wPaSSU (Wang 1046) intron with other introns inserted at the same site indicate that they are orthologs, although their sequences and lengths vary [Comparison of the SSU Wang 046 introths vary ,13. GrouSSU Wang 46 intronths vary  and thusPhialophora species are monophyletic, presumably having originated from a single mutational event. All are similar to the functional putative group I intron from P. americana isolate Wang 1046, in that each possess a P1, P7 and P10. Although their primary sequences have diverged, they all have a G·U pair at the 5' splice site, a G-C pair in the P7 guanosine site, an unpaired A on the 5' side of P7, and a U as the final nucleotide of the intron. These introns may represent the minimum size for group I ribozymes.The set of small putative group I introns from the SSU rRNA genes of The authors declare that they have no competing interests.LH and SR designed the study, produced the secondary structure models, and contributed equally in writing the manuscript. LH performed all of the laboratory work and initial sequence analysis. All authors have read and approved the final manuscript.


Patient registries are often a helpful first step in estimating the impact and understanding the etiology of rare diseases - both requisites for the development of new diagnostics and therapeutics. The value and utility of patient registries rely on the use of both well-constructed structured research questions and relevant answer sets accompanying them. There are currently no clear standards or specifications for developing registry questions, and there are no banks of existing questions to support registry developers.This paper introduces the [Rare Disease] PRISM (Patient Registry Item Specifications and Metadata for Rare Disease) project, a library of standardized questions covering a broad spectrum of rare diseases that can be used to support the development of new registries, including Internet-based registries.A convenience sample of questions was identified from well-established (>5 years) natural history studies in various diseases and from several existing registries. Face validity of the questions was determined by review by many experts (both terminology experts at the College of American Pathologists (CAP) and research and informatics experts at the University of South Florida (USF)) for commonality, clarity, and organization. Questions were re-worded slightly, as needed, to make the full semantics of the question clear and to make the questions generalizable to multiple diseases where possible. Questions were indexed with metadata (structured and descriptive information) using a standard metadata framework to record such information as context, format, question asker and responder, and data standards information.At present, PRISM contains over 2,200 questions, with content of PRISM relevant to virtually all rare diseases. While the inclusion of disease-specific questions for thousands of rare disease organizations seeking to develop registries would present a challenge for traditional standards development organizations, the PRISM library could serve as a platform to liaison between rare disease communities and existing standardized controlled terminologies, item banks, and coding systems.If widely used, PRISM will enable the re-use of questions across registries, reduce variation in registry data collection, and facilitate a bottom-up standardization of patient registries. Although it was initially developed to fulfill an urgent need in the rare disease community for shared resources, the PRISM library of patient-directed registry questions can be a valuable resource for registries in any disease – whether common or rare.N/A If widely used, PRISM will enable the re-use of questions across registries, hence reducing the variation in registry data collection and facilitating a bottom-up standardization of patient registries. Although it was developed to support the rare disease community’s urgent need for shared resources, the PRISM library of patient-directed registry questions can be a valuable resource to registries for all diseases.The value and utility of patient registries are largely contingent upon the use of both high-quality research questions and structured and relevant answer sets accompanying them. The interoperability of registries or registry data—including the feeding of registry data into more rigorous clinical studies and regulatory submissions for new agents—depends upon the use of data standards; yet, there is currently no clear specification for developing registry questions nor are there banks of existing questions to support registry developers. The diverse nature of registries, sponsors, and disease-specific data requirements complicate efforts at standardization in registry applications. This paper introduces the Often patient registries are a helpful first step in estimating the impact and understanding the etiology of rare diseases—both requisites for the development of new diagnostics and therapeutics. Because of the small numbers of patients affected by rare diseases, these registries present unique challenges related to registry design, enrollment of patients, and data collection -5, as weThere is tremendous variability in the type of data and specific questions that patient registries collect, due in part to the lack of registry-specific data standards and also to the heterogeneity of registries’ purposes and sponsors. Patient registries can be designed for many purposes, including public health surveillance, epidemiologic and longitudinal research, patient education, research recruitment, and population monitoring for the safety of post-marketed drugs and devices. Patient registries can include data reported by patients, researchers, or clinicians. (A characterization of registry types is summarized in Richesson and Vehik .) SponsoThere is also a clear role for data standards to promote shared efficiencies in registry development and enable opportunities for data sharing. These needs are particularly pronounced for rare diseases, which have sparse resources and significantly fewer—and highly distributed—domain experts and affected patients. An important standards challenge is the fact that there is no central control of patient registries—there is no single funding or regulatory agency that can oversee all the different registry types and implementations. Because registries are developed by many sponsors to address distinct functions, a top-down standards effort would require countless stakeholders and is not feasible. Additionally, there is no central authority to monitor or enforce standards compliance once developed. Given the tremendous need for standards and the scope of data collected across disease-specific registries, and given that there is no incentive or regulatory means to develop standards or enforce compliance, alternatives to complement traditional Standards Development Organizations (SDOs) are needed. Non-traditional strategies for developing and promoting standards can be effective and embraced across various rare diseases if these various research communities perceive them as accessible, useful, helpful, and easy to adopt.bottom-up development of standards. In addition, the utility and scalability of PRISM is based on the notion that the PRISM content is accessible and open to any registry sponsor, regardless of prior standards knowledge or experience. The authors are engaged with various standards and informatics organizations and deliberately designed PRISM to facilitate linkage with other standards and research resources as appropriate. The PRISM project, therefore, provides not only a library of questions but a vehicle for the registry developers and rare disease organizations to interact, learn, and develop consensus requirements, which can in turn be directed to various standards development organizations  grant administered through the National Library of Medicine (NIH), NLM Grant Number 1RC1LM010455-01, and supported by the Office of Rare Diseases Research) was developed to provide a useful resource to promote the efficient development of patient registries and standardized quality data collection by supporting the sharing and re-use of existing registry questions and data standards. The fundamental idea behind PRISM is that if registry developers could access questions used by other rare disease registries, they could consider and likely re-use these questions, thereby reducing the variation in questions/data collection across various patient registries, and leading to a g, CDISC , HL7 8]bottom-upg, CDISC ), researg, CDISC ,12, Pheng, CDISC ), and nag, CDISC ,15.As a demonstration project, PRISM explored foundational issues related to the types of questions included in the bank (relative to other standards and question repositories) and the inclusion of metadata that will facilitate their search and retrieval. We describe our methodological approach to developing the PRISM library in the Methods section and present the resulting library structure, features, and composition afterward in the Results section.The first questions identified for PRISM included a convenience sample of questions from well-established (>5 years) natural history studies in various diseases  that might not be ideally constructed or might actually conflict with another value set. Regardless, the liberal acceptance policy of PRISM increases the breadth and volume of questions and ultimately increases the value of PRISM as a central resource for questions (which should be supplemented by advice on selection and use). We hope that others use PRISM as a resource for standards development or build applications that can facilitate the ranking or endorsement of certain PRISM questions over others in specific diseases or contexts.As mentioned in the previous section, the growth of the library brought with it challenges for curation and use. Internally, a process was developed to add new content without duplicating questions and ensure that related questions or variants could be indexed for effective retrieval and comparison. We operated under the assumption that PRISM needed to be useful to provide value. We explored user roles and searching techniques to determine the best method for indexing question and answer sets (QAS). Our indexing scheme (including the use of controlled terminology) is described later. A key strategy for identifying the search and retrieval requirements was employing use cases.use cases  that have informed our indexing approaches. For our indexing strategy, PRISM also developed use cases of retrieval requirements as a guide of needed functionality and detail, and leveraged existing metadata and controlled vocabularies. The use cases describe the development of new registries using PRISM as a resource for the questions. Specifically, the use cases describe the development of a prospective vasculitis and pregnancy registry—including the re-use of PRISM questions and the submission of new questions to PRISM—and the subsequent development of another registry using these same pregnancy-related questions. Finally, the use cases articulate a demonstration and initial proof of the interoperability of data collected from different registries and data sources. These functional use cases ultimately will also support the evaluation of PRISM. The use cases were developed by the authors and not widely vetted in the rare disease community. They can be found on PRISM website [The PRISM team developed narrative  website .A critical and largely unaddressed problem for registries  is the need for patient registry questions and answers to be indexed in such a way that they can be retrieved for re-use, for example, to support rapid development of another related rare disease registry. In essence, indexing is the practice of applying metadata (structured and descriptive information) to items in a database for efficient and accurate retrieval . An impo1. Context 2. Format of questions and location of semantics3. Who is asking the question ?4. Audience or person being asked the question 5. Relevant data standards for specific answer setsMetadata are used to describe information resource-type features of questions, such as terms and attributes, and controlled vocabularies represent the actual content. Both are important for retrieval and, ultimately, interoperability. PRISM uses Dublin Core (DC) as a metadata framework for indexing PRISM QAS, and within DC metadata uses controlled terminology to reflect the semantics for each question. Our approach is described in detail in . TerminoThe use of Dublin Core metadata to annotate various QAS in PRISM offers a way to employ the most appropriate controlled vocabulary(s) for the content, while preserving retrievability. In addition to selected Dublin Core metadata elements and controlled terminologies, other decisions were made to ensure that these elements and vocabularies were used appropriately and consistently. Specifically, assuring each QAS is usable, reproducible, and understandable on its own merit. For example, a form that addresses gynecological issues may include a QAS addressing menstrual symptoms, such as “Do you experience cramps?” From the context of the form, this can refer only to menstrual cramps and may be coded with or without the “menstrual semantics”. However, when a later user searches the registry for library questions about abdominal, leg or other body site cramps, this question may be inappropriately selected. The QAS metadata (including the embedded SNOMED CT codes and the narrative definition of the question that PRISM includes) can be used to easily disambiguate this term.Under the leadership of terminology experts from the College of American Pathologists, guidelines for using SNOMED CT were developed related to post-coordination, selection of hierarchies, and level of specificity. Guidelines for the use of SCT within the PRISM Library data model were developed collaboratively by the PRISM team and included the following decisions:1. Assigning codes for entire question and answer set groups vs. discrete codes for questions and answers2. An approach to take the  closest available SNOMED CT concept rather than creating a new one3. Consequently, we considered but rejected the idea to create a SNOMED extension (“Ref Set”) mechanism4. Versioning and change protocols were developed between USF and CAP partnersand retrieve relevant registry questions from PRISM before they re-create their own variation. As new content is introduced to the PRISM outreach team at USF, they first determine if the content is in scope of PRISM  For PRISM to remain a useful resource to rare disease registry developers, and for PRISM to support the goal to reduce question variation across rare disease registries, the library should not contain obvious duplicates and the library indexing model should be sufficient for users to search One of the biggest challenges of the PRISM project has been to keep the scope reasonable and practical. Since the targeted audience for PRISM is researchers and registry developers (including PAGs with non-research backgrounds), we took a minimalist approach to coding with the goal of easy retrieval. It is our expectation that easy retrieval will drive increased usage, which will cultivate de facto standards, and those standards will ultimately support interoperability see .We recognize that interoperability between registry questions and other data  would require more sophisticated coding with SNOMED CT and other data standards. Given the short duration of this project and the desire for maximum retrievability, we determined that this level of coding would be out of the scope for PRISM at this time.It is not clear at this point how large a corpus of sharable items that there is among disparate and different rare diseases. Likewise, it is still unclear whether items for rare diseases are likely to be similar to items for more common diseases, and if so, whether there would be value in finding a way to include and reuse those items via PRISM as well. The future use and evaluation of PRISM content by multiple disease representatives will yield information on the reusability of the questions in PRISM within and across rare and common diseases, as well as provide practical examples of cross-disease standards and determination of standards gaps. To understand the reusability and generalizability of PRISM questions, additional work needs to explore the validity and reproducibility of the categorization and indexing of questions.PRISM fills a void for the rare diseases research and registry development communities. The PRISM library resource can support standardized data collection in patient registries by reducing unnecessary variation. PRISM is free and available to search through the project website . AuthorsAuthors are hopeful for future funding that will allow PRISM content to grow to meet the needs of the thousands of rare diseases registry applications and to allow computer mediated methods for adding and presenting content. The notion of using a distributed community of registry developers to curate this resource by commenting on and ranking items—as with the demonstration of caDSR content that is described in —is very The PRISM developers, with the cooperation and support of the National Organization for Rare Disorders (NORD), are working to make the PRISM resource available and useful to registry developers representing all rare diseases and all countries. Currently, several rare disease patient advocacy organizations are participating in focus groups and expert interviews to inform the development of best interfaces and retrieval strategies to ensure that PRISM is a useful and accessible community-driven resource. In addition, we are developing international collaborations to explore the translation of items to support global rare disease research. Our overarching goal is that—given the sheer number of rare diseases, the variety of registry designs, and the number of languages that might need to be addressed—the PRISM leadership seizes and implements standards opportunities without burdening resource-strapped rare disease communities.The lack of a clear set of standards and specifications for data collection using patient registries represents a significant data standards gap in an explosively growing application area—important to both drug development and patient-directed health communities. Standardization of patient registries can enable the interoperability of health and research data, as registries should be able to receive data from health care system or transmit data into various clinical research or pharmacovigilance applications. PRISM can be used to facilitate interoperability of existing and newly developed registries and to ensure that moving forward, registries use standard sets of questions. Without the use of such a resource, the proliferation of patient registries and variation of data collection questions will be inevitable. This central resource, the PRISM library, will support a bottom-up and incremental standards promulgation. By using a standard set of metadata elements and SNOMED CT to facilitate the retrieval and re-use of existing questions and standards, PRISM will reduce variation in the rare diseases registry community and assist registry implementers to produce high quality registries much more efficiently than ever before. Once variation in patient registries is reduced , then issues related to harmonizing, mapping, and relating to the different standards communities for health care  and research  can be addressed in an efficient manner. In this approach, the standardization of patient registry questions can serve to improve efficiencies, collaboration, and resource sharing across the entire drug development process.Information regarding the development of PRISM and access can be found on the PRISM website  .


Our somatosensory system deals with not only spatial but also temporal imprecision, resulting in characteristic spatiotemporal illusions. Repeated rapid stimulation at the wrist, then near the elbow, can create the illusion of touch at intervening locations along the arm . This is known as the “cutaneous rabbit effect” (CRE). Previous studies have suggested that the CRE involves not only an intrinsic somatotopic representation but also the representation of an extended body schema that includes causality or animacy perception upon the skin. On the other hand, unlike other multi-modal causality couplings, it is possible that the CRE is not affected by concurrent auditory temporal information. The present study examined the effect of a simple visual flash on the CRE, which has both temporal and spatial information. Here, stronger cross-modal causality or correspondence could be provided. We presented three successive tactile stimuli on the inside of a participant’s left arm. Stimuli were presented on the wrist, elbow, and midway between the two. Results from our five experimental manipulations suggest that a one-shot flash enhances or attenuates the CRE depending on its congruency with cutaneous rabbit saltation. Our results reflect that (1) our brain interprets successive stimuli on the skin as motion in terms of time and space  and that (2) the concurrent signals from other modalities provide clues for creating unified representations of this external motion  as to the extent that “spatiotemporal” synchronicity among modalities is provided. Our daily lives are rich with information from the physical world. While some limits are imposed by sensorineural imprecision , we might assume that unimodal causality perception (like the tau and kappa effects or the CRE) could be modulated under multi-modal presentation; however, this is controversial . The present study has suggested that unimodal causality perception would be enhanced but might not be attenuated by cross-modal causality. This could indicate that our brain is tuned to detect the movement of an external agent on the skin since an essential, evolutionarily stable feature of brain function is the detection of animate entities for survival  and an amplifier  connected to a PC. The tactile stimuli were presented through vibrators , and visual stimuli were presented using LEDs (3-mm diameter). Two vibrators  and one red LED were combined using Velcro fastenings onto a band device  – blank – signal (t2) – blank – signal (t3). Thus, the three signals for each trial were presented over a 500-ms duration. The CRE is subject to temporal parameters such as stimulus duration and ISI. According to the results of previous studies  at t1 and for the elbow (L3) at t3. At t2, tactile and visual stimuli (though one or the other of these was not present under some conditions) were presented somewhere between L1 and L3, including the midpoint (L2) between the wrist and elbow.Visual and tactile stimuli were controlled by a sound signal (300 Hz sine wave) using MATLAB . The stimulus duration was 100 ms, and we presented three successive signals with a 100-ms ISI . For instance, one sequence was as follows: signal , then the CRE would be enhanced. This manipulation would provide causal, spatiotemporal correspondence between the visual (the flash) and tactile  senses. We expected that L1–L1–L3 tactile stimuli would be felt as L1–L2–L3 to some extent. Furthermore, a flash on L2 at t2  participated in a 3 (patterns of tactile stimuli) × 2 -factor experiment. For the patterns of tactile stimuli, we presented three successive signals through vibrators: L1–L2–L3, L1–L1–L3, and L1-(blank)-L3. In the visual-stimuli-provided conditions, we presented a one-shot visual signal on the LED located on L2 at F = 6.65, p < 0.01. We also observed a simple main effect of flash under the L1–L1–L3 tactile condition, F = 17.12, p < 0.01, a simple main effect of tactile pattern under the flash condition, F = 65.47, p < 0.01, and a simple main effect of tactile pattern in the no-flash condition, F = 69.79, p < 0.01. These results were analyzed further using Ryan’s method of multiple comparisons . Under both flash conditions, each tactile pattern was significantly different from the others (ps < 0.05). Conversely, for RT, a similar two-way ANOVA with multiple comparisons conducted using Ryan’s method revealed only a significant main effect of tactile patterns, F = 7.28, p < 0.01. There were significant differences between L1–L2–L3 (average RT = 478.1 ms) and L1–L1–L3 (573.7 ms) and between L1–L2–L3 and L1-blank-L3  in terms of RT, indicating that regardless of the flash, participants more rapidly reacted to L1–L2–L3 stimuli.The response ratios of L1–L2–L3 tactile sensation and the RTs under each condition were averaged across participants , L1–L3–L3 tactile stimuli with a flash would be felt as L1–L2–L3 to a lesser extent than L1–L1–L3 tactile stimuli with a flash.Since the previous experiment suggested a cross-modal impact of forward displacement in the CRE, the current experiment examined the possibility of backward displacement. We predicted that as far as a visual flash is congruent with the CRE saltation , it should capture tactile location. A flash could also induce backward displacement just as easily as forward displacement. We expected that L1–L3–L3 tactile stimuli would also be felt as L1–L2–L3 to some extent. Furthermore, a flash on L2 at t2  participated in a 3 × 2-factor experiment similar to Experiment 1A. We changed one tactile condition in this experiment. For the patterns of tactile stimuli, we presented three successive signals through vibrators: L1–L2–L3, L1–L1–L3, and L1–L3–L3 (only the last condition was replaced). Along with the same visual stimuli conditions used in Experiment 1A, we formed a sixth condition by presenting a one-shot signal on the LED on L2 at F = 9.69, p < 0.01, a simple main effect of flash under the L1–L1–L3 tactile condition, F = 9.64, p < 0.01, a simple main effect of flash under the L1–L3–L3 tactile condition, F = 19.0, p < 0.01, a simple main effect of tactile condition in trials including a one-shot flash, F = 6.08, p < 0.01, and a simple main effect of tactile condition without a flash, F = 26.0, p < 0.01 ; further, the difference between the L1–L2–L3 and L1–L1–L3 conditions was not significant when flash was presented. For RT, a similar two-way ANOVA with multiple comparisons conducted using Ryan’s method revealed similar results to those in Experiment 1A: there was only a significant main effect of tactile pattern, F = 4.40, p < 0.05. There were significant differences in terms of RT between L1–L2–L3 (average RT = 642.6 ms) and L1–L1–L3 (739.8 ms) and between L1–L2–L3 and L1-blank-L3 . This indicated that regardless of whether a flash was presented, participants most rapidly reacted to L1–L2–L3 stimuli.For the response ratios, a 3 (tactile patterns) × 2 (with vs. without flash) two-way ANOVA revealed a significant interaction between tactile pattern and the presence vs. absence of visual feedback, t2. In this case, the sensation enhancement was almost the same as in the baseline condition (L1–L2–L3 tactile stimuli). It seems that participants made slightly more miss responses (approximately 10%) under the baseline condition in this experiment, while participants in Experiment 1A made hit responses almost perfectly. Since participants felt L1–L2–L3 tactile sensations under all conditions, they seem not to have been sufficiently conservative in their judgments. This might account for why RTs in the current experiment were approximately 150 ms longer than in the previous experiment. Furthermore, the L1–L3–L3 tactile pattern was also felt weakly compared with the L1–L2–L3 pattern. The CRE can occur when the second tactile sensation is subject to forward displacement  participated in a 3 × 2-factor experiment similar to Experiments 1A and B. The tactile stimulus patterns were identical to those presented in Experiment 1B, but we changed the location of the flash in the current experiment: the one-shot signal from the LED was located on L1 and presented at F = 20.37, p < 0.01. Multiple comparisons using Ryan’s method revealed that each tactile pattern was significantly different from every other pattern in terms of response ratio  = 5.97, p < 0.01; further, there were also significant differences between L1–L2–L3 (average RT = 513.6 ms) and L1–L1–L3 (620.9 ms) and between L1–L2–L3 and L1-L3-L3  in terms of RT, indicating that regardless of the presence vs. absence of the flash, the participants reacted most rapidly to the L1–L2–L3 stimuli.Using the response ratios as the dependent variable, a 3 (tactile patterns) × 2 (with vs. without flash) two-way ANOVA revealed no significant effects except for a significant main effect of tactile pattern, t2 must be spatially incongruent with the L1–L2–L3 tactile sensation. As for the L1–L2–L3 tactile conditions, if a flash captures and causes backward displacement of L2, participants would feel less of an L1–L2–L3 sensation. The fact that a flash did not reduce the response ratio indicates that it is difficult to observe simple spatial visuo-tactile ventriloquism (see t2). Although the flash indicated the correct location of a second tactile stimulus, participants felt the CRE just as well as when no-flash was present. The effect of the flash also was not observed under the L1–L3–L3 tactile conditions. These results indicate that a spatially incongruent flash did not modulate the CRE. The tactile sensation seems to be processed separately from the visual system in the present experiment, indicating that a flash could modulate not a single tactile location but the CRE as a whole to the extent that the correspondence between the visual and tactile senses is maintained. To further examine the present results, we conducted Experiment 2B, wherein we fixed the tactile pattern as L1–L1–L3 and examined the effects of manipulating the flash location at t2.These results suggest that visual stimuli might not reduce the CRE though a flash on L1 at uism see . The flat2 by fixing the tactile pattern as L1–L1–L3 . While a spatially congruent L2 flash enhances the L1–L2–L3 tactile sensation (as we showed in Experiments 1A and B), L1 and L3 flashes are spatially incongruent with the CRE saltation. Although the results of Experiment 2A suggested that the L1 flash did not attenuate the CRE, there is a difference between L1 and L3 flashes: whereas an L1 flash might pull the illusory second tactile location  back to L1, the L3 flash might push it forward to L3. In the present experiment, congruency between displacement of the flash and the direction of CRE saltation was manipulated. As in Experiment 1B, we examined whether a flash at L3 could push the illusory L2 forward to L3  and attenuate the CRE, where the correspondence between a flash and the CRE saltation might not be realized in this condition.In order to examine the possibility that a flash could attenuate the CRE, we varied the flash location at t2  participated in the current experiment. The tactile stimulus pattern was fixed as L1–L1–L3, and we varied the flash location at F = 21,12, p < 0.01, and multiple comparisons (Ryan’s method) revealed that each tactile pattern was significantly different from the others (ps < 0.05), except for the difference between both with/without flash conditions on L1  = 5.48, p < 0.01. Multiple comparisons revealed significant differences between the no-flash (average RT = 615.7 ms) and flash conditions (713.6 ms) on L3 and between the flash on L2 (590.2 ms) and flash-on-L3 conditions .A one-way ANOVA using the response ratios as the dependent variable revealed a significant main effect of condition, F, 39 = 21These results essentially replicated the results of the previous experiments. A flash on L2 enhanced the CRE, but a flash on L1 did not attenuate the CRE as compared to the no-flash condition . We examined what participants felt when they did not feel the L1–L2–L3 tactile sensation under conditions favoring forward or backward displacement.t2 on L1 or L3 , the number of participants was increased. This was done in order to account for the several potential variations in responses. Sixteen university students  participated in a 3 × 2 factorial experiment: the tactile stimulus patterns were L1–L2–L3, L1–L1–L3, or L1–L3–L3, and a flash was presented at F = 9,17, p < 0.01, significant simple main effects of flash condition under the L1–L2–L3 and L1–L3–L3 tactile conditions (ps < 0.01), and significant simple main effects of tactile pattern under both flash conditions (ps < 0.01). Multiple comparisons using Ryan’s method revealed that the report ratio of the L1–L2–L3 tactile pattern was significantly different from those of the L1–L1–L3 and L1–L3–L3 patterns under both flash conditions (ps < 0.01).We summarized the participants’ raw reports. Figure First, we need to consider the potential effects of the requirements placed on participants. In the current experiment, participants reported the tactile sensation pattern they felt. Compared with the results of Experiments 2A and B, the report ratios for the L1–L2–L3 sensation were generally reduced. This might be because participants were placed under time pressure during the previous experiments; thus, they might have encoded other potential pattern identifications, like L2–L1–L3 or L1–L3–L2, as L1–L2–L3. That might also explain the lack of significance of flash condition under the L1–L1–L3 tactile conditions. It seems that the feeling of the L2–L1–L3 pattern could be judged as L1–L2–L3 when responses are made quickly . This is also true when the flash modulates the tactile location. The flash caused forward displacement (attenuating the CRE) but not backward displacement (no attenuation of the CRE) in Experiment 2. On the other hand, the flash caused both forward and reverse displacement (both enhancing the CRE) in Experiment 1. Although the reason for this asymmetry in the ease of enhancement vs. attenuation is still unclear, the brain might intrinsically expect consistently moving tactile sensations, such as something hopping along the skin  and CRE saltation and the existence of cross-modal correspondence between visual and tactile cues seem to play key roles in this ability. First, Experiment 1B suggested that the CRE itself is susceptible to modulation by movement direction, indicating that forward displacement would be more acceptable than backward displacement in the CRE. The results of Experiments 1A and B suggest that when a flash is temporally and spatially congruent with the CRE , and cross-modal correspondence is provided, participants feel the CRE robustly regardless of the direction of tactile displacement. Conversely, the results of Experiments 2A, B, and C suggest that when a flash is spatially incongruent with the CRE saltation, and cross-modal correspondence is not provided, participants experience an attenuated CRE under conditions favoring forward displacement, but not under conditions favoring backward displacement. The results of Experiment 2C also indicate that the combination of these two factors creates various tactile sensations and that temporal order judgment might be modulated under some conditions. Our results provide theoretically important information on unimodal and multi-modal causality perception: the former indicates that our brain needs to detect motion from successive stimuli, and the latter indicates that multi-modal presentation creates a unified, cross-modal representation of motion. Though a previous study suggested that the CRE is the spatiotemporal dynamics of an early, unimodal, sensory map  refers to the effect of watching a rubber hand being stroked synchronously with one’s own, unseen hand. Viewing this for a short time causes the observer to perceptually assimilate the rubber hand into his or her own body  might be possible. The current results indicate that a flash captures the tactile location, except when the flash is presented on L1. However, even if a flash on L1 captured the tactile location, it would not change the current results. If we feel the sensation of the flash location, the L1–L2–L3 tactile stimuli with flash on L1 at ults see . AlthougThe current paradigm has some limitations, and further research is needed to expand our knowledge on the effects of visual flash on the CRE. First, visual capture of the CRE might be susceptible to response bias in Experiments 1 and 2. For instance, previous studies examining the CRE required participants to judge whether or not the tactile stimuli were presented on L2 (e.g., Blankenburg et al., The CRE is an attractive phenomenon, as postdictive processing is one of the key concepts that characterizes our conscious perception (Miyazaki et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The programmed cell death 1 (PD-1)/programmed death-1 ligand 1 (PD-L1) system may play a role in the negative regulation of T cell functions in hepatitis B virus (HBV) infection. Thus, it is important to study its role in the widely used animal model for HBV infection of woodchucks with woodchuck hepatitis virus (WHV).Woodchuck PD-L1 (wPD-L1) and -L2 (wPD-L2) were cloned and characterized. The levels of wPD-L1 expression in primary woodchuck hepatocytes (PWH), peripheral blood mononuclear cells (PBMCs), and liver tissue of naive and WHV-infected woodchucks were examined by real time reverse transcription (RT)-PCR and flow cytometry. Using antibodies against wPD-L1 and -L2, the effect of blocking PD-1/PD-L1/PD-L2 interaction on the proliferation and degranulation of woodchuck PBMCs was examined.In vitro blockade with antibodies against wPD-L1 and -L2 partially enhanced proliferation and degranulation of PBMCs from WHV-infected woodchucks.Both wPD-L1 and -L2 showed a high homology to their counterparts of other mammalian species and humans. WPD-L1 expression in PWH and PBMCs of naive animals was low but could be stimulated by Toll-like receptor (TLR) ligands and interferons (IFN). WPD-L1 expression in liver tissue was significantly higher than that measured in PWHs and was slightly elevated during acute and chronic WHV infection. However, wPD-1 and wPD-L1 expression on PBMCs was strongly up-regulated during acute and chronic infection. Our results demonstrated that wPD-1/wPD-L1 expression in hepatocytes and PBMCs can be induced by different inflammatory stimuli and is up-regulated mainly on PBMCs during WHV infection. A blockade of the woodchuck PD-1/PD-L pathway could partially enhance T cell functions in WHV infection. Hepatitis B virus (HBV) infection is a global health problem due to its high prevalence in the world. It is generally accepted that an appropriate T cell response to HBV proteins is crucial for viral elimination In vitro blockade of the PD-1/PD-L1 pathway using anti-PD-1 or PD-L1 antibodies could partially restore the function of exhausted cytotoxic T lymphocytes (CTLs) in vivo blockade in mice infected with lymphocytic choriomeningitis virus and HBV-transgenic mice could improve the expansion, cytokine production, and the cytolytic activity of virus-specific T cells, and lead to a rapid decrease of viral loads in vitro improved the functions of HBV-specific T cells derived from peripheral blood and liver tissue Programmed cell death-1 (PD-1) and its ligands (PD-L1 and PD-L2) belongs to the B7/CD28 family of co-stimulatory molecules and have important functions concerning the regulation of T cell responses. Normally, PD-1 is expressed on lymphocytes at a low level but up-regulated for a short term after activation. The engagement of PD-1 to its ligands during this period leads to the negative regulation of T cells, which plays an important role in the maintenance of peripheral tolerance against immunopathological tissue damage in vitro by using specific antibodies improved the proliferation of woodchuck PBMCs and CTL functions in some but not in all animals with acute and chronic WHV infection.The woodchuck model is a useful animal model for studies on HBV infection. Woodchucks can be infected with woodchuck hepatitis virus (WHV), a genetically closely related virus to HBV. Like in humans, the specific T cell responses to WHV proteins are strong during the acute WHV infection and weak or absent in chronically WHV-infected woodchucks. The woodchuck model has been used to test different approaches for immunomodulation and immunotherapy of chronic HBV infection Woodchucks were purchased from Northwest Wildlife  and kept at the animal facilities at the University Hospital of Essen. Experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the local Animal Care and Use Committee  with a Permit Number 8.87–50.10.34.08.281. All procedures were performed on woodchucks anesthetized with ketamine-xylazine and all efforts were made to minimize suffering.7 WHV genome equivalents (GE). Adult woodchucks developed mainly acute self-limiting WHV infections after inoculation and all WHV markers were cleared within 6 months. During the acute infection, the peak of viremia appeared around week 4–8 post infection. Therefore, week 8 was considered as demarcation of early and late phase of WHV infection in this study.Chronically WHV-infected woodchucks were positive for WHV DNA, WHsAg, anti-WHc, but negative for anti-WHs. The chronic carriers were directly purchased from Northwest Wildlife. Naïve woodchucks were inoculated intravenously with10Baby hamster kidney (BHK) cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Woodchuck PBMCs and PWH were prepared by Ficoll or liver perfusion and cultured as described previously TLR1-9 ligands were purchased from Invivogen (USA). wIFN-α and wIFN-γ were expressed in eukaryotic cells after tranfection. WHcAg protein was expressed in E. coli and purified by chromatography through Sepharose 6 and sucrose gradient centrifugation. WHcAg-derived peptide was purchased from EMC . HRP labeled goat-anti-rabbit or goat-anti-mouse IgG, fluorescent labeled antibodies including goat-anti-rabbit IgG-FITC, mouse-anti-human PD-1-FITC, rat-anti-mouse PD-L1-PE, rat-anti-mouse CD3-PE, and corresponding isotype antibodies were purchased from eBioscience (USA). Rat-anti-mouse CD107a-FITC, mouse-anti-human CD4-APC, and 7-amino-actinomycin D (7aad) were purchased from BD bioscience (USA). Polyclonal rabbit anti-wPD-L1 or -L2 antibodies were prepared and tested by our group  and S2. The following sequences with the corresponding GenBank accession numbers were included: human PD-L1 (AY254342), woodchuck PD-L1 (EU306520), porcine PD-L1 (AY837780), mouse PD-L1 (NM_021893), cattle PD-L1 (AB510902), human PD-L2 (AF344424), woodchuck PD-L2 (EU306521), chimpanzee PD-L2 (NM_001083599), porcine PD-L2 (NM_001025220), mouse PD-L2 (NM_021396).http://www.ebi.ac.uk). The secondary structure of protein sequences data was predicted by an online service of Swiss Institute of Bioinformatics (http://swissmodel.expasy.org). Recombinant wPD-L1 and -L2 proteins were expressed in E. coli and mammalian cells , TLR7 , and TLR9 (CpG ODN 12.5 µg/ml) by transfection with lipofectamin2000  or direct administration of ligands of TLR1/2 , TLR2/6 , TLR4 , 12.5 µg/ml), and TLR5 , woodchuck IFN-α and -γ (500 U/ml) for 6 h. Total RNA of cells or woodchuck liver samples were prepared for real time RT-PCR to detect the wPD-L1 mRNA as described previously Two commercially available, cross-reactive monoclonal antibodies, mouse-anti-human PD-1-FITC  and rat-anti-mouse PD-L1-PE , were used for FACS staining of PWHs and woodchuck PBMCs, mouse IgGλ, rat IgG1κ-PE were used as isotype contorls. Dead cells were excluded by live staining with 7aad . PWHs were harvested at 20 h post stimulation by incubation with trypsin-free cell dissociation buffer and stained with anti-PD-L1/7aad. Woodchuck PBMCs were stained with anti-CD3/anti-CD4/7aad/anti-PD-1 or anti-CD4/7aad/anti-PD-L1. A detailed description of flow cytometry analysis is given in To test the recognition of wPD-L1 and -L2 by rabbit anti-woodchuck specific antibodies generated by our group , IF stai4 per well for the proliferation assay and 1×106 per well for the CD107a degranulation assay, and cultured in flat-bottom 96-well microtiter plates, as optimized previously. WHcAg was added to PBMCs at a final concentration of 1 µg/ml for the proliferation assay. For proliferation assay, stimulation index (SI) is calculated with the formula: (stimulated cpm - blank cpm)/(unstimulated cpm - blank cpm). To test the effect of the blockade of wPD-1/PD-L1/PD-L2 pathway, woodchuck PBMCs were incubated with different dilutions of anti-wPD-L1 (stock concentration 2.7 mg/ml), anti-wPD-L2 (stock concentration 2.5 mg/ml), anti-wPD-L1 plus -L2, or control antibody, respectively. A negative control without antibodies was included. A WHcAg-derived peptide aa 96–110, which was identified as a CD8 epitope widely recognized by a great number of woodchucks, was used for stimulation in degranulation assay. Due to the lack of available anti-CD8 antibody, woodcheuck PBMCs were stained with cross-reactive antibodies to CD3 and CD4. The CD3+CD4− lymphocytes were gated and regarded as mainly CD8+ cells, with minor fractions of CD3+CD4−CD8− cells. In this study, the CD3+CD4− T cell population were analyzed. Normally, CD3+CD4−T cell response detected by degranulation assays ranged between 0–10%, with highest values in the peak of acute WHV infection. The improvement of T-cell degranulation by the blockade of wPD-1/PD-L1/PD-L2 pathway was expressed as the fold change calculated with measured percentages of CD107+ cells in the assays with the formula: (“anti-PD-L1 plus peptide” - “peptide”)/(“control antibody plus peptide” - “peptide”). A fold change >2 was regarded as a positive result.The PBMCs proliferation and CD107a degranulation assay were performed as described previously The statistical analysis was automatically carried out by GraphPad software . Significant differences between the groups were determined by nonparametric Student's t-test. P<0.05 was considered as statistically significant. Data are presented as means ± standard deviation.A comparison of the complete coding regions of wPD-L1 and -L2 obtained by RT-PCR revealed a high homology at the nucleotide (nt) and amino acid (aa) levels to the counterparts of mammalian species . The put6 wß-actin on average. Similar to human hepatoma cells, the expression of wPD-L1 was upregulated by TLR3 (14.9-fold) and TLR4 (4.3-fold) ligands, woodchuck IFN-α (4.3-fold) and -γ (4.6-fold) in PWHs , woodchucks with acute (n = 5) and chronic (n = 7) WHV infection. Three available woodchuck HCC and peritumoural tissue sets were included for the quantitation of wPD-L1 transcripts. Total RNAs were prepared from liver tissue and subjected to real time RT-PCR for wβ-actin, wPD-L1, and wMxA.6 wβ-actin mRNAs on average and significantly higher than the level in cultured PWHs. Taken the expression level of wPD-L1 in naïve woodchucks as the base line, only a slight up-regulation of wPD-L1 was measured in acutely WHV-infected woodchucks. The expression levels of wPD-L1 in chronically WHV-infected woodchucks were comparable to those of uninfected woodchucks. In addition, wPD-L1 was expressed in HCC tissue at a lower level than the base line , acutely (n = 8), and chronically WHV-infected (n = 14), and stimulated with WHcAg or WHcAg-derived peptides in the presence or absence of anti-wPD-L1 or anti-wPD-L2 antibodies . The proAn increase of PBMC proliferation or degranulation after treatment with antibodies to wPD-L1 and -L2 was found with PBMCs from different woodchucks . Firstlyin vitro blockade of the woodchuck PD-1/PD-Ls pathway using antibodies to wPD-L1 and -L2. PBMCs from some chronically as well as acutely WHV-infected woodchucks showed higher proliferative responses and CD107 degranulation activities after the blockade with antibodies to wPD-L1 and -L2.In the present work, we examined the woodchuck PD-1/PD-L1 in relation to WHV infection. We could show that both wPD-1 and wPD-L1 expression in woodchuck PBMCs was upregulated during WHV infection though the change of wPD-L1 expression in woodchuck liver tissue was not significant. An enhancement of T cell function was evident in an Recent research indicates that the expression of PD-L1 could be upregulated by different stimuli such as TLR agonists and inflammatory cytokines such as IFNs It was rather surprising, that the wPD-L1 mRNA level was not elevated in the liver tissue from chronically WHV-infected woodchucks, as IFNs were produced by various immune cells in the liver tissue during the hepadnaviral infections and stimulated wMxA expression. Thus, the wPD-L1 expression is differently regulated to that in ISGs and may be only enhanced at high concentrations of IFNs. Another possible explanation is the restricted high expression of PD-L1 in hepatic cells, mainly in liver sinusoidal endothelial cells and Kuppfer cells in vitro. Barber et al. showed in the LCMV infection model that a blockade of PD-1/PD-L1 did indeed lead to a restoration of T cell functions and the suppression of viral infection in vivoin vivo blockade using monoclonal antibodies in the Simian immunodeficiency virus-infected macaques results in the rapid expansion of virus-specific CD8+ T cells with a decrease of viral replication. However, several weeks after the blockade, either the T cells or the viral load returned to the previous status et al. showed that a blockade with anti-PD-L1 promoted the function of HBV-specific T cells derived from chronic HBV patients by affecting the phenotype and function of both peripheral and intrahepatic T cells The proliferative capacity and functions like cytokine production of specific T cells to viral antigens may be improved by blocking PD-L1 or PD-L2 with specific antibodies In our study, blockade of the wPD-1/PD-L1 pathway could enhance the proliferation and degranulation of woodchuck PBMCs in response to WHcAg in some of the chronic WHV infected woodchucks. A blockade with anti-wPD-L1 antibody enhanced proliferation in two different chronically WHV-infected animals even without WHcAg. Since antigen-presenting cells from chronically WHV-infected animals may already present some unrelated antigens to T-cells, a blockade of PD-1/PD-L1 pathway will generally enhance T-cell proliferation without exogenous proteins. The addition of exogenous WHcAg may preferentially enhance the presentation of that antigen and therefore confer the specificity of stimulation.in vivo will clarify whether a blockade of the PD-1/PD-L1 signalling may break the immune tolerance in chronic HBV infections and have therapeutic effects.However, not every chronic woodchuck was responsive to the blockade, which indicates that the wPD-1/PD-L1 system may be not the only cause for low T cell responses in chronic WHV infection. It is known that the viral specific T cell response was strongly influenced by the viral loads Text S1Expression of recombinant wPD-L1 and -L2 proteins in E. coli and mammalian cells.(DOCX)Click here for additional data file.Text S2Generation of specific antibodies to wPD-L1 and -L2.(DOCX)Click here for additional data file.Figure S1A summary of the RT-PCR strategy for amplification of wPD-L1 and -L2. Primers used to amplify the complete coding sequences of wPD-L1 and -L2 were designed according to the conserved regions among the known PD-L1 and -L2 sequences of other mammalian species. Woodchuck RNAs were extracted from woodchuck liver samples using the TRIZOL reagents and subjected to RT-PCR for amplification of cDNAs of wPD-L1 and -L2. The positions of primers are indicated according to the reference sequences of wPD-L1 (EU306520) and -L2 (EU306521). The secondary structure of wPD-L1 and wPD-L2 proteins was predicted by online analysis and compared with the structure features of PD-L1 and L2 of other mammalian species. Sig, the predicated signal peptide; Ig-V-like, the immunoglobulin V-like domain; Ig-C-like, the immunoglobulin C-like domain; TM, the transmembrane region; Intra, the intracellular domain.(TIF)Click here for additional data file.Figure S2Flow cytometry analysis of wPD-L1 expression of woodchuck PWHs. Woodchuck PWHs were treated with TLR ligands, woodchuck IFN-α and -γ for 20 h, detached by trypsin-free cell dissociation buffer, and stained with a commercially available, cross-reactive monoclonal antibody, anti-PD-L1-PE  and 7aad. For the analysis, hepatocytes were gated and dead cells were excluded as 7aad-positive cells.(TIF)Click here for additional data file.Figure S3Flow cytometry analysis of wPD-1 and wPD-L1 expression on woodchuck PBMCs. Woodchuck PBMCs were treated with ligands of TLR3  and TLR7  by transfection with lipofectamin 2000 or direct administration of ligands of TLR1/6 , TLR4 (lipopolysaccharid (LPS), 12.5 µg/ml), and woodchuck IFN-γ (500 U/ml) for 20 h. Two commercially available, cross-reactive monoclonal antibodies, anti-PD-1-FITC  and anti-PD-L1-PE , were used for FACS staining. Woodchuck PBMCs were stained with anti-CD3/anti-CD4/7-amino-actinomycin D (7aad)/anti-PD-1 or anti-CD4/7aad/anti-PD-L1. (A) Fresh woodchuck PBMCs were divided in three populations R1, R2, and R3. R1 was excluded as cell debris and erythrocytes for analysis. R2 contained lymphocytes, as stained with anti-CD3 and anti-CD4. R3 contained CD3− cells with high granularity, representing mixed non-T cell populations. (B) For the analysis of wPD-1 and wPD-L1, woodchuck PBMCs were divided in non-lymphocytes and lymphocytes, further in CD3+CD4− and CD3+CD4+ cells. Dead cells were excluded as 7aad+ cells. (C) Analysis of wPD-L1 expression on woodchuck non-lymphocytes without and after stimulation with TLR ligands. (D) Analysis of wPD-L1 expression on woodchuck lymphocytes without and after stimulation with TLR ligands.(TIF)Click here for additional data file.Figure S4Detection of recombinant wPD-L1 and -L2 expressed by transient transfection by specific antibodies. BHK cells were transiently transfected with pxf3H-wL1 or pxf3H-wL2 plasmids. Transfected cells were fixed for IF staining after 48 h. Western blotting of transfected cells stained by control rabbit sera, anti-HA monoclonal antibody, anti-wPD-L1 or anti-wPD-L2 antisera. In SDS-PAGE and western blotting analysis, cells transfected with pxf3H-wL1 and pxf3H-wL2 expressed specific protein bands at the molecular weight of about 30 kD that were detected by anti-HA and antisera to wPD-L1 and -L2, respectively, corresponding to HA-wPD-L1 and -L2 with HA-tag MYP YDV PDY ANS PYP YDV PDY AEF. No band was recognized when cells were transfected with an empty vector.(TIF)Click here for additional data file.Figure S5Blocking the PD-1/PDLs pathway in vitro enhanced the antigen specific proliferation of woodchuck PBMCs. Woodchuck PBMCs were stimulated with WHcAg or ConA for 5 days with or without blockage with anti-PDL1, anti-PDL2, or anti-PDL1 plus anti-PDL2 at different concentrations. Proliferation of woodchuck PBMCs was measured by 2-[3H] adenine incorporation. In naïve animals and some of the chronically infected animals, the blockage with antibodies to wPD-L1 and –L2 had no effect on lymphoproliferation (A) while an enhancement of the antigen specific proliferation at different level was measured with PBMCs from some other chronic carriers (B).(TIF)Click here for additional data file.Figure S6Blocking the PD-1/PDLs pathway in vitro enhanced the antigen specific CD107a degranulation of woodchuck PBMCs. Woodchuck PBMCs were stimulated with WHcAg derived peptide or control peptide for 2 days with or without blockage with anti-PDL1, anti-PDL2, or unrelated antibody preparations. Antigen-specific CD107a degranulation was detected by CD107a staining for PBMCs from naïve, acutely and chronically WHV-infected woodchucks. In naïve animals and some of the chronically infected animals, the blockage with antibodies to wPD-L1 and –L2 had no effect on CD107a degranulation (A) while an enhancement of the antigen-specific CD107a degranulation at different levels was measured with PBMCs from some other chronic carriers (B).(TIF)Click here for additional data file.Table S1The primers used for RT-PCR amplification of wPDL1 and wPDL2.(DOCX)Click here for additional data file.Table S2Homology of wPD-L1 and wPD-L2 to the counterparts of other mammalian species on the nucleotide and amino-acid level.(DOCX)Click here for additional data file.Table S3Raw data of wPD-L1 expression in PWH after TLR stimulation.(DOCX)Click here for additional data file.Table S4Raw data of wPD-L1 expression in PBMCs after TLR stimulation.(DOCX)Click here for additional data file.Table S5Blockage with antibodies to wPD-L1 and -L2 enhances the antigen specific CD107a degranulation in some woodchucks.(DOCX)Click here for additional data file.


Microbial communities carry out the majority of the biochemical activity on the planet, and they play integral roles in processes such as metabolism and immune homeostasis in the human microbiome. Whole genome shotgun sequencing of such communities’ metagenomes is becoming an increasingly feasible complement to obtaining organismal information from taxonomic markers. However, the resultant dataset typically comprises short reads from hundreds of different organisms, making it challenging to assemble and functionally annotate these sequences in the standard manner for single-organism genomes.We describe an alternative to this approach to infer the functional and metabolic potential of a microbial community metagenome by determining whether gene families and pathways are present or absent, as well as their relative abundances, directly from short sequence reads. We validated this methodology using synthetic metagenomes, recovering the presence and abundance of large pathways and of small functional modules with high accuracy. We subsequently applied this approach to the microbial communities of 649 metagenomes drawn from 7 primary body sites on 102 individuals as part of the Human Microbiome Project (HMP), demonstrating the scalability of our methodology and the critical importance of microbial metabolism in the human microbiota. This provided a framework in which to define functional diversity in comparison to organismal ecology, including an example of microbial metabolism linked to specific organisms and to host phenotype  in the posterior fornix. We provide profiles of 168 functional modules and 196 metabolic pathways that were determined to be specific to one or more niches within the human microbiome, including details of glycosaminoglycan degradation in the gut.Bifidobacterium in this disease and suggests the involvement of additional clades, including the Clostridia and Metascardovia. A quantitative validation of LEfSe highlights a lower false positive rate, consistent ranking of biomarker relevance, and concise representations of taxonomic and functional shifts in microbial communities associated with environmental conditions or disease phenotypes.Understanding how and why these biomolecular activities differ among environmental conditions or disease phenotypes is, more broadly, one of the central questions addressed by high-throughput biology. We have thus developed the linear discriminant analysis (LDA) effect size algorithm (LEfSe) to discover and explain microbial and functional biomarkers in the human microbiota and other microbiomes. We demonstrate this method to be effective for mining human microbiomes for metagenomic biomarkers associated with mucosal tissues and with different levels of oxygen availability. Similarly, when applied to 16S rRNA gene data from a murine ulcerative colitis gut community, LEfSe confirms the key role played by Implementations of both methodologies are available at the Huttenhower laboratory’s website ,2. Toget