Spatial and temporal expectations act synergistically to facilitate visual perception. In the current study, we sought to investigate the anticipatory oscillatory markers of combined spatial-temporal orienting and to test whether these decline with ageing. We examined anticipatory neural dynamics associated with joint spatial-temporal orienting of attention using magnetoencephalography (MEG) in both younger and older adults. Participants performed a cued covert spatial-temporal orienting task requiring the discrimination of a visual target. Cues indicated both where and when targets would appear. In both age groups, valid spatial-temporal cues significantly enhanced perceptual sensitivity and reduced reaction times. In the MEG data, the main effect of spatial orienting was the lateralised anticipatory modulation of posterior alpha and beta oscillations. In contrast to previous reports, this modulation was not attenuated in older adults; instead it was even more pronounced. The main effect of temporal orienting was a bilateral suppression of posterior alpha and beta oscillations. This effect was restricted to younger adults. Our results also revealed a striking interaction between anticipatory spatial and temporal orienting in the gamma-band (60–75 Hz). When considering both age groups separately, this effect was only clearly evident and only survived statistical evaluation in the older adults. Together, these observations provide several new insights into the neural dynamics supporting separate as well as combined effects of spatial and temporal orienting of attention, and suggest that different neural dynamics associated with attentional orienting appear differentially sensitive to ageing.  •Both younger and older adults benefit from cued spatial-temporal expectation.•Alpha and beta lateralisation linked to spatial expectation is preserved and even strengthened in older adults.•Temporal expectation modulates decreases in alpha oscillations in younger but not older adults.•Combined spatial-temporal expectation increases contralateral gamma oscillations in older adults.•Different anticipatory neural dynamics may be differentially sensitive to ageing. Our interactions with the world are adaptively shaped by our ability to anticipate relevant sensory events and orient our attention towards them. While such attentional orienting is classically studied with regard to how we orient our attention in space  and beta (15–30 Hz) band oscillations in relevant sensory areas has been repeatedly reported, and proposed as a central mechanism for selectively regulating cortical receptivity based on spatial receptive fields . HoweverFurthermore, recent studies have started exploring how attentional orienting and its neuronal underpinnings change over the lifespan see , showingIn the current study, we sought to investigate the anticipatory oscillatory markers of combined spatial-temporal orienting and to explore whether and how these decline with ageing. Younger and older adults engaged in a joint spatial-temporal orienting task, requiring a difficult perceptual discrimination following a spatial-temporal cue cf. , while wTwenty younger adults  and twenty-one older adults  completed the study. All but one participant were right-handed, according to self-report. All participants were healthy, had no history of neurological, psychiatric or vascular disease and had normal or corrected-to-normal vision. Data of three older adults had to be excluded due to large structural abnormalities visible on the structural magnetic resonance imaging (MRI) scan, technical problems during the recording and excessive artefacts in the MEG data, respectively. Data of two additional older adults had to be excluded because they performed below our cut-off threshold (<26) on the Montreal Cognitive Assessment (MoCA), which is indicative of a mild cognitive impairment . A totalThe older participants' data were collected as part of a larger “Cognitive Health in Ageing” research project. Therefore, in addition to the MEG study, older adults completed a set of demographic surveys, standardised clinical assessment questionnaires, and neuropsychological tasks to assess psychological health and basic cognitive function. This session took approximately 90 min. After the exclusion of two older adults who scored below 26 on the Montreal Cognitive Assessment  MEG system. A magnetic Polhemus FastTrak 3D system  was used for head localisation. Relative positions of three anatomical landmarks  were measured in addition to relative positions of four head-position indicator coils.MEG data were recorded in three (older adults) or four (younger adults) separate blocks of 12–15 min each, that were presented back to back with short breaks in between during which participants remained seated in the MEG chair. Data were sampled at 1000 Hz, and a bandpass filter between 0.03 and 300 Hz was applied during digitisation of the signal. ECG and horizontal and vertical EOG were recorded. In addition, eye movements were recorded with a video-based eye tracker with a sampling frequency of 500 Hz . Fiber-optic response pads  were used to collect manual responses.Stimuli were presented using MATLAB v.7.10  and Psychtoolbox v.3.0 for MATLAB . The stiBoth groups performed the same spatial-temporal orienting task, but there were some minor differences between the number of sessions, and the type and amount of data collected for younger and older adults. Younger adults took part in three sessions: a behavioural training session, an MEG session involving 800 trials of the spatiotemporal orienting task (see 2.4.2 MEG paradigm and procedure), and an MRI session in which an MRI T1 structural scan was obtained. Older adults took part in four sessions: a behavioural training visit, a second session involving MEG with 600 trials of the spatiotemporal orienting task and a resting-state recording , a third session including MRI T1 and T2 structural scans, a functional localiser scan, a resting-state scan and a diffusion tensor imaging scan (results of which will not be discussed in the current manuscript), and a fourth session involving an extensive clinical and neuropsychological evaluation .The experimental task is depicted in Luminance pedestals positioned at 4.78° below the horizontal meridian and 3.38° from the vertical meridian (10% contrast) were presented throughout the experiment, indicating the two possible target positions. Cues were presented for 200 ms and consisted of blue or pink coloured arrows. Arrow direction indicated target location and the colour indicated when the target was most likely to appear. Colour-interval mappings were counterbalanced across participants. Both target locations and interval lengths were equiprobable and randomised across trials. Targets replaced the luminance pedestals for 50 ms. They were followed by a 283-ms backwards mask after a 117-ms target-mask SOA. Targets consisted of horizontally or vertically oriented Gabor patches with a diameter of 1.96° of visual angle and a spatial frequency of two cycles per degree of visual angle. The target contrast was adjusted individually using the adaptive staircase procedure (see MEG practice and staircase). Backward-mask stimuli were constructed by applying a Gaussian-vignette to the convolution of 100% square-wave gratings at the two possible target orientations. Responses were made with the left or right index finger, depending on target orientation. The response mapping was also counterbalanced across participants. A total of 800/600 (younger/older) trials were presented in randomised order , in three blocks of about 15 min each. Self-paced rest breaks were inserted every 20 trials.The MEG session was preceded by a 90-min practice and staircase session, taking place on a separate day  values and reaction times (RTs). Perceptual sensitivity was calculated according to the following formula:The behavioural data were analysed using MATLAB, and statistics were performed in SPSS version 22 . The MEG behavioural data were analysed with respect to perceptual sensitivity /N, where N reflects the number of trials with a horizontal or vertical stimulus for that condition, thereby penalising for conditions with fewer trials . Trials with RTs smaller or larger than a participant's mean RT ± 3 times the standard deviation were excluded from the behavioural analysis. The behavioural results were analysed statistically using repeated-measures analysis of variance (ANOVA). Within subject effects were further evaluated using pairwise t-tests, while differences between both groups were evaluated using independent-samples t-tests.z[percent correct horizontal] and z[percent correct vertical] correspond to the inverse normal (z-score) transformations of the participants' proportions of correct responses to horizontal and vertical stimuli see . In caseMEG data analyses were performed using custom-written MATLAB code, the in-house OHBA Software Library (OSL) version 1.4 and Fieldtrip . First, After using MaxFilter, the data were checked manually to ensure no problems occurred during the MaxFilter stage and down-sampled to 250 Hz. A 0.1-Hz high-pass filter was applied to the data to remove low-frequency drift. Because most datasets included periods with excessive noise recorded during rest-periods, the data were epoched before Independent Component Analysis (ICA) was performed, thereby ensuring that these periods with excessive noise would not negatively impact the ICA. ICA was used to reject artefacts associated with eye blinks, eye movements and heartbeat. All components were inspected manually and component time courses were compared with ECG and EOG time courses before removing them from the data. After ICA, the data were inspected manually to remove any remaining artefacts. Trials with eye blinks occurring during target presentation were excluded as well. On average 12% of trials were removed from the analysis. Trials with incorrect responses were not excluded from the MEG analysis because we were mainly interested in the anticipatory period before target appearance, i.e. before responses were being made. We note that when we repeated our MEG analysis using trials with correct responses only, results were comparable to the results reported here for all trials. Before analysis of high-frequency (gamma) oscillations, Fieldtrip's visual artefact rejection function showing summary statistics was run on data bandpass filtered between 40 and 100 Hz, to remove any remaining artefacts that were specific to the gamma-range.To avoid overlap with the main time period of interest (0–800 ms after cue onset) or main measure of interest (time-frequency data between 4 and 100 Hz), visual ROIs were selected based on target-evoked ERFs. ERFs were calculated separately for all four combinations of target location and interval . ERFs were baseline-corrected with the average of the window 100 ms before target presentation . Data for the planar gradiometer pairs were combined, resulting in a 102-channel combined planar gradiometer map in sensor space. Subsequently, ERFs for short and long targets were aligned at target presentation, and averaged separately for left and right targets. Finally, a left-right difference ERF was calculated for each participant and averaged across all participants. This difference ERF topography was plotted 200–400 ms after target presentation, where the difference was maximal. Based on the topography see  six  and higher (40–100 Hz) frequencies. For both analyses, we used a fixed sliding time window of 300 ms that was advanced over the data in steps of 25 ms. For the lower frequencies, we used a Hanning taper, and estimated power between 4 and 40 Hz, in 0.5 Hz steps. For the higher frequencies, we used a multi-taper approach  using ± For both lower and higher frequencies, the power spectra were averaged separately over trials for each cue condition. The power time series in the planar gradiometer pairs were then combined (Cartesian sum), resulting in a 102-channel combined planar gradiometer map in sensor space. All relevant time-frequency contrasts were computed as the average of the selected ROI channels  and subsequently averaged for ROIs contralateral and ipsilateral to the cue direction. All contrasts were computed as relative contrasts. For example, the contrast between short vs. long cues was calculated as follows :Contrasts of interest were evaluated during the short interval (0–800 ms). This is the time period during which temporal expectations differ between cueing conditions. After the first interval passes, it becomes evident that the target will appear after a long interval irrespective of the initial cue, so temporal cueing benefits dissipate as it is now. Differences between conditions were investigated by means of cluster-based non-parametric permutation testing in Fieldtrip using 1000 permutations with a cluster alpha of 0.05. This approach circumvents the multiple-comparisons problem . The resEye-tracking data were absent or not usable for six younger adults and two older adults. Due to the way the recording was set up, we always got an eye tracker signal, even when the eye tracker was not pointed at the eyes, or when the eye tracker partially or fully “lost” the eye during the recording. The eight non-usable recordings either had a flat signal with lots of 50Hz noise and some drift (6 participants), or a very unstable signal (2 participants). We believe the flat recordings reflect missing data, while the unstable signal reflects the eye tracker “losing” the eye, e.g. because the participant was wearing thick glasses that made it difficult to record, or the eye tracker not being optimally positioned in the first place. Therefore, data from the remaining fourteen younger adults and fourteen older adults were included in the eye-tracking analysis. Eye-tracking analysis was performed on the x-position eye-tracker data  during the short interval, recorded in Volt as a separate channel on the MEG system. All trials that were rejected during pre-processing (see 2.6.1 Pre-processing and artefact rejection) were rejected from the eye-tracking analysis as well. Participant averages were calculated separately for all four combinations of cued target location and interval  and averaged separately for younger and older adults. Data were baselined using the 500-ms window before cue presentation.In a series of post-hoc analyses we investigated the link between our MEG results and behaviour. First, we conducted a logistic regression between accuracy and average MEG power between 300 and 650 ms after short cue onset , separately for the alpha (8–14 Hz), beta (15–28 Hz) and gamma (60–75 Hz) bands. Second, we computed the trial-wise correlation between power in the same time-frequency windows and RT . The results of these analyses are presented in We measured magnetoencephalographic brain activity in healthy human volunteers, while they engaged in a joint spatial-temporal orienting task A. Colourd’) and reaction time (RT) are shown in d’, a main effect of cued interval  = 9.33, p = .004, partial η2 = 0.22) and a main effect of validity  = 7.39, p = .01, partial η2 = 0.18) were found. The interaction between cued interval and validity showed a trend, but did not reach significance  = 3.51, p = .07, partial η2 = 0.09). When, however, testing the validity effect separately for both intervals, we could confirm a significant benefit of valid temporal cues at the short, but not at the long interval (short: t(35) = 2.87, p = .007, Cohen's d = 0.48; long: t(35) = .87, p = .39, Cohen's d = 0.14), in line with a number of previous studies (see e.g. 2 = 0.04) or three-way interaction with group  = .02, p = .90, partial η2 = 0.00) was present.Behavioural results for perceptual sensitivity (see e.g. . No main2 = 0.41), a main effect of validity  = 19.77, p < .0001, partial η2 = 0.37) and an interaction between cued interval and validity  = 7.27, p = .011, partial η2 = 0.18). There was no main effect of group  = 1.36, p = .252, partial η2 = 0.04), but there was a three-way interaction between cued interval, validity and group  = 4.166, p = .049, partial η2 = 0.11). Pairwise t-tests revealed that for both older and younger adults a validity effect was again present for the short (older: t(15) = 2.54, p = .023, Cohen's d = −0.63; younger: t(19) = 6.13, p < .0001, Cohen's d = −1.37), but not for the long interval (older: t(15) = 1.72, p = .105, Cohen's d = −0.43; younger: t(19) = 0.17, p = .87, Cohen's d = −0.04). An independent samples t-test showed that the magnitude of the temporal validity effect for short cues was larger in younger, than in older adults (t(34) = 2.09, p = .045, Cohen's d = 0.70).For RT, we found a main effect of cued interval  = 23.60, p < .0001, partial ηd’ and RT, performance was better following valid, compared to invalid temporal cues. These performance benefits were present following short, but not following long intervals. Based on these findings, we thus expected the largest neural differences due to temporal expectations  to occur in the short interval, and therefore focused on this interval in the MEG analysis on which we report below.To summarise, for both Associated topographies of the spatial lateralisation effect (i.e. left vs. right cues) are shown in We next separated the trials further by also separating “short” and “long” temporal cues. We again focused on the short interval (0–800 ms post-cue). Based on the behavioural data, it is expected that, in this short interval, anticipation of the target is stronger when the trial had started with a “short” cue, compared to when it had started with a “long” cue. Focusing on the lower frequencies . We could not find compelling effects, which is likely due to relatively low numbers of trials and participants. For transparency, results of these analyses are presented in We evaluated the eye-tracking data to investigate whether the spatial-temporal orienting effects may have been driven by gaze differences between conditions (or groups), i.e. by more gaze shifts in the direction of the cued target location after short, compared to long cues. The left graph in Returning to the spatial gaze shift, we were concerned that the stronger lower frequency lateralisation observed with spatial cueing in the older adults may have been driven by a larger spatial gaze bias in this group. We therefore decided also to evaluate the difference in spatial gaze bias between older and younger adults see B. As befWe investigated anticipatory neural dynamics related to spatial-temporal orienting of attention using MEG, in both younger and older adults. In both age groups, we replicated well known effects of spatial orienting by showing lateralisation of posterior alpha and beta oscillatory power . In contSpatial and temporal orienting of attention are typically studied in isolation and have each been associated with anticipatory attenuation of alpha and beta oscillations spatial: . Such atIn older adults, an interaction between spatial and temporal orienting was found in the gamma band, expressed in an anticipatory amplification of 60–75 Hz oscillations in contralateral parietal channels, with stronger gamma power in the short interval when targets were expected to occur early. In younger adults the topographies hinted at a possibly similar  modulation.Anticipatory modulations of gamma-band activity related to temporal orienting have previously been reported by In humans, reports of anticipatory gamma modulations are rare but see  and suchThere are at least three possible reasons why most previous studies may not have found anticipatory modulations of gamma-band activity. First, a constant, driving, visual input might be necessary for detectable gamma-band modulations to occur, like the continuously visible luminance pedestals in the current study, or the constant grating in Our findings highlight that when gamma oscillations are amplified, alpha and beta oscillations do not always concurrently decrease in power. This is also in line with research by Older adults are usually found to maintain the ability to orient attention to specific spatial locations to improve performance . HoweverContrary to these findings, and more in line with the current results, a recent study using a lateralised working-memory task  showed sThe degree to which the benefits of temporal orienting are affected by ageing, has also been a matter of recent debate. Our data have revealed that different neural dynamics associated with attentional orienting may be differentially sensitive to ageing, with some effects appearing exclusively in younger adults , whereas others appear most clearly or even exclusively in older adults . In future work it will be interesting to assess to what extent these changes with age must be attributed to changes in physiology, different strategies, or compensatory processes.The main insights from the current work are threefold. First, when visual spatial and temporal attention join forces, we find certain anticipatory neural dynamics (i.e. in the gamma band) that are sensitive to joint spatial-temporal expectations, while others  are mainly sensitive to spatial and temporal expectations by themselves, irrespective of concurrent expectations in the other dimension. Second, we demonstrate the involvement of anticipatory neural dynamics in the gamma band in older humans and show that it is possible to study this non-invasively using extracranial MEG recordings. Finally, we find that different anticipatory neural dynamics are not always diminished, but sometimes are even more pronounced in older adults.


Sugar beet pectin is rich in rhamnogalacturonan-I (RG-I) region, which is a potential source of prebiotics. RG-I pectin cannot be extracted the same way as commercial homogalacturan-rich pectin using hot acid. Therefore, this study has explored several alternative methods, including microwave-assisted extraction (MAE) and conventional-solvent extraction (CSE) at atmospheric pressure using different solvents, and microwave-assisted hydrothermal extraction (MAHE) under pressure using water. No conclusive differences in microwave and conventional heating were found with heating rate controlled. The optimum treatment times of both MAE and CSE at 90 °C atmospheric pressure and regardless of the solvents used were 120 min; however, MAHE at 130 °C under pressure can dramatically reduce the time to 10 min. Alcohol-insoluble solids (AIS) extracted using pH13 solvent by MAE had both the highest RG-I yield at 25.3% and purity at 260.2 mg/g AIS, followed by AIS extracts using water by MAHE with 7.5% and 166.7 mg/g AIS respectively. Due to its highly branched nature, RG-I is often referred to as “hairy” pectin  and rhamnogalacturonoan-I (RG-I), which are covalently linked to one another . HGA is  degrees  and is r” pectin . HGA-ric” pectin . RG-I-ri” pectin , which h” pectin .Sugar beet pulp was chosen in this present study because of its prevalence in industry and promising RG-I pectin content. 7.5 million tonnes of sugar beet are grown each year in the UK . Sugar bRG-I pectin cannot be extracted the same way as commercial HGA-rich pectin, which uses a mineral acid at pH 1–3 and 80–90 °C , becauseFirstly, the extractions of pectin using other solvents have been analysed. Dilute alkaline solution was considered to be efficient to extract intact RG-I with high neutral side chains while it may destroy the HGA backbone  and thisSecondly, apart from conventional heating, other heating methods have been explored, including microwave heating. Microwave-assisted extraction (MAE) is a novel extraction process that is widely reported to accelerate or enhance the solvent extraction of pectin . MicrowaFinally, hydrothermal treatment, carried out with hot compressed water, has been successfully employed for producing oligosaccharides from a variety of raw materials . This me1.Compare the yield, purity and extraction time of RG-I pectin extracted using atmospheric pressure microwave and conventional solvent extraction (MAE and CSE respectively) using pH 1 to pH 13 and a chelator, controlling all other independent factors including stirring speed, vessel geometry, solid to liquid ratio (S/L) and heating rate.2.Understand the effect of solvent choice (pH 1 – pH 13 and use of a chelator) on the yield and purity of RG-I pectin extracts.3.Understand the effect of processing temperature and time on the yield and purity of RG-I extracted in water from 90 °C to 190 °C.Although many studies have already explored one or several extraction techniques and conditions mentioned above, due to the many experimental variables that need to be controlled, meaningful comparison of different laboratory-scale set-ups is usually not possible. In particular, the control of heating rate in heating methods comparison was emphasised in the work conducted by 22.1Micronised sugar beet pulp powder (particle size range 20–200 2.2The optimum solid to liquid ratio (S/L) was determined in scoping experiments (not presented here) to be 21.5 mL/g. Based on the maximum vessel size of the Miniflow SS200, all experiments at atmospheric pressure were therefore carried out using 43 mL extracting solvent added to 2 g of dry micronised sugar beet powder. Solutions used were: deionised (D.I.) water, a 1% (w/v) sodium hexametaphosphate chelator solution, dilute acid HCl solution at pH 1, 1.5 and 2 as well as dilute alkaline NaOH solution at pH 11, 12 and 13.MAE was carried out using a Miniflow 200SS  single mode microwave heating system at 2.45 GHz. A fibre optic temperature probe was inserted into the reaction vessel. A maximum incident power of 200 W was applied to the system to achieve a temperature set-point (90 °C). The reflected power was adjusted to zero before the treatment, so that the absorbed power can be as close as the incident power. CSE was carried out by adding the extraction vessel to a water bath preheated to the set temperature.After heating, solid beet pulp residue was removed by filtration. The resulting liquid was centrifuged at 3900 rpm for 40 min and an equal volume of IPA was added to the supernatant for the precipitation of alcohol insoluble solid (AIS). Samples were stored overnight at 4 °C. Then the samples were centrifuged for another 40 min at 3900 rpm and the supernatant was discarded. The pellets were either oven-dried overnight at 60 °C to determine yield or freeze-dried for sugar analysis. All experiments were performed in triplicate. 2.3MAHE was carried out using a Monowave 200 microwave . Although the Monowave vessels are smaller than those of Miniflow, for comparison at same S:L ratio of 21.5 mL/g, 0.8 g of dry sugar beet powder was added to 17.2 mL of D.I. water for Monowave experiments. A maximum power of 800 W was applied to the system, but there is no facility in the Monowave system to monitor reflected power, and so the absorbed power is unknown. The samples were heated to a desired temperature (90–190 °C) and held at this temperature for the required amount of time . Stirrer speed was set at 800 rpms. The same procedures, as described above, was then used to purify the sugar beet extracts.2.4The extract quantity was determined by yields using the following equation:AIS yields and error bars were calculated based on the average and standard deviation of triplicate runs. Varying only one variable a time, the optimum conditions of different extraction methods were found when their optimum AIS yields were reached.The yields of RG-I pectin extracted was determined using the following equation  is also referred as RG-I region extractability ):(2)RG-Iwhere the percentage of RG-I region in AIS is also called RG-I purity (mg/g AIS), which is the sum of the sugar contents of rhamnose, arabinose and galactose  in AIS extracts.2.5Soluble pectin extracts, lyophilised using a LyoDry freeze dryer , were diluted to give an estimated concentration of 125 2.6l-rhamnose, l-arabinose, d-galactose, d-glucose and d-xylose) at various concentrations (1–20 mg/L) were used as external standards for identification and quantification. The results and error bars of neutral sugar composition were determined by 4 repeats ((2 freeze-dried samples) The freeze-dried pectin extract was hydrolysed with concentrated sulphuric acid using the same method as in the GalA assay. After the pre-hydrolysis, 100 33.1The purpose of this paper is to understand the effect of a wide range of processing conditions on the yield and purity of RG-I extraction conditions from sugar beet pulp. We have as far as possible held all experimental conditions constant while changing only one variable at a time . To keep the size of the study manageable, the following experimental parameters  were not the focus of this study: stirrer speed, solid to liquid (S/L) ratio, microwave power and extraction vessel geometry. The first two were set using values determined in scoping experiments (not presented here) and not changed during any of the experiments. Unfortunately, it was not possible to hold the latter two (microwave power and vessel geometry) constant between the Miniflow (set temperature experiments) and Monowave (variable temperature experiments). This is because of differences in vessel geometry, electric field intensity (due to differences in microwave cavity design and power) and features of the two microwave power sources. The Miniflow was selected for the constant temperature experiments as the microwave power can be tuned (i.e. the proportion of the incident power that is absorbed by the sample can be maximised) and the incident and reflected power can be recorded, hence enabling the absorbed power to be calculated. In order to carry out experiments above 100 °C, however, the Monowave was required as it has the ability to monitor the temperature of samples under pressure. This has a higher maximum incident power but no facility to measure the reflected power or adjust the tuning, and the extraction vessels are smaller. Therefore, the latter two “set” variables (absorbed power and vessel geometry) were different between the two different sets of experiments. However, comparing the results for the Miniflow (90 °C) with water as the solvent on D with thThe first optimisation step . The difference in extraction characteristics when using the chelator was better understood after characterisation of the AIS extracts, and so is discussed further in 3.2.2As stated above, for each hold time the yield decreased sharply when the temperature was increased above the optimal point. This correlated with visible degradation of the extract; at 190 °C for 0 min hold time and 170 °C for 5, 10 and 15 min, the extracted AIS was in black colour and had a caramelised odour. This demonstrates that higher temperatures can quickly release pectin from the cell wall structure but when it is in solution will rapidly degrade. In this case, microwave heating shows its advantage when applying hydrothermal extractions as microwave allows quicker heating.3.2.3During thermal processing, pectins are subjected to depolymerisation and demethoxylation reactions. Acid hydrolysis and e pectin . β-elimiposition . Demethomination . It is beactions . The parIt should be noted that the above pectin degradation mechanisms were primarily studied on HGA pectin. More work needs to be done to fully understand the impact of thermal processing on the degradation of RG-I pectin side chains.3.3The optimal AIS yield points  were selected for sugar analysis. Results are listed in 3.3.1Water was used as the solvent for both atmospheric pressure extractions and hydrothermal extractions. The optimal AIS yields of hydrothermal extraction was 10.8% achieved at 10 min treatment time and 130 °C compared to atmospheric pressure extraction AIS yields 5.6% for MAE at 120 min and 90 °C; however, the chemical compositions are very similar between atmospheric pressure and hydrothermal extractions. This indicates that the extract chemical composition is dominantly influenced by different solvent natures rather than extraction temperature and pressure; while extract AIS yields can be influenced by solvent as well as temperature and pressure. Although as mentioned above at hydrothermal condition, the property of water might become more like dilute acid, the chemical composition of hydrothermal water extracts are not very similar to atmospheric acid extracts. This is because the change of water pH in hydrothermal process is minor , which 3.3.2The extraction AIS yield using a chelator as the solvent at 10 min was double that of water at 120 min; however, the sugar analysis results showed that the total sugar content of using chelator as the solvent for 10 min were only 321.3 mg/g AIS and for 120 min were 603.4 mg/g AIS, which were both much lower than that of using water 954.7 mg/g AIS. This suggests that the better AIS yields achieved using chelator as the solvent may not be due to more pectin extracted but chelator itself remaining in the extract. This same issue was also discovered by 3.3.3GalA is the main component of pectin HGA region, which is a straight backbone. In terms of GalA compositions, the lower the pH the higher the GalA content. The highest GalA content was 859.9 mg/g AIS for MAE using pH = 1 solvent. Increasing the GalA content also means the increase of the pectin linearity. The sugar ratio 1 that represents linearity of pectin reaches 6.6 at solvent pH = 1 compared to 0.9 at solvent pH = 13. This is because acid often promotes the extraction of GalA while alkali degrades the GalA . SimilarRha is the main component in RG-I region backbone, connecting to RG-I neutral side chains that are primarily made of Ara and Gal. In terms of pectin neutral sugar contents, the lower the pH, the higher the Rha, Ara and Gal contents, with the most RG-I region extracted being 260.2 mg/g AIS for MAE achieved using pH = 13 solvent. Furthermore, sugar ratio 2, which represents the contribution of the RG region of the whole pectin population increased with increasing pH. It reached 0.075 at solvent pH = 13 compared to 0.009 at pH = 1. It is because acid attacks the pectin neutral side chains while on the other hand, alkaline conditions often preserve the side chains . MoreoveHigher alkaline pH was also found to result in extracts with higher Glu and Xyl contents. This is because Glu and Xyl are mainly from hemicellulose that is insoluble in water but soluble in alkaline solutions and higher the alkaline condition better the hemicellulose extraction . Those h3.4Different RG-I pectin extraction methods are compared depending on the yields and the purity of the RG-I pectin extracted. RG-I pectin yield represents how much RG-I pectin could be extracted from raw sugar beet pulp and it was calculated based on Eq. As discussed in However, constraints including process safety concerns, environmental impact and cycle time also need to be taken into account. Firstly, using strong alkaline solvents can cause danger in process operation as well as posing waste disposal issues and pollution if not treated properly. The use of strong alkaline conditions may also require a further purification/neutralisation step. However, water is a cheap, environmentally friendly and food-grade solvent, which does not require further treatment before disposal. All atmospheric pressure extractions were conducted at 90 °C; while hydrothermal extractions would be under a temperature of 130 °C and a vapour pressure of 2.7 bar. Atmospheric pressure extractions can be considered safer, but hydrothermal of 2.7 bar is not very high in the context of industrial processes. For example, a common industrial process of nitric acid production has operating pressure in the range of 1.7–13 bar . SecondlIt worth noting that as different raw materials have huge pectin content variations and structural diversity , the eff4In this work, the influences of different extraction techniques and conditions on the yield and purity of total AIS and RG-I recovered from sugar beet pulp were analysed in a systematic manner, with the aim of informing the design of future RG-I pectin extraction processes. Those extraction techniques were atmospheric pressure microwave-assisted, conventional solvent extraction (MAE and CSE respectively) as well as pressurised microwave-assisted hydrothermal extraction (MAHE). The extraction conditions studied were different solvents (pH 1–pH 13 and use of a chelator), treatment time and processing temperatures. In order to achieve meaningful comparison of different experiment set-ups, all other independent factors including stirring speed, vessel geometry, solid to liquid ratio (S/L) and most importantly heating rate were controlled.No conclusive difference in AIS yield and quality between microwave and conventional heating were observed. This is because the effects of microwave volumetric heating here nullified by using similar heating rates for the conventional and microwave heating experiments, and also indicates that in the case of micronised sugar beet pulp, there are no discernible microwave selective heating advantages for the electric field intensities achieved in these experiments. This is not to say that MAE will not pose an advantage in every extraction.Optimum AIS yields were achieved at 120 min for all solvents in atmospheric pressure. At reaction times greater than 120 min AIS yields decreased due to pectin degradation. Using a chelator as the solvent achieved much better AIS yields but the chemical composition analysis showed that the better yields may not be due to more pectin extracted but chelator itself remaining in the extract, which suggested that chelator may not be suitable for pectin extraction. Low pH favoured HGA region extraction; at pH = 1, the AIS yields were 22.6% for MAE extractions, giving the highest GalA contents of 859.9 mg/g AIS, although it degraded RG-I side chains. High pHs also lead to high AIS yields, and alkaline conditions helped to preserve the neutral side chains; at pH = 13, the yields were 23.4% with total hairy pectin contents of 260.2 mg/g AIS. MAHE at 130 °C in a much shorter treatment time of 10 min using only the green solvent water can achieve both good AIS yield 10.8% and hairy pectin content 166.7 mg/g AIS in extracting sugar beet pectin. Although rapid pectin degradation may occur in high temperatures, microwave allows quicker heating.RG-I pectin recovery was assessed using RG-I pectin yield (how much RG-I pectin could be extracted from raw sugar beet pulp) and RG-I pectin purity (how many grams of RG-I region was present in each gram of AIS extracted). pH = 13 alkaline extracts had both the highest RG-I pectin purity at 260.2 mg/g AIS and the highest yields at 25.3% by MAE. Using pressurised MAHE, extracts obtained by using water at 130 °C and 10 min also had relatively high RG-I pectin yields at 7.5% and purity at 166.7 mg/g AIS, which was similar to pH = 12 alkaline extracts. Extracts by other solvents at atmospheric pressure were low either in RG-I pectin yields, RG-I pectin purity or both. Therefore, pH = 13 alkaline at atmospheric pressure appears to be the best options based on yield and purity. However, the design of future RG-I pectin extraction processes should not only include the considerations of novel pectin-based products requirements  but also industrial process constraints. For example, water as a solvent is more preferable than strong acid or alkaline in terms of environmental impacts and water extracts do not require any further purification steps to remove the remaining solvent. Atmospheric pressure extractions at 90 °C are safer compared to pressurised hydrothermal extractions, although the optimal MAHE conditions of 130 °C and vapour pressure 2.7 bar are not very high in the context of industrial processes. Furthermore, MAHE has much shorter cycle time of 10 min compared with MAE and CSE of 120 min, and it can also potentially achieve 0 min treatment time, which allows design of a continuous system with a smaller plant footprint.


This case uniquely reports a connection between endodontically infected teeth and systemic disease, and additionally presents ozone therapy as a unique therapy and immune system modulator. It is the world’s first such reported case and the treatment holds invaluable lessons in assessing the “unknown” causes of autoimmunity and inflammation. Additionally, it presents ozone therapy as a most needed unique, non-toxic and powerful anti-infective agent, anti-inflammatory and immune modulator.The patient was a Mexican male field laborer, age 48 years, in inflammatory crisis with a confirmed case of dermatomyositis. He had received massive prednisone, and powerful immune suppressing drugs just to function, while disease still raged. I encountered him in the field in June 2012 with severe muscle pain, weakness, and diffuse generalized skin rash, essentially unable to do his work. Creatine kinase peaked at 9293 U/L. History and physical examination findings caused suspicion of subclinical infections in endodontically treated teeth. This impression was confirmed in subsequent dental evaluation. He fully recovered after dental infections were confirmed and surgically removed, while receiving ozone therapy until all symptoms and laboratory abnormalities normalized.Dental focus of occult infection may be a prime cause/trigger of autoimmune disorders and inflammatory disorders, requiring surgical intervention to remove. Ozone therapy, little known in conventional medicine, has been shown in the literature and in this case to be a powerful and safe immune modulator and anti-infective agent. This case has significant relevance across the entire spectrum of both medical and dental practice. It also emphasizes the need for individualized assessment and treatment rather than symptomatic pharmacological approaches treating a “disease” rather than the patient. Subclinical dental infection and ozone therapy are reviewed. Dermatomyositis is a disease belonging to the rheumatologic class, long considered an “autoimmune disease” of idiopathic cause. New research is now linking “autoimmune” and other diseases to heretofore unknown occult infection arising from or translocating from the mouth  and alim3, the strongest naturally occurring oxidant, generated by lightning and ultraviolet irradiation. Medical ozone is made similarly. Oxygen passes through a high voltage corona arc discharge. Medical “ozone” is actually a mixture of oxygen (95–99%) and ozone (1–5%).Ozone is an allotrope of oxygen, OOzone has been in continuous medical use for over 100 years, used by German soldiers in the trenches to disinfect wounds in WWI. Recent research both in vitro and in vivo demonstrate ozone to have powerful immune modulating and healing effects. In addition, it virtually instantly destroys bacteria , 5 and v2/O3 (“ozone”) gas mixture, and returned.Modern methods of ozone delivery include a direct intravenous (DIV) gas protocol, rectal insufflation of gas, and major autohemotherapy method (MAH) developed by Germans. In MAH, an aliquot of blood is removed from the body, heparinized, treated with O3/cc) for nearly 30 years and developed a protocol for administration [Direct intravenous oxygen gas has been long used in medicine. Regelsberger reported it provides powerful immunological effects, and improves blood rheology and oxygenation . It alsostration .2 and the blood then returned to the patient under this pressure. A single pass of 200 cc blood plus gas delivers 14,000 mcg O3. A “double pass” would deliver 28,000 mcg O3 into 400 cc blood. This form of MAH is called “hyperbaric ozone” (HBO3) therapy.In the form of MAH used herein, an aliquot of venous blood is removed from the patient (~ 200 cc at a time) in a vacuum glass bottle. Oxygen/ozone gas mixture, 200 cc at 70 mcg/cc ozone, is pumped into the bottle and swirled, mixing the gas with blood. Pressure in the bottle is maintained at 1.9–2.0 ATA (atmospheres absolute) by pumping additional OThe patient, a muscular Mexican field hand, husband and father, was 48 years old when I saw him attempting to work on 12/6/2012 looking extraordinarily ill. He had exceptionally severe muscle pain involving all extremities.He related recent diagnosis of dermatomyositis, taking several drugs and still rapidly worsening . A skin biopsy 12/2011 showed infiltration of lymphocytes and macrophages, with basement membrane thickening). He had been diagnosed with dermatomyositis on 8/2/12 after several misdiagnoses were made of “sun rash” and eczema. He had no family history of autoimmune disorders. The prognosis given by his conventional physicians was “poor”.He had quickly become extremely weak and found it all but impossible to do his field chores and earn a living. His skin had confluent brown macular rash with violet hue, almost ecchymotic on the extensor surfaces of the forearms and left ankle. He had an inflamed facial butterfly facial rash, moon facies, Gottron’s papules on the dorsal surface of hands, and nail cuticle telangiectasias. His muscles were very weak and tender. He exhibited classic signs of iatrogenic Cushing’s disease Figs. , 2, 3.FiDrugs for immunosuppression: prednisone 80 mg daily, methotrexate 10 mg/week, Plaquenil 200 mg twice daily, azathioprine 150 mg daily (methotrexate was discontinued after a short course due to spike in liver enzymes. AST rose to 130 on 28/8/12); acetaminophen/codeine twice daily for pain.Over several months, his doctors repeatedly attempted weaning to 60 mg prednisone. However, every time he reached that level, his disease sharply flared. In March 2012, his CK was 2088 (44–96 U/L), CK (MB) 103.99, ANA 1:128, ESR 1. On 6-6-13, CK peaked at 9293 U/L as his treating physicians struggled to control his deteriorating condition with immunosuppressive drugs. Prednisone dose was intermittently raised to 120 mg/daily. I was not involved in any aspect of his drug regimen .Helicobacter pylori receiving amoxicillin, metronidazole, bismuth subsalicylate, and proton pump inhibitors. He also had a course of INH and rifampin for “latent” tuberculosis during this period, without change in the dermatomyositis.Additionally, early in his disease, he was treated for Based on history and physical examination actually performed in his agricultural workplace, I suspected underlying and asymptomatic dental infections and sent him for dental X-rays of incisors and an extraction site. I also immediately started ozone therapy (described below) after obtaining full informed consent.Dental X-rays confirmed my suspicions: failing endodontics at numbers 7, 8, and 9, and osteonecrosis at extraction site 32. I suggested that he have the infected teeth removed, but he refused through May 2013. However, by then he was up to 120 mg prednisone and his field labor was made possible by use of ozone therapy.In July 2013, he finally underwent extraction of the failing root canals at 7, 8, and 9, with limited improvement. I suspected more subclinical dental infections and implored him to seek further treatment. However, cost was a major factor. He finally had more definitive dental work  and had teeth 4, 15, and 30 extracted. Following this, in conjunction with ozone therapy, I weaned his prednisone without incident or exacerbation (about 5 mg every other week). Prednisone was discontinued by 1/15. Plaquenil was discontinued the following month (2/15). He suffered no skin or muscle symptoms during this period or any clinical signs of inflammation.His ozone therapy was as follows:3 beginning June 12, 2012 for a total of 13 treatments through 3-13-15 as follows:6-12-12: 12,000 mcg; 2-2-13: 24,000 mcg; 10-15-13: 9000 mcg;10-17-13: 9000 mcg; 1-11-14: 14,000 mcg; 5-10-14: 28,000 mcg;8-2-14: 28,000 mcg; 11-13-14: 14,000 mcg; 3-13-15: 28,000 mcg;8-1-15: 28,000 mcg; 3-18-16: 28,000 mcg; 6-24-16: 28,000 mcg;1-17-17: 28,000 mcg (dates: month-day-year).HBOOzone treatments improved his strength, vitality, and pain virtually immediately, even before the removal of oral infections, but results were partial only. Ozone therapy did appear to be the key factor in keeping him productive in the field, earning his living, during the entire period. He had been rapidly deteriorating clinically on drug therapy alone and was unable to work until receiving ozone therapy.2/O3 gas at 25 mcg O3/cc.He also received DIV ozone averaging once every 2 weeks for a total of 30 sessions between 11/19/14 and 4-27-15. This consisted of 20 cc of O3 therapy quarterly as a courtesy in ozone training classes. He remained totally asymptomatic until March 2017, telling me every time I saw him (weekly) “I never felt better”.Plaquenil was discontinued in 2-15. He continues to receive HBOIn March 2017, he noted slight achiness with faint skin blotches. His CK (3/2/17) was 523 U/L. His doctors were prepared to restart the powerful immunosuppressive drugs. My examination suggested at least two more occult infected dental sites. I suggested he return to his dentist. Within 2 weeks, his dentist removed two more infected teeth, numbers 2 and 15 and his symptoms were gone virtually immediately. On June 10, 2017, his CK had dropped to 313 and he was again “never feeling better”. He suffered no adverse of unanticipated events Figs. , 5, 6.FiOral infection, including occult cavitations and endodontically treated teeth, may be key sources of bacterial seeding in the body that is routinely overlooked. Weston Price, DDS, a renowned researcher of the last century, published his findings on the systemic risks of endodontic infections . He founPorphyromonas gingivalis was the most abundant species detected in coronary and femoral arteries [This case also supports long held concerns about the systemic effects of oral infections. Chan, et al. described a mechanism of post-infection autoimmunity: “postinfection production of cross-reactive tissue-specific autoantibodies” . There harteries . Oral sparteries . It is tAdditionally, German brothers, Huneke, described relationships between specific teeth and acupuncture meridians/organs, and joints to which they are related . They prOzone therapy, in continuous worldwide use for 150 years, is a modality that antedates the modern “FDA approved” category of therapies. “Its effects are proven, consistent and with minimal side effects…. and well documented”, for infections, wounds and multiple diseases . Under URegarding ozone therapy, several reports have emerged demonstrating ozone therapy’s ability to significantly reduce inflammatory mediators and joint damage , 22. FahOzone therapy has other highly desirable circulatory and immunological effects. Basic published research (scores of articles) from Italy  and CubaAll of these effects would be expected to positively influence the course of any disorder. The immune modulating effects would be particularly beneficial in inflammatory conditions. Ozone therapy has not been reported to induce harm, dependence, injury or untoward effects when properly administered.Ozone therapy has significant activity against microorganisms. It nearly instantly destroys pathogens exposed to the gas. Scripps recently discovered ozone synthesis in the body  as part Ozone gas and ozonized water cut through biofilms , well esDIV oxygen has long been administered in Europe, found to stimulate the production of the anti-inflammatory enzyme 15-Lox-1 from eosinophils  and impr3 method of ozone delivery may have a similar intravenous O2 gas like effect, in addition to the ozone effect itself. Boyle’s law of gas diffusion into fluids predicts about 8.8 cc of oxygen gas dissolved into the 200 cc blood by the HBO3 method described, which may well cause a “Regelsberger” effect [The HBO” effect .This patient credits ozone therapy keeping him able to earn a living while in the long process of resolving the dental issues. Neither ozone therapy nor any drug therapy would be expected to singularly remedy closed foci of infection, such as in this case, essentially abscesses.CK values need discussion. In a review article on CK levels, the authors argue that the current accepted “normal” levels of CK are low. CK levels vary by sex, race, body mass and physical activity . “While “Spontaneous remission” is highly unlikely as every effort to lower immunosuppressive drugs was met with failure (severe exacerbation) until immediately after dental attention and receiving ozone therapy, permitting steady and uneventful withdrawal of long-term high-dose steroids, which would have fully dampened his pituitary–adrenal axis.The strength of this case is that it showed high value in addressing a medically unsuspected cause of systemic disease  and in tailoring a specific treatment plan to the individual patient and not the disease model. The limitation is that autoimmune disease likely has multiple causes  and the next case may be totally different. It is important to go beyond a conventional history and physical examination, which may arrive merely at a descriptive diagnosis, to elucidate the cause of the malady.This author has seen many “unrelated” systemic or distant symptoms remit when suspect endodontically treated teeth were removed or jaw cavitations surgically treated. With the modern discoveries of oral bacterial products present in distant inflamed tissue, and the discoveries of alimentary canal organisms in inflamed tissue, medicine/dentistry should revisit the research of the pioneering Weston Price, DDS, to examine undiscovered links between teeth, the oral cavity and the body.This patient has publicly expressed extreme gratitude for his recovery by the combination of the two approaches—dental treatment and ozone therapy—after he rapidly deteriorated while on aggressive chemical therapy. At the outset, he was incapable of performing his field duties. After treatment, he consistently related, “I’ve never felt better.”This report appears to be the world’s first describing total resolution of an autoimmune disease in crisis with removal of oral infection complemented by ozone therapy. It re-presents to the medical world a critical, overlooked and relatively unknown factor and treatment approach in the management of chronic inflammation—occult dental infection—to which both the medical and dental professions must become aware.Inflammatory conditions are notoriously difficult to treat, often requiring highly toxic drugs. This report is also valuable across the board in medicine, bringing forward a novel and non-toxic therapy (ozone) as an alternative and/or adjunctive treatment to drugs. Ozone therapy could advance medicine’s currently limited ability to safely treat inflammatory (and infective) disorders. It is the author’s hope that this report will stimulate institutional study, which, in today’s modern medical world, may be difficult due to ozone’s non-patentability for financial returns.


Sequencing of the T cell receptor (TCR) repertoire is a powerful tool for deeper study of immune response, but the unique structure of this type of data makes its meaningful quantification challenging. We introduce a new method, the Gamma-GPD spliced threshold model, to address this difficulty. This biologically interpretable model captures the distribution of the TCR repertoire, demonstrates stability across varying sequencing depths, and permits comparative analysis across any number of sampled individuals. We apply our method to several datasets and obtain insights regarding the differentiating features in the T cell receptor repertoire among sampled individuals across conditions. We have implemented our method in the open-source R package powerTCR. A more detailed understanding of the immune response can unlock critical information concerning diagnosis and treatment of disease. Here, in particular, we study T cells through T cell receptor sequencing, as T cells play a vital role in immune response. One important feature of T cell receptor sequencing data is the frequencies of each receptor in a given sample. These frequencies harbor global information about the landscape of the immune response. We introduce a flexible method that extracts this information by modeling the distribution of these frequencies, and show that it can be used to quantify differences in samples from individuals of different biological conditions. PLoS Computational Biology Methods paper.This is a 18 different TCRs u⋆ =  was constructed by considering every unique clone size in the repertoire. Then, for each ui, the shape parameter is estimated asni is the number of clones with size larger than the threshold ui. Once this value is computed for every threshold in u⋆, the threshold and corresponding The Desponds et al. model was fit as previously described . BrieflyX, let S(X) be the sample richness, defined as the number of unique clonotypes in X, and let pi be the number of cells of clonotype i normalized by the total number of cells in the sample. Then, the Shannon entropy of X isX isThe ecological estimators , 22 werey, where y ∼ GPD, asThe Desponds et al. model, which is a type-I Pareto distribution, and the “tail” part of our model, which is a GPD, are closely related. In fact, the GPD contains the type-I Pareto distribution as a special case. We can write the distribution function of u and shape αd  implies a heavier-tailed distribution, while a smaller ξ (larger αd) implies a lighter-tailed distribution.Now, let shape αd . Of courξ, the shape parameter of the GPD, positively correlates with clonality. We numerically validated this claim using a simulated cohort of 48 clone size distributions. That is, we generated samples of n = 20, 000 clonotypes, where our 48 parameter settings were derived from every combination of α ∈ {3, 5, 10}, ξ ∈ {.25, .5, .75, 1.1}, and ϕ ∈ {0.1, 0.15, 0.2, 0.25}. We chose β = 0.15, u = ⌊Qα,β(1 − ϕ)⌋ in each simulation, where Qα,β is the quantile function of the Gamma distribution with mean We conjecture that The relationship between a pair of TCR repertoires can be elucidated by evaluating the distance between their fitted spliced threshold models. Several methods to compare densities are available. We propose measuring the distance between each pair of distributions using Jensen-Shannon distance (JSD) . This meP and Q, the JSD between P and Q isGiven discrete distributions X ∼ discrete GPD and denote its distribution function as F with Fc as its continuous analog. Then we can writeThe threshold stability property of the GPD is well-established . Here, wX ∼ discrete GPD, then X − u ∼ discrete GPD. Or, for our application, consider a clone size distribution, where clones larger than some threshold u are distributed according to the discrete GPD. At decreasing sequencing depths, this estimated u decreases, implying naturally that the size a clone in the sample must achieve to be considered “expanded” decreases. Still, while u shrinks, the threshold stability property states that ξ remains constant.This states that if S1 TextFor each real data sample, a plot analagous to (PDF)Click here for additional data file.S2 TextDetails on simulation study comparing the discrete model and Desponds et al. model, and simulation study comparing the discrete and continuous models.(PDF)Click here for additional data file.S3 Textξ and the clonality estimator.Information on simulation study that finds strong positive relationship between (PDF)Click here for additional data file.S4 TextTables summarizing number of unique clonotypes per sample, total reads sequenced per sample, and other patient-specific information for the Sarcoidosis and glioblastoma datasets. Additionally, tables containing fitted model parameter estimates for both our model and the competing model, as well as ecological estimator values, computed for every sample.(PDF)Click here for additional data file.S5 TextBy comparing results from our full model to those from only our tail model, we observe empirically the gains from including the full clone size distribution.(PDF)Click here for additional data file.S6 TextWe downsampled mouse tumor data to 80, 60, 40, and 20% of total reads. We used JSD to compute pairwise distances between the samples for our model fits and the Desponds et al. model fits at each downsample level and did hierarchical clustering using Ward’s method. The dendrograms for each model at each downsample level are presented here.(PDF)Click here for additional data file.S7 TextClustering dendrograms generated on pre- and post-treatment glioblastoma samples. Groupings presented for the post-treatment samples here correspond to the colored groupings in (PDF)Click here for additional data file.S8 TextThis plot calls out patients 33296 and 17232 from the glioblastoma patients. Patient 33296 incorrectly clustered with the individuals with favorable clinical outcome, while patient 17232 incorrectly clustered with individuals with unfavorable clinical outcome.(PDF)Click here for additional data file.


Circulating immune cells, which are recruited to the site of injury/disease, secrete various inflammatory mediators that are critical to nociception and pain. The role of tissue-resident immune cells, however, remains poorly characterized. One of the first cells to be activated in peripheral tissues following injury are γδT cells, which serve important roles in infection, disease, and wound healing. Using a mouse line lacking these cells, we sought to identify their contribution to inflammatory pain. Three distinct models of peripheral inflammatory pain were used: intraplantar injection of formalin (spontaneous inflammatory pain), incisional wound (acute inflammatory pain), and intraplantar injection of complete Freund's adjuvant (chronic inflammatory pain). Our results show that absence of γδT cells does not alter baseline sensitivity, nor does it result in changes to mechanical or thermal hypersensitivity after tissue injury. Myeloid cell recruitment did show differential changes between models of acute and chronic inflammatory pain. These results were consistent in both male and female mice, suggesting that there are no sex differences in these outcomes. This comprehensive characterization suggests that γδT cells do not contribute to basal sensitivity or the development and maintenance of inflammatory pain. The immune and nervous systems are intimately connected, particularly during inflammatory pain. Immune cells and their secreted mediators act on nociceptors in the periphery while neurons can modulate the inflammatory response –3. PeripVarious components of the immune system can bring about peripheral sensitization and pain hypersensitivity , 5, 8–10−/− mice) and observed no role for these cells in inflammatory pain  were backcrossed to C57BL/6J mice (Jackson Laboratory), generating wildtype, heterozygous and knockout littermates. All experiments were carried out using mice between 6 and 12 weeks of age, kept in a temperature- and humidity-controlled room with food and water provided ad libitum.B6.129P2-TcrdMale and female TCRδ littermates were used for all experiments. Mice either received intraplantar injections with 20 μl of a 5% formalin solution, as described , intraplLicking/biting of the formalin-injected hindpaw was assessed in 5 min intervals over 60 min. Mechanical and thermal sensitivity, measured using von Frey, acetone, and Hargreaves tests, were carried out as described . ThermalMice were anesthetized and sacrificed by transcardial perfusion with 2% paraformaldehyde in 0.1 M phosphate buffer. Ears were removed, post-fixed for 1 h, and cryoprotected in 30% sucrose. Serial 15 μm cryostat sections were incubated with anti-mouse TCRδ  and anti-hamster IgG-FITC , and coverslipped for visualization using an AxioSkop2 fluorescent microscope .n = 4/genotype/group) were stained using the following antibodies : FITC anti–CD11b, PE-Cy7 anti–Ly6G, and APC/Fire750 anti–CD45. Flow cytometry was conducted on a CytoFLEX cytometer  and analyzed using CytExpert software (Beckman Coulter).Immune cell infiltration/recruitment was assessed, as described . Footpadpost-hoc Tukey tests (P < 0.05).All statistical analyses were carried out using SigmaPlot . Data are expressed as mean±SEM. One-way analysis of variance (ANOVA) was used for direct comparison between two or more groups, and two-way repeated-measures (RM) ANOVA used to assess change between groups over time, with +/− and TCRδ+/+ littermates but not in TCRδ−/− mice, as we have previously shown (n = 14–22) or female (n = 10–18) mice , and thermal sensitivity, assessed using the acetone  and Hargreaves radiant heat tests , respectively, did not show any difference between TCRδ littermates in males or females. The hot/cold plate test was used to identify differences in noxious thermal response using fixed temperatures between 0 and 55°C. Male (n = 7–15) and female (n = 6–17) littermates did not show any significant differences in time to first response, while there was a group effect in females between all three strains at 55°C (P = 0.039), no significant differences were observed between genotypes (post-hoc Tukey test P = 0.063).γδT cells were visualized using immunohistochemistry and found to be present in the skin epidermal layer  of TCRδ+ly shown . We firs18) mice . Mechanin = 6–9) and female (n = 8–13) TCRδ mice when measured in 5 min intervals , nor in total response time during the acute and tonic stages . We next considered the contribution of γδT cells to the development and maintenance of acute inflammatory pain following plantar incisional wound, a model of post-surgical pain that often resolves within 3–4 days. Male (n = 7–10) and female (n = 8–12) littermates showed no difference between mechanical  and heat  hypersensitivity, assessed over 7 days  or female (n = 6–9) littermates when assessed for mechanical  and thermal  hypersensitivity over 7 days (−/− mice showed a trend toward decreased myeloid cells compared littermate controls, though this was not significant (n = 4/group, P ≥ 0.336, one-way ANOVA; We finally carried out intraplantar injection of CFA to determine whether γδT cells contribute to chronic inflammatory pain, an example of granulomatous inflammation that does not resolve . Similarr 7 days . Behavior 7 days . The infUnderstanding how immune cells contribute to the development and maintenance of pain will be crucial to the development of safe and efficacious therapeutics for the treatment of inflammatory pain. Our group previously identified the contribution of lymphocytes to inflammatory pain, using cell-specific strategies to deplete neutrophils, non-neutrophil myeloid, and αβ T cells . While oOur work started by showing that loss of γδT cells did not have an effect on basal sensitivity, an important finding given that these cells are known to interact with sensory fibers during inflammation . Using t−/− mice treated with CFA. Other studies, however, have found that these cells are either not required or negatively regulate skin inflammation (−/− mice helps to initiate the proliferative phase of wound healing (While there are conflicting results in the literature as to the function of γδT cells in the modulation of inflammation, we now show a divergent role for these cells in the recruitment of myeloid cells in the footpad following injury/inflammation. Our results suggest the immunomodulatory role of γδT cells depends on the type of immune response necessary: a strong inflammatory response is present in the CFA-injected footpad, while a less pronounced inflammation is found following incisional wound. Recent evidence now points to an important role for γδT cells in the recruitment of myeloid cells, including neutrophils, monocytes, and macrophages , 56–58, ammation , 59, 60, healing ; it is t healing , keratin healing , 62, 63, healing , 65, itc healing , 67, and healing , 69. WhiThe datasets generated for this study are available on request to the corresponding author.JP, JRS, IG, and NG contributed to the conception and design of the study. JP performed all behavioral analysis. JRS, CAB, JPS, ASM, and CMH performed histology and flow cytometry experiments. JP and NG performed the statistical analysis. JP wrote the first draft of the manuscript. JRS, IG, and NG wrote sections of the manuscript. All authors contributed to manuscript revisions, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Further examination revealed that the menadione-mediated loss of respiratory reserve capacity in aged hepatocytes was from significant inhibition of Complex I activity and increased proton leak, for which an increase in Complex II activity was not able to compensate. These data demonstrate an age-related increase in mitochondrial susceptibility to a redox-cycling challenge, particularly in regards to Complex I activity, and provide a plausible mechanism to link this vulnerability to mGSH perturbations.The role of mitochondrial GSH (mGSH) in the enhanced age-related susceptibility to xenobiotic toxicity is not well defined. We determined mGSH status and indices of mitochondrial bioenergetics in hepatocytes from young and old F344 rats treated with 300 μM menadione, a concentration that causes 50% cell death in old. At this concentration, mGSH was significantly lost only in hepatocytes from old rats, and with near total depletion due to lower basal mGSH in aged cells. In old hepatocytes, menadione caused mitochondrial membrane potential to collapse, as well as significant deficits in maximal O  Mitochondria from aged tissues display increases in oxidant leakage, mitochondrial DNA damage, and susceptibility to the formation of the mitochondrial permeability transition pore (MPTP), leading to apoptotic initiation [2 generation and electron flow through the electron transport chain (ETC) [m collapses, cells become sensitized to oxidative insult, and intrinsic apoptotic and necrotic pathways are instigated. Thus, the mGSH pool constitutes a unique subcellular fraction that must be maintained in a narrowly defined equilibrium for proper mitochondrial and cellular function.Aging is characterized by a decline in cellular redox homeostasis and detoxification capacity, which is coincident with an increased risk for age-related pathophysiologies . Mitochoitiation . Mitochoin (ETC) ,15. Compin (ETC) . Finallyin (ETC) ,13,20,21l-cysteine). While our prior work demonstrated that increased hepatocellular susceptibility to menadione was mediated through age-related losses of GSH per se, it did not investigate the role of the distinct mGSH pool in contributing to the hepatic vulnerability to redox cycling agents. To fill this important gap in knowledge, the objective of the present work is to determine whether age-related deficits in mGSH specifically contribute to vulnerability to menadione-mediated insults. Based on this objective, we hypothesized that age-related increases in vulnerability to an acute redox cycling challenge are due to a failure of mGSH capacity to maintain mitochondrial ETC function.In spite of the important linkage between maintenance of mGSH and normal cell metabolism, our lab and others have shown that mGSH decreases by as much as 50% with age . While tm, and induces formation of the MPTP [2 consumption and RRC, and finally, collapse of Δψm.In order to test this hypothesis, we utilized an acute menadione dose (300 μM) in hepatocytes isolated from young and old male Fischer 344 rats (F344) and evaluated age-related differences in measures of mitochondrial function. In addition to the aforementioned increased vulnerability with age, other studies show that menadione directly interacts with Complex I in the mitochondria, depletes mGSH, decreases Δψthe MPTP . As such22.150 young: 405 μM; old: 275 μM) with significant differences in GSH loss and increased markers of lipid peroxidation within minutes [50 for old) and assayed mGSH levels over a 20 min time-course. Basal mGSH level  electrode, we showed that there was an 18.7% age-related loss of baseline Δψm (26.9 ± 3.1 mV) in old versus young cells, indicating that losses in this key driving force for ATP synthesis correlated both with mGSH deficits and age. Menadione administration caused a collapse in Δψm in old but not young rat hepatocytes. As shown in m in old rat hepatocytes was 24.1% lower than baseline following menadione addition (−28.3 ± 1.5 mV), but cells from young animals showed no appreciable deficits relative to controls. In fact, the menadione-mediated decline in Δψm in aged rat hepatocytes breached the threshold known to induce mitochondrial permeability transition pore opening (∼−100 mV) [Because previous studies have shown that mGSH depletion adversely affects mitochondrial bioenergetics and initiates cell death , we askeat liver ,34. By m−100 mV) .Fig. 2Chm in young rat hepatocytes that had been treated with menadione remained at initial values approximately 1 h after treatment; however, no recovery in Δψm was evident in old rat hepatocytes. These results demonstrate a striking age-associated sensitivity of Δψm to menadione in both the magnitude of membrane potential decline and in its limited recovery potential with age.Further analysis revealed that Δψ2.3m, we hypothesized that this should translate to decrements in mitochondrial O2 consumption characteristics. An examination of O2 consumption characteristics in young and old rat hepatocytes in the presence and absence of various substrates and with or without menadione addition was undertaken  was monitored in digitonin-permeabilized hepatocytes following additions of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), a protonophore. Consistent with results shown in To better define whether menadione's effect on mitochondrial O2.42 consumption was assessed. Complex I-linked (CI), and Complex II-linked (CII) O2 consumption were examined in the presence of glutamate and succinate (CI and CII substrates). Further differentiation of CI and CII O2 consumption was ascertained by addition of rotenone, an inhibitor of Complex I in the ETC. Following assessment of basal O2 consumption, proton leak-linked O2 consumption (Leak) was measured after injection of oligomycin, an ATP synthase inhibitor that blocks the FO subunit of the proton channel. “Leak” is thus defined as proton leak, proton slip, cation cycling, and electron leak and their contribution to a reduction in biochemical coupling efficiency and the dissipation of the proton gradient that is not available for ATP production [2 consumption or O2 consumption linked to CI, CII, or Leak  that decline with age ,40, may ynthesis , and henynthesis . In thism, which limits mGSH redistribution which would normally occur when isolating mitochondria by standard differential centrifugation techniques [The current study focused on susceptibility to mitochondrial dysfunction during acute menadione insult in freshly isolated intact and digitonin-permeabilized hepatocytes from young and old rats. This cell model is appropriate for understanding aging differences in mitochondrial dysfunction because F344 rat hepatocytes reflect the age-related losses of GSH, mGSH, and mitochondrial function that are seen in humans . The usechniques . It alsochniques .We used menadione in the current study because the mechanisms of its toxicity and detoxification have been thoroughly explored. Menadione is a potent redox cycling compound that generates a strong, persistent oxidative stress via the initial generation of superoxide followed by its dismutation into more deleterious ROS/RNS ,54. We hm, loss of RRC, and the inhibition of Complex I electron flux. This suggests that the lower basal level of mGSH plays a critical role in the age-related loss of detoxification resiliency under an acute redox cycling exposure. While a causal relationship was not determined in this work, mGSH involvement in each of these markers of mitochondrial function is well established [Loss of mGSH happens almost immediately after being dosed with menadione in both young and old hepatocytes. However, menadione-induced mGSH loss in cells from young animals was relatively limited, did not rise to statistical significance and remained above the steady-state basal amounts of mGSH found in old rats. This is a critical observation because the rapid mGSH loss in aged rat cells precedes severe declines in Δψablished ,16,35.2 consumption and Δψm with age [Our results further demonstrate that age increases mitochondrial susceptibility to acute redox cycling challenge, and that Complex I in particular is more vulnerable to inhibition when exposed to a redox cycling agent. Previous studies have demonstrated an age-related shift toward a more oxidized mitochondrial environment ,18. Thiswith age . Additiowith age . Other rwith age . CombineAside from exploring the mechanisms discussed above, this work identifies mGSH as a potential target for age-related therapeutics. Our previous work demonstrated that a two-week treatment with lipoic acid was capable of increasing both cytosolic and mGSH. However this approach is not directly applicable to the present acute exploration since lipoic acid upregulated Nrf2-dependent GSH synthesis genes over the course of days to weeks versus the strictly acute conditions applied herein. Nevertheless, lipoic acid as well as other compounds that induce GSH synthesis (e.g. n-acetyl cysteine) may be a long-term option for prophylactic therapies to prevent loss of mGSH with age in order to limit mitochondrial susceptibility to xenobiotics that may ultimately affect the organelle's function. Additionally, we are utilizing a novel compound (patent pending) to direct GSH to mitochondria, thus avoiding slow or limited uptake from the cytosol, which may be limited with age. Such strategies may act as an interventional means to protect mitochondria under acute electrophilic insult.44.1www.bioblast.at/index.php/MiPNet14.13_Medium-MiR06). In brief, MiR05 medium, pH 7.1, contains 0.5 mM EGTA, 3 mM MgCl2-6H2O, 60 mM lactobionic acid, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM d-Sucrose, and 1 g/L BSA . Collagenase, type IV, was purchased from Worthington Biochemical Corporation . High purity digitonin, a non-ionic detergent that interacts specifically with cholesterol and permeabilizes cell membranes, was purchased from Millipore-Sigma (Cat# 300410). Rotenone, oligomycin, FCCP, antimycin A, menadione, and tetraphenylphosphonium chloride was purchased from Sigma-Aldrich. All other chemicals used for this work were purchased from commercial sources at analytical grade.Mitochondrial respiration medium (MiR05) was prepared as described by Gnaiger et al.  provided management and veterinary care. .Both young (4–6 months) and old (24–28 months) male F344 rats were acquired from the National Institute on Aging animal colonies. The rats were housed at the Linus Pauling Institute animal facility at Oregon State University and allowed to acclimatize for at least 1 week prior to any experimentation. Animals were maintained on a 12 h light cycle (7am to 7pm) and fed standard chow 4.3Hepatocyte isolation was performed as described previously . Briefly4.42 consumption rates of young and old rat hepatocytes were determined using an Oxygraph-2k High Resolution Respirometer  at 25 °C. Young and old rat hepatocytes were treated with either vehicle (0.05% dimethylformamide) or 300 μM menadione for 15 min while being rotated on a MACSMIX (Miltenyi Biotec) rotator. Cells were then pelleted by centrifugation at 100×g, washed in Krebs-Henseleit solution to remove excess menadione and set the treatment time. Cells were then pelleted again, the Krebs-Henseleit was removed and cells were resuspended in MiR05 medium to a cellular density of 2.5 × 105 cells/mL before being added to the instrument chamber. Following assessment of basal O2 consumption, proton leak-linked O2 consumption (Leak) was measured after oligomycin injection . Maximal respiratory capacity under uncoupling conditions was measured by successive 0.05 μM additions of the uncoupler FCCP . In other experiments, electron flux through various portions of the electron transport chain was determined. CI O2 consumption was measured by addition of 10 mM glutamate, 2 mM malate, and 2 mM ADP. CI and CII O2 consumption were then measured in conjunction by addition of 5 mM succinate. Further differentiation of CI and CII O2 consumption was ascertained by addition of 0.5 μM rotenone. Electron transport chain coupling and Leak were then determined by addition of 2.5 μM oligomycin . Non-mitochondrial respiration, which was used as a correction factor for O2 consumption, was measured after addition of 2.5 μM antimycin A .O4.5In order to allow direct mitochondrial examination by controlling substrate provision while avoiding issues of mitochondrial isolation such as yield, quality, and subpopulation selection, we chose to use digitonin permeabilized primary hepatocytes as first described by Andersson and Jones , and lat6 cells) over a time course (1–10 min). Permeabilization of the plasma membrane was confirmed via lack of trypan blue exclusion (>90% of cells are blue). Cells were then pelleted and glutamate dehydrogenase (GDH) activity was measured in the supernatant and pellet fractions. Mitochondria were considered to be intact and functional when >90% of GDH activity was in the pellet and the mitochondrial respiratory control ratio (RCR), a useful general measure of mitochondrial coupling and function, was above 5. Only preparations with these characteristics were used in experiments. Concentrations of digitonin that met these criteria varied slightly between young and old (0.1 and 0.08 mg/106 cells respectively). Time of treatment had little effect on plasma membrane permeabilization and only affected mitochondrial membranes at higher concentrations (>0.2 mg/106 cells). This digitonin: cell ratio remained effective over varying cellular concentrations tested (0.25 × 106–1 × 107 cells/mL).Appropriate digitonin concentrations for hepatocytes isolated from young and old rats were determined empirically based on successful permeabilization of the plasma membrane while mitochondrial membranes remained intact. Briefly, hepatocytes were treated with a digitonin concentration gradient . Samples were placed in the dark, at room temperature for 1 h. The resulting S-carboxymethyl derivatives were subjected to further chemical modification by addition of an equal volume of dansyl-chloride solution (20 mg/mL in acetone). The solution was incubated overnight in the dark and at room temperature. The next day, 500 μL of chloroform was added, samples were vortexed and then allowed to incubate for 1 h in the dark, at room temperature. Samples were then centrifuged at 15,000×g for 15 min at 4 °C before removing and assaying the supernatants. Samples (20 μL) were separated by HPLC using a Thermo Scientific  APS-2 Hypersil column and detected on a Shimadzu  SPD-10AVP fluorometric detector set at 335 nm for excitation and 515 nm emission. Quantitation was obtained by integration relative to GSH and GSSG standards.Glutathione (GSH) content of permeabilized cells was determined according to the methods of Jones et al.  and modi4.80) is indicated by a pound sign (#) over each data point in the figure. Differences between samples that resulted in a p-value of ≤0.05 were considered statistically significant.All statistical analyses were performed using Excel  and Prism 6 . Data are represented as mean ± standard error of the mean (SEM). For comparisons between two samples, two-sided Student's t-test was used. Where comparing the means of two or more independent groups with a single independent variable (i.e. between age groups), one-way ANOVAs with Sidak post-hoc analysis for multiple comparisons were used. Where comparing the mean differences between groups with two independent variables (i.e. between age groups and over a time-course), two-way ANOVAs with Tukey's post-hoc analysis for multiple comparisons were used. For two-way ANOVAs, statistical significance between age groups is indicated on the legend of the figures with an asterisk (*), and statistical significance over a time-course (versus tNOT performed most of the experiments, and largely contributed to experimental design and writing of the manuscript. KPS performed part of the experiments and largely contributed to writing of the manuscript. TMH was responsible for experimental design and largely contributed to writing of the manuscript.The authors declare that they have no conflicts of interest.


Hence, the overdispersion pattern implies increasing interspecific competition with decreasing drier and warmer conditions, which is also supported by fewer species occurring at the driest site. Our results are in line with a hypothesis that decreased biomass production limits resource availability for termites, which leads to competition. Along with this comes a diet shift: termites from drier plots had higher δ13C signatures, reflecting higher δ13C values in the litter and more C4 plants. Our study shows how a phylogenetic community approach combined with trait analyses can contribute to gaining the first insights into mechanisms structuring whole termite assemblages.The mechanisms that structure species communities are still debated. We addressed this question for termite assemblages from tropical dry forests in Colombia. These forests are endangered and poorly understood ecosystems and termites are important ecosystem engineers in the tropics. Using biodiversity and environmental data, combined with phylogenetic community analyses, trait mapping, and stable isotopes studies, we investigated the termite community composition of three protected dry forests in Colombia. Our data suggest that the structuring mechanisms differed between sites. Phylogenetic overdispersion of termite assemblages correlated with decreasing rainfall and elevation and increasing temperature. Food niche traits—classified as feeding groups and quantified by δ The drivers that structure communities are still debated ,2,3,4. NAnoplotermes-group) are relatively scarce in richness and abundance [Tropical dry forests are the most threatened of all major tropical forest types ,15. Colobundance . Nitrogebundance ,31 and nbundance ,33,34,35bundance ,36,37. ITermites were studied in the Coraza Forestry Reserve ‘Colosó’ , the regional park ‘El Ceibal Mono Tití’ , and the National Park ‘Tayrona’  in Colombia . All thehttp://www.worldclim.org/). The data layers were generated through the interpolation of average monthly climate data from weather stations on a 30 arc-second resolution grid (often referred to as a “1 km2” resolution). Variables included were monthly total precipitation and monthly mean temperature (for more details see http://www.worldclim.org). A combination of morphological and genetic analyses (molecular barcoding) revealed a total of 32 species for all three sites , which is potential food for the termites. For each termite species, a whole termite was used. As in several other studies [To determine the feeding type and characterize the food niche using δer study . δ13C an studies ,34,45,46 studies ,36. In a®, Wegberg, Germany), and sent to KOSI. Soil samples were collected in each plot following the protocol by Pansu and Gautheyrou  and OsorR sample is the isotopic ratio of the sample (13C/12C or 15N/14N), R standard is the isotopic ratio of the international standard, and X is the respective element (13C or 15N). For 13C V-PDB and 15N, atmospheric nitrogen was used as the standard. Acetanilide  was used for internal calibration. For the amount of animal tissue analyzed per sample, precision of the measurement was about 0.1‰ for 13C and 0.2‰ for 15N. We calculated the mean and the standard deviation (SD) of all samples for each site.Carbon and nitrogen stable isotope ratios were measured on an elemental analyzer  and an isotope ratio mass spectrometer . Stable isotope ratios were expressed using the delta (δ) notation in ‰ according to:The species pool of the three study sites comprises 32 species that have been morphologically and genetically identified .A commonly used index to quantify the phylogenetic structure of a local community is the Net Relatedness Index (NRI) . It measp = 0.05). Depending on whether niche-relevant traits, such as the feeding niche, are evolutionary labile or conserved, the NRI values can hind at different assembly processes : For ins13C and δ15N values calculated over all collection sites for each species as character states and the phylogenetic tree from Casalla & Korb [In order to interpret the results of the phylogenetic community analysis, it is necessary to know whether the studied food niche traits were phylogenetically conserved or labile. To determine this, we conducted two analyses, including one with feeding groups and one for isotope signatures. We used (i) the feeding groups and (ii) the mean of the δa & Korb  . However, NRI values significantly increased with rainfall at a study plot , using site as the random factor, showed significant effects for temperature only . Mixed e15N values of termites were highly variable, ranging from −1.6‰ to 17.8‰ . The termites from both Colosó (mean −27.1‰ +/− 1 SD 1.1) and Ceibal (mean −27.2‰ +/− 1 SD 1.2) had significantly lower δ13C values than those from Tayrona (mean −26.3 ‰ +/− 1 SD 1.1)  and δ13C . (δ 0.391). .15N values differed significantly between all groups  , III (p < 0.001), and IV (p < 0.001). Termites from feeding group II had significantly lower δ15N values than those of the other feeding groups, III (p < 0.001) and IV (p < 0.001). Termites from feeding group III had significantly lower from feeding group IV (p < 0.001). Thus, there was a gradual increase of δ15N over the feeding groups.There were also significant differences in isotope signatures between feeding groups. δ< 0.001) a. Specie13C, there were less strong differences between groups. Only feeding group II had significantly lower values than all other feeding groups  . Within the Termitidae, the δ15N also reflected the different feeding groups. Wood feeders of feeding group II, such as Microcerotermes, generally had lower values than humus and soil feeders, with the highest δ15N values occurring in the Anoplotermes-group  had very high intraspecific variability , which were significantly higher than for species from the Termitidae  had the highest δ13C values, while Nasutitermitinae and Microcerotermes  had the lowest values.Our analyses showed that food niches, measured as feeding group membership and δgnatures a. The δ1Our results imply that mechanisms which structure the termite assemblages differ between sites, with interspecific competition being more important at drier and warmer, lower-altitude plots . This isInferred from the phylogenetic community analyses, the assembly processes in the studied Colombian dry forests seem to differ between sites. The driest and lowest elevation site, Tayrona, had termite assemblages that were phylogenetically more overdispersed than those of the other sites . OverallNasutitermes species and four species of the Anoplotermes-group co-existed in this plot. Several studies from Neotropical rain forests have also shown that these closely related species commonly co-occur [This does not seem to be the case at the two other sites. NRI values close to zero imply that assemblages at Colosó and Ceibal do not differ much from random associations. The fact that the NRI values at the plot scale did not differ significantly from random expectation might be due to the low numbers of species per plot. Note, that the species number is the sample size for these tests and that small sample sizes are associated with low statistical power, hence making it unlikely to detect significant effects. One plot in Colosó even showed signs of phylogenetic clustering, implying that environmental variables select for a certain subset of termites at this specific plot, which was characterized by huge trees with a dense canopy and high humidity. Four co-occur ,63,64.How wide-spread are the implicated structuring mechanisms in termites? Comparable studies are rare. Most research on termite communities has concentrated on describing local or regional assemblages and testing associations between termite diversity and variables such as fire, disturbance, or elevation gradients ,66,67,6815N and δ13C values were significantly higher at this site. Interestingly, the soil signatures did not differ between sites. For δ13C, but not δ15N, the shift in the litter signatures between sites is reflected in the termites’ isotope signal (13C mainly reflect varying proportions of C3 and C4 plants in the food of termites, while differences in δ15N indicate the diverse proportions of variably humified food resources [13C values at Tayrona reflect the presence of more grasses and especially abundant Euphorbiaceae [4 plants. In addition, due to the proximity of the sea and the associated salinity- and water-stress, also C3 plants have higher δ13C values [In our study, also the isotope analyses, which admittedly included the termites’ gut, of the litter support the notion of vegetation differences in Tayrona as the δe signal . Differeesources ,34,36. Trbiaceae , which aC values ,73. 13C values at the upper range and a lower δ15N signature at the lower range, reflecting a higher proportion of C4 grasses in the habitat and a broader food niche spectrum of fungus-growing termites [In general, our isotope signatures were similar to those found for termites in other forests ,32. Howetermites ,37. 15N [15N values between group I and II or between III humus- and group IV soil-feeders [15N signatures (13C, no such gradient was revealed and only feeding type II had δ13C values that were lower and differed significantly from all other feeding types (Reflecting a humification gradient, the four commonly recognized feeding groups identified by Donovan et al.  can gene15N ,37. Neve-feeders . Our curgnatures a, supporgnatures b. Howeveng types b. This iMechanisms that structure termite assemblages in dry forests are complex. Both neutral and deterministic processes seem to be present, with decreasing rainfall probably leading to interspecific competition and a reduction of species caused by limited food availability. More studies are needed that specifically test for these mechanisms. However, our study shows how a phylogenetic community approach combined with trait analyses can contribute to gaining the first insights into mechanisms structuring whole termite assemblages.


The article “The impact of parity on life course blood pressure trajectories: the HUNT study in Norway”, written by Eirin B. Haug, Julie Horn, Amanda Rose Markovitz, Abigail Fraser, Corrie Macdonald-Wallis, Kate Tilling, Pål Richard Romundstad, Janet Wilson Rich-Edwards and Bjørn Olav Åsvold, was originally published electronically on the publisher’s internet portal (currently SpringerLink) on 24 January 2018 without open access.http://creativecommons.org/licenses/by/4.0/), which permits use, duplication, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.With the author(s)’ decision to opt for Open Choice the copyright of the article changed on 30 May 2018 to © The Author(s) 2018 and the article is forthwith distributed under the terms of the Creative Commons Attribution 4.0 International License (


Eight to ten percent of children have a developmental disability (DD). Caring for a child with DD can be rewarding. It can also be difficult, particularly for older parents or other caregivers (hereafter parents). Few studies have examined their experiences. We followed parents for 18 years using the nationally representative Panel Study of Income Dynamics (PSID). We defined DD with criteria used in research and education law, as: (1) autism, intellectual disability, learning disorder, epilepsy with seizures, attention deficit disorder/attention deficit hyperactivity disorder, cerebral palsy, or traumatic brain injury; and (2) qualification for professional services. We used three waves of the PSID’s Childhood Development Study, 1997-2007, linking children’s and parents’ PSID data  to study three outcomes: parents “not very” or “not at all” satisfied with “life as a whole”; nonspecific psychological distress; and reports that depressive feelings interfered with life or activities “a lot.” Discrete-time hazard models controlled for: child disability; children’s and parents’ age, sex, and education; survey design; and repeated measures. Among parents ages 47-61 at baseline (65-79 in 2015), those who cared for a child with DD were significantly more likely to report low satisfaction with life , distress  and, among women, depressive feelings (HR 1.3 CI 1.0-1.8). We found no such differences for parents less than age 65 in 2015. Results highlight the need to provide emotional support, respite, or other services for older adults caring for children with disabilities.