Ninety boys aged 7 to 13 years completed one-item and three-item VWM tasks in which stimuli were coloured bars varying in orientation. On each trial, participants used a rotating dial to reproduce the probed stimulus from memory. Results show linear age-related improvement in recall precision for both one-item and three-item VWM tasks. However, even the youngest age group stored a significant amount of information about all three items on the difficult 3-item VWM task. Importantly, the development of VWM precision was not accounted for by development on a sensorimotor control task. Whereas storage of a single complete item was previously thought to be well within the capacity limitations of the current age range, these results suggest protracted development during childhood and early adolescence in the resolution with which single and multiple items are stored in VWM. Probabilistic modelling of response distribution data suggests that improvement in VWM performance is attributable to a specific decrease in variability of stored feature representations, rather than to a decrease in misbinding or random noise. As such, we highlight a novel, potentially developmentally plausible mechanism that may underlie developmental improvement in VWM performance, independent of any alterations in the maximum number of complete items which can be stored.Visual working memory (VWM) is the facility to hold in mind visual information for brief periods of time. Developmental studies have suggested an increase during childhood in the maximum number of complete items that can simultaneously be stored in VWM. Here, we exploit a recent theoretical and empirical innovation to investigate instead the  Visual working memory (VWM) or visual short-term memory,capacity of working memory, i.e. the number of items that can be held in VWM, using visual change detection paradigms  and estimates of the maximum number of items encoded (e.g. Cowan’s K) increase with age during childhood  or does not differ (‘no change’) from a comparison array presented following a brief retention period. Test and comparison arrays are either identical, or differ by a feature of one item . Proportion correct judgments, sensitivity  of error in response.Recently, an alternative theoretical and empirical approach to VWM has been developed in studies of adult participants. This approach investigates working memory d in VWM . In precd in VWM . Precisiresource that can be distributed flexibly among memoranda, but without any limit to the number of complete items that can be stored  the maximum capacity of VWM is limited to between two and three complete items  preparatory school. Boys were selected randomly from each school year by a teacher, with the caveat that the developmental disorders dyspraxia and attention-deficit/hyperactivity disorder be excluded. Parental consent was given for each participant. The study was approved by the local ethics committee.SD 1.48; see We excluded three boys with suspected colour blindness who could not perform a colour-naming control task or commented that ‘two bars were the same colour’ in the three-item VWM task. The final sample consisted of 87 participants aged 7.9–13.6  were provided by the school for all but one participant. We used these scores to estimate full-scale (FS) IQ, as per e is estimated FSIQ and CAT-Av is average CAT-3 score calculated by combining standardized scores on verbal, non-verbal and quantitative reasoning subtests . Therefore, we used FSIQe as a covariate in subsequent analyses.FSIQA colour naming task, sensorimotor control task and two visual working memory (VWM) tasks were administered on a laptop computer with the screen at a viewing distance of 60 cm. Participants were tested individually by one of two experimenters in one of two quiet rooms in school, during school hours. The duration of the experimental session was 35 minutes per participant, timed to correspond to a single 35-minute school lesson (period).A colour naming task was administered at the start of the experimental session. Participants were shown five screenshots from the VWM task, each containing a bar in one of five stimulus colours: red, yellow, green, blue and pink. Participants named aloud each of the five colours. Any participant who did not give 5/5 correct responses was excluded from all analyses.Participants completed 25 trials of a sensorimotor control task , directlBar colour was drawn at random on each trial from the possible colour set . Target and probe orientation were independently randomized across π rad on each trial. The inter-trial interval (ITI) was 500 ms.remembered orientation of the target.Participants completed 30 trials of a one-item VWM task , subsequParticipants were prompted every 15 trials by the task programme to take a short break. During this break participants were encouraged to focus on a far point in the room, to minimize ocular fatigue.Participants completed 90 trials of a three-item VWM task see , subsequParticipants were encouraged every 15 trials to take a break. Four participants did not complete all 90 trials of the three-item task due to lack of time.N in year group 3 (see We investigated our dependent variables (see below) for effects of participant age and in some cases school year group (i.e. grade). Year groups 3 and 4 were collapsed due to low up 3 see . The patSD from the sample mean of each dependent variable were excluded for that variable only . Statistical significance is p <.05 two-tailed unless otherwise specified.Outliers > 2.5 SD). Since the parameter space for orientation is circular, we used Fisher’s definition of SD for circular data  interaction in precision using repeated measures ANOVA, since precision is not expected to show additive effects across group and task . Rather, it is more reasonable to assume additivity of variance, i.e. that sensorimotor and recall errors from individual and multiple items contribute independently to response variability. However, variance is not distributed normally in our tasks, so we describe below methods implemented to compare age effects across task and serial position, i.e. interactions.SD2) and sensorimotor error variance, i.e. corrected VWM precision = 1/√(SDVWM2–SDSM2). This correction is implemented throughout.At the outset we tested whether precision in the sensorimotor control task was correlated with age. A positive result entailed correction for sensorimotor precision of raw precision values in the one-item and three-item VWM tasks. Assuming that sensorimotor errors and recall errors contribute independently to response variability on VWM trials, recall precision can be estimated from the difference between VWM error variance .Mean one-item and three-item VWM precision values, corrected for sensorimotor performance, were interrogated for effects of participant age using linear regression and partial correlation controlling for FSIQacross tasks, we calculated precision differences. This was implemented by subtracting raw one-item error variance from raw three-item error variance, and recalculating precision accordingly, i.e. precision difference = 1/√(SDVWM(3)2–SDVWM(1)2). A significant correlation between age and this dependent variable would imply a specific effect of age on storage or recall of multiple items in VWM, i.e. an age effect on the degree of independence of error in the recall of multiple items (e.g. age-sensitive constant attention or decay cost). An absence of association would constitute no evidence for differential rates of development between single- and multiple-item tasks.To compare age effects on VWM precision variance (1/precision2), i.e. independent contribution to response variability of VWM errors from individual and multiple items. However, since response variability is not distributed normally, this violates the assumptions of ANOVA. Hence, our alternative analysis described above, the output of which is conceptually equivalent to the output of a repeated measures ANOVA on group and task.Note that it would not be valid to interpret an interaction resulting from an ANOVA on precision across age group and task in the usual way. This is because ANOVA assumes additivity (i.e. linearity) of the dependent variable across conditions. However, precision does not behave in this way. As such, a significant interaction in repeated measures ANOVA would merely be consistent with our understanding that precision does not behave linearly; it would not necessarily indicate an interesting result. Instead, it is justified to assume additivity of precision differences between the final (SP3) and preceding (SP1/2) items using the method outlined above. A significant result would imply age differences in the degree of independence of error in recall of multiple, serially presented items (e.g. age-sensitive constant attention or decay cost).To evaluate serial order effects, we calculated mean precision for each of the three serial positions (SP) of the three-item VWM task, i.e. first (SP1), second (SP2) and third item in sequence (SP3). Effects across the sample of the serial position of the probed item were evaluated using one-way ANOVA (within-subjects factor: SP). We expected to observe a recency effect, i.e. an advantage for the item presented last . To testPrecision gives an indication of the overall variability in responses. However, we wanted to determine how this variability was distributed across feature space. This could give some indication of sources of error in VWM. Therefore, to visualize these data we plotted the frequency of responses at each of nine arbitrary orientation bins spaced evenly across π rad of response space in the three-item VWM task, first relative to the target orientation and then relative to the non-target (unprobed) stimuli in a sequence.κ (kappa), a concentration parameter encapsulating Gaussian variability in memory for target orientations, and parameters representing the probability of reporting the target orientation, a non-target orientation and a random orientation. Note that reporting a non-target orientation  would constitute a misbinding error  with mean zero and concentration parameter κ (kappa). The probability of reporting the target item, p(T) is given by α, the probability of reporting a non-target item and p(NT) is given by β. {ϕ1, ϕ2…ϕm} are the orientations of the m non-target items, and the probability of responding at random, p(U) is given by γ = 1 –α - β.However, in order to investigate more formally the distribution of responses, and thence to identify potential mechanisms underlying age-related improvement in VWM performance, we fit a probabilistic model (http://www.sobell.ion.ucl.ac.uk/pbays/code/JV10/). Since the four parameters are non-independent, we tested for effects of age on each parameter separately, using linear regression and partial correlation covarying out FSIQe.Maximum likelihood estimates  of the pe and mean three-item VWM precision, corrected for sensorimotor precision and standardized within each year group, using linear regression.We investigated the relationship between FSIQr2adj =.179, p <.001). We therefore factor this out in all subsequent analyses of VWM precision. Sensorimotor precision (1/SD error) was consistently higher than VWM precision . This improvement remained significant after covarying out FSIQe . That is, VWM precision for a singly encoded item continues to develop between age 7 and 13 years. Precision was significantly above chance in all year groups . This improvement remained significant after covarying out FSIQe . Precision was significantly above chance in all year groups . This relationship remained significant after covarying out FSIQe . Thus, whereas we demonstrate significant improvement with age on both the one-item and three-item VWM tasks, the relative magnitude of improvement in VWM performance with age is greater for multiply encoded items.The multiple items in VWM. This could arise, for example, due to an age-sensitive constant attention or decay cost for each item.In other words, the contribution to VWM variability of recall errors from multiple items becomes more independent with age, although this effect is modest. This implies a specific effect of age on storage or recall of r2adj =.120, p =.001; SP2: r2adj =.112, p =.001; SP3: r2adj =.124, p =.001). This improvement remained significant after covarying out FSIQe . Remarkably, VWM precision at each SP was above chance in all age groups  and SP1 ; items presented in SP1 and SP2 did not differ significantly in precision from one another (p >.05). Thus, the last item in a sequence was remembered with greater precision than previous items, as expected.There was an effect of target serial position (SP) on the precision of recall .The difference in precision between SP3 and SP1/2 mean showed a significant effect of age .This was the only significant effect of age on the four model parameters. Thus there was no significant effect of age on the probability of responding to targets, responding to non-targets or making random guesses , but again this was not the case for the remaining model parameters .Mean precision on the three-item VWM task, corrected for sensorimotor precision and standardized within each year group, accounted for a significant proportion of variance in FSIQprecision in VWM during middle childhood and early adolescence. Results show that the precision with which items are recalled from VWM increases with age in a cross-sectional sample aged 7–13 years.The current study was conducted to investigate the development of Importantly, this improvement in precision was observed for items presented individually (one-item VWM task), as well as in sequences of three (three-item VWM task). These effects withstood correction for the effects of age on a control task requiring fine hand–eye co-ordination, so are not readily explicable on the basis of improvement in sensorimotor factors. Thus, regardless of any putative increase in the maximum number of items that can be stored, we have shown an age-associated increase in the precision with which items – and even a single item – are stored. This is consistent with an increase in resolution of a continuous, dynamic memory resource.Applying a probabilistic model  remained somewhat lower than that observed previously in adults, using a similar paradigm (see −1 at x = 3 items). This raises the intriguing possibility that VWM precision may continue to develop beyond the upper limit of the age range in the current study. The extent to which this may be due to developmental changes in attention, VWM decay or metacognition is an empirical question. Importantly, the age effects on VWM precision observed here were not accounted for by concurrent improvement on a sensorimotor control task.The effect of age on precision in the three-item VWM task amounted to a substantial decrease in SD error between the ages of 7 and 13 years see . Howeverindividual items, whether presented individually or in sequence. An implication of this finding is that childhood VWM development is best characterized not as a discrete increase in the capacity to store complete items, but by an increase in the precision with which these items are encoded, stored or retrieved.Our results are consistent with findings from change detection paradigms, which show continuing development during middle childhood in estimates of the maximum number of items that may be held in VWM , they complement earlier findings by providing evidence within a distinctly different empirical and conceptual framework to that of capacity, including recent reformulations of slot models .As a next step, potential effects of target presentation time on precision in each age group might be investigated. It is possible that younger children were detrimentally affected by the short presentation times (500 ms) of each stimulus. Whereas evidence from change detection paradigms suggests comparable childhood performance when viewing objects for 500 ms vs. 1000 ms .precision difference between one-item and three-item VWM tasks, conceptually equivalent to an age by task interaction. Thus, whereas we demonstrate significant improvement with age on both one-item and three-item VWM tasks, consistent with age-associated development in the resolution of VWM, the relative age-associated improvement on the three-item task was more substantial. Further empirical studies are needed to identify the cognitive mechanism underlying this result. Possibilities include serial order effects (see below), emerging metacognitive capability, and the development of attention  succeed at encoding information about each of the three items. Consistent with this interpretation, there was no effect of age on the model parameter p(U), which indicates a lack of evidence that younger children were more likely than older children to respond at random on the difficult three-item task. Note that if younger children were able to store < 3 discrete items, whereas older children were able to store ≥ 3 items, for example, then this component would be expected to show an effect of age (since un-encoded items would give rise to uniformly distributed response errors).Whether VWM capacity is discrete (slot models) or continuous (dynamic resource models) is debated . Intermeeach item from a set of three, as well as development in the ability to precisely recall an item presented individually. This demonstrates that information is not stored in a fully discrete, all-or-none manner. Independent of any putative increase in the maximum number of complete items that can be stored, we have shown an age-associated increase in the precision with which even a single item is stored, consistent with an increase in resolution of a dynamic resource.Second, we observed robust and substantial development between age 7 and 13 years in the ability to precisely recall precision of VWM develops throughout middle childhood and early adolescence. This development is attributable to an increase in the resolution of feature representations held in memory, without the need to invoke alterations in the capacity to store discrete items. As such, these results demonstrate protracted development in VWM performance, and shed new light on the potential mechanisms that may underlie this development. Measuring precision, rather than estimating the maximum number of complete items that can be stored in VWM, may provide a more sensitive metric for assessing childhood VWM development. Questions remain as to whether this development reflects an increase in the ‘amount’ of VWM resource, or an increase in the extent to which VWM can be flexibly distributed across targets (Visual working memory is the facility to hold in mind visual objects for brief periods of time. In this study, we have shown evidence that the  targets .


Thorny catfishes exhibit large variations in swim bladder morphology. These organs are of different sizes, forms and may have simple or branched diverticula. The swim bladder plays an important role in otophysans because it enhances their hearing sensitivity by transmitting sound pressure fluctuations via ossicles to the inner ear.Hemidoras morrisi in which it was ramp-like. Mean hearing thresholds of species possessing smaller rSBLs were slightly lower (maximum 8.5 dB) than those of species having larger rSBLs.To investigate if a form-function relationship exists, the swim bladder morphology and hearing ability were analyzed in six species. The morphology was quantified by measuring the length, width and height and calculating a standardized swim bladder length (sSBL), which was then used to calculate the relative swim bladder length (rSBL). Hearing was measured using the auditory evoked potential (AEP) recording technique. Two species had simple apple-shaped and four species heart-shaped (cordiform) bladders. One of the latter species had short unbranched diverticula on the terminal margin, two had a secondary bladder and two had many long, branched diverticula. The rSBL differed significantly between most of the species. All species were able to detect frequencies between 70 Hz and 6 kHz, with lowest thresholds found between 0.5 and 1 kHz (60 dB re 1 µPa). Hearing curves were U-shaped except in The current findings reveal a relationship between swim bladder form and its function among thorny catfishes. Relatively smaller swim bladders resulted in relatively better hearing. This is in contrast to a prior inter-familial study on catfishes in which species with large unpaired bladders possessed higher sensitivity at higher frequencies than species having tiny paired and encapsulated bladders. The swim bladder in fishes plays an important role in buoyancy, in respiration, in the detection of sounds and in sound production In otophysines, in contrast, swim bladders are connected via a chain of bony ossicles to the inner ear. These Weberian ossicles are found in all representatives of Cypriniformes (carps and minnows), Siluriformes (catfishes), Characiformes (tetras) and Gymnotiformes (South American knifefishes). A complete loss of swim bladders or ossicles has not been reported in any representative of otophysines. Otophysines, which comprise more than 8000 species, evolved an impressive variation in swim bladder and Weberian ossicles morphology, in particular in Siluriformes and to a lesser extent in Cypriniformes Elimination of swim bladders or Weberian ossicles demonstrated that ancillary hearing structures efficiently enhance the hearing sensitivity in otophysines Relationships between the morphological variations in accessory hearing structures and hearing abilities were analysed in non-related holocentrids (squirrelfishes), sciaenids (drums or croakers) and in cichlids This intrafamilial study was designed to determine if larger swim bladders result in higher auditory sensitivity in thorny catfish (family Doradidae). Thorny catfishes are a potential model to investigate the role of the diversity in swim bladder morphology on hearing within one family. This is because, in contrast to non-otophysines such as cichlids, the factor distance is negligible: bladders are always directly connected to the inner ears via ossicles. Six doradid species were dissected, swim bladder dimensions determined and fish hearing abilities measured.Acanthodoras spinosissimus  , Agamyxis pectinifrons (whitebarred catfish) , Amblydoras affinis , Hemidoras morrisi , Megalodoras uranoscopus  and Oxydoras niger (ripsaw catfish) . Three specimens of each species were used for morphological investigations and three to nine for hearing measurements.Six species of the catfish family Doradidae were used for this study: All fish were purchased from a tropical fish supplier . Fish were kept in aquaria equipped with sand on the bottom, plants, roots, and various shelters. The tanks were between 70 × 35 × 40 cm (width × height × depth) and 100 × 50 × 50 cm in size. In order to reduce noise within aquaria, water was maintained by external filters. Temperature was kept at 25±1°C and a 12·h:12·h L:D cycle was provided. Fish were fed four to six times per week with frozen chironomid larvae or artificial food.The study protocol was approved by the Austrian Federal Ministry of Science and Research, permit number GZ 66.006/0023-II/10b/2008.Acanthodoras spinosissimus: NMW-98200 , Aquarium purchase. Agamyxis pectinifrons: NMW-98201  Aquarium purchase. Amblydoras affinis: NMW-98202  Aquarium purchase. Hemidoras morrisi: NMW-98203  Aquarium purchase. Megalodoras uranoscopus: NMW-98204  Aquarium purchase. Oxydoras niger NMW-98205  Aquarium purchase.Specimen of each species have been deposited in the Naturhistorisches Museum, Wien ( = Vienna) (NMW). Fish were euthanized using an overdose of tricaine methanesulfonate (MS 222) and were directly fixed in alcohol (70%) for conservation. Dissections were performed under a dissecting microscope (Wild M7). Fish length and swim bladder measures were taken using digital callipers. All measures were taken including the secondary bladders but excluding the diverticula. The swim bladder form was classified following the terminology by Standardized swim bladder length (sSBL) was calculated using the formula sSBL = (l+h+w)/3, where l is length, h is height and w is width according to Hearing sensitivity was measured using the auditory evoked potentials (AEP) recording technique developed by −1 for Acanthodoras spinosissimus, 3.99–5.38 µg g−1 for Agamyxis pectinifrons, 0.86–3.26 µg g−1 for Amblydoras affinis, 1.73–2.82 µg g−1 for Hemidoras morrisi, 3.8–7.01 µg g−1 for Megalodoras uranoscopus, and 1.73–2.06 µg g−1 for Oxydoras niger. The lowest dosage that immobilized fish while enabling slight movement of the opercula during the experiments was applied. All auditory measurements were carried out in a bowl-shaped plastic tub  which was lined inside with acoustically absorbent material (air-filled packing wrap) to decrease resonances and reflections. The tub was positioned on an air table , which rested on a vibration-isolated plate of concrete. A sound proof chamber, constructed as a Faraday cage (interior dimensions: 3.2 m × 3.2 m × 2.4 m), enclosed the whole setup.The thorny catfish were mildly immobilized with Flaxedil . The dosage used was 3.07–3.27 µg gTest subjects were positioned in the centre of the tub, so that the nape of the head was at the water surface. For respiration a pipette was inserted into the fish’s mouth and respiration was effected by a simple, temperature-controlled (25±1°C), gravity-fed water system. The area of the head above the water surface was covered with a small piece of Kimwipes ® tissue paper to keep it moist. Silver wire electrodes (diameter 0.38 mm) were used for recording AEPs. The recording electrode was placed in the midline of the skull over the region of the medulla, the reference electrode cranially between the nares. Both electrodes were pressed firmly against the skin.Both presentation of sound stimuli and AEP waveform recording were achieved using a modular rack-mount system (Trucker-Davis Technologies (TDT) System 3, Gainesville, FL, USA) controlled by a PC containing a TDT digital signal processing board and running TDT BioSig RP software.−1) was placed 2 cm from the right side of the animal to determine absolute sound pressure levels (SPLs) under water in the immediate vicinity of the test subject. A second custom-built preamplifier was used to amplify the hydrophone signal (1000×). Sound stimuli consisted of tone bursts played at a repetition rate of 21 s−1 and at opposite polarities (90° and 270°). One thousand stimuli of each polarity were presented and the corresponding AEPs averaged by BioSig RP software to eliminate stimulus artefacts. The SPL was reduced in 4 dB steps until the AEP waveform was no longer identifiable. By overlaying replicate traces, the lowest SPL yielding a repeatable AEP trace was determined and regarded as threshold.Hearing thresholds were determined for the following frequencies: 0.07, 0.1, 0.3, 0.5, 1, 2, 3, 4, 5 and 6 kHz. Sound stimuli waveforms were created using TDT SigGen RP software. For tone bursts, two speakers  installed 0.5 m above the fish were used. Tone bursts at different frequencies were presented in random order. A hydrophone . For statistic analysis of the morphological data, a one-way ANOVA was calculated to determine whether rSBLs differ between the species. Differences between the hearing sensitivities of the six species were calculated using a two-way ANOVA followed by a Bonferroni post hoc test. In order to determine whether rSBL affects hearing sensitivities two calculations were carried out. First, the mean thresholds of the three species having smaller rSBLs than 0.165 were calculated for each frequency and compared with those three species having large rSBLs than 0.165 by calculating a two-way ANOVA. Second, Pearson’s correlation coefficient was calculated to correlate the mean hearing thresholds of each specimen at each frequency to rSBL of this species.All statistical tests were run using PASW 18.0 .Acanthodoras spinosissimus and Amblydoras affinis had apple-shaped swim bladders without any diverticula . They were classified as being apple-shaped, heart-shaped (cordiform) or abbreviated cordiform . Acanthoerticula . Agamyxi bladder .Hemidoras morrisi and 0.201 in Acanthodoras spinosissimus and differed significantly between species  (The rSBL varied between 0.129 in p<0.001) . Bonferrp<0.001) .Oxydoras niger and Amblydoras affinis showed lowest thresholds at 0.5 kHz, whereas Acanthodoras spinosissimus, Agamyxis pectinifrons and Megalodoras uranoscopus had best hearing sensitivity at 1 kHz. Hearing thresholds decreased by approximately 15–20 dB from 70 Hz to 0.5/1 kHz and increased at higher frequencies. In contrast, the hearing thresholds of Hemidoras morrisi showed an almost constant increase in sensitivity up to 6 kHz. Thus, Hemidoras morrisi had better hearing abilities at low and high frequencies compared to the other five species and a lower sensitivity in the mid-frequency range from 0.5–1 kHz. The lowest threshold was found in Amblydoras affinis (59.6 dB re 1 µPa at 0.5 kHz)  with best hearing sensitivity located at 0.5 or 1 kHz , Table 20.5 kHz) , Table 25, 320 = 50.913, p<0.001) and a significant interaction between species and frequency . This indicates that auditory sensitivities showed different trends at different frequencies. Hemidoras morrisi differed from all other species in sensitivity.Comparison of hearing curves revealed a significant difference between most of the species    . Differe p>0.05) .The rSBL was positively correlated to the hearing thresholds at 70 and 100 Hz   and at 4Amblydoras affinis is similar to that of Amblydoras bolivarensis  Acanthodoras spinosissimus, which is comparable to the organ of Acanthodoras cataphractus. The swim bladder of Megalodoras uranoscopus from the current study resembles that of M. uranoscopus and Megalodoras guayoenis, i.e. cordiform with a secondary bladder and many well-developed and often branched diverticula. Birindelli et al. (2009) Megalodoras uranoscopus possess diverticula all over the swim bladder, whereas diverticula are lacking anteriorly in larger specimens. The size of the secondary chamber and the number of diverticula increase with growth. Hemidoras morrisi exhibited a cordiform swim bladder with many branched diverticula. Birindelli et al. (2009) Hemidoras species. In Agamyxis pectinifrons there were three to five small posterolateral diverticula, whereas in Agamyxis albomaculatus there was only a single posterolateral diverticulum on either side of the terminal diverticulum according to Agamyxis pectinifrons) to longer diverticula about 1/3 of the swim bladder length (e.g. Oxydoras niger) Thorny catfish comprise 92 species in 31 genera Glanidium, Pseudauchenipterus), almost apple-shaped bladders (Centromochlus spp.) or bladders reduced in size and partially or completely ossified (Ageneiosus inermis). Tocantinsia piresi and Asterophysus batrachus had distinctive-looking bladders with either two or many diverticula. Variations were also found within genera: Trachycorystes menezesi had a simple cordiform organ, whereas Trachycorystes trachycorystes had a pair of lateral diverticula and a well-developed terminal diverticulum. The bladder of the genus Auchenipterichthys was characterized by having a secondary bladder Bagre); a few species have well-developed secondary bladders (Sciades spp.) and some have apple-shaped bladders  Aspistor quadriscutis). In contrast to the diversity in the swim bladder form in thorny catfishes, these organs in pseudopimelodids varied only minimally. The organ in pseudopimelodids is large and cordiform  or moderately sized and apple-shaped . Representatives of the genera Pseudopimelodus and Cruciglanis have diminutive organs partially divided into two lateral sacs, where the parapophyses of the fourth vertebra partially cover the bladder anteroventrally Recent studies on swim bladder morphology in other catfish families revealed further differences in gross morphology, but these intrafamilial differences were smaller than in thorny catfishes Acanthodoras spinosissimus (0.201), the smallest in Hemidoras morrisi. (0.129). Quantitative data on relative swim bladder lengths are found only in Malapterurus beninensis  to 0.152 in Synodontis schoutedeni (Mochokidae). In catfish species with tiny paired and encapsulated bladders, the rSBL was much smaller and varied between 0.016 in Hemiodontichthys acipenserinus and 0.057 in Ancistrus ranunculus (Loricariidae) The relative size of swim bladders varied between different species of thorny catfishes. The largest rSBl was found in Hemidoras morrisi differed from all other species because of its ramp-like shape with an almost constant decline towards higher frequencies. The U-shaped hearing curves of Acanthodoras spinosissimus, Agamyxis pectinifrons, Amblydoras affinis, Megalodoras uranoscopus and Oxydoras niger resemble the audiogram previously gained in the striped Raphael catfish Platydoras armatulus (formerly P. costatus), another representative of thorny catfishes Agamyxis pectinifrons was 10–30 dB higher in the previous than in the current study Overall hearing abilities of the investigated doradids differed between most species, but showed similar general characteristics. The hearing curves where U-shaped with best sensitivities between 0.5 and 1 kHz in five out of six species The hearing curve of Corydoras sodalis had higher hearing thresholds  than Dianema urostriatum, which resembles difference between Acanthodoras spinossisimus and Amblydoras affinis in the current study. Similar differences were found among loricariids, where Ancistrus ranunculus had higher thresholds than Hemiodontichthys acipenserinus and Hypoptopoma thoracatumPimelodus pictus and P. blochii belong to the same genus.Differences in hearing sensitivities were also found in other catfish families such as callichthyids and loricariids. Lechner and Ladich Pimelodus blochii and P. pictus) and doradids (Platydoras armatulus), whereas there was no difference between the latter two families. Lechner and Ladich (2008) Comparing different families with regard to hearing abilities reveals a general trend, namely that relative swim bladder size affects hearing sensitivities. Ladich (1999) Acanthodoras spinosissimus, Agamyxis pectinifrons and Megalodoras uranoscopus) and fish with smaller swim bladders . Surprisingly, fish with smaller swim bladders had slightly better hearing abilities. This finding is in contrast to former studies. Lechner and Ladich (2008) Amblydoras affinis, with its simple apple-shaped swim bladder without any diverticula had the lowest auditory threshold, whereas species with a cordiform gas bladder and diverticula had poorer sensitivities. None of the swim bladders in this study were covered by bones, enabling them to vibrate freely. The differences in hearing abilities may be explainable by differences in accessory hearing structures , by inner ear morphology or by ontogenetic development The present study investigated whether the variation in swim bladder morphology affects hearing sensitivity in thorny catfishes. Regarding their rSBLs the doradids were divided into two groups: fish with larger  were not significantly lower than those of fishes lacking these projections  Etroplus maculatus and in Paratilapia polleniHemichromis guttatus, which lacks anterior extensions but possesses a large swim bladder, has an auditory sensitivity similar to Etroplus maculatus and Paratilapia polleni up to 3 kHz. In contrast, Steatocranus tinanti, which has a tiny swim bladder, detects sounds frequencies only up to 700 Hz.The effects of different swim bladder size on hearing have not been studied in fishes except in catfishes. Typically, investigators concentrated on the distance between swim bladder (including anterior extensions) and inner ear. Smaller distances positively affect the hearing sensitivities in holocentrids and to some degree in sciaenids. The Hawaiian squirrelfish The present investigation showed that thorny catfish with smaller swim bladders had slightly better hearing abilities than species with larger ones. Although this result is unexpected underlines that the relationship between swim bladder morphology and auditory sensitivity is not always straightforward: larger swim bladders and shorter distances between swim bladders and inners ear result in improved hearing. Horodysky et al. (2008)


Borrelia burgdorferi, vary considerably in different patients, possibly due to infection by strains with varying pathogenicity. Both rRNA intergenic spacer and ospC typing methods have proven to be useful tools for categorizing B. burgdorferi strains that vary in their tendency to disseminate in humans. Neither method, however, is suitable for inferring intraspecific relationships among strains that are important for understanding the evolution of pathogenicity and the geographic spread of disease. In this study, multilocus sequence typing (MLST) was employed to investigate the population structure of B. burgdorferi recovered from human Lyme disease patients. A total of 146 clinical isolates from patients in New York and Wisconsin were divided into 53 sequence types (STs). A goeBURST analysis, that also included previously published STs from the northeastern and upper Midwestern US and adjoining areas of Canada, identified 11 major and 3 minor clonal complexes, as well as 14 singletons. The data revealed that patients from New York and Wisconsin were infected with two distinct, but genetically and phylogenetically closely related, populations of B. burgdorferi. Importantly, the data suggest the existence of B. burgdorferi lineages with differential capabilities for dissemination in humans. Interestingly, the data also indicate that MLST is better able to predict the outcome of localized or disseminated infection than is ospC typing.The clinical manifestations of Lyme disease, caused by  Borrelia burgdorferi sensu stricto (hereafter referred to as B. burgdorferi) which is transmitted to humans through the bite of infected Ixodes spp. ticks Lyme disease is a multisystem illness that, in North America, is caused by the spirochete B. burgdorferiospC) and the 16 S–23 S (rrs-rrl) rRNA intergenic spacer (IGS) have been the most commonly used genetic markers for B. burgdorferi strain identification in the US ospC major groups A, B, H, I and K have a stronger tendency for hematogenous dissemination early in the course of disease B. burgdorferi genotypes is responsible for early disseminated infection in humans, suggesting that some degree of differential pathogenicity exists among strains. Both RST and ospC typing methods provide a useful tool for categorizing B. burgdorferi strains that vary in their tendency to disseminate in humans. Neither method, however, is suitable for inferring intraspecific relationships among strains that are important for understanding the evolution of pathogenicity and the geographical spread of disease. While RST typing has limited discriminatory power for this purpose ospC typing may also be restricted since the highly variable ospC gene is subject to recombination and horizontal gene transfer, as well as strong selection by the host immune system Substantial genetic diversity exists within http://www.mlst.net and http://www.pubmlst.org) and has been employed to identify lineages of particular clinical relevance in bacterial pathogens such as Neisseria meningitidisStreptococcus pneumoniaeStaphylococcus aureusCampylobacter jejuniBartonella henselaeB. burgdorferiB. burgdorferi in Ixodes scapularis and Ixodes pacificus, the principal vectors of Lyme disease in North America B. burgdorferi.The most appropriate of the current techniques for large-scale epidemiology, strain identification and understanding of the population structure of bacterial species is multilocus sequence typing (MLST). This method is based on nucleotide sequences of multiple housekeeping genes that are evolving nearly neutrally. MLST analysis has been used successfully to study a number of bacteria   differed at multiple sites , resulting in a total of 11 polymorphisms, which on the tree led to the separation of ST396 from the other STs assigned to CC4. Similarly, the comparison of ST52 (an outlier in CC34) and ST34 (the inferred founder of CC34) revealed seven polymorphisms in the clpA locus, which resulted in the separation of ST52 (and its descendants) from the other STs assigned to CC34.MLST analysis of 146 es (STs) ; 23 havees (STs) . A goeBUes (STs) . Two of es (STs) . CC36, Ces (STs) . FurtherospC major group. In the other two STs (ST3 and ST55) a single isolate possessed an ospC major group different from the other isolates  contained STs of different ospC major groups , I (5 STs) and F (4 STs). In addition, ospC major groups F, I, N and U were shared by isolates of different major clonal complexes  than patients from New York (n = 20). Only two STs (ST4 and ST12) were found in both geographical locations . DespiteospC major groups in Wisconsin patients (65 skin isolates cultured during 1995 to 2003) was compared with 291 previously genotyped skin isolates obtained from New York patients during 1991 to 2005 ospC major groups was observed in skin isolates from Wisconsin (n = 22) than those from New York (n = 17)  . While aisconsin . The fre2 = 65.84, P = 0.066), there was a significant association between type of infection and clonal complexes in each of three separate statistical analyses. The first analysis included major and minor clonal complexes as well as singletons ; in the second analysis, singletons were excluded ; and for the third analysis only major clonal complexes were included  . ospC major groups B, H and K were particularly associated with disseminated infection and ospC major groups F and T were significantly associated with localized infection . Similarly, ospC major group K isolates in CC4 were obtained from patients with disseminated infection; two ospC major group K isolates cultured from patients with localized infection were not members of CC4. An ospC major group B isolate from a patient with localized infection was not part of CC7, a clonal complex that is comprised only of ospC major group B isolates from patients with disseminated infection. ospC major group I isolates were divided between two different major clonal complexes. CC16 contains ospC major group I isolates cultured from patients with disseminated infection and CC228 is comprised of ospC major group I isolates cultured from patients with localized skin infection  . In each= 0.001) . ospC manfection . Furthernfection . The disB. burgdorferi population structure in Ixodes spp. ticks and provided novel insights into the phylogeography of the pathogen B. garinii  B. burgdorferi population structure in patients with Lyme disease, and the results indicate that phylogeny and pathogenicity of B. burgdorferi are correlated.Accurate strain identification of pathogenic bacteria is essential for epidemiological surveillance and permits effective public health decisions. Previously, MLST based on eight housekeeping loci was exploited to examine B. burgdorferi genotypes have been previously characterized among isolates obtained from Lyme disease patients in the northeastern US, primarily using the rrs-rrlA IGS and ospC loci rrs-rrlA and ospC genotypes with early dissemination has been observed B. burgdorferi genotypes have been experimentally corroborated for selected strains in C3H/HeJ mice B. burgdorferi in Lyme disease patients indicating that a wide range of B. burgdorferi strains is capable of infecting humans. Clinical isolates were represented in all the identified clonal complexes suggesting that there is no link between MLST genotype and the propensity to cause human infection per se. However, there was an association of certain clonal complexes with the type of infection  suggesting that some clonal complexes of B. burgdorferi may have a greater propensity for hematogenous dissemination, while others, such as members of CC37, may be capable of only causing localized skin infection. Analysis of the population structure using much larger data sets, in combination with careful epidemiological sampling, is required to better understand the relationship between dissemination potential and clonal complex.Numerous ospC typing, suggesting that the pathogenic traits of B. burgdorferi correlate with the phylogenetic signal. Thus, association between ospC major groups and dissemination properties may be indirect and operate via strong linkage disequilibrium B. burgdorferi strains to cause disseminated infection in humans is a phenotypic characteristic of unknown origin. It is likely that B. burgdorferi interactions with its vectors and natural reservoir hosts throughout a long evolutionary course resulted in genotypes which in the human body display either disseminative or non-disseminative properties. Therefore, further studies of the evolutionary origins of different clonal complexes may help to predict the spatial and temporal occurrence of pathogenic B. burgdorferi strains.Interestingly, the data indicate that the power of MLST to predict the outcome of infection  was greater than that of ospC major groups A, B, H, I and K, isolated from patients in New York, have been identified predominantly as invasive genotypes, while some ospC major groups including T and U have been associated with localized skin infection ospC studies, isolates within CC37, associated with localized skin infection, belonged to either ospC major groups T or U, all but one isolate found in CC4, associated with disseminated infection, belonged to either ospC major groups K or H and all isolates within CC7 and CC16 that were also associated with disseminated infection were identified as members of ospC major groups B and I, respectively. In addition, isolates that belonged to a single ospC major group , but exhibited different dissemination properties, were further divided among distinct clonal complexes or clonal complexes and singletons. These differences could be explained by horizontal gene transfer and recombination at ospC that over time would result in the dissociation of ospC major groups from genes which contribute to the ability to disseminate. A number of studies provide genetic evidence of recombination and horizontal gene transfer at ospCPreviously, B. burgdorferi population structure is dominated by frequency dependent selection acting at ospCospC and other genomic markers, it has been proposed that ospC is a lineage-defining gene ospC and various chromosomal and/or plasmid-encoded loci B. burgdorferi is not subdivided in a manner that corresponds to ospC major groups. First, almost all ospC major groups were associated with multiple STs. Second, although most STs were comprised predominantly of isolates with the same ospC major group, related STs were assembled into clonal complexes that did not reflect a simple clonal descent of ospC major group within B. burgdorferi lineages. Taken together, the data indicate that while ospC major group may be a relatively stable characteristic of a ST, the use of ospC typing as a means of strain identification for epidemiological, ecological and population genetic studies is not reliable.It has been suggested that the B. burgdorferi. Only two STs were identified in patients from both locations. The presence of identical B. burgdorferi STs in I. scapularis ticks from the Northeast and Upper Midwest of North America have been previously explained as either a reminiscence of once overlapping populations of B. burgdorferi or the result of limited gene flow B. burgdorferi in Lyme disease patients from New York and Wisconsin captured by MLST in this study was similar to that previously reported in I. scapularis questing ticks from the Northeast and Upper Midwest of North America B. burgdorferi in humans and I. scapularis ticks are not surprising given that infection is acquired peri-domestically by I. scapularis tick bites ospC did not provide a clear signal of geographical structuring of B. burgdorferiospC major groups between patients from New York and those from Wisconsin, the vast majority of ospC major groups infected patients in both regions. It is conceivable that due to evolution of ospC in ancestral spirochete populations and balancing selection ospC major groups may be more obscure than that observed at MLST loci. Nevertheless, a large-scale geospatial analysis of ospC sequences from I. scapularis ticks demonstrated that despite a substantial overlap of ospC types in northeastern and midwestern populations, differences in frequency distribution allowed a subdivision at the longitude of 83°W B. burgdorferi observed at the MLST and ospC levels and its epidemiological implications warrant further investigation.Patients from New York and Wisconsin were infected with two distinct, but genetically and phylogenetically closely related, populations of B. burgdorferi diversity at the MLST loci. For example, ST52 strains possess a clpA allele that differs from its ancestral clpA allele at seven nucleotide positions and is shared by two STs that are members of different clonal complexes. It is very unlikely that seven independent point mutations would accumulate at a single locus while no changes would be present on the other seven loci, and that two unrelated STs would share alleles that arose by accumulation of seven point mutations. It can, therefore, be assumed that this allele arose through recombination rather than point mutation B. burgdorferi chromosome suggesting that apart from mutation and genetic drift, recombination may also have some impact on the diversification of B. burgdorferiThe MLST analysis performed in this study suggests that recombination was likely to generate some of the B. burgdorferi strains isolated from Lyme disease patients suggests the existence of B. burgdorferi lineages with differential pathogenic properties in humans. The data indicate that MLST strain identification is better suited for epidemiological, ecological and population genetics studies than is ospC typing and suggest that MLST provides finer resolution of differentially pathogenic B. burgdorferi strains than does ospC typing. The results further demonstrate that humans in New York and Wisconsin are infected with two separate, but genetically related, populations of B. burgdorferi. While the data provide new and important insights into the population structure of B. burgdorferi, a much larger collection of diverse isolates must be examined to better understand the full extent of B. burgdorferi strain diversity and population structure in Lyme disease patients, and relationships between human disease, STs and clonal complexes.In conclusion, MLST analysis of All human subjects from New York were enrolled in prospective studies approved by the Institutional Review Board (IRB) of New York Medical College for which they had provided written informed consent at the Lyme Disease Diagnostic Center between 1991 and 2006. Samples and data from Wisconsin patients were collected in the past as a part of routine care/diagnostic testing and were accessed under approved IRB protocols of the Marshfield Clinic Research Foundation. A waiver of informed consent has been granted by the IRB. All data were analyzed anonymously.B. burgdorferi clinical isolates recovered from 146 Lyme disease patients  from New York and Wisconsin  were analyzed in this study. Seventy six isolates were cultured from erythema migrans lesions (n = 60) or blood (n = 16) of patients diagnosed at the Lyme Disease Diagnostic Center at New York Medical College in Valhalla, NY between 1991 and 2005, and 70 isolates were recovered from erythema migrans skin lesions (n = 65) or cerebrospinal fluid specimens (n = 5) of patients diagnosed at the Marshfield Clinic in Wisconsin between 1993 and 2003. Specimens were collected and cultured as described elsewhere A total of 146 rrs-rrlA IGS and ospC loci B. burgdorferi genotypes found in the collection, 3 to 7 isolates per ospC major group were selected for this study. For those ospC major groups that consisted of isolates from skin and blood, representatives from both were included. No preselection was made for the Wisconsin isolates.The New York isolates analyzed in this study belong to a large collection of more than 400 clinical isolates that had been previously typed at the Patients included in this study were diagnosed with early Lyme disease and were classified as having either localized or disseminated infection. Localized infection was defined by a single culture positive erythema migrans skin lesion in the absence of a positive blood culture or clinical and/or microbiological evidence of dissemination to a second site. Disseminated infection was defined by a positive blood or cerebrospinal fluid culture, multiple erythema migrans lesions and/or neurological findings.B. burgdorferi was isolated from the clinical samples with either the IsoQuick kit  or the Gentra PureGene DNA Isolation Kit . Eight housekeeping loci  were amplified by nested PCR and sequenced using PCR primers in both directions  as described previously http://www.borrelia.mlst.net/) hosted at Imperial College London . STs of 23 clinical isolates used in this study have been previously reported DNA from low-passage (passages 1 to 5) ospC genotypes of Wisconsin clinical isolates, the ospC gene was amplified by PCR using OC6 (+) and OC623 (−) primers as described previously ospC groups, the ospC sequences were compared to existing sequences of major ospC groups found worldwide, according to the previously defined criteria To determine the For each unique ST the sequences of eight housekeeping loci were concatenated to produce an in-frame sequence of 4,785 bp. Multiple sequence alignment was generated with the ClustalW algorithm and BioEdit software I. scapularis nymphs and adults included in the analyses performed in this study were collected off vegetation, pets and humans in the northeastern and upper midwestern US and adjoining areas of Canada between 2004–2008 I. scapularis ticks because: (i) there was no temporal overlap between tick and patient samples, (ii) ticks were collected from much wider geographic areas than clinical samples, and (iii) clinical samples from New York were preselected based on their ospC major group.Maximum likelihood (ML) and maximum parsimony (MP) trees were constructed using PhyML 3.0 B. burgdorferi from the two geographic locations (New York and Wisconsin) a permutation test was performed as described previously ospC major groups were significantly (P<0.05) associated with an outcome of localized or disseminated infection. The correspondence analysis of clonal complexes was conducted in three ways using: (i) isolates that were members of major and minor clonal complexes, and isolates that were classified as singletons , (ii) isolates that were members of major and minor clonal complexes only, and (iii) isolates that belonged to major clonal complexes only. To compare the ability of clonal complexes versus ospC major groups to predict disseminated or localized disease, these three analyses were repeated with ospC major groups as the explanatory variables. Correspondence analysis was also used to compare the frequency distribution of ospC major groups from 291 skin isolates obtained from New York patients during 1991–2005 and published previously ospC typing was performed for this set of data. The level of significance was P<0.05 throughout.To test whether there are significant differences between the allelic profiles of Figure S1Results of correspondence analysis using only isolates belonging to major or minor clonal complexes. The x-axis indicates the coordinates of the individual data points. Coordinates for all localized and disseminated infection cases are indicated by the filled diamond and filled square respectively. The strength of association of individual clonal complexes (CCs) (graph A) and ospC major groups (graph B) with disseminated or localized infection is demonstrated by the position of unfilled triangles on the x-axis relative to the filled square and lozenge, respectively. The degree of influence of individual clonal complexes or ospC major groups in the correspondence analysis  is shown by their position along the y-axis. Identities of the particularly influential clonal complexes and ospC major groups are indicated.(TIF)Click here for additional data file.Table S1Properties of B. burgdorferi samples.(DOC)Click here for additional data file.Table S2Results of correspondence analysis assessing differences in frequency of ospC major groups in skin samples of Lyme disease patients from New York and Wisconsin.(DOC)Click here for additional data file.


Lawsonia intracellularis is a common cause of chronic diarrhoea and poor performance in young growing pigs. Diagnosis of this obligate intracellular bacterium is based on the demonstration of the microbe or microbial DNA in tissue specimens or faecal samples, or the demonstration of L. intracellularis-specific antibodies in sera. The aim of the present study was to evaluate a blocking ELISA in the detection of serum antibodies to L. intracellularis, by comparison to the previously widely used immunofluorescent antibody test (IFAT).Sera were collected from 176 pigs aged 8-12 weeks originating from 24 herds with or without problems with diarrhoea and poor performance in young growing pigs. Sera were analyzed by the blocking ELISA and by IFAT. Bayesian modelling techniques were used to account for the absence of a gold standard test and the results of the blocking ELISA was modelled against the IFAT test with a "2 dependent tests, 2 populations, no gold standard" model.At the finally selected cut-off value of percent inhibition (PI) 35, the diagnostic sensitivity of the blocking ELISA was 72% and the diagnostic specificity was 93%. The positive predictive value was 0.82 and the negative predictive value was 0.89, at the observed prevalence of 33.5%.The sensitivity and specificity as evaluated by Bayesian statistic techniques differed from that previously reported. Properties of diagnostic tests may well vary between countries, laboratories and among populations of animals. In the absence of a true gold standard, the importance of validating new methods by appropriate statistical methods and with respect to the target population must be emphasized. Lawsonia intracellularis is a common cause of chronic diarrhoea and poor performance in young growing pigs [L. intracellularis are monitored by serology and used, for instance, in the screening of herd prevalence and herd profiling.ing pigs ,2. In soing pigs . Cultivaing pigs . FurtherL. intracellularis antigen, and the results are visualised following incubation with anti-porcine fluorescein isothiocyanate conjugate. More recently a principally similar method, the immunoperoxidase monolayer assay (IPMA), has been developed [Several serological methods have been described. The first method commercially available was an immunofluorescent antibody test, IFAT , with a eveloped . The latL. intracellularis-specific antigenic epitopes such as LPS [L. intracellularis has been developed and is commercially available [L. intracellularis have been performed employing this ELISA but the results have only been presented in conference proceedings. The method is reported to have a sensitivity of 96.5% and specificity of 98.7% with less than 10% coefficient of variation [Furthermore, several enzyme-linked immunosorbent assays have been described. Such methods include indirect ELISAs employing whole-cell antigens ,8, DOC -h as LPS , LsaA [1h as LPS , or the h as LPS . The stavailable . To ensuariation .However, properties of diagnostic tests may well vary among populations of animals . TherefoL. intracellularis, by comparison to the previously widely used IFAT. Bayesian modelling techniques were used to account for the absence of a gold standard test. In addition, the current infection status of the animals was also estimated by other techniques.The aim of the present study was to evaluate the blocking ELISA in the detection of serum antibodies to The study was approved by the Ethical Committee for Animal Experiments, Uppsala, Sweden.Altogether, 176 pigs from 24 herds were included. Sera had been collected during two previous studies, A , and werTheoretically, the number of samples required validating an assay at 0.95 confidence levels with a 2% error allowed and an estimated diagnostic sensitivity (DSn) of 0.965 should be 324, and the number of samples required to obtain an estimated diagnostic specificity (DSp) of 0.987 should be 123. With a 3% error allowed, the number of samples would be 144 and 55, respectively .Lawsonia intracellularis, Brachyspira spp, Campylobacter spp, Clostridium perfringens, Escherichia coli, Salmonella spp, Yersinia spp, parasites and rotavirus [L. intracellularis and for L. intracellularis DNA by nested PCR, respectively [In study A, the pigs were transported to the laboratory, stunned and exsanguinated, and a blood sample was collected. At necropsy, individual samples were analysed for the presence of otavirus . In studectively .Lawsonia intracellularis monoclonal antibody for one hour at 37°C. Following washing, substrate were added, incubated for 10 minutes at room temperature, stop solution was added and the resulting OD value was read within 15 minutes at 450 nm. As it is a blocking ELISA, a negative result would thus have a high absorbance value. The absorbance values were then converted to the calculated percent inhibition (PI) by the formulaSera were analysed by the blocking ELISA . Positive and negative control sera were supplied by the manufacturer. Test sera were added in duplicate, incubated for one hour at 37°C, washed three times and incubated with horseradish peroxidase-conjugated anti-L. intracellularis by comparison to the positive and negative controls included.Sera were also prepared by the IleiTest and analysed according to the manufacturer's instructions . The slides were read in a fluorescence microscope at 470 nm in 100 × magnification  and all samples were independently examined by three persons and judged as positive or negative with respect to the presence of antibodies to http://www.epi.ucdavis.edu/diagnostictests. This method does not employ a gold standard but requires results from two tests in two populations that differ in the prevalence of the disease. In this analysis, animals in study A and study B were considered as the two populations.The estimation of the diagnostic sensitivity and specificity of the blocking ELISA was performed in WinBUGS version 1.4  using Bae.g. sensitivity and specificity of the tests. Such prior information is often specified either from published papers or expert's best guess and the uncertainty is modelled through the use of beta distributions [i.e. rather diffuse distributions [http://www.epi.ucdavis.edu/diagnostictests.The method also requires prior information about some of the unknown parameters, ibutions . The priibutions . The betEstimates of sensitivity and specificity for the ELISA was first determined at PI-values ranging from 10 to 80 (by increments of 2), with results plotted in a two-graph receiver operating characteristic curve. Subsequently, estimates at PI-values 25 and 35 were also determined. All models were run with 100,000 iterations, where the initial 10,000 iterations were considered as burn-in and discarded from the evaluation.L. intracellularis DNA was demonstrated by PCR in 29 of 54 animals from the poor performance herds. By IFAT, 47 sera from these pigs were judged as positive, while 37 and 34 sera were positive by the ELISA using cut-off levels of PI 30 and 35, respectively. L. intracellularis was not demonstrated in any of the pigs (n = 12) from the good performance herds, and by the ELISA, PI was <21 in all pigs, whereas three pigs were positive by IFAT. In study B, L. intracellularis DNA was demonstrated in 24 of 110 animals from 5 herds. According to the IFAT, 20 animals from 4 herds were seropositive. By employing a cut-off value of PI 30 in the ELISA, 46 animals from 9 herds were seropositive. Using a cut of value of PI 35, 25 animals from 5 herds were seropositive  and an estimated prevalence of 30%. The corresponding negative predictive value would be 0.89.Using PI 30 as cut-off, the IFAT and the blocking ELISA had an observed agreement of 70%. Using PI 35 as cut-off, the agreement was 80% Table . The posi.e. used as gold standard) and the known sensitivity and specificity of the reference test are not a prerequisite.To assess the performance of a new method, the diagnostic sensitivity (DSn) and specificity (DSp) should be determined in reference samples with known history and infection status ,9,22. HoL. intracellularis by PCR on faecal samples and animals from a high health herd where L. intracellularis had not been demonstrated) were analysed by both methods and compared by a 2 × 2 table using the IFAT as gold standard. This resulted in 100% specificity and sensitivity for both methods employed (data not shown).To further illustrate the importance of evaluating the method in the target population with a "natural" distribution of positive and negative animals, reference samples from two populations with known disease status  reported a good reproducibility between different laboratories. Further, in comparison to IFAT, the ELISA provided a higher sensitivity and more unambiguous results. By comparison to an in-house IFA, the DSn and DSp were estimated to 92 and 98%, respectively [Several cross-sectional studies on the serological prevalence of ectively . Howeverectively ,21,24.L. intracellularis-specific antibodies was first detected 2 weeks after challenge [The results partly reflect the difficulties to clinically assess the stage of infection. Based on previous results, >40% of the pigs aged 9 to 15 weeks with clinical signs indicative of proliferative enteropathy were expected to shed the microbe in faeces ,26. Expehallenge . The purhallenge . Study Bhallenge . Furtherhallenge ,26.Several other methods are reported to have a good performance with high DSn and DSp ,9,22. HoL. intracellularis or chronic intestinal lesions are rarely reported [On the other hand, several studies report a high prevalence 90-100%) of seropositive finisher pigs close to slaughter (25-27 weeks of age), despite that shedding of 0-100% ofreported . This mareported ,26, and reported ,21, the reported . This waIn conclusion, the diagnostic sensitivity of the blocking ELISA was 72% and the diagnostic specificity was 93%, as evaluated by Bayesian statistic techniques. This technique allows the validation of a diagnostic method also without a true gold standard. The positive predictive value was 0.82 and the negative predictive value was 0.89, when the analysis was applied on samples from growing pigs in the age of 8-12 weeks. The sensitivity and specificity demonstrated in the present study differed from that previously reported and the importance of validating new methods with respect to the target population was emphasised.The authors declare that they have no competing interests.MJ, PW, AN and MM participated in the design of the study. MJ were responsible for the coordination of the work, for collecting the samples, for the PCR analyses and drafted the manuscript. PW were responsible for the serological analyses. UE were responsible for the statistical analyses and drafted this part of the manuscript. All the authors red and approved the manuscript.


Remodeling of lung tissues during the process of granuloma formation requires significant restructuring of the extra-cellular matrix and cathepsins K, L and S are among the strongest extra-cellular matrix degrading enzymes. Cathepsin K is highly expressed in various pathological granulomatous infiltrates and all three enzymes in their active form are detected in bronchoalveolar lavage fluids from patients with sarcoidosis. Granulomatous inflammation is driven by T-cell response and cathepsins S and L are actively involved in the regulation of antigen presentation and T-cell selection. Here, we show that the disruption of the activities of cathepsins K, L, or S affects the development of lung granulomas in a mouse model of sarcoidosis.Apolipoprotein E-deficient mice lacking cathepsin K or L were fed Paigen diet for 16 weeks and lungs were analyzed and compared with their cathepsin-expressing littermates. The role of cathepsin S in the development of granulomas was evaluated using mice treated for 8 weeks with a potent and selective cathepsin S inhibitor.Apoe-/- mice treated with a selective cathepsin S inhibitor did not develop lung granulomas and only individual epithelioid cells were observed.When compared to wild-type litters, more cathepsin K-deficient mice had lung granulomas, but individually affected mice developed smaller granulomas that were present in lower numbers. The absence of cathepsin K increased the number of multinucleated giant cells and the collagen content in granulomas. Cathepsin L deficiency resulted in decreased size and number of lung granulomas. Cathepsin K deficiency affected mostly the occurrence and composition of lung granulomas, whereas cathepsin L deficiency significantly reduced their number and cathepsin S inhibition prevented the formation of granulomas. Apoe-/- mice fed a cholate-containing high fat diet develop lung granulomas that have many features of human granulomas of sarcoidosis [Cathepsins K, L and S are members of the papain family of cysteine proteases that have been recently implicated in the development of various lung diseases . It is bcoidosis . EpithelSimilarly to cathepsin K, cathepsins L and S are able to degrade major extracellular proteins  but theyApoe-/- mice on Paigen diet.Thus, based on the strong expression of cathepsin K in granulomas and the pivotal role of cathepsins S and L in the antigen presentation and T-cell selection, we hypothesized that the disruption of these protease activities might interfere with lung granuloma formation. In this study, we investigated the effect of cathepsin K and L deficiencies and cathepsin S inhibition on the development of lung granulomas in Apoe-/-) mice (Jackson Laboratories), double knockout mice lacking apolipoprotein E and cathepsin K (Apoe-/- Ctsk-/-) (n = 10) and double knockout mice lacking apolipoprotein E and cathepsin L (Apoe-/- Ctsl-/-) (n = 19). Double knockout Apoe-/- Ctsk-/- and Apoe-/- Ctsl-/- mice were generated by crossing Apoe-/- with the Ctsk-/- or Ctsl-/- mice as previously described [Ctsk-/- and Ctsl-/- mice were kindly provided by Drs. P. Saftig  and C. Peters . The single cathepsin deficiencies have the following phenotypes: Cathepsin K-deficient mice show an osteopetrotic phenotype with excessive trabeculation of the bone-marrow space [Three groups of mice were used: apolipoprotein E-deficient  and another received the same diet plus 0.01% w/w cathepsin S inhibitor (n = 12). The medicated diet was formulated by mixing the chow in meal form with finely dispersed crystalline, orally-active inhibitor to obtain a uniform distribution. The cathepsin S inhibitor  between the two groups. Control Apoe-/- mice used for CD4+ cell quantification in lungs were on normal diet for 22 weeks. Single cathepsin K and L null mice on high fat diet were not evaluated as previous experiments revealed that the induction of lung granuloma formation by high fat diet was dependent on the apolipoprotein E deficiency [The following primer sequences were used for ApoE: 5"-GCCTAGCCGAGGGAGAGCCG-3"; 5"-TGTGAC TTGGGAGCTCTGCAGC-3"; 5"-GCCGCCCCGACTGCATCT-3"; for cathepsin K: 5"-GCCACACCCACACCCTAGAAG-3"; 5"-ACA AGT GTA CAT TCC CGT ACC-3"; and for cathepsin L: 5"-GGAGGGAGAGCGATATGGG-3"; 5"-TTCCTCATTGGTCTTCCG-3"; 5"-CGGAGAACCTGCGTGCAATCC-3". At 6 weeks of age mice were switched from normal to Paigen fat diet (HFD) which contained 18% fat, 1% cholesterol and 0.5% cholate; Test diet 57W4; Purina Mills. LLC, St. Louis, MO). After 16 weeks on HFD, mice were euthanized after overnight fasting by exsanguination under xylazine-ketamine anesthesia. Another two groups of cpd 6 in ) was doscpd 6 in ) which iAfter perfusion with ice-cold phosphate-buffered saline (PBS), lungs and thymuses were fixed in 10% buffered formalin for 5 hours at 4°C, embedded in paraffin, and 5 μm sections were cut and mounted onto Superfrost/Plus slides .Sections were dewaxed, rehydrated, blocked with 10% goat serum in PBS, and incubated overnight at 4°C with the primary antibody in 1% BSA in PBS: affinity purified rabbit polyclonal anti-cathepsin K (1: 50) , mouse mSections were deparaffinized, hydrated with a graded alcohol series and distilled water, and placed in preheated Bouin's solution at 58°C for 10 min. Sections were stained in Weigert's iron hematoxylin solution for 5 min, washed under running water for 30 sec, stained with Gomori's trichrome solution  for 15 min, placed in 0.5% acetic acid for 10 sec, and then mounted for light microscopy.2. Trichrome stained sections were used for the measurement of granuloma size and for the quantification of collagen content. Images were captured with a Leica DMI 6000B microscope  and analyzed with Openlab 3 software.The areas of granulomas stained positively for Mac-3, osteopontin, CD4 and CD8, and collagen were quantified using color threshold and calculated as percentage of granuloma area. The size of granulomas was measured in mmFive sections 200 μm apart from each other were analyzed for each lung. For CD4-positive cell quantification three pictures per lung section were taken using a 40-fold magnification, avoiding areas with dominating tracheal or bronchial tissue. The average number of granulomas and multinucleated giant cells per section and the average number of CD4-positive cells per captured area for each mouse were used for statistical analyses.Student's t-tests were performed when values past test for normal distribution. Data are presented as mean ± standard deviation. Discontinuous data (such as the number of granulomas) were analyzed using the Mann-Whitney U-test. Significance was concluded when P < 0.05.Apoe-/- mice receiving HFD develop epithelioid lung granulomas with characteristic features of granulomas of sarcoidosis [Recently, we described that coidosis . Granulocoidosis . Figure Apoe-/- mice, Apoe-/- Ctsk-/- mice receiving HFD showed fibrotic changes in lung tissues and the presence of such inclusions as Schaumann's bodies and crystalline structures  which are usually induced by inflammatory reactions and are present in bronchovascular bundles [Apoe-/- mice, 41% of the animals displayed lung granulomas, whereas in their cathepsin K-deficient littermates this number reached 88%  and osteopontin positive cells (epithelioid cells marker) in Apoe-/- Ctsk-/- mice when compared to their cathepsin K-expressing littermates  but granulomas were still well-formed  correlates with the increased collagen fibers deposition (10% vs 20%) that may consequently result in a delayed granuloma resolution. Lung granuloma formation and their spontaneous resolution as commonly observed in patients with sarcoidosis [Ctsk-/- mice is due to the lack of the collagenase activity of cathepsin K. This accumulation of collagen fibers might also be responsible for the smaller number and size of granulomas. In our measurements, accumulations of epithelioid cells were considered as granulomas only when they were present as well-formed and ball-shaped structures that are characteristic for sarcoidosis. In Apoe-/- Ctsk-/- mice, epithelioid cells were often observed forming ring-like structures with dispersed collagen fibers, or were randomly distributed in fibrotic areas (Figure The formation of lung granulomas results in an extensive extracellular matrix remodeling. Collagen and elastin are the major components of the pulmonary matrix and it is believed that a cooperative action of multiple enzymes is required for its turnover. Cathepsin K has a unique ability to cleave highly efficiently triple helical collagen at multiple sites and to act as a potent elastase as well . Its strcoidosis  require s Figure .Apoe-/- Ctsk-/- mice, macrophages have an increased size [The cell composition of granulomas in cathepsin K-deficient mice was also changed. They contained a significantly higher number of MGCs. This type of cells is formed by the fusion of cells from the monocyte-macrophage lineage . In athesed size  and micrsed size . Since Csed size , we sugg+ T-cells [+ T cell selection in the thymus [+ cells in thymus and peripheral organs [+ may have a direct influence on sarcoidosis development. T cell activation is mandatory for the development of granulomatous reactions and CD4+ T cells of the Th1-type are essential for the formation and the maintenance of granulomas [+ cells in lungs of mice lacking cathepsin L or treated with cathepsin S inhibitor (Figure Analysis of available literature data suggests that the preventive effect of cathepsin L deficiency or cathepsin S inhibition on lung granuloma development may not only depend on their extracellular matrix-degrading activities, but on their involvement in antigen presentation as well ,38. Cath T-cells . On the e thymus . Cathepsl organs . The rolanulomas -21. The r Figure  supportsApoe-/- Ctsl-/- mice had significantly smaller thymi compared to Apoe-/- mice (Figure In addition to its involvement in antigen presentation, cathepsin L has been described to play a role in T-cell actin polymerization, shape polarization, chemotaxis. Cathepsin L deficiency significantly decreases the expression of laminin, fibronectin, and collagens I and II in thymus of cathepsin L-deficient mice . This mie Figure .In conclusion, our results show that the disruption of cathepsin L and S activities prevents the development of lung granulomas, whereas cathepsin K deficiency results in altered granuloma composition in mice. These results suggest that interventions in cathepsin S and L activities may yield a therapeutic benefit for patients with sarcoidosis, i.e., a potential prevention of disease progression. Such an approach, however, would require extreme caution as both cathepsins have been described as critical participants in antigen presentation and their inhibition may result in increased infection and cancer rates.The authors declare that they have no competing interests.AOS designed and carried out experiments, analysed data and wrote the paper, JYG carried out experiments with cathepsin S inhibitor, MDP designed and carried out experiments with cathepsin S inhibitor and wrote inhibitor-related sections of the paper, DB conceived experiments, analysed data and wrote the paper. All authors have approved the final manuscript.


The goal of the present study was to synthesize mucoadhesive polymer – thiolated chitosan (TCS) from chitosan (CS), then prepared CS/TCS-sodium alginate nanoparticles (CS/TCS-SA NPs), determined which was more potential for ocular drug delivery.A new method for preparing TCS was developed, and the characteristics were determined using Fourier transform infrared spectroscopy and the degree of thiol immobilized was measured by Ellman’s reagent. Human corneal epithelium (HCE) cells were incubated with different concentrations of TCS for 48 h to determine the cell viabilities. CS/TCS-SA NPs were prepared and optimized by a modified ionic gelation method. The particle sizes, zeta potentials, Scanning electron microscopy images, mucoadhesion, in vitro cell uptake and in vivo studies of the two types of NP were compared.The new method enabled a high degree of thiol substitution of TCS, up to 1,411.01±4.02 μmol/g. In vitro cytocompatibility results suggest that TCS is nontoxic. Compared to CS-SA NPs, TCS-SA NPs were more stable, with higher mucoadhesive properties and could deliver greater amounts of drugs into HCE cells in vitro and cornea in vivo.TCS-SA NPs have better delivery capability, suggesting they have good potential for ocular drug delivery applications. Several challenges exist in the treatment of ocular diseases because eyes have special and complex anatomic structures. The main problem is to maintain an effective drug concentration at the site of action for an appropriate period of time, to improve the ocular bioavailability of a drug . For thiChitosan (CS), a cationic polysaccharide, has been widely used in ophthalmic therapy . CS is aTo solve this problem, thiolated chitosan (TCS), comprising a new generation of mucoadhesive polymers – thiolated polymers or thiolmers  – has beMethods for TCS preparation are well known -15. It iSodium alginate (SA) is another interesting polysaccharide that has been used in several ocular delivery systems, either alone or in combination with other materials ,17. The In the present study, we first produced TCS using a new method and measured its characteristic by determining the degree of thiol group substitution using Ellman’s reagent and Fourier transform-infrared (FT-IR) analysis. We then prepared CS/TCS-SA nanoparticles (CS/TCS-SA NPs), optimized the formulation and analyzed nanoparticle characteristics, including particle size, zeta potential, scanning electron microscopy (SEM), and performed mucoadhesion studies with mucin. Furthermore, to confirm that TCS-SA NPs can deliver greater levels of drug into target sites than CS-SA NPs, we chose human corneal epithelium (HCE) cells to measure the degree of in vitro cell uptake, also, in vivo studies were performed in rat corneas by stereomicroscope and confocal laser scanning microscopy (CLSM).Chitosan , and degree of deacetylation 75%–85%), TGA , Ellman's reagent , ≥98%), and mucin  were obtained from the Sigma-Aldrich, St Louis, MO. EDAC·HCl (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, 99%) and NHS  were purchased from J&K China Chemical . FITC  was provided by Alfa Aesar Chemical . DMF  and SA  for a 1% solution at 20 °C) were purchased from Shanghai Chemical Co. Ltd . All other chemicals were reagent grade and were used as received.Male Wistar rats  were used for this study. The rats were kept in standard environment throughout the study as follows: room temperature 25±2 °C, relative humidity 60±10%, and alternating 12 h light-dark cycles (8 AM to 8 PM). All procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Cision Research.Our new method includes two steps. The reaction scheme is shown in The degree of thiol group substitution in the modified polymers was determined spectrophotometrically using Ellman’s reagent, as described in a previous study . Ellman’The FT-IR (Fourier transform-infrared) spectra of chitosan and TCS were determined on a Nicolet avatar 330 FT-IR  using the KBr (potassium bromide) method. TCS was confirmed by the presence of the characteristic peaks of newly formed amide bonds and thiol groups.Nanoparticles of CS or TCS with SA were prepared by a modified ionic gelation method as reported by Calvo et al. . Based oNanoparticles were formed by interaction between the negative groups of alginate and the positively charged amino groups of CS or TCS. High concentrations of CS/TCS (1.5 mg/ml) and SA  resulted in the formation of a high concentration of fibrous aggregates. In subsequent formulations, by reducing the SA concentration to 0.625 mg/ml and changing the concentration of CS/TCS from 0.5 to 1.5 mg/ml, we found the aggregation was reduced or even eliminated, respectively. Also sizes were significantly decreased and TCS-SA NPs had a smaller size than CS-SA NPs at the same concentration. It was observed that the concentration of CS/TCS (0.5 mg/ml) and SA (0.625 mg/ml) resulted in the formation of particles without aggregation, even after the particles were left overnight with stirring. This formulation was selected for subsequent studies and characterization as well as for entrapment of the model drug.To determine cellular uptake, we chose FITC (fluorescein isothiocyanate) as a model drug encapsulated into the nanoparticles. FITC has excitation and emission peak wavelengths of approximately 494 nm and 520 nm, respectively. It is prone to photobleaching, which is suitable for tracking nanoparticles. The packed dye acts as a probe for NPs and offers a sensitive method for determining their intracellular uptake and retention. For FITC-loaded nanoparticles, 2 mg of FITC was dissolved in 100 μl of DMF, then added into the CS/TCS solution under gentle stirring before SA solution was added dropwise. The resultant NPs were collected by centrifugation , then the pellet was redispersed in water and lyophilized. All the operations were shielded from light because FITC is sensitive to light.The mean size, size distribution and zeta potential of the prepared blank and FITC-loaded CS/TCS-SA NPs were determined by dynamic light scattering using a Zeta Nanosizer .The surface morphology of FITC-loaded nanoparticles was imaged on a LEO 1530 scanning electron microscope  with a field emission electron gun. Nanoparticle suspensions were fixed on an aluminum disk and dried at room temperature. The dried nanoparticles were coated with gold metal using a sputter coater.x, of 494 nm and emitted wavelength, Em, of 520 nm. FITC is a pH-sensitive fluorescent label indicator [The encapsulation efficiency (EE) was determined after isolating the NPs from a nanoparticle suspension containing free FITC by centrifugation . The amount of free FITC in the supernatant was measured by fluorometric analysis  at an excitation wavelength, Endicator  so the sEqual volumes of mucin solution (0.4 mg/ml) and FITC-loaded CS/TCS-SA NPs were vortexed for 1 min and the zeta potential of the mixtures were measured by the Zeta Nanosizer.−1 penicillin and 100 μg/ml streptomycin in 37 °C, 5% CO2.HCE cell line was cultured on a 100 mm cell culture plate in DMEM medium supplemented with 10% calf serum, 100 U ml4 cells per well for 24 h. The medium was replaced by 0.2 ml of cell medium containing desired concentrations of TCS. After incubation for 12 h, cell viabilities were measured by the standard MTT (methyl thiazolyl tetrazolium) assay.TCS was dispersed in a sufficient quantity of culture medium to prepare a series of concentrations from 100 to 1,000 ppm, with normal cell culture wells without polymers used as negative controls. For the in vitro cell toxicity assay, cells were plated in a 96-well plate at a density of about 1×104 cells per well. After incubation in fresh medium for 24 h, cells were incubated with 0.5 ml of medium containing 100 ppm FITC of CS/TCS-SA NPs suspensions for 8 h. The cell medium was removed and the cells were washed before PBS buffer solution was added. The samples were then analyzed by fluorescence microscope .Isolated FITC-loaded CS/TCS-SA NPs were resuspended in culture medium for fluorescence microscopy studies. HCE cells were plated on a 24-well plate at a density of ~0.5×10FITC-loaded CS/TCS-SA NPs were concentrated by centrifugation, and resuspended in a small volume of PBS to obtain 1 mg FITC per mL of the nanoparticle suspension. The naked FITC was dissolved in 10 μl DMF, then diluted with 5 ml PBS (pH 7.4) to gain the same concentration of 1 mg/ml with FITC-loaded CS/TCS-SA NPs suspension. We can ignore the effect of DMF to eyes for its trace amount. Normal conscious rats were randomly divided into three groups (n=5 per group), placed in a restraint box and 4 instillations of 15 μl of the FITC solution, FITC-loaded CS/TCS-SA NPs were topically administrated in the cul-de-sac of the right eye at 20 min intervals, while PBS was instilled in the left eye as control.One and a half hour after the last instillation, the eyes were washed with PBS and examined in a stereomicroscope connected to a digital camera . After that, rats were sacrificed and the corneas were excised, embedd in OCT  medium and freezed in liquid nitrogen, then were performed on cryosections (6 μm thick) of the corneas. Latter, the samples were fixed in acetone at −20 °C for 10 min, blocked with DAPI , mounted, and photographed using a confocal laser scanning microscope .All experiments were repeated at least three times. ANOVA  was used for statistical analysis. All the data are expressed as mean±SD and a p value <0.05 was deemed to be statistically significant.It was demonstrated that TCS synthesized by the new method described above was more efficient than previous methods in which CS, TGA, coupling agent EDAC·HCl and NHS were mixed together in water. The scheme of the one-step reaction is shown in −1, corresponding to the vibration of the S-C bond, C=O double bonds of the amido bond and the H-S bond, respectively, while the peak at 2,923 cm−1 is attributed to stretching of the C-H bond [The FT-IR spectra of CS and TCS are shown in C-H bond ,25.The relative cell viabilities of HCE cells incubated with different concentrations of TCS for 48 h were measured by the MTT assay. As shown in The nanoparticles were formed as a result of the interaction of the positively charged amino groups of CS/TCS and negatively charged SA. For TCS, a portion of primary amine groups were coupled with thiol groups In other words, the resulting positively charged amino groups were decreased. This may explain how SA could interact with more TCS than CS without aggregation . In addiIn the best possible formulation of the nanoparticles, CS/TCS (0.5 mg/ml) and SA (0.625 mg/ml) loaded FITC demonstrated a mean diameter of 471.0±6.4 and 265.7±7.4 nm for CS and TCS, respectively. The formulated particles were positively charged, which may be due to free residual amine groups not neutralized by the carboxyl groups of alginate . For blaPositively charged colloidal particles could increase electrostatic interaction between the particles and the negatively charged mucosal surface, thus leading to improved bioavailability and reduced side-effects. Addition of mucin reduced the zeta potential of the prepared NPs . This reIn contrast to FITC loaded in NPs, free FITC molecules were highly fluorescent. We could take advantage of this property to monitor drug release through fluorescence signals. The release of FITC loaded in CS/TCS-SA NPs was studied in HCE cells. To observe the intracellular release of the drug in living cells by fluorescent microscopy, the cells were seeded on glass coverslips and incubated for 24 h. The cells were then further incubated in a medium containing FITC-loaded CS/TCS-SA NPs for 8 h. As revealed by fluorescence micrographs , intenseAs shown in A new method for preparation of TCS was established, and it achieved a high degree of thiol substitution, of up to 1,411.01±4.02 μmol/g, as determined by Ellman’s reagent. In addition, results of the MTT assay  indicated the safety of TCS toward HCE cells. The prepared CS/TCS-SA NPs were characterized by particle size, zeta potential, SEM and mucoadhesion studies. Overall, the results, including smaller size (265.7±7.4 to 471.0±6.4 nm), higher positive charges (49.2±2.3 to 29.5±4.1 mV) and higher mucoadhesion properties, suggest that TCS-SA NPs are more stable and more effective than CS-SA NPs as an ocular drug delivery system. The in vitro cell uptake study and in vivo experiments provide further evidence to support these conclusions.