Lycium barbarum polysaccharides (LBP), extracts from the wolfberries, are good for “eye health” according to Chinese medicine. The aim of our present study is to explore the use of LBP in retinal I/R injury. Retinal I/R injury was induced by surgical occlusion of the internal carotid artery. Prior to induction of ischemia, mice were treated orally with either vehicle (PBS) or LBP (1 mg/kg) once a day for 1 week. Paraffin-embedded retinal sections were prepared. Viable cells were counted; apoptosis was assessed using TUNEL assay. Expression levels of glial fibrillary acidic protein (GFAP), aquaporin-4 (AQP4), poly(ADP-ribose) (PAR) and nitrotyrosine (NT) were investigated by immunohistochemistry. The integrity of blood-retinal barrier (BRB) was examined by IgG extravasations. Apoptosis and decreased viable cell count were found in the ganglion cell layer (GCL) and the inner nuclear layer (INL) of the vehicle-treated I/R retina. Additionally, increased retinal thickness, GFAP activation, AQP4 up-regulation, IgG extravasations and PAR expression levels were observed in the vehicle-treated I/R retina. Many of these changes were diminished or abolished in the LBP-treated I/R retina. Pre-treatment with LBP for 1 week effectively protected the retina from neuronal death, apoptosis, glial cell activation, aquaporin water channel up-regulation, disruption of BRB and oxidative stress. The present study suggests that LBP may have a neuroprotective role to play in ocular diseases for which I/R is a feature.Neuronal cell death, glial cell activation, retinal swelling and oxidative injury are complications in retinal ischemia/reperfusion (I/R) injuries.  Many ocular diseases such as acute glaucoma and amaurosis fugax may be associated with ischemia/reperfusion (I/R). During I/R, there are breakdown of blood-retinal barrier (BRB), accumulation of fluid within retina, oxidative stress and neuronal death Lycium barbarum , a traditional Chinese medicine, has been used for centuries in the East to maintain “eye health and nourish the liver and kidney”, and to balance “Yin” and Yang” in the body Lycium barbarum has a high content of polysaccharides which is approximately 40% by dry mass; therefore studies have been focused to the liquid fraction of the berries, the Lycium barbarum polysaccharides (LBP) The use of animals in this study was conducted according to the requirements of the Cap. 340 Animals (Control of Experiments) Ordinance and Regulations, and all relevant legislation and Codes of Practice in Hong Kong. All the experimental and animal handling procedures were approved by the Faculty Committee on the Use of Live Animals in Teaching and Research in The University of Hong Kong (CULATR #1870-09).C57BL/6N male mice (10–12 weeks old) were used in the present study. The animals were kept in a temperature-controlled room with 12-hr light/12-hr dark cycle in the Laboratory Animal Unit of The University of Hong Kong.LBP extracts were prepared as described previously 2O/30% O2 for induction and 1% halothane in 70% N2O/30% O2 for maintenance) and the rectal temperature were kept at 37±0.5°C. The internal carotid artery (ICA) is one of the bifurcations of the common carotid artery and provides blood supply to the cerebral regions. It also provides blood supply to the eye as the ophthalmic artery is a branch of the pterygopalatine artery (PPA) which originates from the ICA Animals were anesthetized .2. Viable cells in ganglion cell layer (GCL) of the central and peripheral retina were counted. Cells with pyknotic nuclei were regarded as dead cells and were not counted in the counting. Retinal swelling in our I/R model has been shown to be due to the increase in inner retinal thickness Procedures described in our recently published study were adopted 2. TUNEL-positive nuclei in both GCL and inner nuclear layer (INL) were counted.Apoptosis was examined by TUNEL assay  as previously described Antigen retrieval was performed by incubation with proteinase K following deparaffinization of sections. Non-specific binding sites were blocked with normal goat serum for 1 hr at room temperature and then incubated with primary antibodies listed in Sections were blocked with Mouse on Mouse (MOM) blocking solution  and incubated with mouse biotinylated anti-mouse IgG secondary antibody (MOM kit) as previous described The right eye of each animal was subjected to I/R, and the left eye was used as a non-ischemic control. The retinae of right eyes from animals pre-treated with vehicle were compared with those from the non-ischemic control left eyes of both groups. The retinae of right eyes from animals pre-treated with LBP were compared with those from animals pre-treated with vehicle. A double-blinded approach was used in all the experimental procedures, including cell counting and data analyses. Data were presented as mean ± SEM. One-way Analysis-of-variance (ANOVA) followed by the Bonferroni multiple comparison tests  was used in cell counting and IgG extravasations quantification. Kruskal-Wallis Test followed by the Dunn's multiple comparison tests (Prism v4.0) was performed in the semi-quantitative analyses of IHC scores. Statistically significant difference was set at p<0.05.2-hr ischemia followed by 22-hr reperfusion caused severe tissue damage in the vehicle-treated retina. Many cells in the GCL and INL were pyknotic in the central and peripheral retina . The numThere was an increase in inner retinal thickness of the central retina in the vehicle-treated mice as compared with that in the non-ischemic control mice  . On the Ischemia leads to apoptosis in the retina. More TUNEL-positive cells were observed in the GCL of the vehicle-treated I/R retina compared with that of the non-ischemic control retina   and simiPKC-α is a marker for rod bipolar cells. Normally, the cell bodies of PKC-α-positive bipolar cells are located in the outer border of INL and their axons terminate at the inner border of IPL In non-ischemic control retina, calretinin-positive amacrine cells were present in GCL and the innermost layer of INL. The synaptic terminals of amacrine cells formed three distinct strata in IPL . In the In the normal retina, a small population of amacrine cells expresses nNOS In non-ischemic control retina, the expression of GFAP was confined to astrocytes in GCL . In the In the non-ischemic control retina, there was a weak immunoreactivity of AQP4 expressed in the astrocytic end-feet at the ILM and in the perivascular regions at the outer border of INL . The AQPBlood vessel leakage was assessed by IgG extravasations. In the vehicle-treated I/R retina, when compared with the non-ischemic control, there were greater number of blood vessels with immuno-positive IgG signals located outside the endothelial vessel lining. These vessels were situated at the GCL  and OPL Lipid peroxidation was evaluated using PAR immunohistochemistry. In non-ischemic control retina, PAR expression was limited inside the cytoplasm . A profoNT is a marker for nitrosative stress. Minimal NT immunoreactivity was noted in non-ischemic control retina . An incrBRB disruption, glial cell activation, oxidative stress and neuronal death are major concerns in retinal I/R injury. Neuroprotective agents that can retard or prevent these damages are beneficial in treating many ocular diseases in which retinal I/R is a complication. In our present study, we showed that pre-treating mice with LBP for 1 week could protect the animals from I/R injury by reducing neuronal cell death, retinal swelling, glial activation, BRB disruption and oxidative stress.In the present study, unilateral retinal ischemia was induced by occluding the ICA which supplies blood to the ophthalmic artery Lycium barbarum is a dried fruit that is used as a food or a medicine according to Chinese tradition Retinal I/R induces neuronal death in the inner retina, especially RGC. An extensive loss of cells in the GCL has been seen after retinal I/R injury BRB is a protective barrier which consists of the outer and inner BRB. The role of BRB is to maintain the homeostatic condition of retinal microenvironment and exclude harmful substance getting into the retina + and the movement of water inside cells Disruption of BRB leads to swelling of astrocytes and Muller cells processes associated with the activation of GFAP and AQP4 under ischemic conditions Lycium barbarum, increases glutathione peroxidase activity and glutathione levels, and decreases cystine concentrations in retina of rd1/rd1 mice, indicating a decreased level of oxidative stress in retinal generation Oxidative stress plays a role in retinal I/R injury due to the high content of polyunsaturated fatty acid in retina. Reactive oxygen species (ROS) and free radical formation during I/R facilitates lipid peroxidation of membrane, denature of protein and DNA damage Increasing lines of evidence have demonstrated that modulation of immune responses can affect the degenerative processes of neurons in the CNS Our data indicated that pre-treatment with LBP for 1 week could effectively protect the mouse from retinal I/R injury. The present study hence suggests that LBP may be used as a preventive TCM for diseases associated with I/R such as amaurosis fugax and acute glaucoma. Further studies are warranted to evaluate the therapeutic effects of LBP and other potential neuroprotective mechanisms.


Although knee pain is common in older persons and can cause ambulatory limitation, its impact on self-reliance has rarely been examined in Japan, particularly in a community setting. The aim of this 3-year cohort study was to investigate the association of knee pain with dependence in activities of daily living (ADL) and mortality in community-dwelling older Japanese adults.In 2005, presence of knee pain was assessed by a home visit survey of 1391 older adults aged 65 years or older (participation proportion = 97.3%). A total of 1265 participants who were ADL-independent at baseline were followed for 3 years, and information on outcomes, namely death and dependence in ADL, was collected.Participants who always had knee pain were more likely to become dependent in ADL than those who reported no knee pain ; however, always having knee pain was not associated with mortality or a composite outcome of ADL dependence and death. Further analyses of each component of ADL dependence revealed that knee pain was associated with a need for assistance at home , but not with institutionalization.The participants were highly representative of the target population and the rate of follow-up was almost perfect (99.4%). The results suggest that knee pain is associated with future dependence in ADL, particularly a need for assistance at home. Watanabe et al reported that mobility was significantly lower in patients with knee OA than in control subjects,9 and Kondo et al investigated risk factors associated with knee pain and functional limitation.10,11 In both these studies, the subjects were hospital-based patients with knee OA. However, a recent population survey in the United Kingdom found that among 3023 adults aged 50 years or older who reported knee pain within the previous year, only 33% reported visiting their general practitioner for this complaint during the same period.12 Therefore, community-based studies are desirable to produce less biased estimates of the association between knee pain and dependence in ADL. Moreover, because body composition in Asian and Western populations is very different13 and lifestyle factors including kneeling and squatting in daily life also differ between Japan and Western countries, associations observed in whites may not be applicable to Japanese populations. If knee pain were shown to be associated with dependence in ADL among community-dwelling older populations, intervention in the form of exercise programs, for example, could be aimed at maintaining self-reliance in those with knee pain.The self-reliance of older adults is an increasingly important public health concern as the global population ages. Knee pain can cause ambulatory limitation.Therefore, the aim of this 3-year cohort study, in which the participants were highly representative of the target population, was to investigate the association of knee pain with dependence in ADL and mortality in community-dwelling older Japanese adults.14–16 Briefly, from April through July 2005, trained public health nurses and local welfare commissioners conducted a home-visit health survey of residents aged 65 years or older, using a structured questionnaire with items on many aspects of health. Of the 1429 eligible residents, excepting those admitted to a hospital or an institution at the time of the survey, 1391 (97.3% of 1429) participated in the survey, and 1273 who were independent in ADL at baseline were followed as a cohort until September 2008, a period of approximately 3 years. Eight subjects moved out of the study area during follow-up; therefore, the associations between knee pain and the study outcomes were analyzed in 1265 residents . The median (range) age at baseline was 74 (65–97) years in men and 75 (65–98) years in women. The study was approved by the Medical Ethics Committee of the Keio University School of Medicine, Tokyo, Japan.This study is part of the Kurabuchi study, an ongoing community-based longitudinal study of aging that involves functional assessment of an older population in Kurabuchi Town, Takasaki City, a community approximately 100 km north of Tokyo, Japan. The details of the study have been described elsewhere.17 with answer choices of “never,” “occasionally,” “often,” and “always.” This simple question seemed more suitable in a community setting than a complicated disease-specific instrument for measurement of knee pain, such as the Western Ontario and McMaster Universities Osteoarthritis index (WOMAC).18 Moreover, the English version of this question has been validated,18 although the Japanese version has not. In addition, to determine if subjects had gone to a physician for treatment of their knee pain, we asked the question, “Have you had a medical consultation regarding knee pain in the last year?,” with answer choices of “yes” or “no.”Presence of knee pain was assessed using the following question in the home-visit survey, “Have you had pain in the last year in or around the knee?,”19We defined dependence in ADL as either admission to a nursing home or need of assistance at home during the follow-up period. The latter was defined as long-term care (LTC) eligibility or a need for help in any of 6 basic ADL items on the Katz Index of Independence in ADL.LTC eligibility is a requirement for receiving LTC insurance services in Japan, which began in 2000. In this study, any of the 7 levels of LTC insurance services was considered LTC-eligible. However, not all residents who are dependent in ADL apply for LTC insurance services. We added the Katz ADL measures to address this possibility. The Katz ADL is based on self-reported performance levels for 6 basic ADL items , each of which has 3 answer options . On the Katz ADL, dependence in ADL is defined as a need for partial or full help in performing any of the 6 items.Information on death, nursing home admission, and LTC eligibility was obtained from the Kurabuchi Branch Office of Takasaki City Hall. Information on Katz ADL was obtained from repeat face-to-face home interviews conducted every year , and occurrence of ADL decline in any year was defined as an ADL decline.8,20Information on age, sex, marital status (married vs widowed/separated/single), education (junior high vs high school or higher), support by relatives, neighbors, and friends (yes vs no), smoking status (current vs former/never), alcohol drinking (current vs former/never), and current/past history of major diseases, including stroke, myocardial infarction/angina, chronic obstructive pulmonary disease, diabetes mellitus, and cancer (summary answer of yes or no), was also collected at the baseline survey, because these factors have been reported to be related to the study outcomes.21) and were thus not included in the model. Because there was no interaction by sex, all analyses were carried out with combined data for men and women. This analytic method was repeated for dependence in ADL, for the single outcome of mortality, and for the composite outcome of dependence in ADL and death. In the analysis of dependence in ADL, residents who died during the follow-up period were excluded. Finally, we analyzed each component of ADL dependence, including institutionalization, LTC eligibility, and the 6 items on the Katz basic ADL. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to describe the strength of associations. Goodness of fit of all logistic models was assessed and confirmed by using the Hosmer–Lemeshow goodness-of-fit statistic .22Stata 10.0  was used for all analyses. Analyses were carried out by first conducting crude analyses, followed by multivariate analyses using logistic regression models. The initial basic model included only age (as a continuous variable) and sex. Then, the second and final models were constructed by adding marital status, education, smoking status, and current/past history of major diseases. There was no interaction between knee pain and medical consultation, and this was included in the model as an independent variable. Support by relatives, neighbors, and friends and alcohol drinking did not confound the observed association  in particular, but not with the single endpoint of mortality or the composite outcome of dependence in ADL and death.


The goal of this review is to introduce a theory of task-driven visual attention and working memory (TRAM). Based on a specific biased competition model, the ‘theory of visual attention’ (TVA) and its neural interpretation (NTVA), TRAM introduces the following assumption. First, selective visual processing over time is structured in competition episodes. Within an episode, that is, during its first two phases, a limited number of proto-objects are competitively encoded—modulated by the current task—in activation-based visual working memory (VWM). In processing phase 3, relevant VWM objects are transferred via a short-term consolidation into passive VWM. Second, each time attentional priorities change (e.g. after an eye movement), a new competition episode is initiated. Third, if a phase 3 VWM process (e.g. short-term consolidation) is not finished, whereas a new episode is called, a protective maintenance process allows its completion. After a VWM object change, its protective maintenance process is followed by an encapsulation of the VWM object causing attentional resource costs in trailing competition episodes. Viewed from this perspective, a new explanation of key findings of the attentional blink will be offered. Finally, a new suggestion will be made as to how VWM items might interact with visual search processes. The competition is biased, however, towards information that is currently relevant to behaviour. Attended stimuli make demands on processing capacity, whereas unattended ones often do not’ , p. 194.serial visual attention theories [parallel-and-capacity-limited theories’. They imply not only the computation of attentional priorities . The fitheories ,17, assutheories ) to visutheories . The sectheories ,19,20 isweights; ,19). Theweights; ,19. As sweights; ,19,20.(b)The approach, taken, in this review, for understanding task-driven competitive visual processing, is based on a specific parallel-and-capacity-limited theory of visual attention (TVA), developed by Bundesen , namely phase 1, attentional weights are computed within a priority map. The weights are computed for early visual object representations and they are assumed to be bias object-based competition for VSTM access in phase 2 of visual processing. More precisely, this selective second phase consists of a weight-guided race (competition) of visual objects via their features towards capacity-limited VSTM. Importantly, the higher the attentional weight of an early visual object representation, the better its chance that one of its visual features reaches capacity-limited VSTM in time, that is, before all VSTM slots (a limit of about three to four) are taken or before their visual input is replaced (e.g. in backward masking). In this review, these early visual object representations with attentional weights in priority maps and visual features in cortical maps are called proto-objects [TVA implies a competitive race of sensory visual information towards visual short-term memory (VSTM). VSTM information can be used for task-driven actions such as partial report or deciding whether a target object was present in a visual search display. In NTVA ,21, this-objects –25. The -objects ). In othloop between visual features of an object and its object node within a ‘VSTM map of objects’. As soon as a feature of an object is encoded into the VSTM map of objects, a VSTM slot is reserved for other features of the same object. A slot-based limit of VSTM of about three to four objects is assumed [Besides computing and applying attentional weights by the process of ‘filtering’, TVA and NTVA assume a second attentional process, called ‘pigeonholing’ (see ). It can assumed .s of visual object representations within the visual mind and brain of human and non-human primates. The first form for representing an external visual object refers to visual features/visual categories that are segmented into elementary visual object representations [. The following characteristics are ascribed to these proto-objects. First, following Wischnewski et al. [priority map [priority map region. Each priority map region is temporarily connected to the visual features in cortical maps. Besides retinal location within the priority map, the region has a rough shape [attentional weight [As stated above, TVA and NTVA imply a distinction between successive formntations ,19. Herei et al. ,26, protrity map ) and refgh shape ,26 and, visual working memory (VWM) objects—visual tokens [Once features of proto-objects are encoded into VSTM, a second form of visual object representation emerges. Here, these representations are simply called l tokens  or objecl tokens ,29 mightl tokens  for evidl tokens  for a rel tokens ). At thel tokens ,32,33 ofl tokens ,34.Moreover, the activation-based VSTM conception of NTVA includes visual features within cortical maps  as part of the VSTM process, as part of the loop. Therefore, visual perception and VWM cannot be structurally and functionally completely segregated. They should rely, in part, on overlapping structures and representations such as visual features as parts of online perception and of VWM  and VWM processes, see ,36). In (c)saccade target selection (selection-for-action) and biasing competition for perceptual report of a target (selection-for-perception) are strongly coupled to one common target [Natural vision proceeds as a succession of fixations and intervening saccades. The primate retina with its inhomogeneous structure has a much higher resolution in the centre, the fovea, than in the periphery . This ann target ,43 or evn target .attentional weights of TVA. During the preparation phase of a saccade and just prior to movement execution, the priority map region with the currently highest attentional weight should serve as the next saccade target. This suggestion implies that priority map regions couple selection-for-perception  with selection-of-saccade-target-location. As a consequence of priority map modulation by attentional weights, up to four proto-objects  win the competition for VWM access in perception and, simultaneously, one proto-object with the highest attentional weight determines ‘where to look next’. In summary, a covert attentional weight-guided biased competition process should not only modulate access of proto-objects to perceptual VWM, but should also determine—mediated by the same priority map regions—the target location of the next saccade.These findings on couplings of competitive visual processing for perception and for action are beyond the scope of TVA and NTVA in its current form. These theories explain behavioural and neurophysiological results of selective perception tasks such as visual search but do not make any claims of how selection-for-spatial-motor-action ,27,48 mi2.(a)available for goal-directed behaviour such as space-based sensorimotor actions or verbal report actions [actions controlled by the current task [capacity-limited VSTM, here labelled VWM, should represent the results of biased competition. This suggestion implies that encoding visual information into VSTM makes it available for goal-directed behaviour and cognition. NTVA [et al. [What is biased competition in visual processing for? It should make visual information  actions ,9,19. Onent task ,37,50. Hent task , it is a [et al.  showing  [et al. ).use of VWM/VSTM information for task-driven behaviour and cognition should be labelled phase 3 of competitive visual processing. ‘Use of VWM information’ can mean to initiate an already prepared sensorimotor action or store visual information for the delayed use in later competition episodes—examples will be given in the next subsections. It is suggested that only currently relevant VWM objects should be subject to the third phase of visual processing. For instance, if a non-relevant object is encoded into VWM owing to high visual similarity to a currently relevant object , thermation; ).et al.'s [use of VWM information should be of central importance for understanding vision as a whole. The most recent extension of TVA, namely the ‘theory of temporal visual attention’ (TTVA) by Petersen et al. [Recoding should allow, for instance, verbal report of a VSTM object or a motor response based on a VSTM object (e.g. grasping).In summary, the use of VWM objects for task-driven behaviour and cognition is the key function of phase 3 processing. Understandably, standard theories of visual attention such as TVA, NTVA, feature integration theory ,54, guidet al.'s  computatet al.'s ,48,50,55n et al. , suggestIn the next section, one form of using VWM information, namely short-term consolidation will be the focus of interest. It is a process that generates passive VWM representations that do not suffer from attentional resource costs of active VWM maintenance. Another class of phase 3 processes refers to the use of active VWM information for immediate sensorimotor actions. Action targets in space might already been specified at the level of proto-objects within activity-specific priority maps —‘where act next?’ is settled there. However, for triggering such a prepared motor action, it is suggested that VWM encoding of task-relevant features of the action target must have taken place. A goal-directed action should only be initiated if its triggering conditions are given . In other words, phase 3 of VWM should be necessary in order to allow ‘action initiation’.(b)retention of visual information across saccades for the current task relies at least in part on VWM with a capacity-limit of about four objects [An informative constraint on how VWM information could be used will now be derived from findings of how human and non-human primates sample and retain visual information over time in the service of the current task. Sampling occurs in permanently ongoing successions of fixations and saccades, that is, usually three to four saccades per second are executed ,57. Ther objects ,58,59. M objects .real-world tasks [Second, besides findings from these just mentioned highly controlled laboratory tasks, further important constraints on the transsaccadic use of VWM for task-driven action control should now be derived from studies of ld tasks ,61–63. Tretention-encoding dilemma of VWM is postulated. On the one hand, VWM should be able to retain a limited number of task-relevant visual objects (within its three to four slots) over the course of several fixations for later use in the current fixation. On the other hand, task-driven competitive visual processing in each new fixation requires a VWM that has ‘space’, has slots available for encoding the competition winners.In summary, empirical evidence from transsaccadic memory studies and functional considerations about sport tasks and everyday activities suggest that task-relevant visual information sampled in preceding fixations can be retained at the short-term scale and used in later fixations for the current task step. Combining this observation of task-driven short-term retention with the biased competition architecture of TVA and NTVA, a selective maintenance/selective clearance of objects within VWM comes with substantial costs in terms of attentional capacity.An evident solution for handling this retention-encoding dilemma assumes, on the one hand, the selective retention of task-relevant visual information within some of the VWM slots and, on the other hand the selective clearance of other VWM slots prior to each fixation so that ‘space’ for new competition winners is made available. The existence of a clearance process for VWM (VSTM) has been explicitly postulated by NTVA  prior toattentional weights of actively retained VWM objects from preceding fixations act as further competitors within the biased competition process of the current fixation. Attentional weights of all online items of the current fixation should compete via their attentional weights with maintained items from preceding fixations. In other words, it implies that actively retained VWM objects bind attentional weights from previous fixations. Therefore, the competitive processing of visual information within the current fixation should be substantially slowed down by maintained visual objects from preceding fixations. Therefore, this activation-based transsaccadic form of VWM maintenance implies ongoing attentional costs during the maintenance phase.Actively maintained winners from preceding fixations reduce the number of limited slots for competition winners of the current fixation. Moreover, these maintained winners touch a second visual processing capacity limit, namely in terms of normalized attentional weights. Each maintained previous competition winner has an attentional weight that competes via normalization with other attentional weights of the current fixation. NTVA  states t(c)further form of VWM retention without permanent attentional resource costs exists in the primate brain and that this form of short-term retention is realized by passive VWM. On the basis of a variety of findings and on computational considerations, a number of authors [How might these ongoing attentional costs of selective activation-based VWM maintenance—blocking VWM slots and ongoing binding of attentional resources  during retention—be avoided? It is suggested that a  authors –66 argueobjects within activation-based VWM. Here, the encoding of an object into activation-based VWM implies that the loops between visual features and its pointer within the ‘map of VWM objects’ are set up [. The duration of such a consolidation process could follow an exponential distribution [Encoding into VWM means setting up the activation-based loop for a visual object, whereas consolidation means creating a passive code within VWM as a result of a sufficient looping duration. In summary, a distinction is suggested between activation-based VWM and passive VWM. Short-term consolidation refers to the process of transferring relevant visual objects from active VWM into passive VWM.How could such a passive VWM be created? Following Hebb's  suggestie set up . Such obe set up ,68. Borre set up , this trribution  and it mk-winner-take-all process between object pointers of the VWM map given k might be three to four for young adults. Therefore, within one competition episode of a single fixation, only k winners can be subject to short-term consolidation in phase 3. If so, then many competition winners (many active VWM objects) could be stored across several fixations in passive VWM—much more than k winners of one competition episode of one fixation. Given this analysis, why does a limit of three to four objects emerge? At the beginning of the retrieval process, the k-winner-take-all network of active VWM should be initialized (set to zero). An external signal might start competition between passive VWM objects and only three to four winners can emerge within the activation-based VWM map of objects.Evident questions for this conception of active and passive VWM are: how might the limit of three to four objects emerge? How might retrieval from VWM work? Following NTVA , it is aHow might retrieval from passive VWM be controlled? Importantly, the storage in passive VWM serves the key function of allowing task-driven actions based on objects encoded in preceding fixations, preceding competition episodes. Therefore, a key factor that influences the chance of retrieval competition should come from the current task step. For instance, the task ‘making of a peanut butter sandwich’ requires as one step ‘grasping the jelly glass’. Therefore, during this step, the position of the jelly glass should be retrieved from passive VWM for allowing an eye movement to the jelly glass and a following grasping movement . Lonet al. [et al. [k-winner-take-all VWM map of objects) that should not be reduced to one common limitation. Second, TRAM has a more limited explanatory goal in terms of domains and phenomena. It focuses on competitive visual processing and relies on middle-range theories of visual attention and VWM (NTVA) that attempt to explain findings from vision studies such as visual search or partial report. Third, according to the viewpoint taken here, terms such as ‘focus of attention’ within working memory [et al.'s [Cowan  and Oberet al.  put forwet al.  and Ober [et al.  argue fog memory ,72 implyg memory  or Oberaet al.'s ).3.An interim summary of the theory of TRAM specified so far should be given. At the start of a fixation, phase 1 of visual processing computes visual proto-objects that consist of visual features at different levels of the cortical hierarchy and priority map regions. Each priority map region receives an attentional weight, a measure of attentional priority. Each weight is based on stimulus-driven factors such as sensory evidence for its features and on top-down factors such as ‘pertinence’ (e.g. importance of object for the current task). At the end of processing phase 1, after the computation of relative weights, VWM should be cleared in order to create ‘space’ for encoding new competition winners. At the start of phase 2, visual feature processing is competitively modulated by the normalized weights, the attentional priority settings computed in phase 1. Weight-based feature modulation means that competition between proto-objects is biased. Those proto-objects, their features with higher attentional weights will have a higher chance of winning the competition for being encoded into capacity-limited active VWM than proto-objects with lower attentional weights. For young adults, three to four objects can be encoded in active VWM. Importantly, competition between proto-objects is regulated on the basis of priority-(weight)-modulated features. The faster the ‘race speed’ of a feature, the higher its competition value, the higher its chance to access a VWM slot before all slots are taken. If a feature of a proto-object is encoded into VWM (given there were still free slots), then this feature occupies the slot for all other later competition winning (later arriving) features of the same proto-object. Once features of proto-objects are encoded into VWM, a second form of object representation emerges, namely VWM objects, and phase 3 of visual processing starts for relevant objects . One phase 3 operation is short-term consolidation that transfers the activation-based code (loops between a VWM map and features) into a passive code that might rely on short-term synaptic changes. A key advantage of passively retained VWM objects is the missing attentional resource costs in terms of slots and attentional weights.protective maintenance process. In short, the protective maintenance process allows finishing phase 3 operations on VWM objects during subsequent competition episodes. Protective maintenance simply protects the VWM slot from being cleared. As stated in §2, such non-clearance of VWM should have substantial attentional costs in terms of slots and attentional weights. In the following sections, an analysis of key findings from the ‘AB’ paradigm [This sketch of TRAM has an important implication. A new competition episode generates at the end of phase 1 a clearance signal for active VWM in order to achieve ‘space’ for encoding new competition winners ,20. Phasparadigm –77 will (a)The term ‘competition episode’ has been used in the preceding paragraphs. Now, a more formal definition will be given following TVAs  idea abooccluded—either owing to movements of the occluder and/or of the occluded object [A new competition episode can be triggered internally, by a new task step implying a shift of the attentional set, or it can be triggered externally, by visual input accompanied with weight changes. An example of an internal triggering by an attentional set shift can be illustrated with the ‘peanut butter sandwich making’ example. If the task step ‘grasping the jelly glass’ is finished, then the next step of ‘transporting the glass to the desired location’ should be initiated. For the first step, the current location of the jelly glass is relevant, whereas for the following step, the future location of the glass is relevant and should therefore be part of the changed attentional set. An externally triggered new competition episode occurs if changes in visual stimulation are accompanied by changes in relative attentional weights. If an object is moving into (or out of) the retina, then the new weight (or disappearing weight) of the moving object should lead to recomputation of relative weights. Moreover, if objects within a fixation are d object —then rel(b)phase 3 processing of a competition episode is still going on and is not finished while a new competition episode is triggered by changes of attentional priorities? Is VWM cleared completely so that new competition winners can be encoded? An advantage of this solution would be to have no attentional resource costs . However, such complete VWM clearance would have remarkable costs, namely the use of VWM information in phase 3 for behaviour and cognition would be interrupted. This would imply loosing information from VWM without any chance of recovery. However, as sketched above, even in a simple backward masking experiment with short SOAs with likely unfinished short-term consolidation, targets can be reported clearly above chance level. How is this possible? The suggested answer is a protective maintenance process. A protective maintenance process allows finishing phase 3 operations for a VWM object (e.g. such as short-term consolidation) during subsequent competition episodes. Protective maintenance simply prevents that an active VWM slot with an ongoing phase 3 operation is cleared at the start of a new competition episode. Importantly, it is selective protection of only those slots in which phase 3's short-term consolidation process is still going on. During phase 1 of a new competition episode either updating or encapsulation of a VWM object should occur. On the one hand, updating is issued if a VWM object receives visual input that fits in terms of its priority map region characteristics  to the predicted (expected) region characteristics maintained by the VWM object. In other words, updating is called if the visual system signals for new visual input to a VWM object  object continuity. On the other hand, if the new visual input signals a failure of object continuity of a VWM object then encapsulation should take place. It is suggested that encapsulation implies that the visual features and the attentional weight of the encapsulated object are retained at the current state (current activation level) at the moment of the encapsulation call. Crucially, an abrupt change in terms of the priority map region's expected location, rough shape or attentional weight should trigger encapsulation.Given our definition of competition episodes, the key question of §3 can be tackled: what happens if per se should not call a protective maintenance process with encapsulation and ‘resource locking’. According to TRAM, the process of encapsulation and ‘resource locking’ should only be triggered if phase 3 short-term consolidation for later report could not be finished when a new competition episode, that is, a change of relative attentional weights, and when an object continuity failure has been signalled.The ‘TTVA’  made a s(c)As emphasized in §2, such non-clearance and encapsulation should have substantial attentional costs in terms of occupied slots and retained attentional weights. Highly informative in terms of understanding and specifying the processes behind these attentional costs are findings from the AB paradigm. In the following, a new look will be made at several key findings of the AB paradigm: the SOA-dependent core T2 deficit at short SOAs that recovers over time (the AB proper), lag-one sparing (including sparing for up to three Ts), the effect of a short post-T1 blank (150 ms) in eliminating the AB, as well as the reappearance of an AB within increasing SOA between T1 and T2 without any intervening D (lag-one sparing).if a D is followed by a T. Clearly, this D–T sequence is accompanied by a change of attentional weights, that is, the D has a substantially lower weight than the T. Therefore, a new competition episode is called by the T and as a consequence, the preceding D is cleared from VWM. Ds are usually not subject to phase 3 operations and protective maintenance processes. During the new T competition episode, the features of the T-proto-objects should be encoded into VWM. After VWM encoding of the T, its phase 3 operations, that is, short-term consolidation for later report, starts. The D directly after the T signals an object continuity failure due to local object-specific weight change and again a new competition episode owing to a global change of relative attentional weights. Consequently, a clearance signal for VWM should be issued. Because the T is still subject to an ongoing phase 3 operation, namely short-term consolidation, 100 ms are not sufficient to complete all three processing phases including short-term consolidation. The T will be spared from VWM clearing by the protective maintenance process. Further trailing Ds will have to compete for VWM access with the encapsulated T as long as its short-term consolidation process is going on.The AB paradigm belongs to a class of experiments with rapid serial visual presentation (RSVP). An RSVP stream consists of one target T stimulus among several distractor (D) stimuli. Every stimulus appears at the same location. At the end of the stimulus sequence of a trial, the T stimulus has to be reported without time pressure . The finet al. [A standard AB experiment consists of an RSVP stream within two Ts (T1 and T2) within a stream of Ds . The preet al. , discusset al. .core SOA-dependent T2 deficit, the AB, emerge at all? The first phase of a trial of an AB experiment—successive Ds until the appearance of T1—has already been described above in the RSVP section. Crucially, the trailing D after T1 causes an attentional weight change that issues a new competition episode for the D. The new competition episode of the D calls for clearance of VWM. However, T1 with its still ongoing phase 3 operation of short-term consolidation is spared from clearance by the protective maintenance process and the T trailing D signals a failure of object continuity. Every subsequent stimulus—D or T—will suffer from the encapsulated T1, from its attentional weight, as long as short-term consolidation—a necessary condition for protection—is going on. If T2 appears after a D, then a new competition episode will be started. A change in terms of attentional weights from the preceding D to T2 occurs. Therefore, T2 competes with T1 during its phase 2-based attempt to access VWM. More precisely, as long as phase 3 operation of short-term consolidation for T1 is going on, encapsulated T1 competes with T2 during phase 2 of its competition episode. Decisive for competition is the maintained attentional weight of T1 that is linked to its VWM object representation. In other words, as long as short-term consolidation for T1 is working, its corresponding attentional weight of the priority map region is encapsulated and competes with the weight of T2. The simultaneous presence of the T1 weight with the T2 weight during the T2 competition episode slows the race of T2 towards VWM (TVA) down considerably. Why does a D or pattern mask (interruption masking) after T2 lead to the emergence of an AB? [First, why does the f an AB?  Without f an AB? . ProtectT2 performance improve with SOA? The longer the SOA, the higher the chance that phase 3's short-consolidation of T1 has been already finished so that T1 will not be subject anymore to protective maintenance and encapsulation. If T1 is not protectively maintained, then the trailing item (D or T2) will call a new competition episode and clear the unprotected T1 from VWM. In this case, T1 should not be able to compete anymore with T2. The longer the SOA between T1 and T2, the higher the chance of a finished short-term consolidation for T1 in phase 3, and the less likely the chance of a competition between T1 and T2 and the less pronounced the T2 deficit should be. More formalized and as suggested by Jolicoeur & Dell'Acqua [Why does ll'Acqua , short-tlag-one sparing , or sparing for several consecutive Ts . In blank after T1 of a sufficient duration (100–150 ms) reduces or even eliminates the T2 deficit significantly [Third, a ficantly . This fificantly . The devnature of lag-one sparing by Nieuwenstein et al. [Fourth, this suggestion that an iconically decayed T1 weight is a weaker competitor for T2—compared with standard RSVP conditions—will now be applied in explaining a recently published finding of the n et al. —a findinWhat is the major difference of TRAM's explanation of the AB and other explanations? ,76,82,864.et al. [Here, a few selected implications of TRAM for explaining the enormous and still nonlinearly growing experimental literature on the interaction of visual attention  and working memory  shouet al. , see the(a)In most visual search experiments, the search target (e.g. ‘search for black letter X!’) is constant within a block of trials and sometimes even across the complete experiment . For sucet al. [Given this explanation, an important question emerges: if the search target is retained in active VWM why does it not—instead of biasing the computation for sensory-derived weights of the new episode—bring in its attentional weight as a further element of weight computation? If this would be the case, then massive interference effects—see the AB explanations above—should be observed. Why are no inference costs observed? VWM objects encapsulated with ongoing phase 3 operations create interference effects, but not VWM objects without phase 3 operations or VWM objects after the end of phase 3. As stated above, in visual search experiments with trialwise varying search targets, phase 3 of the search target (STM consolidation) should be clearly finished by the time the search display appears. After finishing, iconic decay of features and the attentional weight of the search target should take place within the ongoing processing episode . Howeveet al. ).(b)et al. [et al.'s [Olivers et al.  offer a et al.'s  study, oet al.'s . As statn) and consolidate it into passive VWM for later comparison in the memory test phase. In the next step, competition episode n + 1 with a new attentional set (‘attend to the search target’), the search target item should be encoded into activation-based VWM. This step clears at the end of its phase 1 the STM item from episode n from active VWM. This order of competition episodes and VWM encoding allows the search target to bias the search process by a powerful activation-based code and it later allows a memory test based on passive VWM traces. Given this chain of events, the memory item should exert no biasing effect in visual search in competition episode n + 2. When the search display appears, the search target template encoded in episode n + 1 is still part of active VWM, whereas the STM item from episode n has already been cleared during episode n + 1. Therefore, no effect of the STM item should be observed.On the other hand, the dual task study of Downing & Dodds  and otheThis selective interpretation of two studies on interaction of VWM retention and visual search should illustrate the suggested processing dynamics of TRAM. However, more published studies and results ,65 have


Trichosanthin (TCS) as a midterm abortifacient medicine has been used clinically in traditional Chinese medicine for centuries. Additionally, TCS manifests a host of pharmacological properties, for instance, anti-HIV and anti-tumor activities. TCS has been reported to inhibit cell growth of a diversity of cancers, including cervical cancer, choriocarcinoma, and leukemia/lymphoma, etc. This article purported to review the various anti-tumor activities of TCS and the mechanism of apoptosis it induced in these tumor cells. These research progresses provide an insight into cancer research and treatment as well as disclose new pharmacological properties of the ancient but popular Chinese medicine. Trichosanthin (TCS) is a renowned traditional Chinese medicine and still used in clinics in China for inducing midterm abortion. TCS belongs to the family of plant proteins known as ribosome-inactivating proteins (RIPs), which can attack the ribosomes of eukaryotic cells by virtue of their rRNA N-glycosidase activities –3. This TCS which possesses only one polypeptide chain is a classic example of a type I RIP. As TCS is devoid of a cell-binding B chain, it has less cytotoxic effects than most of type II RIPs, such as ricin, and more selective toxicity to cultured tumor cells, ovulated oocytes, HIV-infected macrophages, and normal human macrophages. , 8. ReceTrichosanthes kirilowii Maxim belonging to the Cucurbitaceae family. It has been employed for centuries in traditional Chinese medicine as an abortifacient drug in early and mid-gestation. Only in recent decades were its enzymatic activities recognized as characteristic of type I RIPs [Trichosanthin (TCS), or Tin Hua Fen, is obtained from the root tuber of e I RIPs . Althouge I RIPs . The fire I RIPs . Since te I RIPs , 12.TCS is composed of a single polypeptide chain with a molecular weight of 27 kDa. Its amino acid sequence demonstrates homology to the A chains of many type II RIPs, such as ricin A chain –15. It iTCS owns a wide spectrum of biological and pharmacological activities Table . It prodTCS is well known to exert deleterious effects on reproduction and is a clinical medicine for abortion. In the mouse, it engenders follicular atresia and degeneration of ovulated oocytes . In fertAbout two decades ago, Dai and colleagues conjugated colloidal gold to TCS molecules and found that TCS specifically entered cultured trophoblast and choriocarcinoma JAR cells via receptor-mediated endocytosis . The finWhen TCS was added to cultured tumor cells, it brought about a reduction in the uptake of radioactive precursors for protein synthesis . This suOnly one article has reported the effect of TCS on cervical cancer in vivo. Zhang et al.  establisAnother cancer that has been reported to be sensitive to TCS is leukemia/lymphoma. As early as in 1990s, TCS was found to be toxic to leukemia/lymphoma cells in vitro , 44. A dTCS has been reported to be effective against a variety of other tumors, including hepatoma, colon carcinoma, stomach cancer, lung cancer, breast cancer, prostate cancer, and melanoma. Some early research reported that TCS did not exert much toxicity to hepatoma cell lines, including H35 and HepA-H , 31, 32.Furthermore, dexamethasone enhances the effects of TCS on apoptosis in HepG2 cells by inhibiting the NF-κB signaling pathway, which highlights the possibility of combined drug application of TCS and dexamethasone in the clinical treatment of hepatoma . StudiesAn animal model of lung caner was created by administration of A549 cells to nude mice. TCS prevented or inhibited the process of lung tumorigenesis . TCS inhAs a toxic protein, TCS needs to enter cells to inactivate the eukaryotic ribosome through its RNA N-glycosidase activity. The first step of this process is that TCS combines and interacts with phospholipids in the cell membrane, because TCS must get across the cell membrane before it can enter the cytoplasm and exert its RIP function . Xia andOn the other hand, receptors are also regarded to be necessary for TCS to enter cells –24. TCS TCS suppresses adenylyl cyclase activity and thus reduces cyclic AMP (cAMP) levels in HeLa cells . InteresThe transcriptional factor cAMP response element-binding (CREB) protein, a downstream molecule in cAMP/PKA pathway, was found to participate in the TCS-induced cell death pathway in HeLa cells . CREB phFurthermore, TCS induced a rapid decline in nuclear factor kappa B (NF-kB) and cyclooxygenase-2 (COX-2) expression leading to apoptosis in hepatoma HepG2 cells , 54. TheAll abovementioned data infer that TCS inhibits cell proliferation in different tumor cells through different pathways and mechanisms, which merit further in-depth investigations. The mechanisms of TCS are summarized in Fig. TCS treatment induced a transient elevation of intracellular calcium and a slow rise of ROS in human chronic myeloid leukemia cell line K562 . However2−) scavenger superoxide dismutase, the H2O2 scavenger catalase, and the hydroxyl radical (OH−) scavenger mannitol [− formation, diethylenetriaminepentaacetic acid, also markedly inhibited TCS-induced cell death [2−, H2O2, and OH− are involved in TCS-induced ROS formation in JAR cells. The increase in ROS is dependent on the presence of both extracellular and intracellular Ca++ ions, and TCS-induced ROS production may be a consequence of Ca++ signaling. In addition, TCS-induced activation of caspase 3 was initiated within 2 h; however, TCS-induced production of ROS was initiated within 5 min. These findings suggest that the production of ROS precedes the activation of caspase 3. The apoptotic morphological changes of nuclei were also observed by two-photon laser scanning microscopy in this experiment. The finding that ROS is involved in the TCS-induced apoptosis of JAR cells might provide new insight into the anti-tumor and anti-HIV mechanism of TCS [In another cell line, JAR, TCS stimulated the production of ROS, which can be inhibited by the superoxide radical anion (Omannitol . The antll death . All them of TCS , 28.Key caspases in both intrinsic and extrinsic pathways, encompassing caspases 8, 9, and 3, were activated in HeLa-60 cells upon TCS treatment . HoweverTCS administration induced upregulation of the protein chaperone BiP and transcription factor CHOP and also activated caspase 4 in HeLa-60 cells, which for the first time strongly supported the involvement of ER stress pathway in TCS-induced apoptosis . On the TCS, a midterm abortion medicine, is a type I RIP endowed with a multiplicity of biological activities, including anti-HIV and anti-tumor functions. TCS has been found to be active against a variety of tumors, including cervical cancer, choriocarcinoma, leukemia/lymphoma, stomach cancer, colon cancer, hepatoma, breast cancer, and prostate cancer. The toxic mechanisms of TCS on tumor cells include inhibition of the proliferation and induction of apoptosis of tumor cells, and the detailed mechanism varies in different tumor cells. Further research on the anti-tumor activities of TCS may not only shed light on cancer therapy, but also on new pharmacological properties of ancient Chinese medicines.


To determine why incidence of mucormycosis infections was increasing in a large university hospital in Belgium, we examined case data from 2000–2009. We found the increase was not related to voriconazole use but most probably to an increase in high-risk patients, particularly those with underlying hematologic malignancies. In September 2009, Bitar et al. reported an increasing incidence of mucormycosis in France from 1997 through 2006  criteria  from 2000 through 2009 were retrieved from the Sirscan 2000 system . Computer queries were run to retrieve all reports from histologic examinations of tissue specimens to which the Leiden code “fungus” had been assigned  . The incThe number of patients belonging to the risk groups diabetes mellitus, solid organ transplantation, hematologic disorder/malignancy, and hematopoietic stem cell transplantation was determined based on International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM), codes entered in the hospital information system (available up to the end of 2008). An increase in the number of diabetes mellitus (p<0.01) and hematologic disorder/malignancy (p<0.01) patients . Mucormycosis developed in 1 patient while that patient was receiving voriconazole therapy for invasive aspergillosis.Aspergillus spp. was recorded. Five of the 6 patients showing a halo sign on chest computed tomography scan were co-infected with Aspergillus spp.Clinical signs included pulmonary (51.5%), disseminated (23%), cutaneous (13%), sinus (6.5%), cerebral (3%), and mycetoma (3%) mucormycosis. A high percentage (45%) of co-infections with The death rate was 65%. For 48% of the patients, death was directly related to mucormycosis infection.We report an increasing incidence rate of invasive mucormycosis not related to the use of voriconazole, either prophylactically or therapeutically, in a university hospital in Belgium. Pongas et al. calculated that 49% of mucormycosis cases were encountered in the setting of voriconazole prophylaxis , our study reported a notably high percentage (23%) of cases of disseminated disease  but appears to be much higher than rates reported in the study in France. However, this discrepancy might be merely a reflection of differences in case definitions . A decreasing number of published mucormycosis cases since the 1990s in patients with diabetes mellitus as underlying illness was reported in a large review of the literature (Mucorales spp. organisms infect a broader and more heterogeneous population. Thus, invasive mucormycosis also occurs in immunocompetent patients without any host factor as defined by these criteria. As such, invasive procedures are necessary in immunocompetent patients to enable the diagnosis of proven invasive mucormycosis according to the EORTC-MSG criteria. However, we estimate the effect of this limitation to be minor because in immunocompetent patients biopsy procedures are generally feasible, particularly because mucormycosis is most often seen as a cutaneous infection in these patients. Finally, our results are single-institution data and are not necessarily representative of other institutions or other geographic regions.Several limitations of our study can be mentioned. First, 5 cases of mucormycosis were diagnosed on the basis of histopathologic findings only. Second, the numbers of the risk populations were derived from ICD-9-CM codes and are thus subject to all inaccuracies associated with discharge coding of patients. Third, we used the EORTC-MSG criteria for classification of cases, but, unlike other filamentous fungal pathogens that target immunocompromised hosts, In conclusion, the increasing incidence of mucormycosis shown in our study was not related to the prior use of voriconazole. Rather, the increasing incidence was most probably associated with an increasing number of patients who had an underlying hematologic malignancy.


Borrelia burgdorferi dramatically upregulates outer surface protein C (OspC) in response to fresh bloodmeal during transmission from the tick vector to a mammal, and abundantly produces the antigen during early infection. As OspC is an effective immune target, to evade the immune system B. burgdorferi downregulates the antigen once the anti-OspC humoral response has developed, suggesting an important role for OspC during early infection.The Lyme disease spirochete B. burgdorferi against innate elimination. However, the deletion greatly impaired the ability of B. burgdorferi to disseminate to remote tissues after inoculation into mice.In this study, a borrelial mutant producing an OspC antigen with a 5-amino-acid deletion was generated. The deletion didn't significantly increase the 50% infectious dose or reduce the tissue bacterial burden during infection of the murine host, indicating that the truncated OspC can effectively protect B. burgdorferi during mammalian infection.The study indicates that OspC plays an important role in dissemination of  Borrelia burgdorferiB. burgdorferi abundantly produces OspA and OspB, but no OspC B. burgdorferi maintains high OspC synthesis during early mammalian infection B. burgdorferi must downregulate ospC after the specific humoral response has developed Coordinating production of outer surface proteins (Osps) is crucial for the pathogenic strategy of the Lyme disease spirochete, ospC gene completely abolishes infectivity of B. burgdorferiospC copy, which is eventually lost under the immune selection pressure during infection of immunocompetent mice, leading to a conclusion that OspC is required exclusively for initial mammalian infection ospC mutants in mammalian hosts B. burgdorferi indeed undergoes dramatic changes in its surface lipoprotein expression during mammalian infection ospC copies. The ability of an Osp to replace OspC in initial mammalian infection highlights a redundant function of the Osps, which is to protect B. burgdorferi against innate immune elimination ospC mutants with expected dissemination ability Inactivation of the B. burgdorferi against innate elimination, as measured by the 50% infectious dose (ID50) and tissue bacterial loads during murine infection, but failed to efficiently promote dissemination of B. burgdorferi to remote tissues. The study allowed us to conclude that OspC is a dissemination-facilitating factor of B. burgdorferi.As described by some investigators, the protective and dissemination-promoting functions of OspC are more like two sides of the same coin B. burgdorferi B31 clone 13A, the ospC mutant (ΔospC), and the complemented clones ΔospC/FL/1 and ΔospC/FL/2 were generated previously The ospC region and the up- and down-stream sequences was amplified from B. burgdorferi B31 genomic DNA by PCR using primers P1F and P1R  deletion, was cloned into pBBE22 after the vector was digested with BamHI and XbaI. The resulting construct was designated pBBE22-ospCnt5. The insert and its flanking regions within pBBE22 were sequenced to ensure the construct was as designed.As illustrated in  and P1R  and clon and P2R . After dospCnt10. Briefly, pNCO1T-ospC was amplified by using primers P3F and P3R  in BSK-H complete medium at 33°C. Approximately 2×107 cells were harvested from 0.2 ml of culture by centrifugation at 10,000× g for 10 min, gently suspended in 100 µl PBS containing OspC MAb, and incubated for 1 hour at room temperature. After 2 washes with excess volumes of PBS by centrifugation at 16,000× g for 5 min, spirochetes were resuspended in 100 µl of PBS containing 1.0 µg of fluorescein isothiocyanate-conjugated goat anti-mouse IgG , incubated for 1 hour, washed twice with PBS by centrifugation, resuspended in 50 µl PBS, applied to microscopic slides, and analyzed using Axio Imager .5 spirochetes. Mice were sacrificed 1 month post-inoculation; heart, tibiotarsal joint and skin specimens were harvested for spirochete culture in BSK-H complete medium. Cultures were examined for growing spirochetes under a darkfield microscope every week for up to 3 weeks. All animal procedures described here and below were approved by the Institutional Animal Care and Use Committee at Louisiana State University (LSU Protocol No. 07-019).Groups of three C3H SCID mice  were inoculated with 105 spirochetes. The two inoculation sites were at least 2 cm apart. Two animals from each group were euthanized at 24 or 48 hours later; inoculation site skin specimens were harvested for spirochete culture in BSK-H medium.Groups of four C3H SCID mice each received two intradermal/subcutaneous inoculations of 108 cells per ml) in BSK-H complete medium at 33°C and 10-fold serially diluted with fresh medium. C3H SCID mice each received one single intradermal/subcutaneous injection of 100 µl of spirochetal suspension (containing 101 to 104 organisms). Mice were euthanized one month post-inoculation; heart, tibiotarsal joint, and skin (not from inoculation site) specimens were harvested for bacterial isolation. The ID50 value was calculated as described by Reed and Muench Spirochetes were grown to late-log phase  as described previously flaB DNA copies per 106 host cells (2×106 actin DNA copies).C3H SCID mice each were intradermally/subcutaneously inoculated with 103 spirochetes and were euthanized at 1, 2, 3 and 4 weeks post-inoculation. Inoculation site and remote site skin, ear, heart and tiobiotarsal joint specimens were harvested for spirochete isolation as described previously C3H SCID or BALB/c wild-type mice each were given a single intradermal/subcutaneous injection of 10t test was used to calculate a P value for each two groups. A P value ≤0.05 was considered to be significant.A two-tailed Student ospC mutants that can be protected from innate elimination but are unable to efficiently disseminate via mutagenizing the ospC gene. Based on X-ray analyses, OspC is a largely α-helical protein and may be a dimer with a characteristic central four-helical bundle formed by the association of the two longest helices, helices 1 and 5, from each subunit B. burgdorferi expressing OspC with each of these small units being deleted (data not shown). Unfortunately, none of the resulting mutants were infectious, suggesting that these sequences are critical for the functions of OspC.The goal of the study was to dissect the dissemination-facilitating from protective function of OspC. Our strategy was to generate E. coli the so-called “+2” rule determines the cellular location of a lipoprotein. However, the sorting signal of B. burgdorferi can extend up to the +4 position ospCnt5 was generated as illustrated in Next, we focused on the N-terminal sequence. One role of this sequence is to carry the sorting signal, which determines the surface location of OspC. In ospC mutant, ΔospC, which was generated and characterized in our previous study ospC lacks lp25, the plasmid that carries the gene bbe22 coding for a nicotinamidase essential for survival of B. burgdorferi in the mammalian environment, the recombinant plasmid pBBE22, which harbors a copy of bbe22, was used as the shuttle vector ospC/ΔNt5/1, ΔospC/ΔNt5/2, ΔospC/ΔNt10/1 and ΔospC/ΔNt10/2. These clones shared the same plasmid content as ΔospC, which had lost lp25, lp5, lp21, lp56 and cp9 The two constructs were electroporated into the B. burgdorferi. To this end, we used indirect immunofluorescence to locate truncated OspC. As shown in Because the deletion was generated close to the N-terminus, which may harbor the sorting signal, it is important to show that the truncated OspC can be successfully sorted to the outer surface of 5 spirochetes of the clone ΔospC/ΔNt5/1, ΔospC/ΔNt5/2, ΔospC/ΔNt10/1, or ΔospC/ΔNt10/2. As a control, mice were also inoculated with the clone ΔospC/FL/1 or ΔospC/FL/2. The clones ΔospC/FL/1 and ΔospC/FL/2 were generated via introduction of a full-length ospC gene carried by the shuttle vector pBBE22 into ΔospC in our previous study ospC/ΔNt5/1 and ΔospC/ΔNt5/2 spirochetes were grown from each sample of all inoculated mice. In contrast, neither ΔospC/ΔNt10/1 nor ΔospC/ΔNt10/2 bacteria were recovered from any specimens, suggesting that the 10-AA deletion may completely abolish the functions of OspC.Groups of three C3H SCID mice were inoculated with 10ospC/ΔNt10/1 and ΔospC/ΔNt10/2 bacteria might remain at the inoculation sites without dissemination. To rule out this possibility, groups of four SCID mice each received two intradermal/subcutaneous inoculations of the clones ΔospC/ΔNt10/1, ΔospC/ΔNt10/2, ΔospC/FL/1 or ΔospC/FL/2. Animals were euthanized at 24 or 48 hours later. As a positive control, the ΔospC/FL/1 and ΔospC/FL/2 bacteria were consistently grown from each of the 16 inoculation sites from all eight inoculated mice (data not shown). In contrast, neither ΔospC/ΔNt10/1 nor ΔospC/ΔNt10/2 spirochetes were recovered from any of the 8 sites harvested at each time point, confirming that the 10-AA deletion completely destroys the functions of OspC and leads to quick clearance of spirochetes in the murine host.The Δ50 value reflects the smallest number of organisms to sufficiently initiate an infection in 50% of inoculated individuals, it is the best measurable criterion to assess how a pathogen is protected against innate immune elimination, especially when this is measured in SCID mice. To examine whether the 5-AA deletion reduced the ability of OspC to protect B. burgdorferi against innate immunity, groups of three C3H SCID mice each received one single inoculation of 101 to 104 spirochetes of the clone ΔospC/ΔNt5/1, ΔospC/ΔNt5/2, ΔospC/FL/1, or ΔospC/FL/2. All animals were euthanized 1 month post-inoculation; heart, joint and skin specimens were cultured for spirochetes. The ID50 values of the clones ΔospC/ΔNt5/1 and ΔospC/ΔNt5/2 were 18 and 32 organisms, compared to 18 and 32 organisms determined for the clones ΔospC/FL/1 and ΔospC/FL/2, respectively . In the remaining mice, joint swelling did not become apparent until 3 weeks post-inoculation and slowly developed after then. All mice were euthanized 1 month post-inoculation; DNA was extracted from heart, joint and skin specimens and quantified for bacterial burden. Although the ΔospC/ΔNt5 spirochete burden was 6.7-fold lower than that of the ΔospC/FL (P = 2.83×10−10) in the heart tissue, there was no significant difference detected either in joint (P = 0.52) or skin tissue (P = 0.13)  . The stu50 value was examined at one month post-inoculation. To determine whether the deletion caused deficiency in dissemination, we examined infection at different time points. Groups of 10 or 15 SCID mice each received a single intradermal/subcutaneous inoculation of the clone ΔospC/FL/1 or ΔospC/ΔNt5/1. Five animals from each group were euthanized at 1-week intervals; inoculation site and remote site skin, ear, heart, and joint specimens were harvested for spirochete isolation. As a positive control, the ΔospC/FL/1 bacteria were grown from all of the skin, joint and heart specimens but from none of the ear samples at first week; all sites became culture positive at 2 weeks after initial inoculation , and β-sheet 1 (5 AA) and 2 (5 AA), completely destroys its protective function . This leads us to hypothesize that Osps may play a common structural role that is required for the protective function of the borrelial surface lipoproteins. Some deletions on OspC may cause this special structure to collapse and consequently abolish the protective function.Our previous study showed that increasing production of one of four randomly chosen surface lipoproteins can replace OspC to protect B. burgdorferi must first be able to evade initial innate immune elimination and gain a foothold. This ability is best reflected by the ID50 value because this criterion measures the smallest number of organisms that is sufficient to escape initial innate elimination and establish an infection. In spite of large inocula, OspC-deficient spirochetes are quickly cleared after inoculation into murine skin B. burgdorferi with ID50 values similar to the control, indicating that the truncated molecule retains the full protective function of OspC.After inoculated into murine skin, B. burgdorferi producing OspC with 5-AA deletion was able to generate similar bacterial loads in both joint and skin tissues of SCID mice as the control, indicating that the truncated OspC can effectively protect the pathogen against innate immune clearance at least in these tissues. Because many other determinants, such as the interactions of host cells and extracellular matrices with the pathogen mediated by its surface antigens, may significantly influence tissue colonization, the lower ΔospC/ΔNt5 spirochete load in the heart does not necessarily suggest that the truncated OspC cannot effectively provide protection against innate immune elimination. Alternatively, OspC may be an important factor contributing to the ability of B. burgdorferi to colonize the heart tissue. This explanation is also supported by previous studies showing that treatment with OspC antibody shuts off ospC expression and more effectively reduces the bacterial load in the heart than other tissues Although tissue colonization is an extremely complicated event carried out by an extracellular bacterial pathogen during infection and can be affected by many factors, one can envision the ability of the pathogen to evade immune clearance should be one of the major determinants. 50 and tissue bacterial load data demonstrated that the 5-AA deletion does not affect the protective function of OspC, allowing us to dissect the protective from dissemination-facilitating functions. B. burgdorferi producing OspC with 5-AA deletion disseminated to remote tissues, especially to the heart and ear at a remarkably slow pace in immunodeficient mice. In immunocompetent mice, the defect in dissemination caused by deletion was much more severe. Four weeks after inoculation, the mutant was not able to colonize heart or ear tissues, in contrast to the control, which disseminated to all tissues examined within 2 weeks. The study also highlights the importance of quick dissemination during infection of immunocompetent animals. As infection induces immune responses, if B. burgdorferi fails to colonize remote tissues and establish a systemic infection before effective immune responses develop, its infectivity potential would be severely impaired.Both IDFigure S1Deletion of N-terminus 5-AA or 10-AA does not affect the surface location of OspC. The ΔospC/FL/1, ΔospC/ΔNt5/1 or ΔospC/ΔNt10/1 spirochetes were grown to late log phase (108 per ml) in BSK-H medium at 33°C. Spirochetes were incubated with OspC MAb, washed in PBS, probed with FITC-conjugated goat anti-mouse antibody, washed again before being placed onto slides. Differential interference contrast (left panel) and immunofluorescence images (right panel) were taken from the same field. Imagines were taken at ×400 magnification.(PDF)Click here for additional data file.


The timely diagnosis of hearing loss in the pediatric population has significant implications for a child’s development. However, audiological evaluation in this population poses unique challenges due to difficulties with patient cooperation. Though specialized adaptations exist (such as conditioned play audiometry), these methods can be time consuming and costly. The objective of this study was to validate an iPad-based play audiometer that addresses the shortcomings of existing audiometry.We designed a novel, interactive game for the Apple® iPad® that tests pure tone thresholds. In a prospective, randomized study, the efficacy of this tool was compared to standard play audiometry. 85 consecutive patients presenting to the Audiology Clinic at the Children’s Hospital of Eastern Ontario (ages 3 and older) were recruited into this study. Their hearing was evaluated using both tablet and traditional play audiometry.Warble-tone thresholds obtained by both tablet and traditional audiometry.The majority of children in this age group were capable of completing an audiologic assessment using the tablet computer. The data demonstrate no statistically significant difference between warble-tone thresholds obtained by tablet and traditional audiometry (p=0.29). Moreover, the tablet audiometer demonstrates strong sensitivity (93.3%), specificity (94.5%) and negative predictive value (98.1%).The tablet audiometer is a valid and sensitive instrument for screening and assessment of warble-tone thresholds in children. The timely diagnosis of hearing loss in children has significant implications for a child’s social and cognitive development. In young children, hearing loss is often inconspicuous resulting in delayed diagnosis and rehabilitation. As a result, children are at increased risk of delayed speech acquisition and the subsequent long-term sequelae. Speech delay and hearing loss also represents a significant cost to society due to the resources required for their treatment, such as special education, speech therapy, and social services ,2. HowevDefinitive diagnosis of hearing loss is typically made through audiologic assessment of pure-tone air, bone and speech thresholds. Traditionally, pure-tone thresholds are documented by asking the subject if they can hear tones of varying frequency and intensity. While auditory brainstem reflex and otoacoustic emissions testing remain the gold standard of hearing assessment, these investigations pose some difficulty in children. Currently, standard clinical audiometry is performed using non-portable hardware and access is therefore limited in developing countries, where hearing loss is more prevalent .Automation of pure-tone audiometry offers several potential benefits . ParticuPure-tone audiometry, regardless of automation or lack thereof, is a mundane task. It is especially challenging to perform in the pediatric population where short attention span and the extent of cognitive development can be limiting factors. Children with hearing impairment may find audiometric testing even more difficult. Several adaptations of pure-tone audiometry are used to evaluate hearing in children. These techniques include behavioral observation, visual re-enforcement, and conditioned play audiometry. While more successful than conventional pure-tone audiometry, these adaptations are resource intensive and typically require two specially trained audiologists to administer. Naturally, an ideal solution would capitalize on the advantages of automation, while maintaining clinical validity and age appropriateness.With the success of the Apple® iPad® , it has become evident that touch interface computing enables even the youngest of users to interact intuitively with complex systems. It is not surprising then that portable tablet computers have been widely embraced in healthcare and education -19. ThusThe purpose of this study is to (1) determine the accuracy of audiometric thresholds obtained by tablet audiometry compared to the accepted standard, conditioned play audiometry in children; and (2) describe our experiences with administering an iPad-based hearing assessment in the pediatric population.We expect children ages 3 and above to be capable of completing a hearing assessment using the tablet audiometer. Further, we hypothesize that the tablet audiometer will be a sensitive screening test and that the audiometric thresholds obtained will closely correlate with conventional audiometric evaluation.Interactive audiometry is a novel paradigm in the assessment of hearing thresholds, in which the patient controls the presentation and pace of sound stimuli, rather than the audiologist. The tablet play audiometer is engineered as a decision-tree interactive game (yes/no paradigm) or as a two alternative forced choice interaction . The twoIn this test paradigm, the user is sequentially presented with a series of objects  and is tasked to categorize the objects into ‘sound-producing’ or ‘silent’ by dragging the objects into one of two ‘containers’ (i.e. chicken coop or egg carton) Figure . In thisStudy participants were recruited from patients presenting to the Audiology Clinic at the Children’s Hospital of Eastern Ontario. The inclusion criteria for this study comprised children at least 3 years of age with normal or abnormal hearing. The exclusion criteria for the study included patients with previously diagnosed visual impairment, learning disability, or developmental delay. These patients were identified by a staff otolaryngologist (MB) through review of their patient records prior to enrollment. If the patient was eligible, informed consent was obtained from the parent/guardian. We aimed to enroll 80 patients into the study.A prospective, randomized study design was employed. Participants completed two sequential audiometric evaluations in a double walled sound booth, one with the tablet audiometer and one with traditional play audiometry assessing warble-tone thresholds. The order in which participants performed the two assessments was determined randomly by the flip of a coin. For the sake of simplicity and speed, unmasked air conduction was the sole investigation in this study. Sound field testing was performed if the child was not amenable to wearing headphones and binaural testing was performed in all other children. Stimuli were presented using TDH-39 headphones in both the tablet and audiologist test scenarios. When sound field testing was employed, the sound booth speaker system or the tablet speaker were used. An audiologist accompanied participants in both groups to provide motivation during the assessment.This study was approved by the Children’s Hospital of Eastern Ontario Research Ethics Board #11/10x.The tablet computers, headphones and speakers that were used in this study were professionally calibrated by Génie Audio Inc.  to ANSI S3.6-2004 standards .Given the novel nature of both the method and the testing hardware no prior studies were available from which to calculate a sample size. A sample size of 80 consecutive patients was selected based on related publications comparing automated hearing tests to standard audiometry ,10,11. FThe primary outcome measure consisted of warble-tone thresholds obtained by both the tablet audiometer and by standard play audiometry. Normal hearing was defined as a threshold of less than or equal to 25 dB in each of the 4 test frequencies . Hearing loss was defined as audiometric thresholds greater than 25 dB in any of 4 test frequencies. Secondary outcomes related to participant performance were also documented, including time to completion as well as feedback from the audiologists. In the tablet group, test reliability was evaluated by calculating the percentage of correctly sorted silent objects.Participants were excluded from analysis for (1) technical/gameplay issues (2) behavioural issues, or (3) questionable reliability, defined as incorrectly assigning greater than 50% of the silent objects. Descriptive statistics were used to summarize participant characteristics. Student’s t-test with Bonferroni correction was used to compare secondary outcomes between the two groups. Preliminary evaluation of the tablet’s performance was determined using a two-by-two table and calculations of sensitivity, specificity, positive and negative predictive value, and likelihood ratios positive were calculated.A more intricate analysis of the concordance between the two hearing assessments was also performed. In order to test for overall differences between modalities (tablet versus play audiometry), a repeated measures model for the detection threshold in each ear at each frequency was fitted using linear mixed effects modeling. The model included fixed effects for testing modality (tablet versus play audiometry), frequency, and ear. In order to account for the correlation of thresholds within participants, random effects modeling was used for frequency nested within ear nested within participant.85 consecutive patients who met the inclusion criteria were recruited into the study. 15 patients were excluded after hearing assessment, resulting in 70 subjects available for analysis. Fourteen of the 70 patients were tested using a sound field. The remainder (56) had binaural assessments for both hearing evaluations. The mean age of study participants was 5.2 years (range 3–13). The demographics for these individuals are summarized in Table Fifteen subjects were excluded from the analysis. Four were excluded from analysis due to poor reliability (<50%). Four patients were excluded for behavioural issues that prevented successful completion of the one or both hearing assessments. Seven patients were excluded for technical issues related to test administration. Of note, 10 of 11 subjects in the latter two categories had some degree of hearing loss. These results are summarized in Table Overall, 55 patients were identified to have normal hearing by conditioned play audiometry, the traditionally accepted standard test. Of these, 52 were found to have normal hearing by tablet audiometry. The 3 remaining children scored slightly outside the defined parameters of normal hearing, namely a 30 dB threshold, in at least one frequency. This appeared to either be due to the child moving through the game too quickly or the presentation timing out before the child could make a decision.The mean time to complete a binaural hearing test with the tablet was 152 s (SD±114 s) overall. In the group with normal hearing, the time was 109 s (SD±65 s). This increased to 317 s (SD±113 s) in children with abnormal hearing. The difference in test duration was statistically significant (p<0.0001).53 patients in total had normal evaluation by tablet audiometry. One of these children was found to have a true mild hearing loss. This child appeared to understand the game but their results did not correlate well. The reliability score of this patient when using the table was only 75%. However he did not meet reliability criteria for exclusion (<50%). The results of the 2×2 table are summarized in Table For the repeated measures analysis, 56 participants with valid results and binaural testing were available. Patients who were tested with a sound field were excluded from this particular analysis, as ear specific information was not available, making analysis impossible.The model showed no significant effect of testing modality . The modeling was repeated for the abnormal cases (n=15), and for the normal cases (n=41). In both cases, the model showed no significant effect of testing modality. Compared to play audiometry, the mean tablet threshold was 1.13 dB lower  and 0.12 dB higher , respectively.play audiometer to be used in a pediatric setting. The purpose of this study was two-fold, to validate the tablet audiometer as a child-friendly application for hearing assessment, as well as to compare tablet thresholds to the traditionally accepted standard play audiometry.This paper describes the first trial of both a novel play algorithm using interactive audiometry and a new tablet audiometer. It is, to our knowledge, the first tablet-based, semi-automated, The data reveal that the tablet audiometer produces warble-tone thresholds that are in agreement with the accepted standard . This was achieved with narrow confidence intervals, suggesting sufficient statistical power. Audiometric data are acquired in an efficient manner, as demonstrated by a mean test duration of approximately 2.5 minutes. Moreover, the data reveals a high specificity 94.5% with a negative predictive value (NPV) of 98.1%, denoting that tablet audiometry is a robust screening tool. A positive likelihood ratio of 17.1 confirms the tablet audiometer’s capacity to diagnose hearing loss Figure .As the tablet audiometer is by definition an objective test, if calibrated adequately, it is not likely to be subject to issues with inter-rater reliability. However, it will be prudent in subsequent investigations to ensure strong inter-rater and test-retest reliability.Conditioned play audiometry can often be employed with children as young as 2 years of age. The supervision and motivation given to the child by a second audiologist in the sound booth allows these very young children to be tested. In the current study, this second audiologist was present for both assessments  to maximize the successful completion of assessments, although attaining the appropriate level of support from the audiologists required training and experience.Our data suggests that using the tablet interactive audiometry method, the majority  of children as young as 3 years of age are capable of understanding the concept of the game and completing the hearing assessment. In fact, 4 of the 15 patients were unable to complete the assessment due to a technical issue related to the audiologist. Therefore, 82% is a somewhat conservative assessment of the user-friendliness of the tablet audiometer. Despite the supervision of an audiologist however, some children had difficulties with the tablet audiometer. These difficulties resulted from technical deficiencies of the hardware/software  and behaviors of the patient . A number of these difficulties eventually lead to the subject being excluded from the statistical analysis. Failure to complete the assessment also appeared to be more prevalent in children with abnormal hearing.Several technical/gameplay issues were documented during data collection. In particular, children showed signs of fatigue with either test method quite quickly. During standard play audiometry, audiologists often switched games several times during standard play audiometry to keep the child engaged. By contrast, only two games were available when using the tablet, with the current software version. This stresses the importance of maintaining attention in this particular age group.Furthermore, due to the nature of interactive audiometry, whereby the test is user-directed, action is required at each point in the decision tree. This gives the appearance of more decisions as compared to standard audiometry, where users who did not hear a sound were not required to perform an action. This was exacerbated in children with hearing loss, who were required to sort more objects in order to determine exact thresholds. For example, when testing a normal hearing individual, the minimum number of objects to complete an entire assessment was 16. This number increased to a maximum of 113 when hearing loss was present or unreliable results were being obtained. The average number of objects presented in in hearing loss was 67.1 (SD±19.1)  insinuates a high degree of user-friendliness, especially given the potentially difficult patient population. Additionally, this emphasizes the importance of audiologist supervision, as the software is currently unable to determine if a child fully comprehends the game. Further gameplay refinement will likely increase the number of patients who are suitable candidates for tablet audiometry.The goal of our research is to develop a portable, versatile clinical audiometer designed for children. This study is the first to validate an automated play audiometer designed specifically for the pediatric population. In this study, we demonstrated that air conduction thresholds obtained by tablet audiometer are not significantly different from those obtained by standard play audiometry. The device has a strong predictive value for normal hearing and is highly sensitive for hearing loss. Finally, our results demonstrated that the tablet audiometer is a viable platform for testing children as young as 3 years of age. Future directions will focus on gameplay refinement and further validation testing of air vs. bone conduction thresholds and test-retest reliability.At the time of research the authors had no conflicts of interest to disclose, however the idea has since been patented.JY was responsible for study design, statistical analysis and manuscript preparation. HJ was responsible for study design. SH, YB and SC were responsible for administration of audiometric testing and data collection/organization. MB is the senior author and involved in all levels of study design, data analysis and manuscript preparation. The manuscript was read and approved by all authors.CHEO AFP Innovation FundDepartment of Surgery Research Fund