However, former pet and entertainment chimpanzees have received little attention to date. This study aimed to investigate the long-term effects of early life experience on 18 former pet and entertainment chimpanzees, based on social grooming data collected at a primate rescue centre over a 12-year period. Moreover, we also focused on the possible short-term effects that alterations to group composition might have on grooming patterns. For this purpose, we compared stable and unstable periods . We used two individual social network measures to analyse the grooming activity and the distribution of grooming among group mates for each individual. We could show that wild-caught chimpanzees were significantly more selective regarding their grooming partners and spent less time grooming when compared to their captive born companions. We also found that individuals who were predominantly housed without conspecifics during infancy spent less time grooming compared to those who were predominantly housed with conspecifics during infancy. Furthermore, we found that alterations to the group composition had short-term effects on the distribution of social grooming from a more equal distribution during periods with a stable group composition towards a more unequal and selective distribution during unstable periods. Thus, we conclude that the social grooming networks of former pet and entertainment chimpanzees are shaped not only by long-term effects such as early life experience, but also by short-term effects such as alterations to group composition. Remarkably, we found not only captive born chimpanzees but also wild-caught individuals to adjust their grooming to socially challenging situations by modifying their grooming distribution in a similar way.The long-term effects of early life adversities on social capacities have been documented in humans and wild-caught former laboratory chimpanzees  Adverse experiences in early infancy affect the behaviour as well as the physical and mental health of human –3 and noWhile there are studies focusing on the lasting effects of laboratory housing on nonhuman primates , 37, lesSocial network analysis (SNA) allows us to statistically describe, quantify and compare the social relationships of individuals within a group –42. AlthThe recovery from early life adversities is a long lasting process, but can, at least in part, be achieved through social, psychological, emotional and environmental interventions in specialized institutions like primate rescue and rehabilitation centres , 47. TheOur study population consisted of chimpanzees who have been confiscated from circuses and private owners and/or relocated from zoos to the primate rescue centre Fundació MONA. We focused on social grooming because it is one of the most relevant social interactions of chimpanzees , 50. GroWhile high levels of self-grooming frequently have been associated with undesirable behaviours and seen as an indicator of lacking welfare , 64, to Studies conducted on wild populations of chimpanzees report on grooming activities ranging from 5.7 to 11.7 percent of their daily activity budget –75. HoweIn captivity, levels of social interaction are expected to be higher due to crowding produced by small enclosures and the absence of environmental constraints which may cause a more solitary life style in wild-living chimpanzees . In captThe fifth edition of the Diagnostic and Statistical Manual of Mental Disorders defines a traumatic event for children up to six years as direct or indirect exposure to the actual or threatened death, serious injury, or sexual violence or witnessing the event to a primary caregiver . AccordiWhile early life adversities were predicted to have a long lasting effect, still detectable after being introduced and living within a social group of conspecifics for years, we assumed care management activities, i.e. alteration to the group composition, to provoke only a short-term adaptation of the individuals’ grooming interactions. To evaluate the effects of alterations of group composition on social grooming we compared the individual network measures of stable periods (i.e. periods without any changes in group composition) to those of unstable periods  over a 12-year observation period. We supposed to find a difference between stable and unstable periods, as the addition of potential new allies or competitors as well as the loss of such might require a certain modification of the social strategy, though only for a short period, until already existing relationships have been reconfirmed and new ones established.This study is based purely on observational data without any invasive interventions and was conducted in accordance with all national and institutional guidelines for the care and management of primates as established by Fundació MONA, Association for the Study of Animal Behaviour/Animal Behavior Society and the Spanish Government (RD 53/2013). Any decision to alter the group composition of the chimpanzees was based on established care management protocols and at no time was influenced by research related staff members.The study population consisted of a total of 19 former pet and entertainment chimpanzees  housed at the primate rescue centre Fundació MONA in Catalonia, Northern Spain. The centre is a member of the European Alliance of Rescue Centres and Sanctuaries (EARS) and it is rehabilitating chimpanzees since 2001. Behavioural observations were conducted on all of the 19 chimpanzees at MONA, but one male was never observed within a group setting of more than two individuals before being transferred to Stichting AAP, another rescue centre in Holland, and thus was excluded, resulting in a total of 18 chimpanzees included in the analysis of this study. Throughout the 12 years of observation, there were two social groups, but the number of chimpanzees housed at the centre varied due to animals passing away and new arrivals. Biographic information on the 18 individuals is shown in Wild-caught chimpanzees typically arrived as adults  at Fundació MONA (mean age: 23.6 ± 10.4 years), whereas the captive born subjects were mostly sub-adults (except for two adults) upon arrival (mean age: 9.2 ± 7.5 years). Considering that our wild-caught chimpanzees have on average spent more time in the pet and entertainment business than captive born ones, we included the age at arrival at the sanctuary as another variable describing the chimpanzees past. Thus, we assigned all chimpanzees to the respective age category, i.e. adult or sub-adult, at their time of arrival at Fundació MONA.The chimpanzees were living in one of the two different social groups . Over the 12-year observation period, several alterations to the group composition occurred in order to integrate new chimpanzees, transfer animals between groups for welfare reasons or due to the natural death of individuals. The alteration of a group composition is defined as the change of the minority of the individuals of a group by either adding or removing one or several individuals at a time. Within the 12 years of data collection, the group sizes could vary from a minimum of four to a maximum of nine individuals, but was most of the time between five to eight individuals per group .2), equipped with a multitude of climbing structures, which give them the opportunity to exploit natural and artificial resources. The enclosure is divided into two separate areas to accommodate both groups: the first of 3 220 m2 and the second of 2 420 m2, with a total perimeter of 191 m. A steel mesh and an electrified fence surround the enclosure. For more detailed information on the outdoor enclosure see [Observations were conducted only while the chimpanzees had access to a naturalistic outdoor enclosure  and have access to water Data on the chimpanzees’ behaviour was recorded over 146 months from April 2006 to July 2018 by using instantaneous scan sampling  every twAlthough a complete set of behaviours was recorded, for this study we only considered "social grooming given" to group members. For this purpose, we created a directed grooming matrix for each group composition within each observation time period see . More prOver the 12-year observation period we identified a total of 42 time periods where it was possible to define a clear ‘stable’ or ‘unstable’ condition produced by alteration of the group composition. A time period was labelled as ‘stable’ when group composition did not change for at least four months beforehand and did not experience any short- or long-term changes to its composition. A time period was labelled as ‘unstable’ when a permanent composition alteration  or frequent short-term changes of group composition due to active integration activities  have occurred, for a minimum duration of four months. However, not all the data available could be included due to a lack of records or unevenly distributed observation sessions during certain time periods. Therefore, we selected 18 observation time periods (seven stable and 11 unstable) for this study. We excluded the rest due to an insufficient amount of observations  or an uneven distribution of the observation sessions within a time period.For all of the 18 chimpanzees the percent of scans every single individual is grooming each of its group members in any of these 18 time periods are used for further analysis. The number of different time periods per chimpanzee varied from one to 12, varying among others due to the difference in the year of arrival.The percent of scans an individual spent on grooming a group member per time period and group composition was used to calculate two individual social network measures to evaluate the standardized grooming activity of every chimpanzee and his choosiness in the distribution of grooming among group mates. This added up to 119 data points for the standardized grooming activity and 104 data points for the distribution of grooming as distribution of grooming could only be calculated for individuals who were grooming at least one group member in the respective observation time period.Most studies on chimpanzees define the social networks by scoring dyadic grooming interactions , 76, 90,We created our networks in R environment  using IgThe grooming matrices were used to calculate the following two network measures which have been previously described by Kasper and Voelkl  and usedsi by the number of group members -1 (N-1). The vertex strength s of vertex i is given by w being the corresponding weight of the edges connected to a vertex.The vertex strength centrality (Y2(vi)) we are able to compare the distribution of grooming between groups of different group size. This is being obtained by calculating the equal disparity Y2 per group which is 1/(N-1) and computing the deviation from Y2 for each individual by subtracting the group specific Y2 from the individual Y2(vi). Thus, the deviation from the edge weight disparity ranges from 0 to (N-2)/(N-1), where 0 represents a perfectly even distribution of grooming among all possible group members and (N-2)/(N-1) represents the total concentration of all grooming given towards one single group member. Note that the deviation from edge weight disparity could only be calculated for individuals who were grooming at all.The edge weight disparity i to all vertexes other than i (i.e. with j ≠ i).The summations in the formulas of vertex strength and edge weight disparity include the edges extending from vertex The effects of various factors on the two individual weighted network measures, vertex strength centrality (VSC) and deviation from edge weight disparity (DEWD) were assessed using linear mixed models (LMMs). Time-Period-Stability (i.e. stability of group composition: stable vs. unstable), Arrival-Age-Category , Sex f were ranAICc  for DEWD and VSC models, respectively. Thus, all fixed factor combinations were taken into account and models were ranked according to the lowest/best AICc. We further calculated the ΔAICc and the AICw  for all candidate models.We used multi-model interferences to compare and rank all 33 possible candidate models (including the Null-model) according to their respective ΔAICc and considered all models with a ΔAICc lower than 10 compared to the best model as equally possible candidates. We further indicate the RVI (relative variable importance) of all fixed factors of the averaged model, which is calculated as a sum of all AICw over all subset models that include the respective fixed factor. Model fit was assessed via graphical evaluation of the residuals  using the Satterthwaite approximation for degrees of freedom.Post hoc Type III analysis of variance (ANOVA) was performed using the Satterthwaite approximation for degrees of freedom. We ran Linear Mixed Models (LMMs) using the "lme4" package  and all We calculated two different social network measures  for each individual per time period and group composition, and ran LMMs to investigate the effects of an (un-)stable group composition, arrival age, sex, origin and predominant housing condition during infancy on social grooming networks. The vertex strength centrality (VSC) represents the standardized strength of an individual’s grooming activity. The deviation from edge weight disparity (DEWD) reflects an individual’s choosiness/restriction in his distribution of grooming among its group members. As the deviation from edge weight disparity is a value limited between 0 and (N-2)/(N-1) we applied a logit transformation. However, the transformation did not lead to any changes regarding the model selection and outcomes. Therefore, we decided to discuss the results of the original DEWD models without logit transformation.AICc) is considered, but also models with a ΔAICc lower than 10 compared to the best model. These subsets including all model candidates with a ΔAICc lower than 10 are listed in the supplementary material  for the five fixed factors of our final full models ranged between 1.02–1.77 indicating that our fixed factors were not correlated.All post hoc tests were conducted on the full models as all five fixed effects were retained within the averaged subsets.N = 66 data points) to have a significantly higher grooming activity than wild-caught chimpanzees  to have a significantly higher grooming activity than males  showed that Sex was a significant predictor influencing the VSC , and Sex . With reC = 0.99  and ANOVC = 0.86 . We founC = 0.92 . An ANOV the VSC .RVIVSC = 0.26) and failed to show any significant effect on the grooming activity  and Stability of group composition (TPstability) were all retained variables in the best model selection but had a very low relative variable importance  in our VSC model, both with high RVI scores, we ran a separate VSC model based on the full model but added the interaction of these two fixed factors. Due to slightly elevated VIFs (2.1–3.1) detected between the interaction and the separate components (Origin and PHCinfant) we ran this LMM apart from the averaged LMM. We found the interaction to have a significant impact on the grooming strength (N = 65 data points) to have a significantly lower deviation from edge weight disparity than wild-caught chimpanzees , compared to unstable periods  . With reD = 0.83 . A ANOVAD = 0.97 . The chiRVIDEWD = 0.30–0.55 and did not demonstrate any significant influence on the grooming distribution among group mates (The factors Predominant housing condition during infancy (PHCinfant), Arrival age category  and Sex were retained variables in the best model selection, but scored low to medium in their relevant variable importance with up mates .Considering that both, Origin and Stability of the group composition, significantly affected the distribution of grooming in our DEWD model, both with high RVI scores, we decided to run an additional DEWD model, based on the full model containing all five previously described fixed factors and added the interaction between TPstability and Origin. Due to slightly elevated VIFs (1.2–3.4) detected between the interaction and the separate components (TPstability and Origin) we ran this LMM apart from the averaged LMM. We found no significant result for the interaction . NeverthGraphical representations of the weighted social grooming networks of all 18 Observation Time Periods/Group compositions for both chimpanzee groups are presented in chronological order in the supporting information . These nFor this study, we chose two individual social network measures in order to describe the social grooming networks and the individuals’ social capacities: the standardized strength of grooming given and the distribution of grooming given among group mates. The findings of our study show that early life adversity is reflected in the social grooming networks of former pet and entertainment chimpanzees. Two out of the three factors referring to past experiences/conditions, i.e. Origin and Predominant housing conditions during infancy, significantly affected one or both social network measures. With respect to Origin, we found that the wild-caught chimpanzees of our study groups spent less time on "grooming given" and were much more restricted in the selection of grooming partners compared to captive born ones. Regarding Predominant housing conditions during infancy, we could show that chimpanzees who were housed predominantly with conspecifics spent more time grooming their group members compared to individuals that were housed predominantly without conspecifics during infancy. Relating to the third factor, Age at arrival at the rescue centre, we did not find any differences between chimpanzees who arrived as sub-adults and those arriving as adults.Beyond that, alterations to the composition of the group had an impact on social grooming, as the distribution of grooming among group mates was significantly more restricted during the unstable periods where alterations occurred, compared to stable periods without any alterations to the group composition.It is known that chimpanzees are highly social animals who create and live in complex social networks . In ordeBy comparison, none of the chimpanzees of our study population did grow up under species-appropriate conditions. While most of the former pet chimpanzees grew up with humans only, the majority of the former entertainment chimpanzees were at least partially socially reared but were dressed and trained to perform for instance in a circus. Bloomsmith et al.  showed tIn order to gain a central position within a social group it is necessary to invest energy and time in social bonding activities, in chimpanzees this is often achieved through grooming interactions. Thus, lacking social experience and skills might reduce the capacity and/or the desire to engage in social grooming interactions and result in the inability to manage multiple relations at any one time. As such, this reduced social activity was found to have a negative impact on the individual’s social position and possibly welfare in early maternally and socially deprived former laboratory chimpanzees who were all caught from the wild .While we know that all of the chimpanzees of our study sample had harmful experiences in the past and most were separated from their mothers at an early age, we nevertheless believe that the chimpanzees who were caught in the wild had an additional traumatic experience. This traumatic experience consisted of witnessing the killing of their mother and other group members, capture and transportation under extreme and often harmful conditions, as well as a dramatic change in their living conditions from wild to captivity. Although all this occurred in a relatively short amount of time, the extreme conditions, potential injuries and stress experienced by these infants during the most vulnerable period of early infancy often led to death during capture and transportation , 104. AsThe majority of our wild-caught chimpanzees arrived as adults (mean age: 23.6 ± 10.4 years) and most of the captive born individuals were sub-adults (mean age: 9.2 ± 7.5 years) upon arrival at the sanctuary. We were aware of the fact that this difference in age might be a confounding variable affecting origin. Therefore, we also tested for a possible effect of age at arrival at the rehabilitation centre. In view of this, it was surprising, that we did not find any effect of age at arrival at the rehabilitation centre, neither in the chimpanzees’ grooming activity nor in their distribution of grooming among group members. This finding suggests that the significant difference in grooming between wild-caught chimpanzees, who by majority arrived as adults, and captive born chimpanzees, who by majority arrived as sub-adults, seems not to be due to the age at arrival at the rescue centre but due to the origin of the chimpanzees. However, we do believe that an older age at arrival at the rescue centre might result in more difficulties during the initial integration process compared to a younger one. Furthermore, the arrival age category, as the name indicates, only refers to the time the chimpanzees arrived at the sanctuary and started their rehabilitation and social integration into one of the two existing groups. However, several of the individuals were not transferred directly to the sanctuary after their time as pets or entertainment animals, but instead spent some time (in some cases years) without being forced to train or perform or were temporarily relocated to zoological gardens, before arriving at the sanctuary. These in between housing situations were at times marked by improved living conditions, as in some cases the chimpanzees were housed with conspecifics and/or received species-appropriate care.Since Kalcher-Sommersguter et al.  could shOne possible explanation for this finding might be related to the amount of human exposure. Although we do not have detailed information on our study subjects with respect to the amount of human exposure, it seems very likely that chimpanzees predominantly housed without conspecifics during infancy had more interactions with humans compared to those predominantly housed with conspecifics. The social interaction with one or several humans might have helped to develop certain social skills. However, as humans usually do not use grooming to interact with chimpanzees, the slowly developing grooming behaviour  might noThe interaction between Origin and the Predominant housing condition during infancy on the grooming strength revealed that while captive born chimpanzees generally exhibited a far higher grooming activity than wild-caught chimpanzees the former also seemed to be affected more strongly by the Predominant housing condition. This became visible through a steeper decline in the grooming activity between captive born chimpanzees predominantly housed with conspecifics and those housed predominantly without conspecifics compared to the much smaller decline between wild-caught chimpanzees housed predominantly with conspecifics and those housed predominantly without conspecifics.In a study conducted by Kalcher-Sommersguter et al. , the samAccording to our findings, we suggest considering information on the animals´ life history, such as the origin and the predominant housing condition during infancy, when introducing new individuals into already existing groups and during the formation of new groups.Even though the focus of our study was on social conditions, we found sex to have an impact on the social grooming networks as well. Lehmann and Boesch  suggest Living in a social group cannot be compared directly between wild ranging populations and groups living in captivity  due to sOur findings show that unstable periods were characterized by a more selective choice of grooming partners. It is important to note, that we refrain from labelling this mentioned adaption, i.e. the change in the distribution pattern, as either positive or negative, but argue that this indicates a certain capacity to react to a social alteration. More importantly, this adaptation seems to be shown by all of our chimpanzees, regardless of their level of choosiness/restriction. Interestingly, the individuals did not differ in the time they spent on "grooming given" between stable and unstable periods, but seemed to be choosier during unstable periods compared to stable periods.One possible explanation for this might be that the amount of grooming needed to maintain a position during stable periods is similar to the amount of the, then more unevenly distributed, grooming needed to form new relationships beside the maintenance of already formed bonds during unstable periods. Another possible explanation might be that the individual’s grooming activity in stable periods already represents the maximum amount an individual is willing to engage. We can exclude the possibility that adding new individuals produced the more uneven distribution of grooming during unstable periods, as unstable time periods include not only the addition of group members, but also reductions in group size and changes to the routine (temporarily splitting and shuffling of a group).One important finding of our study is that the changes in the distribution of grooming among group mates during unstable periods did not last permanently. This became apparent by the fact that unstable periods alternated with stable periods during the 12-years of observations see  and the Due to the interaction plot between Origin and Time period stability we could further see, that regardless of the differences in origin, the chimpanzees modified their grooming distribution similarly. This means that, at least under attentive caring conditions, provided for example by a sanctuary such as Fundació MONA, even individuals with adverse early life experiences detect and react to socially challenging situations such as group alterations, by adjusting and after a few month slowly readjusting their grooming distribution pattern.We do know that the socio-emotional development is a complex process and retrospective studies focusing on early life history include several risks, which can easily lead to misinterpretations. The relatively small sample size of 18 individuals and the vague information on the early life history and pre-sanctuary experience might have caused that we overlooked other potentially important factors. However, the observation period of 12 years and detailed information on the group management allowed us to analyse the effects of alterations to the group composition in detail. By no means do we suggest that the factors, considered here, are the only factors influencing the social grooming of these study population, but rather wish to emphasise that diverse factors and possibly their combinations could have a potentiating or moderating effect.The wild-caught chimpanzees of our study population are on average older than the captive born chimpanzees and as such, most of the wild-caught individuals arrived at an older age at the sanctuary compared to the captive born ones. We considered this in our analysis and found origin but not age at arrival to influence the chimpanzees’ grooming activity. A study conducted on former laboratory chimpanzees has shown that the age at onset of deprivation but not the age at observation time and the years spent in deprivation accounted for differences found in social behaviour , supportIn conclusion, we could show that early traumatic life events and adverse living conditions during infancy, affect the social grooming of former pet and entertainment chimpanzees in the long term. Wild-caught individuals spent less time on grooming given and were more restricted in whom they groomed compared to captive born ones, and chimpanzees who have been predominantly housed without conspecifics during infancy engaged less in grooming others than those predominantly housed with conspecifics during infancy. Astonishingly, all of these former pet and entertainment chimpanzees reacted in a similar way to alterations of group composition as grooming among group members reverted to a more even distribution in stable periods, after a more restricted distribution during unstable periods, throughout the whole 12 years of observation.We believe that these results might be a valuable addition to the already existing knowledge, especially with respect to care management decisions regarding integration and group formations. For future studies, we suggest using multi-level networks including diverse social behaviours and social proximity as this might give an even better understanding, ideally with a larger sample size and more detailed information on the chimpanzees’ past history.S1 TableAICc. All models considered here have a ΔAICc lower than 10 compared to the best model (first model listed). Fixed effects included in each model candidate are marked with an X.VSC models with stability of time period (TPstability), arrival age category , sex, predominant housing condition during infancy (PHCinfant) and origin as fixed effects and group composition in a certain time period and ID as random factors in all models. Models are ranked according to the best (DOCX)Click here for additional data file.S2 TableAICc. All models considered here have a ΔAICc lower than 10 compared to the best model (first model listed). Fixed effects included in each model candidate are marked with an X.DEWD models with stability of time period (TPstability), arrival age category , sex, predominant housing condition during infancy (PHCinfant) and origin as fixed effects and group composition in a certain time period and ID as random factors in all models. Models are ranked according to the best (DOCX)Click here for additional data file.S3 TableSignif. codes: ‘***’ ≤0.001 ‘**’ ≤0.01 ‘*’ ≤0.05 ‘.’ ≤0.1 ‘ ’ ≤1.(DOCX)Click here for additional data file.S4 TableResults of the Interaction contrast analysis: Results are averaged over the levels of TPstability, ArrivalAgeCat, and Sex with a p-value adjustment based on Tukey method for comparing a family of 4 estimates. Signif. codes: ‘***’ ≤0.001 ‘**’ ≤0.01 ‘*’ ≤0.05 ‘.’ ≤0.1 ‘ ’ ≤1.(DOCX)Click here for additional data file.S5 TableSignif. codes: ‘***’ ≤0.001 ‘**’ ≤0.01 ‘*’ ≤0.05 ‘.’ ≤0.1 ‘ ’ ≤1.(DOCX)Click here for additional data file.S6 TableSignif. codes: ‘***’ ≤0.001 ‘**’ ≤0.01 ‘*’ ≤0.05 ‘.’ ≤0.1 ‘ ’ ≤1.(DOCX)Click here for additional data file.S1 FigNodes represent group members, with green nodes being wild-caught and blue nodes being captive born chimpanzees. Node shape represents the sex, with squares being females and circles being males. Graphs marked with a * are describing stable time periods. The procedures used to create the matrices are described in the method section. Graphs were drawn using the Igraph package in R .(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.


To measure the audiological benefit of the Baha SoundArc, a recently introduced nonimplantable wearing option for bone conduction sound processor, and to compare it with the known softband wearing option in subjects with normal cochlear function and a purely conductive bilateral hearing loss. Both ears of 15 normal hearing subjects were occluded for the time of the measurement, yielding an average unaided threshold of 49 dB HL (0.5 – 4 kHz). Soundfield thresholds, speech understanding in quiet and in noise, and sound localization were measured in unaided conditions and with 1 or 2 Baha 5 sound processors mounted on either a softband or a SoundArc device.nd device and sound localization error by 23° to 28°. No statistically significant difference was found between the SoundArc and the softband wearing options in any of the tests. Soundfield thresholds and speech reception thresholds were improved by 19.5 to 24.8 dB (p<.001), when compared to the unaided condition. Speech reception thresholds in noise were improved by 3.7 to 4.7 dB (p<.001). Using 2 sound processors rather than one improved speech understanding in noise for speech from the direction of the 2 Bone conduction sound processor mounted on a SoundArc or on a softband resulted in considerable improvements in hearing and speech understanding in subjects with a simulated, purely conductive, and bilateral hearing loss. No significant difference between the 2 wearing options was found. Using 2 sound processors improves sound localization and speech understanding in noise in certain spatial settings. Bone anchored hearing systems (BAHS) are an established method for the treatment of conductive and mixed hearing loss –3 and, mThe audiological benefits of BAHS are well documented and the devices are widely used , 5, 6, 9Especially in young children, but also in a growing population of older children, adolescents, and even adults, there is a demand for solutions in which users can benefit from the advantages of a BAHS without having to undergo surgery.Until recently, there have been mainly two different nonsurgical solutions to use BAHS sound processors: headbands and softbands . HeadbanThere are two main disadvantages which are associated with the use of these nonimplantable wearing options. One is the sound attenuation by the skin, which increases with high frequencies and reaches, e.g., approximately 15 dB at 3000 Hz , 16. As Recently, a new wearing method for BAHS sound processors without the need for surgery has become available. The Baha SoundArc  is shown in From a clinical point of view, an important question in this context is whether the audiologic performance with the BAHS sound processor is not compromised by choosing the new wearing option. To our knowledge, this has not been investigated to date. With this study, we would like to start to fill this gap.The primary aim of this investigation is therefore to test the audiological benefit of a current BAHS sound processor worn on a SoundArc in terms of soundfield hearing thresholds, speech understanding in quiet, and speech understanding in noise in persons with a purely conductive hearing loss, as this is the population which is expected to benefit most likely from the new option. The second aim of this study is to compare the audiological benefit with the softband, as this is the most frequently used solution, which is already available today. The third aim is to investigate and to quantify the binaural benefit when using two sound processors on a single SoundArc instead of just one.This prospective study was approved by the local ethical committee of Bern (KEK-BE 2017-00642). All tests were performed at the University of Bern in accordance with the declaration of Helsinki.15 young and normal hearing volunteers  participated in this study. Their air conduction (AC) and bone conduction (BC) thresholds were better than 20 dB HL at all audiometer frequencies between 250 Hz and 6000 Hz. A bilateral conductive hearing loss was simulated for the duration of the tests by blocking both ears with a combination of ear plugs  and an additional filling of the remaining volume within the pinna with silicon mould material . bilateral simulated hearing loss, the binaural benefit can be easily measured. Further implications of this choice of subjects are discussed in The rationale for the choice of normal hearing subjects and a simulated conductive hearing loss rather than real patients is as follows. All currently available nonsurgical wearing options for BAHS sound processors are known to introduce a considerable high frequency skin attenuation at higher frequencies , 16. As A combination of earplugs and silicon mould was used to simulate conductive hearing loss, as either of these blocking methods by itself did not yield a sufficiently high sound attenuation in preliminary tests and circumaural hearing protectors cannot be used together with either the softband or a SoundArc wearing option, because of their size and spatial interference.After otoscopy and pure tone audiometry, both ears of each subject were blocked as described above. Then all participants underwent a total of 5 series of measurements, one in each of the following 5 conditions: (i) unaided, (ii) aided with one BAHS sound processor mounted on a SoundArc, (iii) aided with 2 BAHS sound processors on a single SoundArc, (iv) aided with one BAHS sound processor on a softband, and (v) aided with 2 BAHS sound processors on a single softband. The order of these 5 conditions was varied systematically between subjects to minimize the effects of learning or fatigue.The following measurements were performed in each of these conditions: the sound field thresholds were measured using narrow-band noise at 0.25, 0.5, 1, 2, 3, 4, and 6 kHz. The speech reception threshold (SRT) in quiet, i.e., the presentation level required for 50% word understanding, was measured using German two-digit numbers from the Swiss Version of the Freiburger Test . Speech The SRT in noise, i.e., signal-to-noise ratio required for 50% speech understanding in noise, was measured using the adaptive OLSA test . This teSound localization was measured using 12 loudspeakers spaced 30° apart in a circle with 1 m diameter and centred around the head of the subject. 36 bursts of white noise with a duration of 200 ms were presented in a randomized order and 3 bursts from every loudspeaker at level of 60, 65, and 70 dB SPL, respectively. Subjects indicated from which of the 12 loudspeakers they thought the sound had been presented. Sound localization was measured in all aided conditions.3) with an almost frequency independent reverberation time of approximately 0.14s. JBL Professional Control® 1 PRO loudspeaker  was used in all tests.All of the above measurements were carried out in a sound-attenuated room  were used in all aided conditions. They were programmed for each subject and for each of the test conditions separately, following the manufacturer's recommended fitting procedure for conductive hearing loss, using the most recent fitting software  and BC-direct measurements. The settings “position compensation”, “automatic sound classifier”, and “adaptive microphone directionality” were selected.Baha SoundArcs and softbands were fitted individually for each subject according to the instructions provided by the manufacturer with the products. The force with which the disc with the attached sound processor was pressed to the head was measured 3 times in each subject and for each mounting method using a spring balance  and the results were then averaged.To assess the treatment effects, linear mixed-effects models were implemented for each outcome measure. The treatment condition was included as fixed effect . For SRTs in noise, the test situations  were additionally considered as fixed effects. To account for multiple measures, the subject IDs were included as random effects. Post hoc comparisons between the tested wearing options  and number of devices used  were performed with general linear hypothesis testing using two-tailed tests and Holm correction  for multAided soundfield thresholds are significantly better (p<.001) than unaided by 23.6 to 25.1 dB, when averaged over the same 4 frequencies between 0.5 and 4 kHz, as above. Statistically, results do not differ between any of the 4 wearing options  significantly, as shown in detail in If speech is presented from the front  with the SoundArc wearing option.If speech is presented at the side of the Baha  by 3.7 dB with the softband and by 4.0 dB with the SoundArc, respectively.If a single Baha is used unilaterally and speech is presented from the side of the device  and 28° (SoundArc), respectively (p< .001). The relatively small differences between both wearing options are not statistically significant in either the unilateral or the bilateral situation.The average force needed to lift the holding disc from the skin of the subjects was 1.69 ±0.21 N for the softband and somewhat higher (1.80 ±0.16 N) for the SoundArc. The difference is not statistically significant (p=.178).nd Baha mounted on either a SoundArc or a softband.We believe that these results can be adequately summarized into 3 main findings: (1) there is a clear improvement with a Baha mounted on either a SoundArc or a souftband, when compared to the unaided condition, (2) audiologically, no statistically significant difference between the use of a softband and a SoundArc was found, and (3) there is an added audiologic benefit from an additional, 2The benefit of the aided versus the unaided situation can be seen in the soundfield threshold measurements , but it No statistically significant difference was found between sound processors worn on either a SoundArc or a softband in any of the measurements. This includes sound field thresholds, speech understanding in quiet and in noise, and sound localization. These results seem reasonable, as the same sound processors were used in all tests and the placement as well as the force measured is similar for both mounting methods.nd Baha decreases speech understanding.As bone conduction has a much lower sound attenuation across the head than air conduction , 22, it As a side effect, the results of the tests in noise shown in There are two potential issues with the relevance of the presented work: first, the practical importance of the SoundArc and similar devices as an alternative to the already existing softband and second, the applicability of our results to real patients.Regarding the first of these potential issues, we believe that a new wearing option can be of substantial importance for current and future patients, if it is perceived as attractive by the users. It is known that a significant number of patients chooses not to use or to discontinue the use of bone conduction devices because either a surgical intervention is needed or the nonsurgical wearing option is not aesthetically appealing , 24, 25.All of our tests were performed with normal hearing subjects with bilaterally blocked ears rather than with patients with bilateral conductive hearing loss and with medium-output-power sound processors rather than with power or super-power sound processors.A number of advantages, but also some limits are associated with this choice. Using young, adult, normal hearing subjects allowed us to study the impact of the devices in subjects with bilaterally normal inner ears and a pure conductive hearing loss. We believe that, in this way, the hearing of children with a purely conductive hearing loss and normal inner ears is adequately simulated. Unlike children, these young adults can easily perform all the rather taxing and time consuming tests required for this study. We expect children and adolescents with a purely conductive hearing loss to become one of the important group of potential users of the SoundArc, as this is an important group for BAHS already today at our centre and at other centres worldwide , 28. FurAs subjects with an additional cochlear hearing loss were not included, we cannot directly draw conclusions from our results on the efficacy of the SoudArc or the softband wearing options for BAHS sound processors in mixed hearing loss or single sided deafness. Nevertheless, we feel that the most important target group for these wearing options is reasonably represented. Future studies may shed more light on the benefit in these groups of users.As a by-product of this study, we have presented a method which can reliably, safely, and reversibly simulate a considerable conductive hearing loss in the order of magnitude of 45 dB without having to resort to circumaural hearing protectors, which preclude the use of several types of bone conduction hearing devices.nd sound processor improved sound localization and speech understanding in noise, if the target speech was presented from the side of the 2nd sound processor.The results of our study suggest that in subjects with a bilateral conductive hearing loss and a medium power BAHS sound processor mounted on a SoundArc device can benefit from significant improvements in terms of sound field hearing thresholds, speech understanding in quiet, and speech understanding in noise, when compared to the unaided condition. We have not found any significant difference in the audiological performance when compared to the mounting on a softband. The use of an additional 2


Leishmania (L.) major infection is characterized by different clinical presentations which depend in part on the host factors. In attempt to investigate the impact of the host's immune response in the outcome of the disease, we conducted a prospective study of 453 individuals living in endemic foci of L. major transmission in Central Tunisia. Several factors were assessed at the baseline including (i) the presence of typical scars of ZCL, (ii) in vivo hypersensitivity reaction to leishmanin, and (iii) the in vitro release of granzyme B (Grz B) by peripheral blood mononuclear cells (PBMC) in response to stimulation with live L. major promastigotes. After one season of parasite's transmission, repeated clinical examinations allowed us to diagnose the new emerging ZCL cases. Heterogeneity was observed in terms of number of lesions developed by each individual as well as their size and spontaneous outcome, which led us to establish the parameter “severity of the disease.” The efficacy of the presence of typical ZCL scar, the leishmanin skin test (LST) positive reactivity and the high levels of Grz B (≥2 ng/ml), in the protection against the development of ZCL were 29, 15, and 22%, respectively. However, these factors were more efficient against development of intermediate or severe forms of ZCL. Levels of Grz B >2 ng/ml showed the best efficacy of protection  against development of these forms of ZCL. The association of such parameter with the positivity of the LST exhibited a better efficacy . In conclusion, our results support the involvement of Leishmania-specific cytotoxic cellular immune response in host protection against Leishmania-infection. This factor could be of great interest in monitoring the success of vaccination against human leishmaniasis.Zoonotic cutaneous leishmaniasis (ZCL) caused by  Leishmania parasite and grouping distinct clinical manifestations. In Tunisia, ZCL is due to infection by the parasite L. major zymodeme MON-25 and transmitted by Phlebotomus papatasi , PBMC proliferation, or interferon (IFN)-γ production in response to stimulation with Leishmania antigens. These indicators of Th1 response were usually used for the selection of naïve individuals and as correlate of protection during the clinical trials  living in the endemic region of Kairouan. All of these patients showed a positive LST reactivity.Leishmania antigens (SLA).24 subjects healed from ZCL living in the endemic regions of Sidi Bouzid or Rmada, characterized by the presence of typical ZCL scars and a positive LST reactivity and/or a positive lymphoproliferative response to soluble 11 healthy subjects living in the endemic focus of Rmada selected basing on the absence of ZCL scars and the negative LST reactivity.L. major transmission. These individuals were enrolled in the study basing on the absence of ZCL scars and the negativity of Leishmania-specific lymphoproliferative response . Table 9 healthy subjects  living outside endemic area of A total of 59 subjects have been enrolled in the first part of this study consisting on the optimization of Grz B test. Subjects were categorized on four groups basing on clinical, epidemiological and immunological criteria:L. major (Zymodeme MON-25) infection in Tunisia: an old endemic focus (OEF) situated in the district of El Gtar, governorate of Gafsa (south west Tunisia); and a new endemic focus (NEF) situated in the district of Souk Ejjdid, governorate of Sidi Bouzid . This study was carried out as part of the research project “Advances in epidemic parameters of ZCL to validate tools for surveillance and control.” At enrolment of the study population between April and May 2001, before the parasite transmission season, the donors had a clinical examination looking for active ZCL lesions or typical scars, a blood sampling and an intradermal administration of leishmanin for LST reactivity. Repeated clinical examinations were performed during the season of emergence of lesions (between September 2001 and April 2002) to evaluate the clinical course of L. major infection, occurring after the transmission season  and from three primary schools in the district of Souk Ejjdid (n = 187). Children <6 years old and therefore, of pre-school age, from the region of souk Ejjdid were also recruited . The protocol was approved by the Bio-Medical Ethics Committee (BMEC) of Pasteur Institute of Tunis. The leishmanin test applied to the study population is made of leishmanin approved by WHO norms and regulation. It was previously used for thousands of individuals worldwide without significant hazards.LST was performed by intradermal injection of 100 μL of leishmanin suspension as previously described by Sassi et al. . An induL. major  obtained from a human ZCL lesion  as previously described  supplemented with 2 mM L-glutamine (Sigma), 100 U/mL penicillin (Sigma), 100 μg/mL streptomycin (Sigma), and 10% heat-inactivated human AB serum (Sigma). PBMCs were stimulated with SLA (10 μg/mL) for 5 days and lymphoproliferative response was evaluated after adding 1μCi/well of [3H]-thymidine  for the last 6 h using a liquid scintillation counter . Results were expressed as Δcpm, difference between mean counts of triplicates in SLA-stimulated PBMC and mean counts of triplicates in unstimulated PBMC.PBMCs were separated from heparinized blood samples using Ficoll-Paque  density gradient centrifugation. PBMCs were cultured in 96-well plates at a concentration of 1 × 103 pH 9.6, at 2 μg/ml. Plates were subsequently washed five times with PBS containing 0.02% Tween 20. The free sites were saturated by incubation for 45 min at room temperature (RT) with 150 μl/well of PBS containing 2% tween 20. The recombinant Grz B and the samples, diluted in HPE  and 100 μl were added and incubated for 1 h at RT. After washing, 100 μL of GB10 MoAb diluted at a concentration of 0.5 μg/mL in HPE buffer containing 1% (v/v) of normal murine serum (NMS). After incubation for 1 h, the wells were washed and then incubated for 20 min with 100 μl of streptavidin coupled to peroxidase  diluted at 1/10,000. Five washes were carried out before adding 100 μl/well of TMB solution  at a concentration of 100 μg/mL containing 0.003% (v/v) H2O2, diluted in 0.11M sodium acetate buffer pH 5.5. After stopping the reaction with 2N H 2SO4, optical density was measured using an automated plate reader (Titerteck Multiscan) at 450 nm/540 nm. Grz B levels were determined by reference to the standard curve and results were expressed in pg/mL. Detection threshold was fixed at 1.42 pg/mL.Measurement of Grz B levels was performed using ELISA described by Spaeny-Dekking et al. . The GrzMeasurement of IFN-γ level within supernatant was carried out using OptEIA set ELISA  according to the manufacturer's recommendations. The results were interpolated from the standard curve established using recombinant IFN-γ and expressed in pg/mL. Detection threshold was fixed at 6.02 pg/mL.r > 0.3 with a significance level p < 0.05.Statistical analysis was performed using SPSS  software. The association between two parameters was assessed by the Spearman's Rank Correlation Coefficient test. The correlation is considered statistically significant if t-test (for the cohort study) or by the Mann-Witney U-test .The optimal and recommended cut-off points (where applicable) were used to calculate the numbers of subject positive and negative for each test. The comparison between two groups for a studied parameter was carried out by Student's P-value was also obtained for each association. A positive association is indicated by an odds ratio >1 (significant when p < 0.05 and the 95% confidence limits both exceed 1). Odds ratios significantly <1 indicate a negative association. Estimation of the efficacy of the immune status of the host on the protection against the development of the ZCL was performed through the calculation of the relative risk (RR) and then the preventive fraction . Odds ratios (OR) were then computed to determine the nature of the association between different parameters by stratifying subjects as described by van Belle et al. . A FisheLeishmania-specific cytotoxic immune response. This was done by measurement of Grz B level in supernatants of PBMCs stimulated for 5 days with promastigotes of L. major at a ratio of 3 parasites per cell. The analysis was carried out for 15 subjects with active ZCL, 24 healed ZCL subjects selected basing on the presence of typical scar and positive reactivity to LST (Scar+LST+), and a total of 20 apparently healthy donors. The latter group includes 11 subjects living in endemic areas with no history of ZCL and negative LST (Scar−LST−), and 9 subjects living outside these area (Table p < 0.001). In addition, levels of Grz B measured in culture supernatants of cells from apparently healthy and naïve (Scar−LST−) individuals but living in endemic region of L. major transmission were significantly higher (mean ± SD: 7088 ± 8837.11 pg/mL) compared to those detected in the supernatants of PBMCs from healthy and naïve individuals living outside endemic areas . This result is very important and may suggest that in endemic area, even in the absence of LST reactivity other marker of specific immune response (like Grz B production) could indicate a previous contact with the Leishmania parasite.Before starting the prospective study, we optimized the conditions for the analysis of −LST−) living in endemic area were Grz B+. However, none of the healthy negative controls living outside endemic areas had Grz B levels greater than the cut-off  within donors included in the prospective study (n = 453). Results of the clinical examination as well as those of the LST collected at the baseline showed that 167 subjects had a positive LST, among them 75 individuals exhibited ZCL scars . Likewise, the difference between Grz B levels measured within PBMCs from male subjects and those from female ones was not statistically significant (p = 0.449). Furthermore, no significant difference was found between Grz B levels detected within individuals from El Gtar  and those within subjects from Souk Ejjdid . However, in the new endemic focus, Grz B levels were significantly higher within individuals over 6 years old comparing to those within children <6 years old  of culture supernatants at variable levels ranging from 1.43 to 211 560.4 pg/mL . Production of Grz B by PBMCs stimulated with r = 0.523, p < 0.0001; Figure p < 0.0001; Figure +) and those without scar  and those with a positive LST with scars (LST+ Scar+) or without scars  and the presence of scars .In addition, we found that 138 individuals among the 453 individuals tested showed Grz B levels >2,000 pg /ml. These high levels were specifically found within individuals with positive LST and those with ZCL scars Figures . As desc2 (mean ± SD = 492.7 ± 784.8 mm2) and a mean duration of progression ranging from 71 to 439 days (mean ± SD = 143.7 ± 62 days).To investigate the protective effect of the host's immune status on the development of ZCL, we performed a clinical follow-up of the 453 individuals, between the month of September 2001 and April 2002, to diagnose new lesions of ZCL. We noticed that, after one season of transmission, 90 among the 453 subjects (19.9%) developed new ZCL lesions. Number of lesions ranged from 1 to 9 Figure  and wereTo refine the analysis, we defined a new variable for the expression of the severity of the disease calculated basing on the total number of lesions, the total surface of lesions (taken at their maximum during the various visits) as well as the average duration of evolution of the lesions. The severity score is determined by the formula:n = 84), 45 developed a mild form of the disease (GI) while 39 individuals presented intermediate or severe forms of the disease  with a lesion's severity score comprised within the interval [0–20] and a second group (GII) with a severity score >20. Among the 90 individuals who developed a new ZCL lesion and for whom information on the spontaneous evolution and size of lesions were available . These factors showed a weak efficacy of protection against the development of ZCL lesion. Indeed, protective efficacy was 29, 15, and 22% for the presence of scar, positive LST reactivity and high Grz B levels with a non-significant p . However, all these parameters were highly protective against the development of intermediate or severe leishmaniasis  and presence of scar showed a protective efficacy of 62%  to protect against such forms of leishmaniasis. Interestingly, together, the positivity of the LST and level of Grz B >2,000 pg/ml, leads to a better efficacy of protection against development of intermediate or severe leishmaniasis (efficacy is 83.6%). Indeed, only 2% (2/100) of LST+ individuals, with Grz B levels >2,000 pg/ml, developed intermediate or severe ZCL  according to the lesion's severity score, we showed that none of these parameters was protective against the development of mild ZCL  showed high levels of Grz B. Interestingly, even in the absence of LST reactivity, Grz B induction may constitute an indicator of a previous contact with the parasite and may reflect an eventual role in control of Leishmania infection. This data is contradictory with those described by Cordosa and collaborators showing the involvement of CD8+T cells producing IFN-g rather than those with cytotoxic activity in the control of Leishmania infection , the main source of GrB produced in response to stimulation with Leishmania-antigens is CD4+ T cells, including T regulatory cells. These CD4+ T cells were different from the Th1 cells producing IFN-γ . In American cutaneous leishmaniasis (ACL), the percentage of CD8+ T cells were higher in lesions compared to blood  have been shown in ACL lesions. An enrichment of TEM for both CD4+ and CD8+ T cells sets were observed comparing to blood  the associations between the different parameters indicators of an earlier contact with the Leishmania parasite . Our results showed some discrepancies between production of Grz B and scars or LST reactivity. The discrepancy between production of Grz B and the presence or absence of scars might be explained by the fact that lesions of ZCL could disappear without leaving scars and that scars of other pathologies could be taken for ZCL scars. However, the discrepancy between Grz B and LST reactivity seems to be more relevant. Thus, a large fraction of individuals (n = 134) living in the endemic area of leishmaniasis showed a diameter of induration equals to zero but variable levels of Grz B which are sometimes very high. This is consistent of the striking results found during the validation of the Grz B test. Indeed, we were struck by the differences in Grz B levels between the so-called “naïve” (Scar− LST−) living in an endemic area of leishmaniasis vs. those living outside the endemic areas. These results are extremely important for the selection of naive populations in endemic areas, especially when including adults in vaccination trials. In fact, the selection of individuals Scar−LST− does not exclude an eventual previous contact with the parasite which could be tracked otherwise by the Grz B analysis.In a next step, the measurement of Grz B levels in culture supernatants of PBMCs stimulated with L. major infection, as well as the positive LST reactivity, usually used as a protective correlate. Immune response was evaluated at the baseline (before the parasite transmission season) and the development of new ZCL cases was monitored actively during the season of emergence of the disease. A gradation of the disease was also made by calculating a new parameter, severity of the disease, basing on the size of lesions as well as their total duration of evolution.Secondly, we explored the protective role of the cytotoxic effector against the development of the disease. Since ZCL is heterogeneous in terms of disease severity , we hypothesized that host immune response will not protect against the development of leishmaniasis but rather against the severe forms of it. Our objective was achieved through the monitoring of the cohort included in our prospective study. We analyzed the predictive value of a cytotoxic response (investigated by the Grz B assay) in resistance to n = 45) of individuals that develop a simple ZCL characterized by a unique or low number of lesion(s) and a rapid spontaneous healing (severity score <20). Surprisingly, we found that neither the positivity of the LST, nor the presence of ZCL scar, nor the cytotoxic activity , were protective against the development of the disease. This can be explained by the fact that in our study, patients have been actively monitored allowing the diagnosis of mild forms of ZCL, often ignored by the patient himself. However, the positive LST reactivity, the presence of ZCL scar and also the cytotoxic activity were protective against the development of intermediate or severe forms of ZCL. Accordingly, we showed that a Grz B level >2,000 pg/ml has a protective efficacy of 72.8% against the development of intermediate or severe forms of ZCL. Such efficacy was greater than those obtained with the presence of ZCL scar and the positivity of LST . Interestingly, together positivity of the LST and a cytotoxic response  confer an excellent protection (>80% efficiency) against intermediate or severe forms of ZCL.During the season of emergence of the disease, we diagnosed a total of 90 new cases of ZCL among the 453 individuals tested, with mild, intermediate or high severity score. Interestingly, we noticed a significant number (L.major although the negativity of LST and the absence of scars. Such parameter could be used in vaccination trials for the selection of naive populations living in endemic areas. Moreover, our data show that the preexistence of a parasite-specific cytotoxic immune response may confer protection against the development of intermediate or severe forms of ZCL. These results are of crucial importance for anti-Leishmania vaccine design and more generally for the evaluation of their protective efficacy.Altogether, our study clearly demonstrated that Grz B production may be an indicator of a previous contact with TB, KD, HL, and AB conceived and designed the experiments. TB performed the experiments. MM carried out the clinical monitoring. TB and SC performed the statistical analyses. TB, MB, and HL wrote the manuscript with the input and approval of all other coauthors.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.


Lignocellulose, more generically named simply as plant biomass, represents one of the most abundant renewable resources for biofuels. Lignocellulosic biomass from forest residues, agro‐wastes and energy grasses is extensively exploited for bioenergy production. This renewable biomass source is abundant, highly accessible, relatively cheap and diversifies the energy matrix  and makes up between 20% and 35% of the total cell wall. Arabinofuranose residues (Araf) may be α‐ or α‐ linked to the xylan backbone forming arabinoxylan (AX), which may be further substituted with ferulic (FA) or p‐coumaric acid residues  reduces the cross‐linkages among AX, lignin and cellulose in plant cell walls, decreasing biomass recalcitrance , IRREGULAR XYLEM9 (IRX9) and IRX14 proteins, and one member of GT47, IRX10, are implicated in the biosynthesis of the xylan backbone, but the specific role is not completely established  required for xylan biosynthesis were first identified in IRX9 and IRX14 result in decreased xylan synthase activity and xylose content, accompanied by a shorter xylan backbone  were screened, associating them with a single quantitative trait locus (QTL) for saccharification. The study revealed that RNAi suppression of BdGT43A in Brachypodium decreases xylose and arabinose content and increases stem saccharification relative to the wild type, which is clear genetic evidence that BdGT43A is involved in xylan biosynthesis. In addition, the transgenic lines showed a decrease in FA and an increase in p‐coumaric acid, compared to the wild type. Similarly, plants of hybrid aspen (Populus tremula × tremuloides) down‐regulated simultaneously for PtGT43B and PtGT43C, the orthologues of IRX9 and IRX14, respectively, present reduced xylose content relative to the reducing end sequence in xylan, with slight alteration in the chemical composition of wood, a small decrease in S and H lignin, accompanied by a higher lignocellulose saccharification efficiency , without changing biomass productivity. Therefore, the increase in stem saccharification obtained by Whitehead et al. (Further examination of AX feruloylation (de Souza d et al.  reflectsArabinoxylans are abundant in nature and in the grass cell wall. AX and FA are essential components, cross‐linking polysaccharides to lignin and increasing the cell wall resistance to hydrolysis. Although further elucidation of xylan biosynthesis mechanisms is still necessary, a possible model to explain how it is associated with biomass digestibility is emerging. The advantage of discovering the genes associated with the expression of the enzymes in the biosynthesis of xylan is that there are now more ways to design this structure. Genetic manipulation of xylan biosynthesis and feruloylation raises many interesting questions that should be addressed in the future and is a potential approach to engineering crops that match the industrial requirements for food, cellulosic ethanol and biorefineries.The authors declare no competing interests.


To determine the incidence of hearing loss after acute episode of meningitis in childrenA descriptive study carried out in the Department of Pediatric Medicine of The Children’s Hospital Lahore, Pakistan from January 2014 to July 2016. A total of 175 children one month to 13 years of age admitted with diagnosis of meningitis were included. Complete blood count, CSF cytology, biochemistry and culture sensitivity were sent. CT scan brain was done if required. Hearing assessment was done two weeks after admission using otoacoustic emissions in the patients having normal tympanogram. Hearing impairment was classified as sensorineural if otoacoustic emissions were absent while tympanometry was normal.Of 175 children, 58% were males and 42% were females. Mean age was 2.1 years. Orientation as assessed by Glasgow comma scale (GCS) was normal in 63% while 5% had GCS<8 and 32% had GCS between 8 and 15. Signs of meningeal irritation were seen in 58% while focal signs only in 4%. In 15 % cases CT scan was done, out of which 73% showed abnormal findings. Otoacoustic emissions were absent in 22% of cases. Risk factors of hearing deficit were stay duration of more than 10 days (p=0.04), low GCS at presentation (p=0.009) and meningitis with complications (p=0.008).The frequency of hearing loss is 22% following acute episode of meningitis which necessitates the need for implementation of screening assessment after meningitis in Pakistan. Prolonged stay, low GCS and complicated meningitis are risk factors for hearing impairment. Hearing deficit is a major complication of meningitis in children.1In Pakistan, there are very few studies done on assessment hearing loss in such children. One study yielded hearing loss incidence to be 18.5%.This was a hospital based descriptive cross sectional study, conducted at The Children’s Hospital and The Institute of Child Health Lahore. A total of 175 children one month to 13 years of age admitted with diagnosis of acute bacterial meningitis were included. Positive CSF was defined as white blood cell (WBC) more than 10/ul in CSF. Children with meningomyelocele, prior central nervous system pathology, seizure disorder, hydrocephalus, history of recurrent meningitis and acute head trauma were excluded.A detailed history and thorough clinical examination was undertaken for each patient. Their clinical features focusing mainly age, gender, presenting symptoms, examination findings, duration of illness before presentation, total hospital stay, complications and outcome were documented. The fontanelle was examined for children less than two years. In all patients’ relevant laboratory data including complete blood count (CBC), CSF cell count with differential, protein, glucose, gram staining with CSF culture sensitivity (c/s) was performed. The patients were managed with intravenous antibiotics. Dexamethasone was given at a dosage of 0.6 mg/kg/day at onset within half hour of the first dose of antibiotic above four months of age as per hospital protocol. Informed consent was obtained from parents before enrolling children into the study. The ethical approval was taken from local hospital committee. The antibiotics were continued for a minimum of 10 days and were switched to second line according to CSF C/S report thus treatment was individualized based on each patient’s clinical course. All patients were examined daily for neurological complications such as seizures after four days of treatment, motor and sensory loss and cranial nerve palsy. CT scan brain was used when required and results were documented.Hearing assessment was performed after two weeks of presentation with meningitis using otoacoustic emissions. First tympanometry was done to rule out conductive deafness. Those patients who had abnormal tympanogram were not subjected to otoacoustic test. Those with abnormal tympanogram were sent to ENT for further evaluation and scheduled for follow up later. Hearing impairment was classified as sensorineural if otoacoustic emissions were absent in the presence of normal tympanometry.We used standard deviation for continuous variables and frequency percentages for categorical variables. The Chi Square was applied where applicable. The level of significant was considered p < 0.05.We analyzed outcomes of 175 children admitted with diagnosis of acute bacterial meningitis. Of 175 children 102 (58%) were males and 73 (42%) were females. Majority of children were less than five years i.e. 138(79%) whereas only 37 (21%) children were more than ten years of age. Mean age was 2.1 years. Total stay was more than ten days in 66 (38%) cases while 109 (62%) had stay less than ten days.Nearly all cases i.e. 173 children (99%) presented with fever followed by seizures which were present in 155 (89%) children. We found 9(5%) children had GCS<8 while 56 (32%) children had GCS between 8 and 15 while 110 (63%) had normal score. Signs of meningeal irritation were seen in 101 (58%) while focal signs were observed only in 7(4%) and irritability was seen in 38% cases.CSF count was raised more than 100 cells in 45 (27%) cases while 92 (53%) patients had between 50-100 cells and 37 (20%) had between 10-50 cells. Neutrophil predominance in CSF was seen in 93% cases. Abnormal glucose was seen in 123 (70%) patients while CSF protein was increased in 76 patients (43%).Klebsiella in 8 (4.5%) children followed by Streptococcus pneumonia in 2(1%) patients and then coagulase negative staphylococcus in one (0.5%). Hearing test was abnormal in 38 (22%) children (CSF culture was positive in 11 (6%) patients while complications were seen in 16 (9%) patients. The most frequent early clinical complication was focal deficit in 7 (4%) patients followed by hydrocephalus in 7 (4%) and one each of cranial nerve palsy, and visual loss. The duration of illness before presentation was less than two days in half of cases whereas 21(12%) had illness lasting for more than five days and 66 (38%) had between two and five days. The frequency of clinical parameters assessed is given . CT scanchildren .Hearing deficit was correlated with stay duration of more than 10 days (p=0.04), low GCS at presentation (p=0.009) and meningitis with complications (p=0.008) . IllnessBacterial meningitis is the most common cause of acquired hearing loss in childhood.There have been very few studies in Pakistan on hearing deficit, one study in 2008 showed nearly similar frequency of 20%as ours.10Comparable with other studies gender had no relationship with hearing loss in our data.11The classic triad of fever, fits and signs of meningeal irritation (SOMI) are present in published literature in 50% whereas focal signs in 33% and unconsciousness in 14%.14In our study the duration of illness before presentation was two days which was similar to other studies which suggest that parents approached health care facility within two days. Most studies show that the incidence of hearing loss is higher in children who were more toxic looking and sickHaemophilus influenza B whereas Streptococcus Pneumonia and Neisseria meningitis are common pathogens leading to meningitis. In another study Streptococcus Pneumoniaca used 22% where as Neisseria caused 8% of loss.Klebsiella followed by Streptococcus pneumonia 1% and coagulase negative staph aureus 0.5%. In a recent study of hearing loss in UK Meningococcal is isolated in 75% followed by Streptococcus pneumonia in 15%cases.15Hib immunization has eliminated almost all the cases of meningitis caused by Otoacoustic emission is a noninvasive test which gives flection of functioning of inner ear status and has been used as a screening. It can be affected by middle ear infections. Only patients with normal tympanogram were subjected to otoacoustic emission test. It cannot reflect function of auditory pathways so its presence cannot rule out hearing loss.17We found hearing deficit was strongly correlated with prolonged stay more than 10 days, low GCS and complicated meningitis . Complic20We could not grade the severity of hearing loss because otoacoustic emission is a screening test which represents cochlear function itself and needs more specialized testing to confirm it. We could not perform Auditory Brainstem Response due to our technical limitations so we could only identify the patients at risk of deafness.Keeping in view the high rate of occurrence of hearing loss after meningitis in pediatric population in our study we believe the universal hearing screening protocol after meningitis should be implemented in Pakistan. It will help detect early hearing deficit and timely intervention can be implemented to reduce the social and educational challenges for the affected children.FZ: Main Author designed and did statistical analysis & editing along with writing of manuscript.AB: Did critical review & final edit.MN and FS: Involved in methadology & lab investigaitons.