Lice are socially-transmitted ectoparasites. Transmission depends upon their host’s degree of contact with conspecifics. While grooming facilitates ectoparasite transmission via body contact, it also constrains their spread through parasite removal. We investigated relations between parasite burden and sociality in female Japanese macaques following two opposing predictions: i) central females in contact/grooming networks harbour more lice, related to their numerous contacts; ii) central females harbour fewer lice, related to receiving more grooming. We estimated lice load non-invasively using the conspicuous louse egg-picking behaviour performed by macaques during grooming. We tested for covariation in several centrality measures and lice load, controlling for season, female reproductive state and dominance rank. Results show that the interaction between degree centrality (number of partners) and seasonality predicted lice load: females interacting with more partners had fewer lice than those interacting with fewer partners in winter and summer, whereas there was no relationship between lice load and centrality in spring and fall. This is counter to the prediction that increased contact leads to greater louse burden but fits the prediction that social grooming limits louse burden. Interactions between environmental seasonality and both parasite and host biology appeared to mediate the role of social processes in louse burden. Many parasites, i.e. organisms that live and feed exclusively within or on other living organisms (hosts), are socially-transmitted, either directly through contact between individuals or indirectly through spatial overlap and resource sharing3From this perspective, social network analysis (SNA) provides a useful tool that captures such heterogeneity by taking into account direct and indirect connections (edges) between individuals (nodes), allowing for multilevel analyses from individuals to populations32One common type of network relevant to social animals derives from social grooming . Grooming is often highlighted as a mechanism of establishing and maintaining group cohesion and social bonds7911121415161719Rickettsia prowazekii, Bartonella quintana, and B. recurrentisRickettsia, Anaplasma, and Bartonella spp. in various animals2025One common ectoparasite of mammals is the louse, which feeds on blood and requires a host during each stage of its life cycle. Adult lice typically lay one to several eggs (nitts) every few days, which are glued to the base of a hair. Like many organisms, lice respond to environmental conditions . In ungulates, for instance, adult louse populations peak in spring and drop in summer202122Lice and their mammalian hosts are thus a good host-parasite system to study the likelihood or risk of disease/parasite transmission within host social networksMacaca fuscata fuscata) is a social primate species living in multi-female multi-male groups, where individuals form linear dominance hierarchies and differentiated affiliative social relationshipsPedicinis obtusus and P. eurygaster). Based on quantification of egg and nymphal/adult stages of lice detected on culled macaques, it was estimated that an average-sized macaque could harbour up to 550 louse eggs17In this study, we investigated the risk of louse infestation within Japanese macaque social networks. The Japanese macaque . Because body contact provides a transmission opportunity for lice, and thus an infection risk for hosts, yet grooming may constrain louse density through parasite removal, we made two opposing predictions: compared to less central females, 1/ more central females will be most infested with lice due to their higher diversity of interacting partners or their higher frequency of body contact in the network; or 2/ more central females will be least infested with lice because they have their louse eggs removed through grooming by a higher diversity of partners and/or more frequently. Evidence for a positive relationship between centrality and lice load would indicate that despite increasing exposure risk to potentially deleterious parasites, being central still presents advantages. Conversely, a negative relationship would speak in favour of social grooming as an efficient antiparasite strategy that can be exploited by females through their grooming networks. To test prediction 1, we investigated the relationship between estimated lice load and centrality measures based on undirected weighted networks of body contacts, including grooming. To test prediction 2, we looked at whether lice load was related to in-centrality measures derived from directed weighted networks of grooming received. Testing different centrality measures allowed us to investigate whether it is the actual number (or diversity) of partners and/or the actual amount of social contact a female has that influences lice load. First, grooming skills and thus louse egg-picking efficiency significantly vary across individualsGrooming represented 27%  of all activity scans of female Japanese macaques of Koshima during this study. Body contact without grooming represented only 9%  of all scans with body contact. Females were in contact with other adult females in 48% of all scans with body contact , with males in 6%  and sub-adults, juveniles and infants in 41% . Total louse egg-picking events averaged 129 events per female over the whole study period , which represented less than one event per grooming minute-scan .The modularity Q of social networks, representing the extent to which a network is partitioned in smaller units, shifted between seasons especially before and after summer where it was highest . RandomiLice load also varied across seasons, being higher in summer and fall compared to spring and winter ; Fig. 2.obs < βrand = 0.043, Overall, only one of the centrality measures from contact networks was related to lice load and only through an interaction with season , 4, 5: fobs < βrand p = 0.087, Models with grooming received centrality measures showed a tendency towards lice load being related to in-degree through an interaction with season and reproductive state separately , 4, 5: fIn animal groups, increased centrality in social networks is often linked to increased parasite load and disease risk151920It must be stated up front that our measure of lice load is indirect, observational and dependent upon observing individuals grooming. Capturing monkeys and marking/collecting lice provides a direct estimate of lice loads and allows testing whether lice actually carry pathogens, but this approach is inconvenient and often impossible to implement in wild populations. That said, Japanese macaques are very conspicuous when picking items from the hair or on the skin of their grooming partners, and 98.9% of what is picked and consumed has been demonstrated to be louse eggs17The study of this louse egg-picking behaviour led researchers to discover that macaques groom for demonstrably longer durations if they find many louse eggs to pick and eat , irrespective of the relationship between groomer and groomee151730Tamias striatus) and mouse lemurs (Microcebus rufus), presumably because of the concomitant increase in both contact between individuals and host reproductive activity2821Spilopsyllus cuniculi), in which the reproductive cycle is triggered by pregnancy and parturition of the host doe. The fleas then migrate en masse to the nest to breed on the immunologically-naïve young50One ecological factor that clearly affects numerous behavioural outcomes is seasonality. We considered seasonality of the environment as well as host and parasite biology, as it is generally highly relevant to infectious disease dynamics3334sensu the effect of shearing on louse density in domestic sheepThese synergies between host and parasite biology also explain the results of this study. Lice load was highest in summer during the periparturient period. Females thus seemed more vulnerable to louse infestation at this time. This was particularly the case for reproductively active females, especially those that were less central in the social networks. This pattern could be due to the combination of several factors, also linked to the effect of environmental seasonality on host and parasite biology. First, the surge of sex hormones in the host around birth could have triggered louse reproductive activity and proliferation202122At Koshima, mean centrality indices generally seemed to vary in the opposite direction of mean lice load across seasons; the season in which mean centrality was lowest corresponded to the season in which mean lice load was highest. However, network modularity did follow somewhat closer to the pattern of mean lice load in that the season in which mean lice load was highest was also the season in which modularity was greatest. Greater modularity in a network means that the network decomposes into more modules than in a network with lower modularity. Greater modularity is hypothesized to negatively impact transmission of infectious agents throughout social groups by breaking down the chain of transmission5355Suricata suricatta)But if the network reflects pathways for parasite transmission, centrality indices such as degree and strength measuring risk of exposure are expected to be positively related to parasite infection patternsIn this evolutionary arms race, parasites effectively use their hosts’ behaviour to increase their own fitness6264In conclusion, our study shows that variation in contact and grooming network centrality in terms of number of connections explains variation in lice load in female Japanese macaques in that less central females have higher lice burden, providing further evidence of grooming as an efficacious anti-parasite strategy. However, our study also demonstrates that this relationship is dependent upon ecological and biological conditions, such as less central females have higher lice burden only during winter  and summer . Our study is the first to estimate lice load from direct observation of wild animals and to link this estimate to the centrality status of individuals in their contact networks. This study also highlights the importance of seasonal variation both in parasite intensity and in host behaviour in explaining variance in infection risk and exposure from socially-transmitted external parasites.2 and covered by evergreen broadleaved forestWe studied Japanese macaques on Koshima islet, Miyazaki prefecture, Japan . The region has a warm-temperate climate with monthly mean temperatures ranging between 9.3 °C in January and 27.2 °C in August, and monthly mean precipitation between 35 mm in January (humidity 60%) and 730 mm in June . Koshima is approximately 0.3 kmWe observed the 19–20 adult females (>7 years old) of the Main group. We focused on females because, in macaque societies, they form the core of the group and dominate dynamics of social networks, males are often few and not very social, and juveniles are difficult to recognise, often have their own subgroup, and usually engage in different age-typical activities than the adults6970Research adheres to the ASAB/ABS guidelines for the use of animals in research and was approved by the ethic committee of Kyoto University Primate Research Institute.ad libitum.Data were collected from January to November 2014  along the length of the hair, and eat itWe divided our dataset in four parts to account for seasonal variations biologically relevant to the studied host-parasite system. The winter dataset included observations between January and March and corresponded to the macaques’ mating seasonTo compute lice load, we used minute-scans of grooming received, the focal female being either the groomer or the groomee, and minute-scans of self-grooming per female as well as minute-counts of louse egg-picking gestures across all grooming received bouts per female. Lice load was then calculated per female as follows:where n is the total number of grooming or self-grooming bouts.undirected weighted networks of body contacts We used SNA to investigate the risk of exposure to louse infestation of females within their social network of female group members. First, we calculated the modularity Q of the contact and grooming received networks  based on eigenvector centrality, which provides a descriptive statistic of detection and characterization of community structure in networkscontacts . The secreceived . Althougad libitum. As there was no change in female hierarchical order across seasons, but individual normDS varied in magnitude, we finally assigned females an ordinal rank with 1 being the highest and 20 the lowest.To assign dominance rank, we calculated normalised David’s scores  while accounting for potential confounding factors, we built General Linear Mixed Models (GLMM) with Gaussian distributions and identity link functions using the function lmer from the lme4 packaged season . In modeTo assess the variability present in host sociality and lice load that may affect the relationship between the two, we built GLMMs with each centrality measure as the response variable, season, reproductive state and rank as predictors, and individual identity as random effects. Variation in lice load was assessed from the null model that was built to test predictions 1 and 2 and that included season, reproductive state, treatment status and individual identity as well (see below).lice load as the response variable, centrality index as the main predictor, season, reproductive state, rank, and treatment status as confounding factors and individual identity as a random effect.To address our main question concerning the relationship between measures of centrality and lice load, we built GLMMs with We also included a three-way interaction between centrality, season, and reproductive state, and its associated two-way interactions . The effect of centrality on lice load could indeed depend on seasonal variations in both lice load and centrality, and lice load could also be differentially influenced by some stages of host reproduction , and these stages depend on season. Thus, it may be that the effect of the interaction between centrality and season on lice load varies according to female reproductive state, such as females more central in the network have more lice in winter and summer but only if they are reproductively active . We usedHow to cite this article: Duboscq, J. et al. Network centrality and seasonality interact to predict lice load in a social primate. Sci. Rep.6, 22095; doi: 10.1038/srep22095 (2016).


The Canadian Hearing and Auditory Research Translation (CHART) group is a newly formed taskforce to develop collaborative research initiatives. Initial discussions centered on diagnostic improvements for middle ear disease, auditory neuropathy spectrum disorder, tinnitus and presbycusis. Central to these discussions was the widely held view that the standard audiogram and its interpretation is inadequate to for describing many forms of hearing problems that we now recognize. This letter is designed to create awareness and to seek feedback from hearing healthcare professionals on their experience regarding the adequacy (or otherwise) of the audiogram in its present form. A new Canada-wide initiative in for research collaborations in audiology/otology was recently (Nov. 2015) established. This is the Canadian Hearing and Auditory Research Translation (CHART) group. The Hearing Foundation of Canada (THFC) aided with some industry sponsorship (Oticon Foundation) held the first workshop in Halifax NS, hosted by Drs. Manohar Bance and Steve Aikin. Leaders in audiology, otology and hearing science from across the country discussed priority research questions, and plans to address them. The focus was on clinical problems rather than basic science issues. Four key areas were chosen, namely middle ear function, auditory neuropathy, tinnitus and presbyacusis.In addition, and the reason for this note to clinicians, is that we also discussed the limitations of clinical diagnostic assessments for patients with hearing difficulties. Importantly we are of the opinion that the standard audiogram, and its interpretation is inadequate to address many forms of hearing problems that we now recognize.Here are three examples:[1] We have long known that threshold measures of hearing (i.e. the audiogram) very often do not correlate with real hearing difficulty. This notion was put into sharp focus when we formally recognized auditory neuropathy spectrum disorder (ANSD). More recently we have become aware of “hidden hearing loss”, from studies showing that noise exposure can deplete more auditory neurons than cochlear haircells. Cochlear thresholds can look normal despite the retro-cochlear lesions. Standard audiometry has no chance of revealing any subtle threshold deficits. However if we used a higher definition testing with 1–2 dB accuracy instead of 5–10 dB approximations, we might identify subtle clues about hidden hearing loss and ANSD problems.[2] Why do we still, in 2016, not measure the whole high-frequency range to our hearing? Why do we only measure up to 8 kHz when we know that most hearing problems typically start in the high frequencies? Ototoxic drug damage, presbycusis, noise trauma can all cause hearing loss at frequencies above those that we routinely test. Some studies in patients with tinnitus that appear to have “normal audiograms” turn out to have very high frequency hearing loss (above 8 kHz), but not usually detected. We need to make high frequency audiometric testing a standard clinical procedure.[3] Given the lack of level definition and frequency range of standard audiometry the interpretation of the audiogram for reporting purposes is very misleading. To suggest that a patient with threshold elevations of 10–15 dB is in the “normal range” is not useful. Statistically, a 5 dB threshold loss is significant! To report that a subject has “normal hearing” when at 16 kHz there may be an unmeasured 50 dB threshold elevation is clearly inaccurate. Our recognition of ANSD and “hidden hearing loss” etc. should, by now, have prompted us to revise our standard audiometric procedures. We need to see some evolution, and that will only come when audiologists and otologists agree that change is necessary.Our CHART group would be very interested to hear your feedback comments on this audiogram issue. Please feel free to contact the group (through myself at: rvh@sickkids.ca).On behalf of the CHART groupRobert V. HarrisonProfessor, and Vice-Chair (research)Department of Otolaryngology – Head & Neck SurgeryUniversity of Toronto.Director, Auditory Science Laboratory,Program in Neuroscience and Mental HealthThe Hospital for Sick Children, TorontoCANADA


By studying the literature about tetracyclines (TCs), it becomes clearly evident that TCs are very dynamic molecules. In some cases, their structure-activity-relationship (SAR) are well known, especially against bacteria, while against other targets, they are virtually unknown. In other diverse fields of research—such as neurology, oncology and virology—the utility and activity of the tetracyclines are being discovered and are also emerging as new technological fronts. The first aim of this paper is to classify the compounds already used in therapy and prepare the schematic structure that includes the next generation of TCs. The second aim of this work is to introduce a new framework for the classification of old and new TCs, using a medicinal chemistry approach to the structure of those drugs. A fully documented Structure-Activity-Relationship (SAR) is presented with the analysis data of antibacterial and nonantibacterial  tetracyclines. The lipophilicity and the conformational interchangeability of the functional groups are employed to develop the rules for TC biological activity. Streptomyces aureofacians [in vitro and in vivo are not yet entirely clear [The number of articles published on tetracycline drugs has reached the threshold of 50,000 papers since 1948. Over the last 10 years, technological fields are emerging in bacteriology and cellular physiology of eukaryotic cells. However, chemical mechanisms of tetracyclines are not completely understood in terms of their function in human cells and, to this day, no (Q)SAR model is validated without doubts. Tetracyclines were first discovered by Dr. Benjamin Dugger of Lederle Laboratories in the mid 1940s as the fermentation product of an unusual golden-colored soil bacterium aptly named ofacians . This meofacians . TCs binly clear .The therapeutic uses are as follows: antibacterial and non-antibacterial. In the literature, these uses fall into five main categories, namely: (I) newer and more potent tetracyclines used in anitibacterial resistance , (II) thCurrently, as a consequence of their overuse, bacteria have developed TC resistance (efflux pump type) as opposed to the oldest compounds. Medicinal chemists with the intention to optimize structure and improve the antibacterial power have successfully introduced an alkaline group on C-9 of minocycline skeleton, starting as a compound from total synthesis: Tigecycline . Searching for new molecules, it is not only important to study the binding of drugs specifically to bacterial ribosomes, but also to understand how the tetracycline skeleton can act as a chelator and ionophore . Moreove2 [Both laboratory and clinical studies have investigated the anti-inflammatory properties of tetracyclines. These include: Acting as an inhibitor forlymphocytic proliferation , suppres2  and acce2 .In recent times, starting from the end of the 1990s , TCs havIn the same way that tetracyclines act as pro-apoptotics in neuronal cells, they also act in peripheral metastasis of generalized tumor cells. Experimental data using various carcinoma cell lines and animal carcinogenesis models showed that doxycycline, minocycline and chemical modified tetracyclines (CMTs) inhibit tumor growth by inhibiting matrix metalloproteinase (MMPs) and by having a direct effect on cell proliferation ,19. The The tetracycline-controllable expression system offers a number of advantages: Strict on/off regulation, high inducibility, short response times, specificity, no interference with the cellular pathway, bioavailability of a non-toxic inducer, and dose dependence. The tet-off system , which uFirst generation if they are obtained by biosynthesis such as: Tetracycline, Chlortetecycline, Oxytetracycline, Demeclocycline. Second generation if they are derivatives of semi-synthesis such as: Doxycycline, Lymecycline, Meclocycline, Methacycline, Minocycline, Rolitetracycline. Third generation if they are obtained from total synthesis such as: Tigecycline. However, some researchers consider Tigecycline to be distinct from other tetracyclines drugs and are considered as a new family of antibacterials called Glycylcyclines.Historically, tetracyclines are considered + and Gram−) Aminomethylcycline derivatives are considered in the same way as Glycylcycline , will be available in therapy. TCs obtained via total synthesis, such as Tigecycline, are considered members of the Third generation if they show wide spectrum activities  much more then monovalent ions . It is known that TC forms complexes in different positions with calcium and magnesium ions that are available in the blood plasma [Hydroxyl groups are a source of radical oxygen species (ROS) that irreversibly damage macromolecules such as DNA, RNA and proteins. All these effects are considered as fundamental to in cellular death for oxidative stress. In the case of antitumor anthracyclines and CMT-3, these act to block the enzymatic transcriptional complex formation on DNA and then induce apoptotic events. Antifungal and antitumor TCs act in a different way from antibacterial TCs. Once passed into the eukaryotic cells, TCs change the electronic balance equilibrium sequestering divalent ions  using PubChem Project. The computational analysis has been performed on a data set of 1325 TCs with a similarity score of 90%, starting from more than 322,000 compounds recorded in PubChem (this paper is in preparation). From that set were chosen the best in class TCs with a similarity score major of 95% . For eacThe role of lipophilicity of TCs is the main factor to explain the biological potency and dynamics of this old family of drugs. It is important to understand the conformational flexibility and the affinity of TCs of metals ions due to the several functional groups present in the molecular skeleton to obtain a significant SAR study. After more than 60 years of scientific investigation, TCs are considered a master example of the pleitropic family of compounds with promising therapeutic properties. Due to the wide use in therapy and their low toxicity, TCs can be considered the first Multi-Target Drugs fully and well discussed in the history of pharmacology.Newer and more potent antibacterial compounds can be expected to fight the tetracycline-resistant pathogens. New, third-generation derivatives have been designated to be more potent, especially against bacteria possessing ribosomal protection and efflux mechanisms. TCs have increased in potency over time compared to other structural classes of antibiotics. TCs have shown a complex mechanism of action towards various targets due to their conformations under physiological conditions. Such conformations, depending on pH and metal ion concentrations, allow TCs to act in a “chameleonic-like” manner in a large number of diseases. As already suggested, the future of TCs will have increased utility as one of the best anti-inflammatory drugs.


As compared with the control group, the number of TCs and Tps in HF decreased more than twofold. There were no differences between HF and control in the number of Ki67-positive TCs. In contrast, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling-positive TCs increased threefold in diseased hearts as compared to control. Significant inverse correlations were found between the amount of mature fibrillar collagen type I and the number of TCs  and Tps . The levels of degraded collagens showed a significant positive relationship with the TCs numbers. It is concluded that in HF the number of TCs are decreased because of higher rates of TCs apoptosis. Moreover, our results indicate that a close relationship exists between TCs and the ECM protein composition such that the number of TCs and Tps correlates negatively with the amount of mature fibrillar collagens and correlates positively with degraded collagens.Telocytes (TCs) are a novel type of interstitial cells only recently described. This study aimed at characterizing and quantifying TCs and telopodes (Tps) in normal and diseased hearts. We have been suggested that TCs are influenced by the extracellular matrix (ECM) composition. We used transmission electron microscopy and c-kit immunolabelling to identify and quantify TCs in explanted human hearts with heart failure (HF) because of dilated, ischemic or inflammatory cardiomyopathy. LV myectomy samples from patients with aortic stenosis with preserved ejection fraction and samples from donor hearts which could not be used for transplantation served as controls. Quantitative immunoconfocal analysis revealed that 1 mm Telocytes (TCs) are a novel type of interstitial cells in vitro study using isolated TCs has shown that the dynamics of cardiac Tps can be influenced in vitro by the protein composition of the extracellular matrix (ECM) In cardiac pathologies, TCs have been described in atrial amyloidosis in human patients n = 6) according to the criteria of the Society for Cardiovascular Pathology n = 6), or ischemic cardiomyopathy . Diagnosis of ICM was based on clinical history, Doppler echocardiography and cardiac catheterization data. Patients were functionally classified according to criteria of the NYHA and were listed for heart transplantation according to the guidelines for organ transplantation of the German Medical Association. Clinical data are summarized in Tablei) LV samples from 4 patients subjected to subvalvular myectomy during surgical aortic valve replacement, and (ii) LV samples from 2 donor hearts with normal LV function which could not be used for transplantation. Patients with aortic valve stenosis showed preserved cardiac function (ejection fraction ≥60%), and the resected myocardial tissue did not present signs of ischemic damage or myocardial hypertrophy. The institutional Ethical Committee approved the study, and all patients have given written informed consent.LV samples were collected from the explanted hearts of patients who underwent orthotopic transplantation because of end-stage HF. All patients were severely symptomatic [New York Heart Association (NYHA) grade IV]. Patients either had normal coronary arteries with histologically proven chronic myocarditis  sections were stained with toluidine blue and viewed in a Leica DM microscope . Ultrathin sections were stained with uranyl acetate and lead citrate, and viewed and photographically recorded under a Philips CM10 electron microscope .® O.C.T.™ compound  and cryosections 5 μm thick were prepared. Cryosections were air-dried and fixed for 10 min. in either acetone (−20°C), 4% paraformaldehyde (room temperature) or Carnoys' solution (room temperature). After washing in PBS sections were incubated with 1% bovine serum albumin for 30 min. to block non-specific binding sites. After rinsing in PBS, the samples were incubated overnight with primary antibodies against c-kit , Ki67  and collagen I . Antibodies against carboxyterminal telopeptide (ICTP), N-terminal propeptide I (PINP) and N-terminal propeptide III (PIIINP) were prepared and used as previously described ® 7.4.2 software .The tissue samples were mounted in Tissue-Tekhttp://rsb.info.nih.gov/ij/). The area occupied by different collagen markers was calculated as percentage of collagen I or of collagen markers labelling per tissue area.Measurements of immunofluorescence for collagen I and markers of collagen metabolism were carried out using a ×40 Planapo objective and a Leica (Leitz DMRB) fluorescent microscope (Leica Microsystems). Cryosections from at least two different tissue blocks in each case were used. For quantitative analysis, all sections were immunolabelled simultaneously using identical dilutions of primary and secondary antibodies and other reagents. Immunofluorescent images were obtained under identical parameters of imaging, zoom, objectives and fluorescence power. Sections exposed to PBS instead of primary antibodies served as negative controls. Image acquisition settings were standardized for all groups to ensure that the image collected demonstrated a full range of fluorescence intensity from 0 to 255 pixel intensity level and were kept constant during all measurements. For each patient, at least 10 random fields of vision were analysed for the quantity of the collagen markers investigated using analysis software (Leica Microsystems) and NIH ImageJ program  and Tps .We have studied the relationship between mature fibrillar collagen type I and TCs using double labelling immunoconfocal microscopy. Images presented in FigureNext, we studied the relationship between TCs and markers of collagen metabolism. Carboxyterminal telopeptides (ICTP) labelling detects a degradation product of cross-linked collagen type I. The collagen propeptides PINP and PIIINP characterize, respectively, the newly synthesized type I and type III collagens. Representative images showing the relationship between TCs and markers of collagen I metabolism are presented in FigureThe coefficients of correlations between the number of TCs and Tps with the markers of collagen metabolism are shown in TableThe ultrastructure of TCs in normal hearts is very well described. An example of a TC in normal human heart is shown in FigureMost probably, both TC degenerative changes and the ECM composition may lead to TC death. FigureThe relationship between the ultrastructure of the ECM and TCs is presented in FigureThe major finding of this study is that TCs are reduced in the diseased and failing human myocardium. The imbalance between TC proliferation and TC apoptotic death, documented in this study, is responsible for the TCs depletion in cardiac diseases leading to HF. Reduced numbers of TCs have been documented in other cardiac diseases including myocardial infarctions in rats We have also demonstrated that in diseased human hearts with HF, TCs display severe ultrastructural degenerative changes and undergo apoptotic cell death. The latter was confirmed using both, TEM and TUNEL staining. Similar ultrastructural alterations of TCs and TC apoptosis have been documented in psoriatic dermis 2 of the normal human myocardium contains 14.9 ± 3.4 TCs and 41.6 ± 5.9 TPs. The values of c-kit-positive TCs found by Zhao et al. 2 in the base part of control hearts. In addition, a recent morphometric study of TEM images revealed 22 ± 2 TCs per 1 mm2 myocardial area of the neonatal human heart and 19 ± 3 TCs per 1 mm2 myocardial area of the adult human heart Currently, there are no specific immunohistochemical markers for the identification of cardiac TCs. However, cell morphology and positive expression of c-kit have been used for the identification of cardiac TCs via homo- or heterocellular cell-to-cell contacts The pathophysiological consequences of reduced TCs observed in our patients with different etiologies of HF are largely unknown. It is, however, tempting to speculate that the reduction in TCs in diseased human hearts could participate in the abnormal three-dimensional spatial organization and disturbed intercellular signalling of the myocardium as TCs are considered to be as structurally connecting cells with other heart cells and given that TCs are involved in intercellular signalling Another major finding of this study is that the number of TCs is markedly influenced by the composition of the ECM. We have found that in zones of replacement fibrosis or in zones with tightly packed fibrillar collagens, the TCs and Tps are significantly reduced or even absent. Similar observations have been documented in the scar tissue after myocardial infarction in vitro study showing that the dynamics of cardiac Tps are also influenced by the ECM proteins: the stronger Tps spreading being produced by fibronectin, while the lowest by laminin in vivo is a more complex process than that in vitro. Despite such a discrepancy, both studies complement each other and add to the conclusion that TCs are influenced by the ECM composition.Our findings demonstrating that TCs are influenced by the ECM protein composition are in good agreement with a recent This study demonstrates for the first time that in diseased and failing human hearts the number of TCs is reduced because of a higher rate of apoptosis than in normal hearts. It is also concluded that TCs and Tps in diseased human hearts respond to any quantitative and qualitative changes in the ECM composition such that the number of TCs and Tps correlates negatively with the amount of mature fibrillar collagens and correlates positively with degraded collagens.


Lawsonia intracellularis is the etiologic agent of proliferative enteropathy, which causes diarrhea in several animal species, including swine. Serology can be used both to determine the prevalence of antibodies against a specific pathogen in a herd and to obtain the serological profile, which is used to determine the dynamics of infection in the herd. The objective of this study was to determine the serological profile and seroprevalence of anti-L. intracellularis antibodies in swine herds from intensive production regions of Minas Gerais, Brazil, and to identify the risk factors related to the herd-level seropositivity.A total of 2999 serum samples were collected for this cross–sectional study in the four major regions of intensive swine production in Minas Gerais, Brazil. To obtain better estimates and increase the external validity of the seroprevalence, the sample data were weighted based on the pig population of each herd, the stratum in which the herd was classified and the swine population of the region where each herd was located. A questionnaire was used to identify potential risk factors related to this herd-level seropositivity. The overall weighted prevalence in Minas Gerais was 34.7 % , and there was no significant difference among the sampled regions, with the seroprevalence rates ranging between 32.06 and 37.66 %. Finishing pigs were the most prevalent among the sampled categories. Among the evaluated risk factors, “cleaning before disinfecting” had a negative impact in the seroprevalence (p < 0.05) and was considered a protective factor.L. intracellularis antibodies were detected in all of the investigated herds in Minas Gerais, which indicated a wide distribution of the agent in the state. The predominant serological profile was consistent with the dynamics of infection previously observed in pig herds in other countries with similar antimicrobial usage, in which the nursery pigs usually show the lowest seroprevalence and the finishing pigs exhibit the highest. Herds that adopt the practice of “cleaning before disinfection” can decrease their L. intracellularis antibody seropositivity.The anti-The online version of this article (doi:10.1186/s12917-015-0618-z) contains supplementary material, which is available to authorized users. Lawsonia intracellularis, is responsible for relevant economic losses in swine production systems due to increased mortality rates, increased drug usage and compromised weight gain  was adopted by 100 % of the properties research at our research. Herd size, pointed as a risk factor by Bronsvoort et al. (2001)  = _b [p p (line number)(column number)].Data were entered into Excel (Microsoft Excel 2007) and analyzed using Stata, (version 12.0). The estimated prevalence, by region and strata, were calculated using the commands svyset < L. intracellularis at the farm level, being selected those who had association with a liberal p-value of 0.20 or less (svy command: regress < answer variable > <independent variable>). After performing each of the univariable analyzes, we utilized a backwards method, manually, maintaining in the final model the variables with p < 0.05 and those that have been identified with multicolinear association with significant factors (variables) .Risk factors considered relevant to the enteric diseases were selected from the questionnaire and used to determine the association with seroprevalence at a herd level. Univariable linear regression was performed initially to predict the association of risk factors with the seroprevalence of


Escallonia species is geographically structured and there is a deep divergence between infraspecific groups that could be associated with ancient evolutionary events like orogeny. We also found evidence of admixture, likely the result of hybridization at the margins of the parental species' distribution range.Mountain orogeny has been a major factor in plant evolution in all continents by changing the landscape and climate, creating new habitats and ecological opportunities. In this study we found that diversity in two southern Andean  Escallonia is known to have diversified in the Andes. Species with similar morphology obscure species delimitation and plants with intermediate characters occur naturally. The aim of this study is to characterize genetic variation and structure of two widespread species of Escallonia: E. alpina and E. rubra. We analyzed the genetic variation of populations of the entire distribution range of the species and we also included those with intermediate morphological characters; a total of 94 accessions from 14 populations were used for the Amplified Fragment Length Polymorphism (AFLP) analysis. Plastid DNA sequences  from sixteen accessions of Escallonia species were used to construct a Statistical Parsimony network. Additionally, we performed a geometric morphometrics analysis on 88 leaves from 35 individuals of the two E. alpina varieties to further study their differences. Wright’s Fst and analysis of molecular variance tests performed on AFLP data showed a significant level of genetic structure at the species and population levels. Intermediate morphology populations showed a mixed genetic contribution from E. alpina var. alpina and E. rubra both in the Principal Coordinates Analysis (PCoA) and STRUCTURE. On the other hand, E. rubra and the two varieties of E. alpina are well differentiated and assigned to different genetic clusters. Moreover, the Statistical Parsimony network showed a high degree of divergence between the varieties of E. alpina: var. alpina is more closely related to E. rubra and other species than to its own counterpart E. alpina var. carmelitana. Geometric morphometrics analysis (Elliptic Fourier descriptors) revealed significant differences in leaf shape between varieties. We found that diversity in Escallonia species analyzed here is geographically structured and deep divergence between varieties of E. alpina could be associated to ancient evolutionary events like orogeny. Admixture in southern populations could be the result of hybridization at the margins of the parental species’ distribution range.Mountain uplifts have generated new ecologic opportunities for plants, and triggered evolutionary processes, favouring an increase on the speciation rate in all continents. Moreover, mountain ranges may act as corridors or barriers for plant lineages and populations. In South America a high rate of diversification has been linked to Andean orogeny during Pliocene/Miocene. More recently, Pleistocene glacial cycles have also shaped species distribution and demography. The endemic genus  Mountain orogeny has been a major factor in plant evolution in all continents, and has been linked to recent diversification and speciation events . The uplIn South America, the Andes mountains have played these roles since their uplift during Pliocene/Miocene (16–5.3 Mya). Moreover, the Andes themselves could have played as a North-South corridor allowing the exchange of plant lineages; or as a barrier promoting vicariance. Consequently, these evolutionary processes triggered by the Andean uplift promoted a high speciation rate conducting to great biological diversity in South America . Moreover, plants with intermediate morphology (IM) between species have been described  and clade D with the remaining nine species sampled . Within this major lineage, two species, E.alpina and E. rubra, are highly polymorphic and share the same distribution range along the Andes in Patagonia, although they only differ in maximum elevation: E. alpina reaches higher altitudes (up to ca. 2200 m) than E. rubra, which occurs up to ca. 1700 m. These species are differentiated mainly by the distribution of glands in leaves and flowers and by the type of inflorescence. For both species some varieties have been described on the basis of morphological characters and geographical distribution. There are two varieties described for E. alpina based on vegetative characters: E. alpina var. carmelitana only differs from the type variety in the colour of the stem and leaf size. A preliminary study on the genetic variability, including five populations, supported species boundaries and one population showing intermediate morphological characters was detected and corroborated by AFLP patterns . Additionally two southernmost populations with intermediate morphological characters between both species are included to investigate the genetic bases of their variation. We further analyze morphological variation in E. alpina  using a geometric morphometrics analysis of leaf shape. We predict that E. rubra, E. alpina var. alpina and E. alpina var. carmelitana will have a high degree of genetic differentiation, while populations with intermediate morphology will show genetic admixture. Finally, leaf shape will be useful to differentiate the two varieties of E. alpina.Our aims are to characterize the genetic variation between E.rubra were collected from 39° to 47° south latitude (S) in Neuquén, Chubut and Santa Cruz provinces, four of E.alpina var. alpina from 42° to 49° S, in Chubut and Santa Cruz provinces, and two of E. alpina var. carmelitana in northern Neuquén province, from 36° to 37° S. Two additional populations with intermediate morphological characters were collected at ca. 50° S in southwestern Santa Cruz province  protocol  with somtrnS-trnG, 3′trnV-ndhC and the ndhF gene Supporting Information 2]. Pearson correlation coefficients between each fragment size and its frequency were calculated in order to assess the possibility of homoplasy  for all populations and for separate species. Additionally a matrix of pairwise Fst genetic distances between all populations was constructed.The AFLP matrix was used to infer genetic structure from Principal Coordinate Analysis PCoA,  was perfE. rubra, E. alpina var. alpina and E. alpina var. carmelitana); and among and within populations, and to test for significant differences.We performed an analysis of molecular variance (AMOVA) using Arlequin v.3.5.1.3 . This anE. alpina, leaf shape of var. alpina and var. carmelitana was described by traditional methods, using specimens from our collections  were cut, hydrated, pressed and photographed using a Cannon PowerShot G11 digital camera over a clear background. The digital files were manipulated to obtain a binary image using Fiji  was carried to show maximum separation among  variety . AdditiotrnS-trnG) and 602 bp (trnV-ndhC), and the amplification of ndhF gene (incomplete sequence) resulted in a 1803 bp-fragment. All accessions, including multiple accessions for one species, showed exclusive haplotypes , E. rubra, and E. florida, which are separated by few mutational steps. The remaining species are separated by a long chain of mutational steps from the main group.In the network , one majE. alpina var. alpina and var. carmelitana were included in the major group together with those downloaded from the GenBank database. Varieties were grouped together, although neither of them shares a unique haplotype. Moreover, all accessions of E. alpina var. alpina are more closely related to other species like E. rubra and E. florida than they are to E. alpina var.carmelitana accessions; both varieties were separated by at least 7 mutational steps.In particular, new sequences of R2 = −0.153; P = 0.07). Escallonia rubra and E. alpina (including both varieties) had, respectively, 7 and 19 exclusive fragments. At the same time E. alpina var. alpina and E. alpina var. carmelitana had seven exclusive fragments each.Non replicated peaks (4.8 %) were eliminated and a matrix with 174 fragments for the 94 individuals sampled was obtained. Linear regression of fragment size and fragment frequency was not significant (P < 0.001). As this Fst value encompassed differences among taxonomic units we performed separate analyses for E. rubra  and E. alpina var. alpina . Escalloniarubra showed a much higher level of genetic structure, although both were significantly different from zero.When comparing all populations, Wright’s fixation index showed a significant degree of genetic structure  but higher values when compared to E. alpina var. carmelitana (0.17–0.22).Pairwise differences among populations are shown in Escallonia.rubra and E. alpina individuals are separated along the first axis, while individuals that share morphological diagnostic characters of both species are spread in an intermediate position . Populations of IM (229 and 233) showed a mixed genetic contribution from E. alpina var. alpina and E. rubra. On the contrary, individuals from E. alpina var. carmelitana (populations 321 and 340) showed almost no signal of genetic admixture.STRUCTURE results are shown in E. rubra, E. alpina var. alpina and E. alpina var. carmelitana (25.67 % P < 0.001). The highest percentage of variation was found within populations .AMOVA analysis  revealedE. alpina var. carmelitana as narrowly obovate to spatulate, lanceolate, tapering gradually to the base, with the apex acute to obtuse, rarely truncate . A MANOVA test also showed significant differences between the two varieties (P < 0.001).Geometric morphometry further reinforces these observations ; the PCAerlapped . The firE.alpina and E. rubra. Two populations , exhibited intermediate genetic characters and low genetic distance from E. rubra and E. alpina var. alpina; accordingly, both collections showed IM between E. alpina  and stem colour (which is affected by age and state of preservation). During the morphological study of collections of E.alpina var. carmelitana we observed differences in leaf shape rather than size. Leaf shape differences were strongly corroborated by geometric morphometrics analysis and this new character will be useful for species identification purposes.According to the protologue , E.alpiE.alpina var. carmelitana is a clearly distinct group, with genetic and morphological differences and with a restricted geographic distribution, which deserves to be considered an independent entity. Populations of E. alpina var. carmelitana are located between 35° and 38° S, 70° W: this region is characterized by two high mountain ranges (the Andes and Cordillera del Viento), volcanoes, e.g. Copahue, and valleys; the zone is well irrigated, with several tributaries of the Neuquén river, although there are only small lakes, e.g. Varvarco Campos and Caviahue  granted to M. Morando.S.S. and S.M. conceived the idea; S.S. collected the samples; S.S. and S.M. performed the molecular analysis; S.M. performed the geometric morphometric analysis; S.S. and S.M. analysed the data and led the writing.None declared.


The aim of this study was to develop an assessment tool for activities of daily living (ADL) from the perspective of patients with Parkinson’s disease (PD) and examine the validity and reliability of the assessment.A preliminary 45-item questionnaire was developed through intensive interviews with 54 patients with PD and administered to another group of 248 patients with PD. Based on clinical and statistical analyses, 20 ADL-items were selected. The final 20-item questionnaire was examined in the other group of 59 patients with PD.The new ADL questionnaire showed high internal consistency  and acceptable test-retest reliability (0.632–0.984). Concurrent validity was shown as a significant positive correlation between the new ADL questionnaire and other ADL or clinical instruments. The Hoehn and Yahr stage showed the highest degree of correlation with the new ADL questionnaire, followed by the other ADL scales . Additionally, a regression analysis was conducted with the disease-specific quality of life questionnaire, and the new ADL questionnaire was the most powerful predictor of quality of life among the clinical instruments.The new ADL questionnaire is a valid tool for assessing ADL from the perspectives of patients with PD. Parkinson’s disease (PD) is a chronic disorder due to the progressive degeneration of dopamine-producing cells in brain structures, including the substantia nigra . PD is cDespite the recent development of medical, surgical, and rehabilitative treatments, PD remains a progressive condition; there are no proven management strategies to reverse or halt its pathological processes. Patients with PD are at increased risk for decreased activities of daily living (ADL) with progression of the disease, particularly those requiring coordination and balance –5. The tSome studies have focused on the ADL of patients with PD. However, these studies have revealed limitations due to a lack of reliable and valid tools for assessing ADL from the patient’s perspective –10. The Improvements in motor symptoms, which are unresponsive to medical and surgical treatments, have been observed in patients with PD with recent advancements in rehabilitation therapy . These sWe developed a new ADL questionnaire from the patient’s perspective and assessed its validity and reliability in this cross-sectional study.Patients with PD were recruited consecutively from an outpatient clinic in a specialized center of a referral hospital. Patients were diagnosed with clinically probable PD according to the United Kingdom Parkinson’s Disease Society Brain Bank clinical diagnostic criteria . PatientMovement disorder and neurorehabilitation specialists reviewed motor aspects of disabilities of daily living in patients with PD with reference to various ADL scales and collected ADL items –25. An oAnother group of 248 patients with PD  were asked to select the items that they found difficult in their daily living and were asked to select the three items that were most important to them using the preliminary 45-item questionnaire.Clinical and statistical analyses were performed to select the ADL items. The frequency of items reflecting patient’s experience and importance and clinical significance for infrequent items, such as swallowing, were included in the clinical analyses. Statistical analyses were conducted using maximum likelihood to estimate the factor structure matrix. The number of items was determined using parallel analysis and the scree test. We used the Promax (Kappa = 2) oblique rotation method to simplify the structure, and each item was selected and classified based on a factor loading > 0.4.All items were given a score on a 5-point scale (0: no impairment of ADL and 5: inability to perform ADL) Appendix .Fifty-nine patients with mild-to-moderate PD were additionally recruited to validate the final 20-item questionnaire Table . We asset-test and Pearson’s chi-square test, as appropriate, to analyze differences between the two groups. A correlation analysis was conducted using Spearman’s method. The statistical analysis was performed using SPSS ver. 19.0 for Windows software . A P-value ≤ 0.05 was considered significant.All data are expressed as mean ± standard deviation for continuous variables and as proportions of patients (%) for categorical variable. The univariate analysis included Student’s P ≤ 0.05).The most common ADL items experienced by patients were getting up (61.7 %), dressing (57.7 %), and moving objects (56.9 %) Table . The mosA positive correlation was observed between the number of items patients found difficult and severity of the disease, as shown by the HY stage Table .Table 3TThe internal consistency of the new questionnaire was acceptable with an item-total correlation  and high consistency, as shown by Cronbach’s α of 0.962–0.966. Test-retest reliability was acceptable, reached at 0.632–0.984. Concurrent validity was detected as significant correlations between scores on the new ADL questionnaire and other ADL scales (SEADL and the ADL subscore of the UPDRS). The other clinical instruments were also correlated with the new ADL questionnaire, except the MMSE .The regression analysis for quality of life showed that the new ADL questionnaire was the most powerful predictor for quality of life in patients with PD followed by the ADL subscore of the UPDRS Table . The newThe number of ADL items that were difficult for patients with PD increased according to the HY stage using the 45-item questionnaire, indicating that progression of the disease increased ADL impairment. This finding suggests that items on the preliminary questionnaire covered ADL throughout the disease stages and comprehensively reflected the patient’s perspectives.Some differences were found in the list of selected items according to HY stage. However, the most common and important items for the patients were similar at all HY stages. Getting up was the most common, and walking outside was the most important activity. These findings suggest that mobility deserves special attention compared to others activities during the daily lives of these patients.Among the most frequently selected 10 items, only four  are present in the ADL section of the UPDRS. These findings suggest that the ADL section of the UPDRS may be insufficient to reflect the daily disabilities of patients with PD.The validity of the new questionnaire was acceptable, as shown by the correlation with other clinical measures. Concurrent validity was reasonable based on the high correlation of the new questionnaire with other ADL and clinical instruments, except the K-MMSE. Given that cognitive function can greatly affect ADL, this result may be distorted due to the limited distribution of the K-MMSE scores based on the exclusion criteria of this study. The new questionnaire showed the highest degree of correlation with HY stage and also had a high degree of correlation with other clinical instruments, such as the SEADL and the ADL section of the UPDRS. Moreover, the motor subscore of the UPDRS was marginally correlated with the new ADL questionnaire, but it was less correlated than depression, autonomic dysfunction, and fatigue. This finding is consistent with a recent report that severity of motor symptoms, as expressed by the motor subscore of the UPDRS, has less impact on the health status of patients with PD than non-motor symptoms .Quality of life may be the most important clinical measure representing the current status of patients with PD. Our results show that the new questionnaire was a strong predictor of quality of life in patients with PD among the clinical instruments evaluated in this study. A regression analysis showed that the new questionnaire and the ADL subscore of the UPDRS had strong predictive value for quality of life in patients with PD after adjusting of for age, disease duration, treatment, and daily levodopa dosage.Several limitations of this study should be discussed. Different importance could be assigned to some items in different cultures, such as “getting up from or sitting on the floor”. Thus, it may be necessary to validate the new questionnaire in different cultural areas. Also, there could be some biases from the predominance women in recruited patients at the evaluation of preliminary 45-item questionnaire. And, the new questionnaire showed relatively weak correlation with motor part of UDPRS, compared to other scales. This finding could suggest that the new questionnaire might not be sensitive enough as an assessment tool for motor changes responsive to physical therapy. Finally, this study was conducted using a cross-sectional design. Therefore, we failed to assess the feasibility of the new questionnaire for detecting changes along the course of the disease. This deserves further longitudinal studies.Despite these limitations, the new questionnaire has several advantages over other clinical instruments; it reflects actual patient disabilities during daily living based on a patient’s perspective. Changes in the ADL of patients could be easily and effectively evaluated, because the questionnaire is designed for self-administration. Thus, the new questionnaire is applicable to long-term follow-up in an actual clinical setting.The new questionnaire developed based on the perspective of patients with PD was found to be a valid tool for assessing ADLs of patients with PD.This study was approved by the Institutional Review Board of Dong-A University Hospital (No. 14–101) and patients submitted written informed consent.Not applicableAll the data supporting our findings is contained within the manuscript.


In October 2014, a hospital in Connecticut notified CDC and the Connecticut Department of Public Health of a fatal case of gastrointestinal mucormycosis in a preterm infant. The infant, born at 29 weeks’ gestation and weighing 1,400 grams (about 3 pounds), had developed signs and symptoms initially consistent with necrotizing enterocolitis approximately 1 week after birth. Exploratory laparotomy revealed complete ischemia of the gastrointestinal tract from the esophagus to the rectum; a portion of necrotic cecum was sent for microscopic examination. Following surgery, the infant developed multiple areas of vascular occlusion, including a large clot in the aorta, findings not usually associated with necrotizing enterocolitis. The infant died soon after. Histopathology results from the resected cecum revealed an angioinvasive fungal infection consistent with mucormycosis. Gastrointestinal mucormycosis is an extremely rare fungal infection caused by mold in the order Mucorales. It occurs predominantly in low birth weight infants, patients with diarrhea and malnutrition, and those receiving peritoneal dialysis; mortality is 85% , and the Connecticut Departments of Health and Consumer Protection initiated an investigation. ABC Dophilus Powder, manufactured by Solgar, Inc., Leonia, New Jersey, is a dietary supplement intended to contain three live bacterial species: a recall  advisingCDC initiated intensive case-finding efforts through several large clinician, laboratory, and public health networks for infants with gastrointestinal mucormycosis or for unexplained infant deaths following receipt of Solgar ABC Dophilus. No additional cases of gastrointestinal mucormycosis in neonates have been identified to date. Case-finding also was conducted by hospital staff members in the neonatal intensive care unit that had cared for the infant; no additional infections were identified.Dietary supplements thought to have probiotic effects have been reported to reduce the incidence of necrotizing enterocolitis and all-cause mortality in preterm infants , althoug


At the heart of most rehabilitation programs for people with hearing loss is the use of amplification. The purpose of hearing aid amplification is to improve a person's access to sound. Depending on the degree and configuration of the individual's hearing loss, the hearing aid is tasked with increasing sound levels at different frequency regions to ensure that incoming speech frequencies are reaching the ear at sufficient levels to compensate for the individual's hearing loss. However, a perceptual event is dependent not only on the audibility of the signal at the level of the ear, but also on how that sound is biologically coded, integrated, and used. As described by Tremblay and Miller in this special issue, this complex ear-brain neural network system starts with sound leaving the hearing aid. At this stage the acoustics of the amplified signal has been altered by the hearing aid. It is this modified signal that is being encoded at subsequent stages of processing: the ear, brainstem, midbrain, and the cortex. The integrity of the signal, and the biological codes, are therefore assumed to contribute to the resultant perceptual event and it is for this reason that the brain can be considered an essential component to rehabilitation. Yet, little is known about how the brain processes amplified sound or how it contributes to perception and the successful use of hearing aid amplification see .The intent of this IJO special edition is to integrate neuroscience and clinical practice to advance hearing health care. Such lines of inquiry can spawn new insight into stimulation-related brain plasticity that might, in turn, help explain why some individuals (but not others) report increased speech understanding while wearing their devices. From a clinical perspective, there is interest in determining if measures of brain activity might be of use to the clinician, during hearing aid selection and fitting, as well as to the engineers who are designing the instruments. However, to move forward, there are unresolved issues that can appear conflicting to the clinician and/or scientist who wish to embark in new research directions or clinical services. For this reason, a call for papers on the topic of Hearing Aids and the Brain was made in an effort to define converging evidence. What resulted is a collection of papers from different laboratories that identify caveats and concerns, as well as potential opportunities.The collection of papers addresses two main questions: (1) Is it possible to use brain measures to determine a person's neural detection of amplified sound? (2) Can brain measures provide information about how the brain is making use of this amplified sound? Before we summarize the answers to the questions, it's important to review the framework for including objective measures to examine the neural representation of amplified sound.When a person is fitted with a hearing aid, there are two categories of test procedures involved in the process: (i) behavioral measures, which require active participation by the patient , and (ii) objective measures, where subjective responses are not required . Here we expand the use of electrophysiology to determine if brain measures can be used as an objective measure to estimate aided thresholds and/or quantify hearing aid transduced signals in the brain for the purpose of guiding device fitting and/or to assess suprathreshold representation of amplified auditory signals in the brain to estimate perceptual performance and/or the related cognitive resources involved. This expanded use of objective measures is relevant to patients of all ages but is particularly germane to the pediatric population where the use of behavioral tools is limited. As described by L. M. Jenstad et al. in this issue, behavioral threshold information is not usually available before age 6 months (and often later), speech testing is unavailable, and subjective questionnaires are limited to caregiver observation of behaviors. Thus, there is greater reliance on objective procedures to measure the effects of amplification beyond the tympanic membrane in infants and young children. One such measure is the use of auditory evoked potentials (AEPs).The use of AEPs to aid in clinical assessment is not new. Click-evoked auditory brainstem responses were tried long ago to estimate unaided and aided thresholds in infants and young children. However they proved to be unsuccessful because the short duration signal  interacted with the hearing aid circuitry in a way that introduced ringing and other artifacts . In thisThere is much literature exploring the role of CAEPs in assessing people with hearing loss, but the inclusion of people with hearing loss who wear hearing aids is still quite sparse. In this special issue, investigators from different laboratories describe some of caveats and concerns when measuring evoked CAEPs in combination with hearing aid amplification. L. M. Jenstad et al. showed that CAEPs P1-N1-P2) do not reliably reflect hearing aid gain, even when different types of hearing aids  and their parameters  are manipulated. As shown in  do not rL. M. Jenstad et al. describe how these issues impact the clinician. It would be possible to appropriately fit a hearing aid (for degree and configuration of the hearing loss) in a client but show no evoked brain response or no improvement from the unaided cortical response, even though, behaviorally, the client shows clear perceptual improvements. In another manuscript, D. Glista et al. also show instances when cortical P1-N1-P2 responses are absent in normal hearing children. Even when cortical evoked potentials are present, their peak morphology may or may not reflect defining characteristics of the brain response. Instead they could reflect aspects of signal processing influenced by the hearing aid.We know very little about this last point, and the research published in this issue attests to this. L. M. Jenstad et al. published examples where differences in the type/brand of hearing aid resulted in substantial differences in cortical activity, even though standard electroacoustic measures indicated no major differences among the hearing aids. In contrast, D. Glista et al. presented a few pilot cases where altering frequency compression hearing aid parameters not only improved audibility of a 4 kHz tone burst but also improved detection of cortical evoked responses. So, it appears that the presence of P1-N1-P2 in response to an amplified signal indicates the neural detection of a sound at the level of the cortex, but the morphology of the response (latency and amplitude) may say more about the signal processed acoustics of the amplified sound than the status of the neural mechanisms generating the response. A dilemma ensues when the CAEP is absent. The absence of a brainstem or cortical response, or the absence of change in the evoked brain activity, may result from a number of variables not yet fully understood and therefore complicating the use of AEPs as a sensitive and specific metric of hearing loss and abnormal brain processing. To summarize then, when examining the effect of hearing aid amplification on brain maturation or plasticity, the presence or absence of change over time might simply reflect changes in signal alterations introduced by the hearing aid within a single recording or between sessions. In all cases, changes to the hearing aid prescription can be presumed to contribute to changes in the evoked neural responses. The same can likely be said for cochlear implants too. For any scientist or clinician to overlook the contribution of the hearing aid-transduced signal to stimulation-related patterns in the brain is to overlook a core essential variable.While the previously expressed concerns highlight the cautionary aspects of combining brain measures and hearing aid amplification, the contribution of device-related changes to evoked brain activity could also be viewed as an opportunity to study the utility of specific hearing aid algorithms in a way that could be used as an outcome measure. This opportunity assumes two things: (1) it is possible to reliably characterize signal processing schemes specific to each device in a way that contributes information to the hearing aid fitting process in an efficient and informative way, and (2) that clinicians (who are reluctant to use existing technology such as probe microphone technology) would be accepting of adding an additional measure to their protocol.However, as this direction of ear-brain testing continues, future research is needed to explore appropriate test stimuli and presentation paradigms when hearing aids are included in research. As described by D. Glista et al, we need a better understanding of the testing conditions and stimuli that yield the most valid measures electrophysiologically in relationship to behavioral measures. Traditional presentations of speech stimuli, with tones or speech syllables being presented in isolation interleaved with silent periods so the necessary average brain responses which can be obtained, might not be a comparable auditory experience to the running speech that is usually delivered to the brain by the hearing aid. For example, in this issue, V. Easwar et al. evaluated the output levels of 10 different hearing aids for phonemes in isolation and in running speech to determine the effects of processing strategies on the output level of each. Their results show remarkable differences in sound level and hearing aid activation, depending on the method of stimulus presentation. This means that different conclusions could be drawn for the same person, depending on the way in which the stimuli interact with the hearing aid. A more optimistic spin on this finding could be that it might be possible to use cortical activity to assess the effects of hearing aid fine-tuning , or other aspects of hearing aid signal processing, such as frequency lowering, as illustrated by some participants reported by D. Glista et al. However, validation of aided findings in larger groups of participants with hearing loss including infants and in young children would be needed to establish valid clinical interpretations both for present and absent aided CAEPs. Particularly for assessing the outcome of an infant's hearing aid fitting, appropriate interpretation of an absent CAEP is critical, so that appropriate communication of results to caregivers can take place.Another important point is that there might be optimal montages for recording brainstem and cortical activity that have not yet been realized. Even though most published data to date show that it is possible to record brainstem and cortical responses using only a small number of electrodes, this need not mean that the routine recording montage typically used in clinical settings is the most sensitive and specific recording approach for research purposes. A recent example of what information can be lost when using a standard vertex recording of the P1-N1-P2 complex in adults can be found in Tremblay et al.. They shAnother potential area of research is to examine the interaction between onset and change responses evoked by the auditory cortex. This information provides an objective quantification of the relationship between the onset of the processed signal versus changes within a between processed sounds. The P1-N1-P2 cortical response is appropriate for this purpose because it is sensitive to the onset of sound as well as to acoustic changes within an ongoing sound , 5. WhenFor example, brain measures could guide manufacturer designs. Knowing how the auditory system works and how it responds to gain, noise reduction, and/or compression circuitry could influence future generations of biologically motivated changes in hearing aid design. Biological codes have been harnessed to drive the motion of an artificial limb/prosthesis, it might therefore be possible one day to design a hearing prosthesis that includes neuromachine interface systems driven by a person's listening effort or attention. At this stage, however, finding ways to quantify a person's listening effort is still being defined. For example, demonstrating the benefits of digital noise reduction algorithms, as implemented in hearing aids, on improved speech recognition in noise has been elusive. Anecdotal reports suggest that such algorithms may provide nonspeech perception benefits such as improved ease of listening. Therefore physiologic measures of stress and effort are being explored to determine if they could provide an objective measure of nonspeech benefits. One such example is pupillometry. In this special issue, T. Koelewijn and colleagues were able to demonstrate a difference in the amount of pupil dilation among normal hearing participants listening to speech masked by fluctuating noise versus a single talker. The authors posit that the degree of pupil dilation may reflect the additional cognitive load resulting from the more difficult listening task and suggest that pupillometry may offer a viable objective measure of the benefits associated with specific hearing aid signal processing features such as digital noise reduction.However, as illustrated by K. L. Tremblay and C. W. Miller, the successful use of hearing aids to improve human communication will ultimately depend on more than just brain measures. Many factors can contribute to aided speech understanding in noisy environments, including device centered  and patient centered variables . Research aimed at exploring one variable in isolation  is likely to fall short when trying to optimize the many interactive stages involved in human communication.To summarize and conclude, the collection of articles that appear in this special issue of IJO show that (1) it is possible to use brain measures to determine a person's neural detection of amplified sound and (2) brain measures can provide information about the use of this amplified signal sound. The use of brain measures to quantify and model neural mechanism associated with the perception of amplified sound is complex and sometimes counter-intuitive. For this reason, it is important to remind clinicians and neuroscientists that the interpretation of aided evoked neural activity cannot simply be based on prior published data acquired from normal hearing or unaided participants.K. L. TremblayK. L. Tremblay S. Scollie S. ScollieH. B. AbramsH. B. AbramsJ. R. SullivanJ. R. SullivanC. M. McMahonC. M. McMahon


Binaural pitch diplacusis refers to a perceptual anomaly whereby the same sound is perceived as having a different pitch depending on whether it is presented in the left or the right ear. Results in the literature suggest that this phenomenon is more prevalent, and larger, in individuals with asymmetric hearing loss than in individuals with symmetric hearing. However, because studies devoted to this effect have thus far involved small samples, the prevalence of the effect, and its relationship with interaural asymmetries in hearing thresholds, remain unclear. In this study, psychometric functions for interaural pitch comparisons were measured in 55 subjects, including 12 normal-hearing and 43 hearing-impaired participants. Statistically significant pitch differences between the left and right ears were observed in normal-hearing participants, but the effect was usually small . For the hearing-impaired participants, statistically significant interaural pitch differences were found in about three-quarters of the cases. Moreover, for about half of these participants, the difference exceeded 1.5/16 octaves and, in some participants, was as large as or larger than 1/4 octave. This was the case even for the lowest frequency tested, 500 Hz. The pitch differences were weakly, but significantly, correlated with the difference in hearing thresholds between the two ears, such that larger threshold asymmetries were statistically associated with larger pitch differences. For the vast majority of the hearing-impaired participants, the direction of the pitch differences was such that pitch was perceived as higher on the side with the higher  hearing thresholds than on the opposite side. These findings are difficult to reconcile with purely temporal models of pitch perception, but may be accounted for by place-based or spectrotemporal models. Binaural pitch diplacusis—also referred to as ‘binaural harmonic diplacusis’ or, in short, ‘binaural diplacusis’–refers to a perceptual anomaly, whereby the same tone is perceived as having a different pitch depending on whether it is presented in the left ear or the right ear of the same listener –11. ResuIn addition to being of theoretical interest, the existence of pitch discrepancies between the two ears may be relevant for understanding the perceptual difficulties experienced by hearing-impaired listeners. Such discrepancies may be indicative of a mismatch between the information received by the left and right ears, which may impact the combination of information across the two ears. In this context, it would be useful to know how prevalent this effect is in hearing-impaired individuals, and how its magnitude can be predicted based on audiometric measures such as hearing thresholds. Although, as mentioned above, binaural diplacusis appears to be more prevalent, and more pronounced, in individuals with unilateral or asymmetric hearing loss than in individuals with normal hearing –11, all In this study, binaural diplacusis was assessed in 55 participants, including 43 individuals with high-frequency hearing-loss of presumed cochlear origin, and 12 individuals with normal hearing thresholds. In the hearing-impaired (HI) participants, binaural diplacusis was assessed at two frequencies, 500 Hz and a higher frequency close to the audiogram ‘cutoff’–defined as the point where the slope of the audiogram became steeper. Based on previous findings, the following predictions were made. Firstly, binaural diplacusis  should be more prevalent, and the size of the pitch difference between the left and right ears should be larger, in individuals with hearing loss than in individuals with normal hearing. Secondly, the magnitude of the pitch difference between the left and right ears should tend to increase with the magnitude of the asymmetry in hearing thresholds. Thirdly, in individuals with asymmetric hearing thresholds, for a given stimulus frequency, the pitch should be higher in the ear with the larger  hearing thresholds. Fourthly, all other things being equal, binaural diplacusis should be less prevalent, and less marked, at 500 Hz than at higher frequencies. The aim of this study was to test these predictions. Results were consistent with the first three predictions, but not with the latter.Twelve  normal-hearing (NH) and 43  HI individuals were tested. The NH participants were aged between 21 and 52 years . The HI participants were aged between 42 and 79 years . All HI participants had hearing loss sloping toward high frequencies. For about half (20) of these participants, the hearing loss was asymmetric across the two ears, with an interaural difference in absolute thresholds of at least 15 dB at the audiogram ‘cutoff frequency’ (Fc) in the participant’s ‘better’ ear, i.e., the ear with lower average absolute thresholds, where the average was computed across the following audiometric frequencies: 500, 1000, 2000, and 4000 Hz. The audiometric frequencies tested were: 250, 500, 1000, 1500, 2000, 3000, 4000, 6000, and 8000 Hz. To determine Fc, we computed the audiogram slope per octave. For most patients, Fc was determined as the lowest of the two consecutive audiometric test frequencies for which the slope of the audiogram (absolute thresholds as a function of frequency) was higher than 10 dB. For 8 patients who had an almost flat audiogram, and in whom the audiogram slope was never higher than 10 dB, Fc was set to 2000 Hz, the mode of the statistical distribution of Fc across HI participants for whom Fc could be determined using the audiogram slope-difference criterion.The study was approved by the institutional review board of the Institut de Sciences et Techniques de Readaptation, Université Lyon I, and was performed in accord with the Declaration of Helsinki. All participants provided written informed consent.Each participant was tested individually as follows. Firstly, pure-tone hearing thresholds were measured using the procedure described in . Hearingf), and the participant was instructed to indicate which of the two tones presented on a trial  was louder than the other. Participants’ responses were recorded by the experimenter on a computer using a screen interface with two response buttons labeled ‘1’ and ‘2’. The level of the tone in one ear (hereafter referred to as the ‘reference’ ear) was fixed at 70% of the dynamic range (defined as the difference between the hearing threshold and the MCL) for the current test frequency (f). The level of the tone in the contralateral ear (the ‘comparison’ ear) was adjusted using an adaptive one-down, one-up procedure. The procedure stopped after two reversals in the direction of the change in level. The adaptive procedure was run twice, first, with the tone in the comparison ear starting at a higher level than the tone in the reference ear, and a second time, with the converse situation. The mean level at the reversal points across these two runs of the procedure was used to determine the level of the comparison tone needed for this tone to have approximately the same loudness as the reference tone. This loudness-comparison test was performed at three frequencies: fref, fref –1/4 octave, and fref +1/4 octave, where fref denotes the ‘reference’ frequency. The reason for testing– 1/4 octave and +1/4 octave around fref is that these span the frequency range around fref tested in the subsequent pitch-comparison test; this made it possible to determine the level needed for the comparison tone to have approximately the same loudness as the reference tone, for all comparison-tone frequencies tested in the pitch-comparison test.For the loudness-comparison test, the two tones had the same frequency  had a higher pitch than the other. Participants’ responses were entered by the experimenter on a computer using a visual interface with two buttons labeled ‘1’ and ‘2’. The level of the reference tone was fixed at 70% of the dynamic range (as in the preceding loudness-comparison test), while the level of the comparison tone was adjusted based on the results of the preceding loudness-comparison test so that, on average, the loudness of the comparison tone was approximately equal to the loudness of the reference tone. In order to further limit any systematic influence of loudness differences on the participant’s responses in the pitch-comparison test, the level of the comparison tone varied randomly across trials over a 10-dB range centered on the nominal level. The frequency of the reference tone was fixed. The frequency of the comparison tone was varied across trials over a half-octave range (+/- 1/4 octave) in 1/16-octave steps. Each combination of reference and comparison frequency was presented 10 times to each participant, in random order.fref’s. For the NH participants, fref’s of 500, 1000, 2000, and 4000 were tested. For HI participants, two fref’s were tested: 500 Hz, and either 3000 Hz , or Fc (for participants who had high-frequency hearing loss with a well-defined edge frequency). Fc was equal to 750 Hz for two HI participants, to 1000 Hz for 6 HI participants, to 1500 Hz for 10 HI participants, to 2000 Hz for 19 HI participants, to 3000 Hz for 5 HI participants, and to 4000 Hz for one HI participant. The 500-Hz fref was included because, for such a low frequency, there is psychoacoustic evidence that human listeners can make use of temporal fine-structure information for perceptual discrimination [The above-described combination of loudness- and pitch-comparison tests was performed at different reference frequencies, mination . Accordifref of 1000 Hz. These fits were used to extract the point of subjective equality (PSE), defined as the frequency for which the psychometric function reaches 50%, which corresponds to the frequency for which the comparison tone should be perceived as having the same pitch as the reference sound. These PSEs were used, in turn, to compute PSE shifts, defined as the difference between the PSE and fref. The PSE shifts reported below are expressed in units of 1/16 octave, the stepsize used in the pitch-comparison test.For each combination of reference and comparison sound frequency, the number of trials for which the participant indicated perceiving the comparison sound as having a higher pitch than the reference sound was computed. These count data were fitted using a logistic regression model with a binomial error distribution. To assess whether the PSE shifts differed significantly from zero (no shift), we computed 95% credible intervals  for each PSE shift. This was done using Markov-Chain Monte Carlo algorithms implemented in the OpenBUGS software .Sennheiser HD 202 headphones.All tests were performed with the participants in an audiometric testing cabin. Hearing thresholds were measured using either an Aurical (Gn Otometrics) or an Affinity (Interacoustics) system with Sennheiser HD 202 headphones. For the pitch-perception tests, stimuli were generated and presented using Matlab. They were converted to analog signals  using a SoundBlaster X-FI HD SB1240 CREATIVE sound card, and delivered to the participants’ ears through fref, left ear). Therefore, when analyzing the data of the HI listeners, pitch shifts smaller than 1.5/16 octave (about 7%) may be regarded as ‘normal’.When looking at these individual data, an important question, is whether any of these pitch shifts is statistically significant, or whether all of these shifts can be confidently imputed to measurement error. To address this question, we examined 95% Bayesian confidence intervals for the individual pitch shifts. These intervals were computed with the assumption that the count data that were used to fit psychometric functions followed a binomial distribution. Whenever the 95% confidence intervals computed in this way did not include zero, we concluded that the pitch shift measured in the considered participant and test condition could not be imputed to limited precision of the psychometric procedure used. Using this approach, we found that 23 out of the 96 pitch shifts (or about 24%) measured in these NH participants could not be imputed simply to measurement error.In order to determine whether PSE shifts varied significantly as a function of the test frequency, or to differ between the left and right ears, we computed mean PSE shifts and their associated 95% Bayesian confidence intervals. The results are shown in Using 1.5/16 octave (or 7%) as a normative boundary, we found that 10 out of 43 HI participants had abnormally large pitch shifts at 500 Hz, while 20 out of 43 had abnormally large shifts at Fc. For a vast majority of these subjects , the direction of the shift was consistent with pitch being perceived as higher on the side with larger  hearing thresholds than on the opposite side.Note that, although the range used to measure psychometric functions in the interaural pitch-comparison test was limited to +/- 1/4 octave, pitch shifts larger than 1/4 octave could be, and sometimes were, obtained using logistic-regression analysis. These large pitch shifts must be interpreted cautiously, since they are based on extrapolation beyond the actual range of the data.To determine whether the pitch shifts that were not statistically significant could be accounted for by shallow psychometric-function slopes, indicating poor or highly variable interaural pitch-matching abilities, we examined the relationship between the psychometric-function slope and the pitch shifts. p = 0.03); for Fc, the Pearson correlation coefficient was -0.45 (p = 0.003). A significant correlation was also found between the pitch shifts at 500 Hz and the pitch shifts at Fc . However, it might be argued that the correlation at Fc is driven to a large extent by one data point, from the participant who had a large negative estimated pitch shift (about 3/4 octave). When this data point was removed, the correlation became subsantially lower, and was no longer statistically significant .Results of previous studies suggest that binaural diplacusis can be observed in individuals with NH, although the effect usually remains small –30. The Also consistent with earlier findings, the results of the present study indicate that binaural diplacusis is more prevalent, and the magnitude of the effect is often larger, in HI participants than in NH participants. For the test condition in which the reference frequency corresponded to the cutoff frequency (Fc) of the audiogram, approximately two thirds of the HI participants showed statistically significant interaural pitch shifts, and for about half of the HI participants, the pitch shifts were abnomally large, i.e., larger than 1.5/16 octave (about 7%). These proportions were somewhat lower for the 500 Hz test frequency; however, even for this low frequency, about one fourth of the HI participants displayed abnormally large interaural pitch shifts.a priori for the HI participants. However, we found no statistically significant difference in the slopes of the psychometric functions for the interaural pitch-comparison task between the HI and the NH participants. Moreover, the small and statistically non-significant pitch shifts in the HI participants were not systematically associated with shallow psychometric-function slopes. Thus, we may conclude that, at least for some HI participants, the absence of significant binaural diplacusis is not due merely to a poor or highly variable interaural pitch-matching ability.The lack of statistically significant binaural diplacusis in some of the HI participants might be due to these individuals having poor or highly variable interaural pitch-matching abilities, which should be reflected in shallower psychometric-function slopes in the interaural pitch-comparison task for these participants than for other participants. In particular, based on data from previous studies showing poorer or more variable performance in monaural or interaural pitch-comparison tasks for HI listeners than for NH listeners , poorer-Based on the findings of earlier studies, we expected the magnitude of binaural diplacusis to increase with the interaural asymmetry in hearing thresholds. The results are partly consistent with this expectation, in that significant correlations were found between the interaural difference in hearing thresholds and the left-right pitch shifts for both test frequencies (500 Hz and Fc). However, these correlations were relatively low, with the interaural asymmetry in hearing thresholds accounting for only 22% of the variance in the interaural pitch shifts, at most. Moreover, the correlations were not very robust; for the condition in which the reference frequency was equal to Fc, the correlation vanished after one participant, who was possibly an outlier, was removed. These results suggest that binaural diplacusis depends on other factors, besides factors related to the interaural asymmetry in hearing thresholds. At present, these other factors remain poorly understood. They could stem from peripheral and/or central modifications in auditory function, which accompany hearing loss, but which do not manifest as shifts in absolute thresholds (see next section).Interesting, for the vast majority of the HI participants, the direction of the pitch shifts was consistent with pitch being perceived as higher on the side on which hearing thresholds were higher (‘worse’), compared to the contralateral side (‘better’ ear). This finding provides some constraints on the nature of the underlying biological mechanisms of binaural diplacusis, as discussed in the following section.An effect, which could have contributed to the results of this study, relates to shifts in pitch with sound intensity. Specifically, previous studies have found that, as stimulus intensity increases, the pitch of pure tones tends to shift toward higher frequencies above 1 kHz, and toward lower frequencies below 1 kHz –32–althobelow the audiogram cutoff frequency. Although the effect was a little less prevalent at 500 Hz than at Fc, the fact that it was observed at 500 Hz as well, is difficult to reconcile with a central-reorganization model in which central reorganization occurs only at, or near, the region in the tonotopic map that corresponds to the audiogram edge.Another effect, which may have contributed to the results of this study, relates to central neural plasticity. Several studies have shown cortical reorganization of tonotopic maps following peripheral damage –38. SpecAn alternative explanation for these results is in terms of changes in cochlear mechanics following cochlear damage. Physiological observations indicate that, following cochlear damage, the peak of excitation on the basilar membrane shifts toward a more basal region . A corolpositive abnormal  pitch shifts, indicating a higher pitch on the side with the lower  hearing thresholds than on the opposite side. For one participant, the positive pitch shift was observed in the condition where fref was equal to 500 Hz; for the other participant, it occurred in the condition where fref was equal to Fc. These positive pitch shifts may have been mediated by a different mechanism than the damage-induced basal shift observed in the physiological studies cited above. Some studies have found upward shifts in the best frequency of auditory nerve fibers with increasing stimulus level for frequencies lower than about 1 kHz, contrasting with downward shifts for higher test frequencies [One limitation of the damage-induced basal-shift explanation outlined in the previous paragraph stems from the fact that not all of the interaural pitch shifts measured in this study were in the same direction. Specifically, two HI participants showed quencies –41. It iAn extreme form of peripheral damage corresponds to a ‘dead cochlear region,’ which is a region of the cochlea with no residual functional hair cells or auditory-nerve fibers. Using pitch matches across the two ears, and monaural octave matches in HI listeners with cochlear dead regions, a previous study found that pure tones with frequencies falling inside a dead region evoked an unclear and/or abnormal . AlthougWhile the results of the present study are, for the most part, consistent with an injury-induced basal-shift in the cochlear mechanics, they support the view that pitch perception depends on a place code –2. HowevNonetheless, in the current state of knowledge, the results of the present study and those of previous studies of binaural diplacusis are difficult to reconcile with purely temporal theories of pitch coding, according to which pitch is encoded in the fine timing of neural spikes evoked by the stimulus ;44–46. WS1 TableOpenBUGS code for the Bayesian model used analyze the pitch-comparison data of the normal-hearing and hearing-impaired participants(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.