The ability to separate two interleaved melodies is an important factor in music appreciation. This ability is greatly reduced in people with hearing impairment, contributing to difficulties in music appreciation. The aim of this study was to assess whether visual cues, musical training or musical context could have an effect on this ability, and potentially improve music appreciation for the hearing impaired.Musicians (N = 18) and non-musicians (N = 19) were asked to rate the difficulty of segregating a four-note repeating melody from interleaved random distracter notes. Visual cues were provided on half the blocks, and two musical contexts were tested, with the overlap between melody and distracter notes either gradually increasing or decreasing.Visual cues, musical training, and musical context all affected the difficulty of extracting the melody from a background of interleaved random distracter notes. Visual cues were effective in reducing the difficulty of segregating the melody from distracter notes, even in individuals with no musical training. These results are consistent with theories that indicate an important role for central (top-down) processes in auditory streaming mechanisms, and suggest that visual cues may help the hearing-impaired enjoy music. Music often contains multiple “streams”–a number of melodic lines for instance–either played on the same or separate instruments. In order to enjoy music, listeners must be able to perceptually separate and group auditory streams. This ability is called auditory stream segregation, and is based partly on perceptual differences (such as pitch and timbre) between the streams However, recent research The power of visual cues to improve auditory perception has long been known, particularly in the case of speech perception in background noise. When a speaker's lip and facial movements are visible, an improvement in performance equivalent to increasing the signal-to-noise ratio by up to 15 dB has been observed Visual stimuli can also influence how sounds are grouped perceptually. In an early study investigating the interplay between auditory and visual grouping, O'Leary and Rhodes et alsegregated perception. Thus, the visual cue was found to improve the ability to segregate two streams of ambiguously organised tones. However, it is unknown whether this benefit extends to segregation in music, such as in the segregation of a melody from background notes.More recently, Rahne Musical education is an intense training activity–professional musicians spend many hours per day listening to and producing multiple streams of auditory information. This intense activity, usually over the course of many years, has a variety of effects on behaviour, brain structure and brain function. A combined magnetoencephalography and structural MRI study Training in music has also been shown to influence auditory stream segregation. The decay of streaming effects occurs more slowly in musicians compared to non-musicians decreased until participants were able to name the melodies . However, when two high-performing participants repeated the experiment with the overlap gradually increasing, they were able to continue following a single melody even when the melodies completely overlapped. Unfortunately, apart from noting this effect in these two participants, Dowling did not test the effect on stream segregation of increasing or decreasing the overlap between the melodies. It would seem reasonable to assume that listeners should be able to better segregate the melody from the background notes in the increasing context compared to the decreasing context due to the longer exposure to the segregated melody. However, recent studies by Snyder et al. et al. should accelerate the fusion likelihood between the melodies.Musical phrases are heard within a specific context. For example, a specific melodic line may be heard against a variety of different backgrounds–sometimes appearing out of the background and sometimes disappearing into it. Depending on the prior knowledge of the listener, and on the specific arrangement of the musical background, a melody can be perceived in different ways. Listeners with more experience may be able to maintain their perception of a melody as it becomes more entangled in accompanying notes and may be able to identify it earlier as it emerges. The emergence of the melody can be influenced by the separation in pitch of the melody and background notes. Dowling In the present experiment, the effect of visual cues, musical training and musical context on musical streaming were examined in normal-hearing listeners. A musical streaming paradigm was employed that involved the extraction of a simple repeating melody from a background of interleaved random distracter notes see . The mel1] Visual cues would reduce the difficulty of extracting a simple melody from random distracter notes2] Musicians would find the task less difficult overall and benefit more than non-musicians from the visual cues3] The musical context would affect the result as it should be easier to segregate a melody in the INC condition than the DEC condition.If visual cues decrease the difficulty of segregating a melody from background notes, it may be possible to design an appropriate visual cue for use by the hearing impaired in order to improve their ability to appreciate music. It is unknown whether musical training will be required to utilise the visual cue. If musicians gain more benefit from the visual cue, training using the device may transfer to everyday listening situations.The criteria for defining ‘musicians’ vs ‘non-musicians’ is variously defined on the basis of years of experience i.e., the melody was perceived as segregated. The difficulty ratings were generally higher with higher levels of overlap. Finally, when the distracter range totally overlapped the melody (the highest distracter note was above midinote 74), the melody was judged as impossible to perceive. All participants showed a similar pattern.p levels and estimates of sphericity (ε) are reported if Mauchley's test was violated. In order to asses the significance of the effects of visual cues, musical training and musical context on the difficulty of extracting the simple melody from the distracter notes, the difficulty ratings were entered into a repeated-measures mixed ANOVA with a between-groups factor Group , and within-groups factors for Vision , Context , Repeat , and Overlap . See As expected, a significant main effect of Overlap was found indicating that as the overlap between the melody and the distracter increased, difficulty ratings increased significantly. A significant main effect of the factor Vision was found, indicating that, overall, difficulty ratings were significantly lower when the visual cue was present. A significant Vision-by-Group interaction was found, indicating that difficulty ratings were significantly reduced to a greater extent for non-musicians than musicians when the visual cue was present. Furthermore, pairwise comparisons found no significant reduction in difficulty for non-musicians when the visual display was present (p = .11), and a highly significant reduction for non-musicians (p<.001).There was also a significant Context-by-Overlap interaction, indicating that difficulty ratings varied across overlap levels differently depending on the context (INC or DEC). This indicates that when the distracter notes overlapped the melody, difficulty ratings were different for a given overlap level, depending on the context. No significant main effect or first-order interaction was found for the factor Repeat, indicating that difficulty ratings were consistent within INC and DEC blocks, no matter whether they were presented first or second.In Experiment 1, listeners were asked to rate their subjective perception of the difficulty of the task. The rating results thus may have included a component related to the response bias of the listeners. It is possible that a listener may over- or under-estimate the true difficulty. In order to validate the method, a control experiment was performed with 9 of the participants. For the sake of brevity, only a summary of the method and results is reported here. The stimuli and procedure were similar to the Experiment 1, except that: 1] an inversion of two notes of the melody was pseudo-randomly inserted, 2] the task was to report detections of the inversion with a button-press, and 3] only Non-Visual conditions were tested. It was assumed that if the listeners were able to segregate the melody, the detection task would be easy when there was no overlap between melody and distracter, and would gradually become difficult as the overlap was increased. Average difficulty ratings from Experiment 1 were compared with the miss rate (number of misses divided by the number of inversions) for melody inversions. In this study, it was demonstrated that the rated difficulty of extracting a simple melody from a background of random distracter notes increased as the overlap between melody and distracter notes increased. It was also shown that the presence of visual cues showing the entire melody, as well as the exact melody note playing at any instant, could reduce the rated difficulty of segregating the melody. It was found that musical training was not necessary to gain an advantage from the visual stimuli. This effect was relatively strong in listeners without musical training, however those with musical training received no additional benefit from the visual cues. These results indicate that visual cues could potentially help to restore part of the musical information degraded by hearing loss, and that no special training may be required to make use of these visual cues. Long-term musical training also reduced the rated difficulty, but provided no extra benefit in utilising the visual cues.The task of separating a melody from a complex musical context is often required in many types of music appreciation. For example, in a piece of solo piano music, there is often a melody line carried by the right hand, and various accompaniments in the left hand. The ability to separate the melody from the accompaniment is vital to appreciating the intent of the composer and perceiving the affective impact of the music. In a pioneering study, Dowling The key finding in the current study was the reduction of melody extraction difficulty when visual cues were provided. The visual cues took the form of a musical stave which was presented on a screen immediately in front of the participants  toward that level, compared to the same overlap level in DEC blocks, where the melody and distracter note range was initially overlapping. This result can be partly explained by the well known build-up effect There has been very little research investigating the effect of context in the segregation of melodies from background notes; however, the concept is similar to the gestalt principle of ‘emergence’. The typical example of emergence is in vision–a photograph of a spotty Dalmatian is degraded such that all the elements, including the dog, are made up of black or white spots et al.et al., this previous exposure increases the likelihood of fusion between each sequence and therefore increase the difficulty to perceive the melody.On the other hand, when the difference between the melody and the distracter notes was within a “bistable region,” when the listener's perception could switch easily between one or two streams, ratings were higher for INC blocks compared with DEC blocks. This result is consistent with the “contrastive context effect” found by Snyder The current study was undertaken in order to assess whether visual cues may improve the ability to segregate musical sources for the hearing impaired, and whether training would be required to use these visual cues. When no visual cues were present. it was shown that those with musical training found the melody extraction task easier, indicating the effect that top-down processes like training can have on auditory stream segregation. It was also shown that visual cues could indeed reduce the difficulty of segregating a melody from background notes. These results demonstrate the possibility of using visual cues, either as part of an active listening device or as a training device, in order to improve music appreciation for the hearing impaired. More research is required to better understand the types of visual cues that will be most useful in this regard.with musical training, there was no additional benefit of the visual cue, showing that there is no super-additive effect of both training and visual cues. The results also show that long-term training is not necessarily required in order to provide a useful enhancement of melody segregation by a visual cue. Providing a simple visual cue reduced the difficulty of extracting the melody to approximately the same degree as extensive musical training. These results show that there may be two approaches to improving stream segregation ability–the provision of assistive visual cues, and music training. However, training may still be of assistance in combination with visual cues, especially for those with hearing impairment. As hearing impaired listeners are often highly experienced with extracting auditory information from real-time visual cues  it might be possible that they will gain even more from visual cues, and that this type of ‘real life’ training may be more effective than the formal music training tested here. Further studies investigating this effect in listeners with hearing impairment will be helpful in determining the extent of the benefits this approach to improving music appreciation for the hearing impaired may provide.For those The experimental protocol conforms to The Code of Ethics of the World Medical Association (Declaration of Helsinki), and was approved by the Human Research Ethics Committee of the Royal Victorian Eye & Ear Hospital (Project 09-880H). Written informed consent was obtained from all participants involved in the study.Thirty-seven participants  were recruited from the community using social networks and advertisements in music schools. Ages ranged from 18 to 45 years . All participants reported normal hearing and normal or corrected-to-normal colour vision. Travel and lunch expenses were reimbursed $40 AUD. In order to assess participants' musical ability, four measures of musical activity were recorded: self-ratings on 0–5 scales for sight-reading ability and general musical aptitude, the number of hours of musical practice per week, and years of musical training.The melody and distracter notes were constructed using Matlab 7.5 and presented using MAX/MSP 5 through an M-AUDIO Firewire 48-kHz 24-bit sound card. Each note consisted of a 180 ms complex tone with 10 harmonics. Each successive harmonic was attenuated by 3 dB, and each note included a 30 ms raised-cosine onset and 10 ms offset. The notes were played from a loudspeaker (Genelec 8020APM) positioned on a stand at the listener's ear height, 1 m from the listener's head. Each note was equalised in loudness to 65 phons according to a loudness model The participants were exposed to a series of notes with each note onset presented every 200 ms. Within this series of notes was a repeated four-note target melody and interleaved distracter notes. The target melody pitches see  were G, The visual cue was generated with the software MAX/MSP 5. It consisted of a musical staff with the 4-note target melody depicted in standard musical notation see . Each noTwo counterbalanced sessions were run for each participant–one with the visual cue present (Vision) and one without (No-vision). The participants were asked to rate the difficulty of perceiving the four-note melody continuously throughout each block using a variable slider on a midi controller (EDIROL U33). The slider was labelled from 0 (no difficulty hearing melody) to 10 (impossible to hear melody). Participants were instructed to move the slider to the “10” position if the melody was impossible to perceive and to the “0” position if the melody could be easily perceived.Each session was divided into four blocks - two blocks where the overlap of the melody and distracter notes gradually increased (INC) and two where it gradually decreased (DEC). The overlap was varied in 20 levels from no overlap (plus a separation of one octave between the highest distracter note and the lowest melody note) to total overlap, and expressed as the midinote value of the highest note in the distracter range. In INC blocks, distracter notes were initially picked from the range of midinotes 45–56. This starting range provided an octave separation between the highest possible distracter note and the lowest melody note, and was selected to ensure that the melody was easily perceived for every participant. The range of possible distracter notes was then slowly increased until they completely overlapped the melody (midinote range 65 to 76). In each level, the melody was repeated 10 times (lasting 16 seconds). In DEC blocks, the distracter note range initially completely overlapped the melody (midinote range 65 to 76) and was decreased in twenty steps until it reached the minimum level (midinotes 45–56). The INC and DEC blocks provided different musical context for the melody. The procedure is illustrated in Before each test session, the melody was presented 20 times without distracter notes; an INC practice block followed. During testing, each INC/DEC block was repeated twice, with INC-DEC-DEC-INC or DEC-INC-INC-DEC order counterbalanced across participants. The duration of each block was about 5 minutes, and each session lasted about 30 minutes.In order to reduce possible pitch memory effects between Vision and No-vision sessions, a pitch increment, randomly chosen between 0 and 4 semitones, was added to all notes of the same session.


Although reciprocal regulation of protein phosphorylation represents a key aspect of signal transduction, a larger perspective on how these various interactions integrate to contribute towards signal processing is presently unclear. For example, a key unanswered question is that of how phosphatase-mediated regulation of phosphorylation at the individual nodes of the signaling network translates into modulation of the net signal output and, thereby, the cellular phenotypic response.To address the above question we, in the present study, examined the dynamics of signaling from the B cell antigen receptor (BCR) under conditions where individual cellular phosphatases were selectively depleted by siRNA. Results from such experiments revealed a highly enmeshed structure for the signaling network where each signaling node was linked to multiple phosphatases on the one hand, and each phosphatase to several nodes on the other. This resulted in a configuration where individual signaling intermediates could be influenced by a spectrum of regulatory phosphatases, but with the composition of the spectrum differing from one intermediate to another. Consequently, each node differentially experienced perturbations in phosphatase activity, yielding a unique fingerprint of nodal signals characteristic to that perturbation. This heterogeneity in nodal experiences, to a given perturbation, led to combinatorial manipulation of the corresponding signaling axes for the downstream transcription factors.Our cumulative results reveal that it is the tight integration of phosphatases into the signaling network that provides the plasticity by which perturbation-specific information can be transmitted in the form of a multivariate output to the downstream transcription factor network. This output in turn specifies a context-defined response, when translated into the resulting gene expression profile. Reciprocal regulation of protein phosphorylation by kinases and phosphatases represents a key aspect of signal transduction -6. AlthoMurinr B lymphoma, A20, cells were first individually depleted of one of a set of ten selected phosphatases siRNA. The extent of depletion varied 65% to 90% at the protein level  obtained for each intermediate, for each of the various conditions of perturbation. Although a gross approximation, we took this value to represent signal intensity at that particular node, under that specific perturbation condition. To estimate total flux of signal generated, the AUCs of each of the nodes under individual conditions were then summed up. Figure Figure We next selected four target proteins that were stably phosphorylated – upon cell stimulation – to yield a plateau phase that was sustained over an extended period of time . Our aim was to ascertain whether this plateau phase truly described a stably phosphorylated state, or, if it simply identified an alteration in turnover between the phosphorylated and the dephosphorylated states of the protein.32[P]-orthophosphoric acid were chased with excess of non-radioactive phosphate at the time of maximal stimulation with anti-IgG. The target proteins were then immunoprecipitated from cell lysates at various times thereafter, and the extent of phosphorylation determined either by Western blot with specific antibodies, or by autoradiography to monitor the level of radioactive phosphate incorporated. Western blot analysis confirmed that stimulation of cells leads to phosphorylation of all four proteins examined, with the maximally phosphorylated state persisting over the remainder of the experiment  were immunoprecipitated from lysates of either unstimulated cells, or, cells stimulated either for 10 or 30 min. Immunoprecipitates were then subjected to a Western blot analysis with antibodies directed against the seven phosphatases identified in Figure Figure To examine the consequences of phosphatase-induced modulations in signaling behavior, we studied activation of a set of three transcription factors (TFs) as a simple and direct readout for modulations in net signal output. These wFigure max/tmax; measured as the ratio of the peak activation and the time taken to achieve it), the total area under the phosphorylation curve (A), and the rate of subsequent dephosphorylation (Δ)[To extract underlying inter-dependencies between the signaling network and TF activation, we used Partial Least Square Regression (PLSR) analysis -20. We tation (Δ). DetailsWe next enlisted all of the principle component axes obtained in the models for each of the TFs. The corresponding signaling parameters were then arranged along these axes in the descending order of their significance to determine whether this produced segregation between those signaling parameters that correlated positively and negatively, with the activation of that particular TF. PC2 yielded this segregation for the models for NFAT and AP1, whereas it was PC1 for the pp65-derived model  and Q2 (predictive ability).Click here for fileMicroscopy images of AP1 activation under Signaling intermediate knockdown condition. Confocal microscopy images for the activation of AP1 under new set of perturbations.Click here for filePredictive ability of the model for untrained data. Ability of the PLS model to predict AP1 activation under untrained conditions.Click here for fileAlignment of signaling parameters on PC1 and PC2axes and respective correlation with the three TF activation profile. Functional segregation of signaling parameters on principle component axes along specific TF activation.Click here for fileSensitivity of response specific VIPs to perturbations. Sensitivity of response specific VIPs to perturbations.Click here for file


The first two amino acids are not necessary since the eptapeptide 206–212 was equipotent in both assays (IC30 of 12.5 μM and 420 μM). Of the two pentapeptides 204–208 and 208–212 only the latter showed inhibitory activity in both models although the potency was much reduced (IC30 of 170 μM and 630 μM) compared with that of the parent nonapeptide. Comparison of peptide 204–212 effects with those of its analogues on LC1 and LC2 indicate that lysine 208 and aspartic acid 211 are essential in order to maintain a fully active nonapeptide.Peptide 204–212 of lipocortin (LC) 5 inhibited porcine pancreatic phospholipase A


Computational Biology and Bioinformatics (CBB) are indispensable components in the training of life scientists The Guide refers to the preparation, presentation, and assessment of CAI. It should be an aid for those who teach CBB with CAI in class, and it is expected to stimulate student motivation and deeper learning in CBB, thus making class time more effective and improving satisfaction of both students and instructors.CAI activities in a course should not be isolated exercises, but should be embedded in lesson plans and integral to the instructor's goals CAI is the first option, if the goal is developing students' IT skills or other skills difficult to attain in the real world. For instance, computer-based laboratory simulations have been used in place of dangerous, time-consuming, ethically constrained, or expensive experiments Inadequate infrastructure and deficient on-site technical or teaching assistance can limit the effectiveness of CAI applications, so plans should be adjusted to existing conditions.The following items should be included in a checklist: (a) facilities ; (b) characteristics of the computers ; (c) technical support ; (d) onsite teaching assistants; (f) ethical and copyright issues; (g) connectivity and network. All instructors should be familiar with the CAI resources. Therefore, training sessions should be provided for instructors when necessary.Science and engineering students are more motivated and learn better when they are actively engaged than when they are simply watching and listening Student-centered learning in class can be implemented with CAI Providing the appropriate guidance becomes critical when CAI is used with complex problems Make sure that your students read and understand the CAI tasks, the deadlines, and their role in instruction. Present instructional objectives in terms of contents and skills, for example, “at the conclusion of this exercise you should be able to search a database for specific gene sequences.” Adjust the intended conceptual depth and mastery of skills to a feasible level. A good challenge pulls the student ahead and promotes learning, whereas objectives that are out of reach result in frustration. Keep in mind that students often find it difficult to work to achieve instructional objectives. A clear understanding of goals will increase student motivation, independence, and satisfaction with the CAI class The dynamic presentation of processes and theoretical models is a great strength of CAI Always be aware that assessment drives learning. Students tend to ignore instructional activities that make no contributions to marks CAI is not the only solution in education, and your syllabus may be better taught by alternative methodologies. CAI is powerful in achieving educational goals such as the development of skills involved in data searching, integration and analysis, leverage of IT proficiency , and the development of visual literacy. However, merely using keyboards and screens instead of pen and paper does not guarantee improvements in teaching or learning


Signaling networks are designed to sense an environmental stimulus and adapt to it. We propose and study a minimal model of signaling network that can sense and respond to external stimuli of varying strength in an adaptive manner. The structure of this minimal network is derived based on some simple assumptions on its differential response to external stimuli.We employ stochastic differential equations and probability distributions obtained from stochastic simulations to characterize differential signaling response in our minimal network model. Gillespie's stochastic simulation algorithm (SSA) is used in this study.We show that the proposed minimal signaling network displays two distinct types of response as the strength of the stimulus is decreased. The signaling network has a deterministic part that undergoes rapid activation by a strong stimulus in which case cell-to-cell fluctuations can be ignored. As the strength of the stimulus decreases, the stochastic part of the network begins dominating the signaling response where slow activation is observed with characteristic large cell-to-cell stochastic variability. Interestingly, this proposed stochastic signaling network can capture some of the essential signaling behaviors of a complex apoptotic cell death signaling network that has been studied through experiments and large-scale computer simulations. Thus we claim that the proposed signaling network is an appropriate minimal model of apoptosis signaling. Elucidating the fundamental design principles of complex cellular signaling pathways such as apoptosis signaling remains a challenging task. We demonstrate how our proposed minimal model can help elucidate the effect of a specific apoptotic inhibitor Bcl-2 on apoptotic signaling in a cell-type independent manner. We also discuss the implications of our study in elucidating the adaptive strategy of cell death signaling pathways. Cellular signaling networks are designed to sense an environmental stimulus and respond in a strength dependent manner. In this manuscript, we develop and study a minimal model of a signaling network that can respond to an external stimulus in a manner such that the activation is fast under a strong stimulus but slow if the stimulus is weak. We derive the minimal network by assuming that the cell-to-cell variability dominates the slow signaling activation, under weak stimuli, in order to adapt to a fluctuating environment. In such a scenario, population average over many cells cannot capture cell-to-cell variability in signaling. We employ stochastic differential equations and stochastic simulations to study the signaling response in our proposed minimal signaling network. We carry out a sensitivity analysis of the minimal model with respect to parameter variations that also provides simple quantitative relations connecting different parameter values. We further use probability distributions of signaling molecules to characterize differential signaling response in the minimal network and such distributions show very distinct types of behavior depending on the strength of the stimulus. Specifically, for the case of a weak stimulus, a characteristic bimodal distribution is obtained for the activation of a downstream signaling molecule indicating large cell-to-cell fluctuations.Interestingly, the results from our minimal stochastic signaling model capture the essential stochastic signaling behavior observed in simulations of complex apoptotic cell death signaling pathways We designed our proposed minimal signaling network to respond to an external stimulus in a manner dependent on the strength of the stimulus.Under a strong stimulus, activation of the signaling network is fast and there is no need for cell-to-cell stochastic fluctuations;whereas under a weak stimulus, activation of the signaling network is slow with large cell-to-cell variability so that it can adapt to a fluctuating environment.We attempted to find a minimal set of signaling reactions that can achieve the above response, with the nature of signaling reactions being that of irreversible chemical reactions of the type Rapid deterministic activation under a strong stimulus can be achieved through a one-step direct activation of downstream signaling molecules . HoweverWe derive a stochastic differential equation for the one-step irreversible reaction The number of molecules of the activated species corresponding to This is the one step signaling reaction described by the Here we use the three steps signaling reactions described by the equation set (2) with By comparing the two coefficients in the equation for the athway:  . Hence, We use Gillespies Monte Carlo sampling technique left panel). The plot was generated for i.e. variance(left panel) also shows that Fano factor at a given time increases with increasing For a weak external stimulus  results . Decreasignaling . The smamiddle panel). Also note, for pure type B signaling (when the kinetic constant for type A is set to zero), presence of a large number of molecules of an initial stimulus  does not change the observed stochastic signaling behavior.The stochastic signaling observed in response to a weak external stimulus is essentially dominated by the type B pathway, as a similar signaling response is generated if the kinetic constant for the type A pathway is set equal to zero. This result is also consistent with our theoretical analyses of the minimal network that showed the presence of large stochastic fluctuations under a weak stimulus. Stochastic signaling behavior through the type B pathway in response to a weak stimulus is maintained as long as the fast-slow-fast kinetics of signaling reactions is also maintained in the type B pathway . For a sRapid deterministic activation under a strong stimulus: Under a strong external stimulus, i.e. for large values of right panel), as also observed in the theoretical study using stochastic equations. The plot was generated for Large  type A signaling dominates for all times when x0>k320 √k20x10x20/k310, (ii) type B signaling dominates from the beginning when x0<k20x10x20/k310x30. Parameter values used here are x10 = 100, x20 = 100, x30 = 100, k310 = 10−5, and k320 = 1.0.Differential signaling response of the minimal network as k(0.04 MB TIF)Click here for additional data file.


We use data from the Demographic and Health Surveys to examine the patterns of stall in fertility decline in four Eastern African countries. Contrary to patterns of fertility transition in Africa that cut across various socio-economic and geographical groups within countries, we find strong selectivity of fertility stall across different groups and regions in all four countries. In both Kenya and Tanzania where fertility decline has stalled at the national level, it continued to decline among the most educated women and in some regions. While fertility has remained at pre-transition level in Uganda over the past 20 years, there are signs of decline with specific groups of women  taking the lead. For Zimbabwe, although fertility has continued to decline at the national level, stall is observed among women with less than secondary education and those in some of the regions. We link these intra-country variations to differential changes in socio-economic variables, family planning programme environment and reproductive behaviour models. The results suggest that declines in contraceptive use, increases in unmet need for family planning, increasing preferences for larger families, and increases in adolescent fertility were consistently associated with stalls in subgroup fertility across all four countries. These results are consistent with models that emphasize the role of declines in national and international commitments to family planning programmes in the premature stall in sub-Saharan fertility transition. The persistent high fertility in sub-Saharan Africa (SSA) and its potential adverse effects on the region's development efforts led to substantial research, policy and action focused on identifying and addressing the various social, cultural and economic factors that served to maintain fertility at high levels in the region . Apart f1 in fertility decline  and within subregions and subgroups in each country. In particular, we examine why specific subgroups within a country experience an increase or stagnation in their fertility behaviour while other subgroups within the same country continue to experience a decline in their fertility. We draw implications of our findings for the future of the region's population size and policy directions.In seeking answers to these questions, we reiterate what is known about stalled fertility decline in SSA, underscoring issues relating to patterns and drivers of the stall. We quantify the magnitude of the fertility change, highlighting specificities and commonalities across four2.describe fertility trends, patterns and magnitude of stalled fertility decline in four Eastern African countries at the national levels and among subgroups within the countries;identify unique and common factors associated with stalling or continuing fertility decline among subgroups within each country;highlight the implications of the patterns, magnitude and drivers of the stall for the future population size of the region; andidentify possible policy and programmatic strategies for addressing fertility stall in the region.Following from our stated research questions, this study specifically seeks to address the following objectives:3.reproductive behaviour model, the socio-economic model and the institutional model. Changes in fertility, including both fertility transition and stall in the transition, have also been explained by changes associated with these models. Changes in fertility may result from changes in the reproductive preferences or behaviour of individual women; changes in the compositional distribution of women by different socio-economic characteristics that are associated with fertility; or changes in the institutional policy or programme environment that may constrain childbearing decisions of individual women or limit their access to reproductive health services. The effects of changes in the institutional policy or programme environment may operate through changes in individual preferences or behaviour. We summarize below the main propositions of each of these models with special focus on stalled fertility transition, how we operationalize them in the current study, and their respective importance in understanding stall in fertility decline in the Eastern Africa region. Again, we analyse these changes at the regional level within each country.Most of the literature seeking to explain fertility patterns in SSA can be classified into one of three models in terms of the factors they hold as important in explaining observed levels, differentials or trends in fertility. These are the (a)fertility stalls. The model primarily focuses on changes in fertility preferences or behaviour. In general, declining or steady age at marriage, earlier start of motherhood, shorter birth intervals, increasing out-of-wedlock childbearing, decreasing contraceptive use and/or declining infertility rates could slow or stall fertility decline  is directly related to fertility outcomes, it also captures both the changing tempo and changing onset of childbearing which are known to influence overall fertility rates .(b)3 reported that donor support for family planning commodities and service delivery fell from US $560 million to US $460 million between 1995 and 2003. Other estimates covering longer periods show how the proportion of donor assistance for family planning dwindled relative to other competing new challenges such as HIV/AIDS since the mid 1990s. For instance, One of the conclusions that has received far more consensus among scholars of sub-Saharan fertility transition is the role played by organized family planning programmes in initiating fertility decline in the region. Growing interest by many developing country governments to adopt national population policies between the late 1960s and mid 1990s was matched by phenomenal growth in international funding assistance for family planning programmes. Although only two developing country governments had official policies to support family planning programmes in 1960, this number had increased to 74 by 1975 and 115 by 1996 . BetweenScholars adopting the institutional model have specifically linked the stall in fertility decline to shifts in the observed level of support for family planning programmes either at the local, national or international levels. Under this perspective, changing patterns of unmet need for family planning, increasing levels of unwanted childbearing, declining knowledge of family planning methods or sources, increasing negative attitudes towards family planning, decreasing importance of public institutions as a key source of family planning services, etc., have all been pointed to as consequences of this shift in priority and hence, as drivers of the stall in fertility decline. Other related measures of the strength of this hypothesis include the proportion of national budgets allocated to family planning services, the proportion of international development assistance going into family planning programmes, number of organizations and personnel working in the area of family planning, and the amount of media coverage for family planning programmes ((c)Perhaps the most dominant theme in the explanation of fertility levels, differentials and trends is the socioeconomic model. Both within and across countries, differentials and changing fertility patterns have been explained largely as resulting from socio-economic and sometimes socio-cultural differences among groups. At the risk of oversimplification, the socio-economic approach to fertility analyses largely assumes that high fertility is an economically rational response . There aAlthough available evidence suggests that groups defined by several of these factors participated in the initial fertility decline in SSA, they have varied in their experiences of the stall in the fertility decline. We re-evaluate the contribution of these socio-economic factors to initial declines in fertility and, focusing on each characteristic, we assess how specific behavioural and institutional shifts have affected the relative contribution of each characteristic to the stall in fertility decline. We argue that shifts in family planning programme environment may affect different subgroups of women differently. This will lead to different behavioural outcomes and hence, differential contributions to stall in fertility decline. In our analyses, we focus on female education, place of residence and household living standard. Further, we examine changes in the proportion of women in professional employment in each region. We examine differentials and trends in fertility rates by each of these characteristics and then assess how the various institutional and behavioural factors explain the observed trends in each region.Following from the framework discussed above and availability of data, we summarize the analytical framework for this study in 4.(a)We use secondary data from the Demographic and Health Surveys (DHS) programme for four East African countries that have had four rounds of national surveys as described in Overall, data from 16 surveys were analysed for 27 regions of the four countries. TFR and other reproductive behaviour indicators  were calculated for each survey by the defined socio-economic, demographic and reproductive health service environment variables and by region. Changes in each indicator were defined by comparing estimates between two consecutive surveys. Whereas the majority of previous studies have focused on national level analyses, we based our analysis at the regional level. This approach allows us, following Generally, female education, urban residence and household wealth have been identified with initial adoption of contraceptive use and small family size values in SSA. These factors have also been linked to explanations of the stall in fertility decline in the region ((b)Predictors: changes in reproductive health service environment, socio-economic development and reproductive behaviour factors are our three key predictors of stalled fertility decline.We use change in the proportion of women who got their last contraceptive method from a public facility, change in the proportion of women who received family planning information through the mass media, and change in unwanted TFRs as proxies for changes in the family planning service environment. Other variables considered under this model include changes in attitudes towards family planning (measured by changes in the proportion of women and men who approve of family planning) and changes in proportion of women with unmet need for family planning. As national and international support for family planning programmes waned, the public sector may become less important as the main provider of family planning services; family planning information, education and communication services may suffer leading to less favourable dispositions towards family planning; and unwanted childbearing may increase as demand for services outstrip supply. Although, some of these variables are missing in some of the survey years, especially for the most recent period for family planning attitude variables, we examine their bivariate relationships with stall in fertility decline for the countries where they are available.4 to capture changes in socio-economic and demographic characteristics of women that influence fertility outcomes. Both the family planning service environment and the socio-economic characteristics operate through a set of proximate factors to influence fertility outcomes.We use changes in proportion of the most educated women (secondary level or higher), changes in proportion of females living in urban areas, changes in proportion of women in professional employment, and change in proportion of women living in poor households(i)For changes in reproductive behaviour, we examined changes in contraceptive prevalence, adolescent fertility, family size preferences and proportion of women in union. Changes in prevalence of modern contraceptive use and ideal number of children measure changes in the motivation for fertility control, while change in adolescent fertility captures the change in the tempo of reproductive behaviour beyond age at first marriage and age at first birth, as unmarried childbearing continues to assume increasing significance in the region . We incl(ii)Stall in fertility decline is our main outcome variable. ‘Stall’ is a dummy variable created by comparing fertility levels in the three most recent surveys. If change during the third period is equal to or greater than zero  while change in the previous two periods is negative (meaning that TFR has been declining in the prior surveys), ‘stall’ equals 1. ‘Stall’ equals 0 in all other cases. This definition of stall is consistent with that used by previous studies that defined stall as an interruption period in an ongoing fertility transition before a country reaches the end of the transition To identify the factors associated with stalled fertility decline, we use descriptive and multivariate methods. We examine the relationships between observed changes in institutional, socio-economic and reproductive behaviour variables and change in TFR across the four countries and all the regions within each country. Descriptive method includes analysis of change in fertility for the last inter-survey period compared with change in each explanatory variable for the same period. For the multivariate analysis, we employ a probit regression model.(i)pi is the probability that iy = 1 and Φ−1(pi) is the inverse of the cumulative distribution function of a standard normal variable. As probability ranges between 0 and 1, the probit function ranges between −∞ and +∞; and (ii) xi1, … ,xik are independent variables.Given that our main outcome is a dummy variable  and predictors are continuous, we implemented probit models whose likelihood function follows the binomial distribution. We estimated the probit model using a maximum likelihood function in the form:b, is that a one-unit increase in the predictor leads to an increase in the probit score by b standard deviations.The interpretation of a probit coefficient, (ii)The analysis is performed in three stages. First, we assess the crude effect of each independent variable on the outcome . Second, we performed analysis by the theoretical model presented in the analytical framework . We ran a separate model for each set of explanatory variables to identify the best predictors among the selected independent variables. We examined the independent variables for multicollinearity, and in cases where two or more independent variables were highly correlated, we only use one of the variables. Lastly, we included all significant variables from each model in the final model to capture their net effect.5.(a)Two distinct fertility trends are evident across the four countries. Uganda and Tanzania which, by the most recent survey period, still had TFRs of 6.7 and 5.7 children per woman, respectively, experienced the least annual per cent change in TFR at 0.5 and 0.7 per cent, respectively, while Kenya and Zimbabwe experienced annual rates of decline that were 2.5 to 3.8 times larger.Focusing on the most recent inter-survey period, however, Kenya and Tanzania show clear reversals in their fertility decline with TFR in their latest survey edging higher than in the penultimate survey. Uganda and Zimbabwe continued to experience a decline in TFR, albeit at different fertility levels. For Zimbabwe, the rate of decline has continued to decrease over the past 15 years, while in the case of Uganda, fertility only slightly declined in most recent periods, but changed very little over the 19-year period covered by the surveys. In fact, TFR remains at pre-transitional level at 6.7 children per woman by 2007 in Uganda.These four countries indeed represent three fertility regimes: Uganda represents a country at pre-transitional fertility level but one that is poised to enter the phase of incipient demographic transition; Kenya and Tanzania started their fertility transition much earlier but are currently experiencing clear stall in their fertility decline; and Zimbabwe represents a country where fertility decline, though ebbing, has continued to progress.(b)Regional analysis of fertility trends shows different patterns within the same country . Of the This pattern of wide regional variation in fertility trends within the same country is evident across the four Eastern African countries. In Kenya, Central region experienced an 8 per cent decline in fertility between 1998 and 2003 while Nyanza, Rift valley and Eastern provinces experienced increases in fertility rates of between 9 and 12 per cent. In Tanzania, fertility declined in Coastal region by 7 per cent while Southern Highlands and Central regions experienced increases in fertility rates of 14 and 13 per cent, respectively. Irrespective of fertility trends at the national level, each country contains regions that are experiencing continued declines in fertility as well as those experiencing stalls in their fertility decline. Regional analysis of fertility trends may therefore provide better understanding and explanations of stalls in fertility declines than analysis at the national level. Understanding what changes in family planning service environment, socio-economic and demographic characteristics and behavioural factors that drive these regional patterns is essential to explaining and addressing the stall in fertility decline in SSA. In the bivariate analysis, we focus attention on the regions within each country that have widely contrasting fertility patterns to assess changes in the contextual and proximate fertility determinants between the third and fourth DHS surveys.The differences in fertility patterns observed across regions within the same country may also exist among other subgroups defined by socio-economic and other characteristics. In The selectivity of fertility change observed across different regions, socio-economic groups and urban and rural areas within and across countries support findings from previous studies in the region . For insThese findings raise one key question: what factors are driving regional and other subgroup differentials in fertility trends within and across countries? To answer this question, we first compare changes in institutional and socio-economic factors associated with fertility trends in SSA as well as changes in proximate determinants of fertility as defined in our analytical framework with changes in TFR at the regional level. Through this process, we identify both unique and common factors associated with stall (or continuing fertility decline) in each region across all four countries. We further identify the common factors that determine overall fertility change at regional level across all four countries in our study by implementing multivariate regression models to assess the net effect of each of the factors defined in our framework. The results of the relationships between change in TFR and the institutional, socio-economic and reproductive behaviour factors in all regions of the four countries discussed in the subsequent sections are summarized in tables S1–S3, respectively in the electronic supplementary material.(c)(i)Tables S1–S3 in the electronic supplementary material provide trends in each of the variables included in our explanatory models. We use logarithmic scale to compute change in each of the variables within our three explanatory models of stalled fertility decline and present these in tables 5How do these national patterns differ across regions with divergent trends in fertility? Again, as In Tanzania, Coastal region experienced the largest decline in TFR between 1999 and 2004. In this region, unwanted fertility and unmet need for family planning declined significantly by 22 and 32 per cent, respectively. Approval of family planning and access to family planning information on the radio also increased significantly by 7 and 3 per cent, respectively. In the two regions that drove the increase in fertility in Tanzania , unwanted fertility increased substantially by 59–61%. However, while unmet need for family planning significantly increased in Central region, it declined significantly in Southern Highlands. In Zimbabwe, the role of the public sector in the provision of family planning services and access to family planning information on the radio declined across all provinces, suggesting an overall deterioration in the family planning service environment. The only exception is Matabeleland North where the role of the public sector increased significantly by 6 per cent. However, while unmet need for family planning increased significantly in Matabeleland North, it decreased in Midlands. These results suggest that changes in the family planning service environment may be a significant determinant of stall in fertility transition in Eastern Africa.(d)We use four variables to assess the impact of changes in socio-economic factors on the stall in fertility decline in Eastern Africa: change in the per cent of women living in urban areas, change in per cent with secondary or higher education, change in the proportion working in professional jobs and change in the proportion living in poor households. Across all four countries, percentage living in urban areas increased significantly in Kenya and Tanzania. Percentage of women with secondary or higher education increased in all countries except Kenya while the percentage working in professional jobs decline significantly in Kenya and Zimbabwe, while it increased in Tanzania and Uganda. The percentage of women living in poor households declined significantly in all countries except Zimbabwe although this may not reflect improved economic status as the variables used to define poverty, especially in urban areas may reflect more the differential availability of certain services and amenities in some geographical locations.Part of the difficulty of modelling the impact of changes in these explanatory variables on observed fertility patterns at the regional level is that regions vary enormously on these indicators, with some approximating the maximum observable value while others are at very low levels. For instance, while the percentage of urban has been 100 per cent in Nairobi region across all surveys (meaning change is zero), the percentage of urban in Eastern region was less than 1 per cent in 1988, increasing to 9 per cent by 2003. Again, focusing on regions with divergent fertility trends, we assess the extent to which observed changes in these socio-economic factors are related to observed changes in fertility trends in each region.In Kenya, Central region experienced substantial increases in percentage of women living in urban areas, proportion with secondary or higher education, and percentage working in professional jobs. Indeed, it is the only region where the proportion of women working in professional jobs actually increased given a significant decrease of 19 per cent at the national level. In addition, the proportion of women living in poor households in Central region declined by 68 per cent between 1998 and 2003. Nyanza and Eastern regions experienced significant declines in the proportion living in urban areas while Eastern and Western regions experienced significant declines in the proportion of women with secondary or higher education.In Tanzania, Coastal region which experienced the largest decline in fertility recorded significant increases in the proportion of women living in urban areas and in those with secondary or higher education between 1999 and 2004, as well as an increase in the proportion working in professional jobs. These factors declined in Southern Highlands which experienced the highest increases in fertility during the same period. In Uganda, Eastern region where fertility remained high with signs of increasing trends, the proportion of women living in urban areas significantly declined between 2000 and 2007. In Zimbabwe, the proportion of women with secondary or higher education increased significantly at the national level between 1999 and 2006. This increase was also significant in almost all regions except those experiencing a stall in their fertility decline. The proportion of Zimbabwean women in professional jobs declined significantly at the national level but this decline is only significant in two of the three regions experiencing a stall in fertility decline.(e)As noted earlier, changes in socio-economic and family planning service environment may influence fertility stall through its influence on the reproductive behaviour of individual women. Limited programmatic emphasis on family planning could reverse gains in promoting small family size values and use of contraception. Adolescents may be disproportionately affected as available services are biased towards women in union. We examine, at both the national and regional levels, how changes in these reproductive behaviour factors are related to observed changes in fertility trends. Overall, there were no significant changes in desired family size across countries and regions. Contraceptive prevalence increased significantly in Tanzania and Zimbabwe but did not change in Kenya and Uganda. While adolescent fertility increased in Kenya, it declined significantly in Uganda and only marginally in Tanzania and Zimbabwe. The proportion of women in union declined significantly in all countries except Tanzania where a marginal increase was observed.Again, we assess how these national trends operate at regional levels in each country given observed differentials in fertility trends at the regional level. Central region of Kenya is the only region in Kenya that experienced a significant decline in the proportion of women in union. Contraceptive prevalence increased in the region and in Western region while it declined in all the other regions. Nyanza province, which recorded the highest increase in fertility, also experienced a significant decline in contraceptive prevalence at 17 per cent. The fertility increase in Nairobi appears to have been driven largely by increases in adolescent childbearing, where the proportion of 15–19 year olds who have become mothers increased by 50 per cent.In Tanzania, Southern Highlands region, which recorded the highest increases in fertility between 1999 and 2004, is the only region that experienced a decline in contraceptive prevalence. Adolescent childbearing also increased significantly in Southern Highlands region by 43 per cent while Coastal region which recorded the highest decline in fertility experienced a significant decline in adolescent childbearing at 36 per cent. In Zimbabwe, although contraceptive prevalence increased significantly at the national level and across all regions, marginal declines were observed in two of the three regions (Masvingo and Matabeleland North) where fertility decline stalled. In addition, adolescent childbearing increased significantly in these two regions, despite a marginal decrease nationally.The foregoing outcomes underscore the point that a significant predictor of fertility in one country or region of a country may not necessarily be relevant in another country or regions within the same country. Following from the above, we identified three behavioural variables: increases in adolescent fertility, decreases in contraceptive use, and changing proportions of women in union as the most consistent variables associated with stall in fertility at the regional level in Eastern Africa. Also important are factors associated with family planning service environment . Changes in these variables vary not only across countries but also across regions within the same country and are generally related to observed trends in fertility patterns.(f)To identify the common predictors of stall in fertility decline across all regions in the four countries, we further interrogated the institutional, socio-economic and reproductive behaviour variables that indicated association with stall in fertility decline using the probit regression model as explained previously in §4. All variables belonging to each of the three theoretical models are included in the same model to identify the best predictors among the selected independent variables. We analysed the institutional, socio-economic and reproductive behaviour determinants of stall in fertility decline at the regional level. Our outcome variable is whether or not a region experienced a stall in its fertility decline between the third and fourth national demographic and health surveys in each country.(i)(Bivariate models ). Four variables have significant effect on the likelihood of stalled fertility decline at the regional level in the four Eastern African countries under study: change in proportion of women who approve of family planning, change in adolescent fertility, change in ideal number of children, and change in proportion of women in union.The likelihood of stalled fertility increases if ideal number of children, adolescent childbearing and proportion of women in union increase. The same trend is observed with respect to increase in unwanted fertility and unmet need but the results are not statistically significant. By contrast, the likelihood of stall is decreased if the proportion of women who approve of family planning increases. Likewise, positive change in proportion of women living in urban areas, women with secondary or higher education, and women in professional employment, as well as increases in contraceptive use are all associated with decrease in the likelihood of stalled fertility decline. However, although these results are theoretically and empirically intuitive, they are not statistically significant.Multivariate models. Our multivariate models include family planning service environment, socioeconomic and reproductive behaviour variables separately and jointly. The results are presented in subsequent columns of (g)Researchers have suggested that the future of fertility transition in SSA will depend on the future of key socio-economic indicators, including, among others, changes in women's education. Accordingly, if improvements in these socio-economic and family planning service environment indicators that have been realized in the past do not continue throughout the region, then the stalling phenomenon may well spread to more countries. Conversely, stronger progress in improving these indicators in the future would be expected to enhance the likelihood of a second wave of fertility decline in the countries currently experiencing a stall .Despite the acute awareness of the uncertainty intrinsic in population projections (Casterline Casterline a,b had s6.Our findings are generally consistent with previous studies and raise a number of programmatic and policy relevant questions. In most of the countries, contraceptive prevalence stalled or increasing trends were reversed. Even in places where prevalence has not declined, the percentage increase decelerated from one survey period to the next over the last decade. In Kenya, The observed increase in desire for large families evokes an important question. We found stall in fertility decline is positively correlated with the proportion of women with no education. This outcome is congruent with the conclusion that positive shifts in educational attainment contributed substantially to the decline in fertility in Kenya . This fiIn conclusion, the stall in fertility in Kenya and Tanzania, and the marginal fertility decline in Uganda at pre-transition levels sums the view of a truly dramatic population growth in these countries over the next 20 years. Given the strong linkages between rapid population growth and poor economic growth and development, it is evident that many countries in SSA will not meet the MDG targets. Initiating a second wave of fertility transition in the region, however, will require a refocusing of attention to family planning programmes as a critical part of development agenda in the region.7.Our analysis identifies some potential opportunities for addressing stalled fertility in the region. First, there is the potential to look beyond national level fertility indicators, with particular attention on complex contextual nuances and regional differentials within different countries and across subgroups. Second, is the need to reinvigorate campaigns on contraceptive use and engaging policy makers with evidence to increase budgetary allocation towards increasing contraceptive prevalence. It is important that a new MDG target on universal access to reproductive health services has been introduced. Given the lag in the introduction of this target, conscientious efforts are needed to reverse the impact of the many years of neglect of family planning programmes in the region. Efforts to achieving universal access to reproductive health services must emphasize the rebuilding of systems of family planning service delivery that crumbled under years of neglect. The public sector must be strengthened to respond effectively to addressing the high levels of unwanted childbearing and unmet need for family planning.Finally, the finding of increased adolescent childbearing is consistent with the prognoses that Africa's adolescent reproductive health yielded the worst indicators relative to other regions of the world and advances further evidence that the onset of sexual encounters in the region happens in the context of low levels of contraception and protective measures . Followi


A skin-derived cytokine with high systemic availability provides a mechanistic explanation for atopic march and highlights a potential therapeutic target for preventing the development of asthma among people with atopic dermatitis. tgK14-TSLP mice that maintained high systemic levels of TSLP without developing any skin pathology. Importantly, epidermal-derived TSLP was sufficient to trigger the atopic march, sensitizing the lung airways to inhaled allergens in the absence of epicutaneous sensitization. Based on these findings, we propose that in addition to early treatment of the primary skin-barrier defects, selective inhibition of systemic TSLP may be the key to blocking the development of asthma in AD patients.Asthma is a common allergic lung disease frequently affecting individuals with a prior history of eczema/atopic dermatitis (AD); however, the mechanism underlying the progression from AD to asthma  is unclear. Here we show that, like humans with AD, mice with skin-barrier defects develop AD-like skin inflammation and are susceptible to allergic asthma. Furthermore, we show that thymic stromal lymphopoietin (TSLP), overexpressed by skin keratinocytes, is the systemic driver of this bronchial hyper-responsiveness. As an AD-like model, we used mice with keratinocyte-specific deletion of RBP-j that sustained high systemic levels of TSLP. Antigen-induced allergic challenge to the lung airways of RBP-j–deficient animals resulted in a severe asthmatic phenotype not seen in similarly treated wild-type littermates. Elimination of TSLP signaling in these animals blocked the atopic march, demonstrating that high serum TSLP levels were required to sensitize the lung to allergic inflammation. Furthermore, we analyzed outbred  Eczema (atopic dermatitis) is a common allergic skin inflammation that has a particularly high prevalence among children. Importantly, a large proportion of people suffering from eczema go on to develop asthma later in life. Although the susceptibility of eczema patients to asthma is well documented, the mechanism that mediates “atopic march”—the progression from eczema to asthma—is unclear. We used genetic engineering to generate mice with chronic skin-barrier defects and a subsequent eczema-like disorder. With these mice, we were able to investigate how skin-specific defects predisposed the lungs to allergic asthma. We identified thymic stromal lymphopoietin (TSLP), a cytokine that is secreted by barrier-defective skin into the systemic circulation, as the agent sensitizing the lung to allergens. We demonstrated that high systemic levels of skin-derived TSLP were both required and sufficient to render lung airways hypersensitive to allergens. Thus, these data suggest that early treatment of skin-barrier defects to prevent TSLP overexpression, and systemic inhibition of TSLP, may be crucial in preventing the progression from eczema to asthma. Allergic asthma is a chronic lung disease characterized by T-helper2 (Th2)–mediated inflammation and airway obstruction in response to allergen exposure A survey of the literature has identified several possible mechanisms underlying the clinical link between AD and asthma. These include: (a) a systemic immune system disorder leading to excessive Th2 response at epithelial surfaces exposed to allergens To address this question, we studied mice lacking Notch signaling in the skin. Skin keratinocytes are organized in highly interconnected basal, spinous, granular, and cornified layers, forming an elaborate barrier protecting the organism from the outside environment. One of the major molecular regulators of this structure is Notch signaling Msx2-Cre transgene . Msx2-Cre is ectopically expressed at embryonic day 9.5 (E9.5) in clusters of ectodermal cells, resulting in a chimeric pattern of RBP-j deletion in the skin Identifying a clear mechanism for the atopic march in the complex network of genetic, immunological, and environmental factors that contribute to AD has been a challenge to the field and has resulted in much debate as to the right therapeutic approach for preventing asthma The ablation of Notch signaling in skin keratinocytes by removing RBP-j severely impairs the differentiation of basal layer keratinocytes and maintenance of upper spinous and granular cell layers RBP-j is not deleted in the lung  did develop spontaneous lung inflammation . To detethe lung  and S7, It is possible that atopic skin lesions are the essential components downstream of skin-barrier defects that initiate systemic atopy in RBP-jCKO mice IL7Rα subunit of TSLP receptor in RBP-jCKO animals , IL7rα-/-, and wild-type littermates with OVA. To prevent RBP-jCKO lethality, all the animals in this experiment received intranasal OVA challenges only twice. Despite this reduced exposure, RBP-jCKO animals developed a severe asthmatic response, which was absent in RBP-jCKO;IL7rα-/- mice . HistoloAirway hyper-responsiveness is a hallmark of allergic asthma tgK14-TSLP mice tgK14-TSLP mice confirmed that in this outbred background and in the absence of skin-barrier defects, TSLP overproduction was insufficient to attract mast cells to the skin . Thus, tgK14-TSLP mice developed an intense Th2 response upon allergen exposure. In the airway reactivity test, OVA-treated tgK14-TSLP mice showed a severe airway hyper-responsiveness to low doses of nebulized methacholine, which was not seen among similarly treated wild-type littermates  results in the sensitization of the lung airways to allergens. Similar to the clinical cases, barrier-defective mice require a secondary insult by allergens to expose their predisposition to asthma. Importantly, we show that epicutaneous sensitization with a common allergen tgK14-TSLP mice contribute to the phenotype, they do so by adding to the already high levels of skin-derived serum TSLP. Because TSLP can activate dendritic cells Importantly, we were able to show that TSLP was necessary to predispose RBP-j–deficient animals to the asthmatic phenotype. Concomitant removal of IL7Rα subunit of the TSLP receptor in RBP-jCKO mice prevented the atopic march despite persistent AD-like pathology and elevated serum IgE levels. Complementing this experiment is the observation that overexpression of TSLP by skin keratinocytes leads to high serum levels of TSLP that are sufficient to sensitize the lung airways to allergic inflammation in the absence of any skin pathology. If the low levels of TSLP in the trachea of The findings outlined in this report have a potentially important implication for human health. They provide a plausible explanation of why a large number of AD patients develop asthma and other allergic disorders later in life flox/floxMsx2-Cre/+;RBP-j (RBP-jCKO), flox/flox; IL7rMsx2-Cre/+;RBP-jα-/- , and K14-TSLPtg mice were generated as previously described The allergic sensitization and lung airways challenge with OVA was carried out as previously outlined tgK14-TSLP and wild type; The airway responsiveness to aerosolized methacholine was determined in OVA-treated animals by measuring total lung resistance and dynamic compliance as previously outlined Dorsal skin samples were harvested from the mice and fixed in 4% PFA at 4 °C overnight. Lungs were inflated through the trachea to 25-cm water pressure with 4% PFA prior to excision from the chest and fixation. These lungs were PFA-fixed for 24 hr at 4 °C and embedded in paraffin. The paraffin-embedded tissues were sectioned at 5–6 µm and stained with hematoxylin and eosin (H&E), toluidine blue, or periodic acid-Schiff (PAS). For RBP-j immunohistochemical staining, anti–RBP-j antibody  and biotinylated anti–rat secondary antibody were used. HRP-conjugated streptavidin and DAB substrate kit (Pierce) were used to visualize the signal. Hematoxylin was used to counterstain the sections. For TSLP immunostaining, paraffin-embedded tissue samples and biotinylated anti-TSLP antibody (R&D Systems) were used. Sections were counterstained with DAPI nuclear stain.BAL fluid was collected by infusing the lungs of the anesthetized mouse with 1-ml PBS through tracheal insertion of a Surflo catheter . Leukocyte count in BAL fluid was determined using a Hemavet 950 analyzer (Drew Scientific). BAL fluid was spun down and the supernatant was stored for cytokine/immunoglobulin analysis. The cell pellet was resuspended in PBS and used for differential cell count after Giemsa staining on the slide.Serum TSLP levels were determined using Quantikine mouse TSLP kit according to manufacturer's instructions (R&D Systems). Serum and BAL fluid IgE levels were measured using Mouse IgE ELISA kit (Immunology Consultants Laboratory).Intracellular cytokine staining was conducted to estimate Th2 cell census as previously described RBP-j allele was performed on genomic DNA isolated from skin and lung of adult RBP-jCKO mice using KlenTaq10 (DNA Polymerase Technology) supplemented with 1.3 M final concentration of betaine (amplification cycles = 32). The following primers were used to distinguish between deleted (ΔRBP-j) and floxed (floxRBP-j) alleles of RBP-j: Deleted allele: 5′-TGTTTGCCACCAGAATCTGTTTGTTATTTGC-3′ and 5′-ATTTGCTTGAGGCTTGATGTTCTGTATTGC-3′.Conventional PCR for the 5′-TGTTTGCCACCAGAATCTGTTTGTTATTTGC-3′ and 5′-AGGTACCTGGTACTAACTGTCTGGGACCG-3′.Floxed allele: TSLP were: 5′-CCAGGCTACCCTGAAACTGA-3′ and 5′-TCTGGAGATTGCATGAAGGA-3′.mRNA isolated from P4 epidermis and lung of RBP-jCKO and wild-type littermates was used to perform qRT-PCR analysis as previously described t-test as the test of significance and presented as mean ± standard deviation in bar graph format. Our studies were conducted in outbred cohorts of animals; therefore, we used nested ANOVA to exclude any effect of between-family differences and make certain the significant differences observed were solely attributable to the gene removed  or overexpressed (tgK14-TSLP).The quantitative measurements were assessed using Student http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for proteins discussed in this article are as follows: RBP-j, NM_009035; TSLP, NM_021367.The Genbank . A few mutant mice that survive up to one year (red circles), however, develop spontaneous lung inflammation.RBP-jCKO animals die prematurely due to their severe skin phenotype ((78 KB TIF)Click here for additional data file.Figure S2n = 4 for each group; *p<0.01, comparing the mutants to the wild-type littermates).Serum TSLP levels in RBP-jCKO mice are highly elevated. TSLP overproduction is evident in RBP-jCKO serum at 1 wk after birth, reaching extreme levels in the adult animals ((55 KB TIF)Click here for additional data file.Figure S3flox/floxMsx2-Cre/+;RBP-j) and wild-type (flox/floxRBP-j) skin and lung shows that RBP-j locus is intact  in the lung . (B) Immunohistochemical analysis for RBP-j protein confirms that RBP-j is present in the lung airway epithelium. Skin sections stained under the same condition are presented as controls .Lung epithelium is normal in RBP-jCKO animals. (A) PCR analysis of DNA isolated from adult RBP-jCKO ((7.35 MB TIF)Click here for additional data file.Figure S4RBP-j in epidermal keratinocytes persist in the absence of IL7Rα . Note that serum TSLP levels in RBP-jCKO;IL7rα-/- mice are consistently and significantly higher than in RBP-jCKO animals. We are currently investigating the underlying reason for this surge.Serum TSLP levels of 10-wk-old RBP-jCKO;IL7rα-/- mice are highly elevated. This indicates that skin-barrier defects caused by the loss of (50 KB TIF)Click here for additional data file.Figure S5tgK14-TSLP mice appear normal at P120. This emphasizes that in an outbred genetic background (C57BL/6 and CD1 mix) these transgenic animals do not develop any skin inflammation under normal conditions.The ear and skin of (1.3 MB TIF)Click here for additional data file.Figure S6tgK14-TSLP lung airways or parenchyma. Because the K14 gene is expressed in basal cells located in the trachea, we analyzed mRNA levels by qRT-PCR on samples isolated from epidermis, trachea and lung of tgK14-TSLP and wild-type mice. This analysis shows that the K14-TSLP transgene is active in basal cells within the trachea, but the overall levels are 200-fold lower than those made by epidermal keratinocytes (***p<0.0000001 and *p<0.01 compared to wild type).There is no TSLP overexpression detectable in (145 KB TIF)Click here for additional data file.Figure S7tgK14-TSLP, RBP-jCKO, or RBP-jCKO;IL7rα-/- animals. Immunofluorescence staining for TSLP protein (red) confirms that TSLP is overexpressed only in the epidermal keratinocytes of the mutant mice. All sections are stained under the same conditions. Dotted lines outline the basement membrane and asterisks highlight the epidermal keratin cysts present in RBP-jCKO skin .Lung epithelium does not overexpress TSLP in adult (5.3 MB TIF)Click here for additional data file.Figure S8tgK14-TSLP mice remains normal. H&E and toluidine blue staining of tgK14-TSLP and wild-type skin show no significant signs of cutaneous inflammation in the transgenic mice .The skin of OVA-treated (1.3 MB TIF)Click here for additional data file.Table S1Major antimicrobial peptides are overexpressed in postnatal epidermis of RBP-jCKO animals. This overexpression indicates the persistence of the skin-barrier defect in RBP-jCKO mice after birth (37 KB PDF)Click here for additional data file.


Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of iron-sulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX), a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1) as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosomes.Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of  Microsporidia are obligate intracellular parasites responsible for a number of diseases in commercially important animals (e.g. bees) and of significant medical concern, in particular in immunocompromised humans. Though related to fungi, microsporidia have undergone a rapid phase of adaption to the intracellular environment and have in the process reduced many aspects of their biology. Notably, microsporidia have highly reduced mitochondria (powerhouses of the cell) reflected in reduced energy metabolic pathways. Thus they likely produce ATP only through the process of glycolysis. In some parasites, this glycolytic pathway is dependent on an additional step involving a protein called the “alternative oxidase”. We have shown that this protein is also present in several species of microsporidia. Crucially, this protein is absent from humans and so can potentially be exploited as a drug target. Our experiments show that this protein is likely widespread in microsporidia, and is sensitive to the antibiotic ascofuranone, which is currently being tested as a potential treatment for the agent causing sleeping sickness. Our results suggest that knowledge gleaned from drug trials on sleeping sickness is potentially transferrable to the treatment of some cases of microsporidiosis. Microsporidia are a large and diverse group of eukaryotic intracellular parasites that infect a wide variety of animal lineages, including humans 1-ATP synthase complex. In the absence of the ability to synthesize ATP through oxidative phosphorylation, microsporidia appear to import ATP directly from their host cell via ATP translocases located in the cell membrane Chlamydia and RickettsiaEncephalitozoon cuniculi, together with immunolocalization studies in E. cuniculi and Trachipleistophora hominis, suggest that the major functional role for the mitosome is not in energy generation, but instead the assembly of iron-sulphur clusters for export to the cytoplasm In particular, microsporidian mitochondria have been severely reduced into biochemically and physically streamlined “mitosomes” E. cuniculi mitochondrial glycerol-3-phosphate dehydrogenase does not appear to be located in the mitochondrion any longer E. cuniculiTrypanosoma brucei parasites, the mitochondrial glycerol-3-phosphate dehydrogenase is coupled to an alternative oxidase (AOX) that together achieve this process Cryptosporidium parvumBiochemical and genomic evidence all generally point to glycolysis as the major route of energy generation in most microsporidia Acanthamoeba castellanii, the heterokont Blastocystis hominis, and the trypanosomes. In the alveolates, it is found in the apicomplexan Cryptosporidium and some other distantly related alveolates including some ciliates, but absent from the more closely related Plasmodium parasites AOX is a cyanide-insensitive terminal oxidase that is typically located on the inner surface of the inner mitochondrial membrane. It branches from the main respiratory chain at the level of the ubiquinone pool, results in the net reduction of oxygen to water, and is non-protonmotive E. cuniculi and from the recent large-scale genome surveys of Nosema ceranae and Enterocytozoon bieneusiAntonospora locustae, demonstrating the pattern of retention versus loss is also uneven within the microsporidia, despite their otherwise common mode of intracellular parasitism and apparently similar metabolism. The possible presence and function of AOX in microsporidia is of practical interest as well, because the absence of AOX in mammals, including humans, renders it a potential therapeutic target for the treatment of microsporidiosis, as is the case in a number of protisitan parasites V. corneum and E. bieneusi are resistant and in these cases fumagillin, which is mildly toxic, has to be used In fungi, the protein also has a wide but discontinuous distribution T. hominis and the insect parasite A. locustae. Phylogenetically the microsporidian AOX is weakly related to mitochondrial homologues from other eukaryotes, and both A. locustae and T. hominis AOX proteins include an N-terminal leader that was demonstrated by confocal microscopy to target the proteins to mitochondria in yeast, altogether suggesting the enzyme is likely derived from the mitosome and may be localized in the organelle still, though direct co-localization would be required to give a definitive location of function. Enzyme assays with recombinant proteins demonstrated both possess cyanide-resistant oxidase activities sensitive to inhibition by the very specific trypanosome AOX inhibitor ascofuranone Here, we characterize the phylogenetic distribution of microsporidian AOX, and examine the functional activity of AOX enzymes from the human parasite E. cuniculi lacks any gene resembling AOX, but we identified a full-length homologue of the AOX gene in the partial genome of A. locustae . To determine the distribution of this gene, degenerate PCR was used to amplify a short fragment of AOX from other species of microsporidia, Glugea plecoglossi (233 bp), Spraguea lophii (235 bp), and T. hominis (236 bp). To examine the complete sequence of an AOX from a human parasite, the ends of the T. hominis AOX gene were also sequenced using 5′ RACE and splinkerette protocols A. locustae AOX, which had a length of 831 bp).The complete genome of A. locustae and T. hominis sequences contain all conserved sites consistent with AOX activity. Specifically, both genes encode the six conserved di-iron binding ligands that are essential for AOX activity  is considered to be critical for the net reduction of oxygen to water and probably plays a key role in enzyme catalysis (A. locustae and T. hominis sequences encode AOX proteins is the finding that a putative substrate binding site (residues 242–263) A. locustae the tryptophan has been replaced by serine whilst in T. hominis it has been replaced by alanine. Similar to other parasite AOXs however, none of the cysteines postulated to play a role in the regulation of AOX activity in plants A. locustae or T. hominis.Hypothetical translations of activity , which aatalysis  [19]. FuT. hominis AOX protein was also predicted by Predotar and TargetP 1.1 to have a mitochondrial targeting peptide. In order to test the degree of conservation and functionality of potential targeting signals, full-length proteins were expressed in S. cerevisiae cells fused to a green fluorescent reporter protein. Expression in yeast shows that GFP overlays mitotracker fluorescence, indicating successful heterologous targeting for both proteins , ubiquinol-1 oxidase activity indicates that the activities of both proteins are as expected for AOX  algerae are less conclusive , but for other human parasites (e.g. T. hominis) the AOX is clearly a potential target, and may also be in other unexplored lineages .The demonstration that Trypanosoma brucei, where glucose is abundant and there is no conventional respiratory chain With respect to the potential function of AOX in microsporidia a possible role may be similar to that proposed in the bloodstream form of some trypanosomes. In the bloodstream form of E. cuniculi show no evidence that the enzyme is confined to mitochondria or specifically localized there, unlike ferredoxin, frataxin, ISCU and ISCS E. bieneusi the gene appears to be absent altogether It is plausible that most microsporidia rely on a similar system and that AOX fulfils the role of the terminal oxidase, as shown in A. locustae alternative oxidase sequence was retrieved from the GMOD MBL A. locustae database and used to design degenerate primers to amplify a fragment of the alternative oxidase gene from T. hominis, G. plecoglossi and S. lophii . Degenerate PCR was carried out on DNA from E. cuniculi to exclude the possibility that the AOX gene is present in the genome within the subtelomeric regions that have not been fully assembled Brachiola algerae and Edhazardia aedis. The full-length gene was amplified from T. hominis DNA and RNA obtained from purified spores from cultures maintained in rabbit kidney cells at Rutgers, State University of New Jersey. The 5′ prime end of the gene was amplified using RLM-RACE using primers designed from within the fragment amplified by degenerate PCR. The first round of PCR yielded a product truncated at the 5′ end. Primers were then designed from within that fragment to obtain the presumed full-length gene. A splinkerette strategy was used to obtain 3′ end of the gene The A. locustae and T. hominis AOX open reading frames were amplified by PCR by using primers that generated in-frame restriction sites. PCR products were cloned upstream of green fluorescent protein (GFP)-S65T under the control of the MET25 promoter leu2-3,122/leu2-3,122 ura3-52/ura3-52 rme1/rme1 trp1/trp1 his4/his4 GAL+/GAL+ HMLa/HMLa), and plated on uracil and methionine deficient SD plates (2% (w/v) agar, 2% (w/v) glucose and 0.67% (w/v) yeast nitrogen base supplemented with the relevant amino acids). Positive colonies were grown overnight in SD medium lacking uracil and methionine and stained with MitoTracker (MitoTracker Red CM-H2XRos) according to the manufacturer's protocol (Molecular Probes). Yeast cells were visualized using the Zeiss meta confocal microscope.DNA fragments corresponding to S. guttatum AOX Separation of yeast mitochondrial proteins on non-reducing SDS-polyacrylamide gels, transfer to nitrocellulose membranes, and detection of AOX protein using monoclonal antibodies raised against the A. locustae and T. hominis gene sequences were amplified using Phusion High-Fidelity Taq (New England Biolabs) and cloned into the pet14b expression vector. Both constructs were used to transform E. coli strain C41, which is especially suited to the expression of transmembrane proteins. Bacterial membranes were prepared using 2.5 L Luria broth cultures, adapted from Berthold et al 2009 4 and 50 µgml−1 ampicillin were inoculated with 10 mlL−1 starter culture, and incubated at 37°C for 4 hours. The temperature was reduced to 18°C, and the cultures were incubated for one hour prior to induction with 100 µM IPTG. After induction, the cultures were incubated for 18 hours at 18°C. Cells were then harvested using centrifugation at 11,000×g for 10 minutes. After initial centrifugation, cells were resuspended in 60 mM Tris-HCl (pH 7.5), 5 mM DTT, 300 mM NaCl and 0.1M PMSF and then sonicated for 8 minutes at 14 microns. After sonication, cell debris was removed by centrifugation at 12,000×g for 15 minutes, and clear supernatant was further refined by a 2-hour ultracentrifugation step at 200,000×g. Pellets from final spin were resuspended in 60 mM Tris-HCl (pH 7.5), 5 mM DTT, 300 mM NaCl and used for subsequent gel and assay analysis. Ubiquinol oxidase activity (AOX activity) was measured by recording the absorbance change of ubiquinol-1 at 278 nm (Cary UV/vis -400 Scan spectrophotometer). Reactions were started by the addition of ubiquinol-1  after 2 min preincubation at 25°C in the presence of rAlAOX and rThAOX in 50 mM Tris-HCl (pH 7.4). Endogenous ubiquinol activities were inhibited by inclusion of 1 µM antimycin A, 2 µM myxothiazol and 1 mM potassium cyanide in the assay medium.The A. locustae and T. hominis AOX amino acid sequences were aligned to 47 diverse proteins sequences with representatives from animal, kinetoplastid, fungal, heterokont, plant and proteobacterial lineages. Sequences were aligned using ClustalW www.bioportal.uio.no). A burn-in of 400 trees was removed from each run and a consensus created from remaining trees. Five hundred bootstrapped data matrices were also analysed by maximum likelihood using PhyML 3.0 S. lophii, G. plecoglossi was also analysed. Trees were inferred and 100 bootstrap datasets analysed from this short alignment using PhyML, using the parameters described above. The SSU rRNA backbone phylogeny was based on available SSU sequences from NCBI, which were aligned using ClustalW, manually edited and masked and analysed using PhyML 3.0 with a JC69 nucleotide substitution model with estimated gamma parameter and 4 categories of rate change.The