Clinical observations suggest that tinnitus may interfere with programmingcochlear implants (CIs), the process of optimizing the transmission of acousticinformation to support speech perception with a CI. Despite tinnitus beinghighly prevalent among CI users, its effects and impact on CI programming areobscure. This study characterized the nature, time-course, and impact oftinnitus effects encountered by audiologists and patients during programmingappointments. Semistructured interviews with six CI audiologists were analyzedthematically to identify tinnitus effects on programming and related copingstrategies. Cross-sectional surveys with 67 adult CI patients with tinnitus and20 CI audiologists in the United Kingdom examined the prevalence and time-courseof those effects. Programming parameters established at CI activationappointments of 10 patients with tinnitus were compared with those of 10patients without tinnitus. On average, 80% of audiologists and 45% of patientsreported that tinnitus makes measurements of threshold (T) levels more difficultbecause patients confuse their tinnitus with CI stimulation. Difficultiesappeared most common at CI activation appointments, at which T levels weresignificantly higher in patients with tinnitus. On average, 26% of patientsreported being afraid of “loud” CI stimulation worsening tinnitus, affectingmeasurements of loudest comfortable (C) stimulation levels, and 34% ofaudiologists reported observing similar effects. Patients and audiologistsreported that tinnitus makes programming appointments more difficult andtiresome for patients. The findings suggest that specific programming strategiesmay be needed during CI programming with tinnitus, but further research isrequired to assess the potential impact on outcomes including speechperception. Patients and clinicians have identified the treatment of tinnitus in people withsevere-to-profound deafness as one of priorities for further research . TinnituTinnitus occurs in approximately 80% of candidates for implantation and is rTinnitus effects on measuring behavioral thresholds are not unique to CI programming.It has long been known that tinnitus can also cause artifacts when measuringconventional air-conduction hearing thresholds  differ in patients with tinnitus from those without tinnitus.The study comprised three parts. In the first part, six audiologists from twomajor implant centers in the United Kingdom, the Nottingham Auditory ImplantProgramme (NAIP) and the Midlands Hearing Implant Programme, were interviewed tocharacterize tinnitus-related effects during CI programming appointments.Purposive sampling was used to recruit senior audiologists who specialized inthe postoperative management of adult CI recipients. The interviews lasted forapproximately one hour, and audio recordings were made for later transcriptionand then destroyed. Interviews were conducted in person wherever possible or bytelephone where the clinical schedule of the interviewee did not allow for aface-to-face meeting to be held within the time frame of the study.Two cross-sectional surveys were conducted in the second part of the study tofurther capture whether audiologists and patients experience tinnitus-relatedeffects that cause difficulties during CI programming appointments, howfrequently they occur, and over what time-course. A national survey targetingaudiologists with experience of managing adult CI patients across all 18auditory implant centers that provide services to adults in the United Kingdomwas advertised through the British Cochlear Implant Group. CI audiologists inthe United Kingdom would typically have experience of programming devices fromthree major manufacturers: Cochlear Ltd., Advanced Bionics, and Med-El. Twentyaudiologists from nine centers, representing 50% of adult CI services in theUnited Kingdom, took part in the survey. An invitation to participate in apostal survey to explore the potential burden arising from tinnitus afterimplantation was also sent to all adult CI recipients from the NAIP of whom 128(20%) responded to the survey. Of the 128 respondents, 67 (52%) patientsreported experiencing tinnitus. The proportion of survey respondents withtinnitus was almost identical to and representative of the proportion of CIusers experiencing tinnitus (50%) found in a population study in the UnitedKingdom . The 67 In the third part, programming parameter data  wereextracted from the clinical notes of a group of 20 CI users who had givenwritten consent for their clinical records to be accessed. Only data fromrecipients of Cochlear Ltd. devices were extracted as both T and C levels aremeasured routinely as part of the recommended programming procedure of thosedevices, rather than set automatically during programming appointments cf., and theThe study followed the principles of the Declaration of Helsinki. Audiologistsconsenting to participate in the interviews also gave written consent to includetheir data in the study. Completion of the survey was taken as informed consentto participate. Separate written consent was obtained to access the clinicalnotes of CI patients participating in the postal survey. The study was approvedby the National Research Ethics Service Committee South East Coast—Surrey,United Kingdom.A schematic of the interview schedule is shown in Information about programming difficulties related to tinnitus was collected fromaudiologists and CI users as part of surveys exploring the burden from tinnitusafter cochlear implantation in the U.K. population . SurveyThe survey asked respondents to agree or disagree with statements about thepotential effects on the measurement of T and C levels due to tinnitus.Respondents were then asked to judge whether these effects, if present, had animpact in terms of creating difficulties with programming and increasing thelevel of tiredness experienced by the CI patient. Audiologists were asked toreport if they experienced these effects and impacts at three points in time: atthe first CI activation, at follow-up appointments between 6 and 12 months afterfirst activation, and at follow-up appointments >12 months after firstactivation. CI recipients were asked to report whether they personallyexperienced these effects and impacts at first activation, at follow-upappointments between 6 and 12 months after first activation, and at their mostrecent appointment. The 6- and 12-month cutoffs were chosen as routine timepoints for appointments following CI activation and because the alleviation oftinnitus and related symptoms has been found to occur on average within 6 to 12months following implantation .Two additional questions were included in the audiologist survey to assess theirviews on the need to provide advice on managing tinnitus to patients and theneed to use specific programming procedures for patients with tinnitus. Theresponses to these questions were compared with the analogous findings from theinterview data.… it’s almost like a distraction technique really … But thereare some people you can do loads of [different techniques toovercome tinnitus effects] and I would say there’s many a time whenyou can’t be a hundred percent sure that you’ve got an accurate Tlevel.In the next phase of the analysis, the codes were organized intothemes and reviewed alongside the entire data set by the same researcher usingthe following criteria: (a) the collated interview extracts are representativeof and support the themes, (b) the themes are consistent with the overallnarrative when compared against the entire data set, and (c) the assessment ofpotential relationships between the themes confirms a theme’s independence orsuggests the emergence of a dominant, overarching theme or a set of relatedsubthemes. The themes and subthemes were then reviewed by two other researchers,one of whom had also undertaken an independent familiarization with the data setby an in-depth reading of the interview transcripts. The final choice and namingof themes was arrived at by consensus among the three researchers.The interviews were coded using the NVivo 10 software  and analyzed thematically following the methodologyoutlined by Descriptive statistics were used to summarize the prevalence of tinnitus-relatedeffects and impacts on programming appointments reported in the surveys,separately for audiologists and CI users. The comments from audiologists on thereported tinnitus-related advice and specific programming strategies used toovercome programming difficulties due to tinnitus were compared with those foundin the interview data.p < .05.For the analysis of programming data, T and C levels for each active electrode ofeach patient were extracted from their earliest programming MAP. Their earliestMAP was defined as that which was measured at their first CI activationappointment and subsequently programmed into their speech processor. The T and Clevels reported by the programming software Custom Sound  were first converted into microamperes using conversion formulaeobtained from the manufacturer and then to decibels or more commonly as a reduced awareness of tinnitus.The reduced awareness was also associated with the presence ofelectric stimulation .However, audiologists also reported that tinnitus can bebothersome despite using a CI and that tinnitus can worsen in some CI recipients afterimplantation .A common backdrop narrative throughout all interviews related to therecognition by the audiologists that they commonly observe the alleviationof tinnitus following cochlear implantation. This benefit to tinnitus wasexpressed in different ways, either as a degree of suppression of thetinnitus sound itself or as expressed by audiologist A3:you’re trying to get [patients] to focus on a sound … ,you’re getting them to sit in silence … Most of the day they’rehaving their implant [switched] on. You … hook them up to thecomputer, they’ve got no other sound, they’re profoundly deaf,it’s completely silent and you’re asking them to focus on[programming stimuli] bleeps  … it doesn’t help tinnitus thatsort of situation where they’re putting all their focus onlistening to little [soft] sounds …  it seems to make it worse[for tinnitus].However, some audiologists also reported specific issues withprogramming in CI recipients who perceive their tinnitus during programmingappointments even though their CI is switched on . The audiologists also described situations inwhich restored awareness of tinnitus during T level measurements can lead toconfusion between tinnitus and the stimuli being presented:[patients are] tapping away [with a pen to signal hearing thestimulus] and I’m not doing anything [with stimuluspresentation] … either their tinnitus will mirror the sound thatyou’re playing or tinnitus [sounds the] same … so [tinnitus]just keeps going and going, and going so it can be moredifficult. (A4)Distinguishing between the stimulus and the patient’s tinnitusalso appeared to be more difficult when the pitch of the presented stimulusresembles the perceived pitch of tinnitus (A5: you’ll oftenfind … certain electrodes which match up nicely to the tinnitus pitchand [patients] find those ones difficult). However, oneaudiologist was of the opinion that difficulties are not specific to aparticular pitch or electrode .All audiologists reported experiencing difficulties due to tinnitus during CIprogramming. The audiologists focused primarily on the disruptive effectsthat a patient’s awareness of their tinnitus can have on T levelmeasurements. They reported that their patients become aware of theirtinnitus during appointments when the implant is switched off and they areencouraged to focus on soft sounds during measurements of T levels:I’m not sure that [tinnitus] does necessarily [affectC levels]; A2: I wouldn’t say [measuring C levels] isparticularly different when they have tinnitus; A6:I’ve never known tinnitus to have an effect on comfort levels).Two audiologists did acknowledge that an effect on C levels may be possible,but this was not supported by examples of particular experiences duringappointments .Evidence of tinnitus-related effects on the measurement of C levels waslimited. When asked about C level measurements directly, the audiologistsdid not feel that this aspect of programming was particularly affected bytinnitus (A3: tinnitus is less of a problem if you switch from T to Clevels) and that they would also alternate between thresholdand suprathreshold stimulation to overcome tinnitus-related difficulties(A1: I would move away from the threshold levels and onto the louderlevels … and then go back to the threshold levels to see ifyou’ve … moved their attention away [from tinnitus] slightly).Other audiologists reported repeating stimulus bursts and asking patients tonot only report when but also how many sounds they hear, for example . Some audiologistsreported that asking patients to count stimulus bursts could also be used toascertain the accuracy of measurements (A2: if you want to be sureabout a T level … then yes you would ask to count the beeps so you knowit’s a true threshold … that’s standard practice). Threeaudiologists reported alternating stimulation between the low- andhigh-frequency electrodes as an effective strategy to disrupt the patient’sfocus on their typically high-pitched tinnitus:if tinnitus, say, is particularly high-pitched and [patients]are having a lot of trouble with the high-pitched T levels thenI might change … the frequency to a more apical electrode whereit’s a deeper sound … to try and get their attention away fromthat continual sound they’re hearing. (A1)or audiologists would move across different parts of theelectrode array:I would certainly move to a different part of the electrodearray … start in the middle and instead of then laboriously gothrough [the electrode array], move around and then [patients]are getting a novel stimulation from … a differentpitch. (A2)However, some audiologists were of the opinion that changingthe electrode channels would not make a difference.[The tinnitus effect] seems to be consistent across all[electrodes] so I’m not sure whether jumping around would makeany difference … you sometimes do that anyway as part of themapping but it doesn’t seem to have a big influence.(A3)Audiologists described various ways of turning the patient’s attention awayfrom tinnitus as effective strategies for dealing with tinnitus-relateddifficulties during T level measurements. For example, one audiologistcommented that alternating between different assessments is helpful . Some audiologists acknowledged potential issueswith the accuracy of measured T levels , but this effect of tinnitus was notalways regarded as a major issue during CI programming (A2: I’m notruling [tinnitus] out it’s just [tinnitus] is not that often that itwould become an issue).The audiologists admitted that the difficulties they experience withmeasuring thresholds in the presence of distracting tinnitus are common inaudiology and viewed them as “normal.” Although they reported thatstrategies to redirect attention can help overcome these difficulties, theystill voiced concerns over the reliability and accuracy of the resultingthreshold measurements. The potential effect of tinnitus on the reliabilityof thresholds was recognized as a known issue in pure-tone audiometry . They identified effectivetime management as a key skill that is necessary to obtain MAPs within theallocated appointment time and to avoid the need to make repeatedmeasurements.When asked about other effects of tinnitus on programming appointments, theaudiologists reported that tinnitus-related difficulties have the potentialto increase the length of time required to program a CI, for example, due tothe false responses associated with a patient confusing their tinnituspercept with stimulus presentations  and potentially tiringthe patients and aggravating their tinnitus (A4: the longer you keepgoing at it the more tired the patient gets or the worse the tinnitusplays up).The discussion around effective time management strategies was summarized ina subtheme on the necessity to complete programming of MAPs within theappointment. Repeating measurements or extending appointments were regardedas counterproductive strategies  but emphasized the importance ofprioritizing to address other patient’s issues during the appointment .Despite pressures on their time, audiologists seemed to be confident in theirtime management strategies (A1: people [withtinnitus] can take longer to program … if I can see that they’re havinga lot of trouble then I will go to interpolated levels … it’s probablythe best you can do given the inconsistency in responses). Theaudiologists agreed that CI recipients with tinnitus would gain more from anextended hearing therapy than from extended or additional programmingappointments .Audiologists also considered the interpolation of intermediate T levelsbetween those actually measured as a useful strategy to complete theprogramming process within the available time . Theavailability of a written guide was seen as particularly useful for lessexperienced audiologists (A2: it would be helpful if there wasmore … written down … for [new audiologists] … so they’re not completelybaffled by [tinnitus-related difficulties]). The role ofguidance was viewed consistently as an “ideas bank” that could supportclinical practice or judgment .Four audiologists agreed that guidance on programming of CI recipients withtinnitus would be of some benefit. Guidance was seen as a potentially usefulmethod of sharing approaches to patient management among both new andexperienced audiologists (A1: I suppose it’s a way of standardizing), theaudiologists felt strongly that any programming guidelines should notrestrict clinical autonomy required to address the individual patient’s needs:I think if you contrast [guidelines] with something like[British Society of Audiology] recommended proceduresfor … [non-implant] audiometry, I would expect people to adhereto those because that’s … the agreed standard. Cochlear implantsare slightly different and there’s no recommended procedure … sopeople would find it useful to have guidelines, but I thinkthey’d still find it useful to be able to use their ownexperience. Every patient’s different … so you’ve got to be veryadaptable. (A2)In the opinion of one audiologist, patient heterogeneity wouldmake the task of creating guidance infeasible . The task of providing usefuladvice that could be generalized to all patients with tinnitus was alsothought to be hindered by differing programming recommendations andprogramming strategies across CI makes and models (A2:  … they might have different opinionsabout what to do about tinnitus).because theway we program different implants is slightly different [with differentCI manufacturers] The importance of theaudiologists’ contribution and experience to the programming process wasemphasized as crucial in formulating any form of guidance (A2: themanufacturers need to be included in the consultation but it’s got tocome from experienced clinicians).The potential benefits of guidance were contrasted with a need to retain aflexibility in how the audiologists choose to manage individual patients.Although guidelines have the potential to standardize clinical practice (A4:SD = 14.7). About 84% of patients reported that theonset of their tinnitus occurred before CI surgery, while 5% of patientsreported that tinnitus started after implantation but before implantactivation, and about 11% of patients reported to have developed tinnitusafter CI activation. The demographics of CI users with tinnitus participating in the patientsurvey are listed in The reported levels of perceived difficulty with programming due to tinnitusdiffered substantially between audiologists and CI users. Nearly allaudiologists (88% to 94%) agreed that tinnitus makes programming moredifficult at all appointments. However, only 28% to 30% of patients withtinnitus agreed that tinnitus makes CI programming more difficult. Whenasked whether tinnitus increases the level of tiredness in patients, about33% of audiologists and 22% of CI users agreed that even experienced implantusers (>12 post CI activation) can feel more tired at the end of theirprogramming appointments due to tinnitus.The majority of audiologists (89%) reported providing specific advice abouttinnitus to their CI patients during routine appointments. The adviceincluded tips on how to reduce the impact of tinnitus and various tinnitusmanagement strategies, for example, sound enrichment therapy, stressmanagement, and relaxation. About 29% of those audiologists also consideredreferring their patients for further advice or therapy for tinnitus such ascounseling or a visit to a specialist tinnitus clinic.Almost all (95%) of audiologists also reported using specific CI programmingstrategies to overcome tinnitus-related difficulties during programmingappointments. The reported strategies are summarized and compared againstthose reported in the interview data in p > .05). p = .048). This finding was consistent with thetinnitus-related difficulties during T level measurements reported byaudiologists and adult CI patients in parts 1 and 2 of the current study. Themean C level was also found to be higher by about 1.6 dB in patients withtinnitus than in patients without tinnitus, but this difference was notstatistically significant (p > .51). The size of the EDRs inthe two groups was also not significantly different(p > .74).The GEE model results showed that T levels were significantly higher on averageby 1.7 dB in patients with tinnitus than in those without or at reducingtinnitus-related effects by frequent task switching, for example, by alternatingbetween near- and suprathreshold levels, T and C level measurements, or differentelectrodes. The audiologists also endeavor to avoid spurious measurements and obtainthe required programming parameters as quickly as possible, for example, by usinginterpolation for electrodes eliciting pitch percepts similar to the patient’stinnitus pitch. These strategies somewhat resemble more general troubleshootingmethods used in CI programming practice . DespiteIt is not clear whether the strategies for obtaining reliable T levels would beequally effective for different tinnitus percepts. For example, the method ofinterpolation or switching electrodes may not overcome or alleviate interferencefrom more spectrally complex or multiple tinnitus percepts . If tinnThe use of distraction techniques that involve changing the patient’s task fromreporting of “when” a stimulus is heard to “how many” repeated stimulus bursts areheard may have a detrimental effect on the reliability and accuracy of measured Tlevels in CI patients with attention problems . IrregulStudies in other areas of medicine such as fear and pain management  have alsFurther research should assess the reproducibility of T levels to confirm or rejectaudiologists’ and patients’ perceptions that tinnitus interferes with themeasurement of T levels at different appointments over time and to replicate thecurrent finding that T levels are significantly higher in those with tinnitus. Alongitudinal study of tinnitus-related effects may be particularly insightful forclinical practice as the programming process to establish the programming MAPs andEDRs may extend in CI users over about 12-month period after CI activation . ThatreClick here for additional data file.Supplemental Material for Effects of Tinnitus on Cochlear Implant Programming byRobert H. Pierzycki, Charlotte Corner, Claire A. Fielden and Pádraig T.Kitterick in Trends in Hearing


Tumor stroma associates with prostate cancer (PCa) progression, but its specific cellular composition and association to patient survival outcome have not been characterized.We analyzed stromal composition in human PCa using multiplex immunohistochemistry and quantitative, high‐resolution image analysis in two retrospective, formalin‐fixed paraffin embedded observational clinical cohorts  using PCa‐specific mortality as outcome measurement.A high proportion of fibroblasts associated with aggressive disease and castration‐resistant prostate cancer (CRPC). In a multivariate analysis, increase in fibroblast proportion predicted poor cancer‐specific outcome independently in the two clinical cohorts studied.Fibroblasts were the most important cell type in determining prognosis in PCa and associated with CRPC. Thus, the stromal composition could be critically important in developing diagnostic and therapeutic approaches to aggressive prostate cancer. VIM is expressed in fibroblasts and myofibroblasts, whereas aSMA is expressed in smooth muscle and in myofibroblasts.Vimentin  and unbiased automatic image analysis to determine the different stromal cells including fibroblasts, myofibroblasts, and smooth muscle cells in situ in human PCa tissue samples. We show that a stromal phenotype with an increased number of fibroblasts associates with aggressive disease and castration resistance. The proportion of fibroblasts independently predicts poor cancer‐specific outcome in two independent patient cohorts.22.1Study cohorts were formalin‐fixed paraffin embedded (FFPE) samples from transurethral resection of prostate  and retrospective radical prostatectomy . Tissue microarrays (TMA) were constructed for Cohort I by taking 1 to 5 cores per patient and for Cohort II as described earlier.2.2CAV2) were optimized for mIHC using immunohistochemistry in prostatectomy samples as described earlier.CDH1) [clone 36]) were optimized earlier for mIHC.Antibodies used in the study are listed in Table S1. Antibodies for vimentin (VIM), alpha‐smooth muscle actin (aSMA), and CAV2  was measured as the area of positive pixels or cell counts within stroma in each TMA core. For a given cell class, the average relative area or relative cell count in the stroma in all TMA cores per patient was used as the final metric. Cell class variables were categorized in quartiles when appropriate.2.5Demographics for patients with high‐quality image data are presented in Table S2. Patients with missing clinicopathological data were excluded from final analyses where the missing data were considered relevant. For example, in multivariate survival analyses, only patients with complete data for all the variables were included.t test for normally distributed continuous variables. An independent Kruskal‐Wallis H test with pairwise Dunn's post hoc test was used to test differences between three or more non‐normally distributed continuous or categorical variable groups. Normality of data was tested using Shapiro–Wilk tests. Correlations between continuous variables were calculated using the two‐tailed Pearson product moment correlation coefficient function, and P value using two‐tailed Student t test. Unsupervised hierarchical clustering and data plotting were performed using ClustVisThe Mann‐Whitney U test was used to test differences between two groups of non‐normally distributed continuous or categorial variables, and For survival analyses, we used Cox proportional hazard regression model and Kaplan‐Meier plots with the Wald test and log‐rank, respectively. Proportional hazard assumption was tested for given variables using the Schoenfeld test. The percentage proportions of the stromal cell classes were multiplied by 10 for the Cox proportional hazard regression in order to yield hazard ratios for a ten percent change in the proportion.P values were controlled for 20% false discovery rate using the Benjamini‐Hochberg step‐up procedure.P < .05 were considered statistically significant. All statistical analyses and data plotting were performed using the IBM SPSS 24 .If multiple tests were performed, 2.6Ethical approval and consent to participate: Ethical approval for the use of tissue material and clinicopathological data was obtained from the Institutional Ethics Committee of Hospital District of Helsinki and Uusimaa (D:no 446/13/03/02/2009) and from the National Institute for Health and Welfare (D:no THL490.5.05.00/2016) according to the national legislation. A permission for retrospective use of patient data and the archived tissue blocks was approved by the National Supervisory Authority for Welfare and Health .33.1P < .001) and aSMA lower (P = .018) in the stroma of patients dying of PCa compared with those of who did not die during the follow‐up time  had high‐quality image data. We systematically assessed VIM and aSMA expression in full TMA cores or in the epithelium and the stromal compartment of each core. Representative IHC staining examples of VIM and aSMA in PCa are provided as supplementary data Figure S2). VIM expression was significantly higher . Kaplan‐Meier analysis showed that patients in Cluster 6 had very poor cancer‐specific outcome compared with patients in Clusters 1‐5 , myofibroblasts (VIM‐pos and aSMA‐pos), and smooth muscle cells (aSMA‐pos and VIM‐neg). Unsupervised hierarchical clustering of these stromal cell subtypes resulted in six major phenotype clusters Figure A. Tumors3.3P = 9.23E‐7) or a low proportion of smooth muscle cells hazard ratio  separately predicted poor clinical outcome whereas the proportion of myofibroblasts was not predictive of outcome  . Further, we validated the prognostic effect of fibroblast counts by using an alternative image analysis method based on digital stromal cell detection by nuclear segmentation . Moreover, the proportion of fibroblasts, smooth muscle, or myofibroblasts was not significantly different in tumors with different grade , although high‐grade tumors had significantly less stroma compared with low‐grade tumors (P < .001). We observed an inverse association between the proportion of fibroblast and the total stromal area, whereas the proportion of smooth muscle cells weakly correlated with the total stromal area .As the “fibroblast‐high, smooth muscle‐low” phenotype of Cluster 6 showed clear association with poor survival and aggressive cancer, we then studied how individual cell types predict outcome. Kaplan‐Meier analysis and univariate Cox regression analysis showed that both having a high proportion of fibroblasts  and in Cox regression univariate  and multivariate  analyses . Interestingly, the proportion of fibroblasts in matched benign cores (from the same patients adjacent to tumor) also predicted PCSM when comparing the highest and lowest quartiles in Kaplan‐Meier analysis . However, the Cox regression analysis did not reach statistical significance  for the proportion of fibroblasts in the benign TMA cores. Benign cores were available only in Cohort II. We found no significant associations between the proportion of fibroblasts and the expression of AR or PTEN proteins .The prognostic effect of fibroblasts was validated in Cohort II which comprised radical prostatectomy samples from primary PCa. In Cohort II, 340 patients (97%) had high‐quality image data and the distribution of the proportion of fibroblasts was similar to that of Cohort I . Univariate Cox regression analysis of the proportion of fibroblasts in the stroma showed a significantly worse cancer‐specific outcome with increasing proportion of fibroblasts . The average difference between fibroblast count before and after endothelium exclusion was negligible . The effect of immune cells was controlled by visually assessing the amount of stromal VIM‐pos immune cells in IHC‐stained sections in Cohort II. We found no difference in the proportion of fibroblasts between patients with different amounts of stromal immune cells . Together, the effect of endothelium and immune cells on the proportion of fibroblasts was small.4We quantified the cell subtype composition of human PCa stroma by using mIHC, as well as systematic and quantitative digital image analysis. To our knowledge, this is the first quantitative study showing that a high proportion of IHC‐classified fibroblasts predicts poor cancer‐specific outcome. A high proportion of fibroblasts was also associated with CRPC. Tuxhorn et alVIM gene expression predicts metastatic potential in primary PCa. Moreover, we show that fibroblast‐rich stroma associated with CRPC, an observation supported by Thalmann et alOur data expand beyond previous evidence suggesting the clinical significance of stroma in PCa. Our results of fibroblast‐rich stroma predicting poor survival in hormone‐naive PCa is in line with Mo et alTogether, these data suggest that fibroblast‐rich stroma is not only a reaction to the tumor but is an active component in the tumor development. Quantitation of fibroblasts should, therefore, be considered in developing preventive, diagnostic, predictive, and prognostic tests for prostate cancer. Our data indicate that fibroblasts are strongly associated with aggressive PCa, therefore being a potential target for the development of therapeutic approaches.mIHC and automatic, unbiased image analysis is a powerful tool to study solid tumors, allowing quantitative studies of single cells in situ and excluding observer biases inherent in previous studies. To test the robustness of our cell classification, we assessed the effect of immune cells and endothelium in fibroblast classification. We observed a low proportion of immune cells in PCa stroma, which is in agreement with the RNA expression data from The Cancer Genome Atlas.While both clinical cohorts have many cancer‐specific events, the cohorts are different in terms of clinical disease profile as well as sample collection period. As a limitation, both cohorts are observational retrospective cohorts, where therapy after TURP or prostatectomy was not pre‐determined and therefore may introduce confounding bias in patient survival. However, the fact that the same conclusions were obtained from both cohorts suggest that our key observations are robust and validated. Importantly, the fibroblast‐rich stroma was prognostic in both cohorts independent of tumor grade and patient age in a Cox regression model.5Comprehensive multiplexed mIHC and quantitative digital image analysis facilitates high‐resolution and unbiased analysis of the cell subtypes in the cancer‐associated stroma in human PCa. We conclude that a fibroblast‐rich stroma associates with aggressive disease and adverse clinical outcome. The study provides evidence for the clinical significance of fibroblast‐rich stroma in human PCa that could have implications for prevention, diagnosis, prognostication and therapy of prostate cancer.Sami Blom is an employee of Aiforia Technologies Oy, a company providing image analysis products and services. Other authors declare no conflict of interests.Supporting informationClick here for additional data file.


Hybridization is increasingly recognized as a creative evolutionary force contributing to adaptation and speciation. Homoploid hybrid speciation—the process in which hybridization results in a stable, fertile, and reproductively isolated hybrid lineage where there is no change in ploidy—has been documented in several taxa. Hybridization can directly contribute to reproductive isolation or reinforce it at a later stage. Alternatively, hybridization might not be related to the evolution of reproductive isolation. To account for these different scenarios, I propose to discriminate between two types of hybrid speciation: type I where reproductive isolation is a direct consequence of hybridization and type II where it is the by‐product of other processes. I illustrate the applicability of this classification scheme with avian examples. To my knowledge, seven hybrid bird species have been proposed: Italian sparrow, Audubon's warbler, Genovesa mockingbird, Hawaiian duck, red‐breasted goose, golden‐crowned manakin, and a recent lineage of Darwin's finches on the island of Daphne Major (“Big Bird”). All studies provide convincing evidence for hybridization, but do not always confidently discriminate between scenarios of hybrid speciation and recurrent introgressive hybridization. The build‐up of reproductive isolation between the hybrid species and their parental taxa is mainly driven by premating isolation mechanisms and comparable to classical speciation events. One hybrid species can be classified as type I (“Big Bird”) while three species constitute type II hybrid species . The diversity in hybrid bird species across a range of divergence times also provides an excellent opportunity to study the evolution of hybrid genomes in terms of genome stabilization and adaptation. In the following paragraphs, I discuss the evidence supporting the hybrid origin of these species.Here, I will illustrate the applicability of the type I and type II classification scheme for hybrid species by exploring the hybrid speciation continuum in birds, which are prone to hybridize  and Spanish sparrow (P. hispaniolensis) so resemble the Italian sparrow  that it was hypothesized to be of hybrid origin  complex comprises four distinct taxa—coronata, auduboni, nigrifrons and goldmani—of which the taxonomic relationships remain controversial  is a hybrid lineage between myrtle warbler (S. coronata) and black‐fronted warbler (S. nigrifrons)  shares mitochondrial haplotypes and certain autosomal loci with the San Cristobal Mockingbird (M. melanotis). At other autosomal loci, it clusters closely with another species, the Galapagos Mockingbird (M. parvulus). These observations suggest that the Genovesa Mockingbird represents a lineage of hybrid ancestry  complex, a phylogenetically challenging group of ducks , whereas mtDNA clusters the Hawaiian duck with the mallard. This pattern of morphological‐nuclear‐mitochondrial discordance can be explained by incomplete lineage sorting or introgressive hybridization.The Hawaiian Duck is one of the 14 taxa within the mallard  and the ancestor of the white‐cheeked geese . The putative hybrid speciation event happened at least 3.5 million years ago, before the diversification of the white‐cheeked geese clade. The red‐breasted goose seems partially reproductively isolated from its parental species; some hybrids have been documented, but their fertility has not been directly assessed  is located between the ranges of opal‐crowned (Lepidothrix iris) and snow‐capped manakin (L. nattereri). Genetic analyses suggest that the golden‐crowned manakin is the outcome of a hybrid speciation event between the latter two species about 158,000 years ago  arrived on the island and mated with a female medium ground finch (G. fortis). The resulting offspring only bred with each other and as such established a new lineage on the island  due to differences in song and beak morphology. Whether it is also reproductively isolated from the other parental species (G. conistrostris), which resides on the island of Española, remains to be tested. This case shows that reproductive isolation can develop in few generations  and waterfowl (Anseriformes). Given the incidence of hybridization in birds  hybrids showed that alleles from the minor parent are more common in regions of high recombination where they can become uncoupled from deleterious alleles  over thousands of years  to millions of years old (red‐breasted goose). This spectrum of divergence times allows for the comparison of hybrid genome stabilization and adaptation over time, while taking into account species‐specific processes. In the initial stages of first‐generation hybrids and backcrosses, there are many possible outcomes (Nolte & Tautz, Introgressive hybridization can introduce new genetic variation or create novel allelic combinations Hedrick, . The mosJO conceived the idea, conducted the literature search and wrote the manuscript.The manuscript is solely based on a literature review. All data can be found in the reference list below.


The recognition of intrinsically disordered proteins (IDPs) is highly dependent on dynamics owing to the lack of structure. Here we studied the interplay between dynamics and molecular recognition in IDPs with a combination of time‐resolving tools on timescales ranging from femtoseconds to nanoseconds. We interrogated conformational dynamics and surface water dynamics and its attenuation upon partner binding using two IDPs, IBB and Nup153FG, both of central relevance to the nucleocytoplasmic transport machinery. These proteins bind the same nuclear transport receptor (Importinβ) with drastically different binding mechanisms, coupled folding–binding and fuzzy complex formation, respectively. Solvent fluctuations in the dynamic interface of the Nup153FG‐Importinβ fuzzy complex were largely unperturbed and slightly accelerated relative to the unbound state. In the IBB‐Importinβ complex, on the other hand, substantial relative slowdown of water dynamics was seen in a more rigid interface. These results show a correlation between interfacial water dynamics and the plasticity of IDP complexes, implicating functional relevance for such differential modulation in cellular processes, including nuclear transport. Hydration is crucial to the expression of bio‐molecular functionality.EFRET≈0.8 which changed to ≈0.3 upon Importinβ binding  and unbound states in line with our previous studies.Nup153FG is a 600aa long IDP domain that belongs to the class of FGNups, which are F‐ and G‐rich IDPs that constitute the permeability barrier of the nuclear pore complex (NPC) and facilitate transport across the NPC by binding nuclear transport receptors (NTRs).To characterize the interface of Nup153FG and IBB in complex with Importinβ, we next employed steady‐state and picosecond‐resolved fluorescence encoded by site‐specifically labeled acrylodan, a thiol‐selective fluorophore that is highly sensitive to microenvironment polarity.We next employed picosecond‐resolved anisotropy experiments, which probe the depolarization kinetics of a fluorophore on a picosecond–nanosecond timescale. For IDPs this provides information about segmental motion, that is, the conformational motion of a segment of the chain , and information about very local dynamics in the immediate vicinity of the labeling site (very few amino acids), as we have shown before.We next set out to probe the modulation of surface water dynamics at the interface of IBB and Nup153FG complexed with Importinβ, compared to the unbound proteins. We employed dipolar relaxation to probe water dynamics from femtoseconds to picoseconds by monitoring the time‐dependent Stokes shift (TDSS) of the site‐specifically labeled acrylodan.The relaxation functions shown in Figure S(t)(free)/S(t)(bound) of normalized relaxation functions S(t) for the bound and unbound scenarios for Nup153FG and IBB, where the ratio quickly approached values larger than one for IBB while for Nup153FG the values stayed close to one and started decreasing slowly at longer times  should be addressed to the authors.SupplementaryClick here for additional data file.


IMPACT study, a multi‐national investigation of targeted prostate cancer (PCa) screening among men with a known pathogenic germline mutation in the BRCA1 or BRCA2 genes.To report the baseline results of a longitudinal psychosocial study that forms part of the IMPACT study were invited to complete a questionnaire at collaborating sites prior to each annual screening visit. The questionnaire included sociodemographic characteristics and the following measures: the Hospital Anxiety and Depression Scale (HADS), Impact of Event Scale (IES), 36‐item short‐form health survey (SF‐36), Memorial Anxiety Scale for Prostate Cancer, Cancer Worry Scale‐Revised, risk perception and knowledge. The results of the baseline questionnaire are presented.Men enrolled in the BRCA1 and BRCA2 genes, respectively, and 174 were controls . Participants’ perception of PCa risk was influenced by genetic status. Knowledge levels were high and unrelated to genetic status. Mean scores for the HADS and SF‐36 were within reported general population norms and mean IES scores were within normal range. IES mean intrusion and avoidance scores were significantly higher in BRCA1/BRCA2 carriers than in controls and were higher in men with increased PCa risk perception. At the multivariate level, risk perception contributed more significantly to variance in IES scores than genetic status.A total of 432 men completed questionnaires: 98 and 160 had mutations in BRCA1/BRCA2 mutations undergoing PCa screening. No clinically concerning levels of general or cancer‐specific distress or poor quality of life were detected in the cohort as a whole. A small subset of participants reported higher levels of distress, suggesting the need for healthcare professionals offering PCa screening to identify these risk factors and offer additional information and support to men seeking PCa screening.This is the first study to report the psychosocial profile of men with  BRCA1 or BRCA2 gene mutations are known to be at an increased risk of PCa. This risk is estimated to be 1.8–3.75‐fold and 2.5–8.6‐fold increased by the age of 65 years for BRCA1 and BRCA2 mutation carriers, respectively BRCA1 mutation carriers, there is solid evidence that BRCA2 mutation carriers present at a younger age and with aggressive disease BRCA2 mutation carriers Prostate cancer (PCa) is the most common non‐melanoma tumour in men worldwide, with an estimated 1.1 million men diagnosed with PCa in 2012 There is controversy about PCa screening using PSA testing in the general population and the benefits and harms of screening have been widely debated BRCA1/2 mutation carriers. The IMPACT study (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted Screening in men at higher genetic risk and controls) is an international, multicentre study evaluating the role of targeted PSA screening in men with BRCA1/2 mutations The limited number of studies evaluating men with a family history of PCa have generally supported the use of screening in this population BRCA1 and BRCA2 mutations does not have a significant long‐term psychological impact on most people tested BRCA1/BRCA2 mutations confer an increased disease risk and psychological distress Evidence supports the theory that genetic testing for BRCA1/BRCA2 mutations, and many factors affect the way in which men react to and use information about their genetic status and risk of developing cancer BRCA1/BRCA2 mutation living with an increased risk of PCa, or in those men who have gone on to develop PCa Many issues arise when counselling men with In the present study, we report the baseline results of a longitudinal HRQoL investigation carried out as part of the IMPACT study. The specific aims of the study were to evaluate the baseline psychosocial profile of men in the IMPACT study and to identify possible predictors of high levels of psychological distress or poor HRQoL.BRCA1 or BRCA2 mutations, with or without the familial mutation, to a programme of annual PCa screening via PSA testing, for a minimum of 5 years. The IMPACT study opened in 2005 and screening will end in 2019. The full design and methods of the IMPACT study have previously been reported The IMPACT study recruited men from families with BRCA1/BRCA2 mutation and were aged 40–69 years. Men who tested negative for their familial mutation constituted the control group. Men were excluded if they were known to have PCa at enrolment or if they had another cancer with a prognosis of <5 years survival.All men eligible for IMPACT were also eligible for the HRQoL study. Men were eligible for participation if they tested either positive, negative or were at 50% risk of inheriting the familial The HRQoL study was added to the IMPACT study protocol in 2009. All sites were invited to participate in this sub‐study. Men enrolled in the IMPACT study at participating sites were approached by letter prior to their next scheduled study appointment inviting them to take part in the HRQoL study. The HRQoL study involves completing a set of questionnaires annually for 5 years, with each assessment taking place prior to the annual PSA test. Men were sent the questionnaires ~4 weeks before their appointment and asked to post it back or bring the completed questionnaire to their appointment. Men were split into two cohorts: (i) a prospective arm, which included men who joined the HRQoL study before their first PSA screening within the IMPACT study; and (ii) a truncated prospective arm, which included men already enrolled in the IMPACT study before joining the HRQoL study. The total target sample was a minimum of 300 men in each arm. In the present analysis, we report the results of the baseline questionnaires in the prospective (not truncated) cohort.Distress was assessed using the Hospital Anxiety and Depression Scale (HADS), the Impact of Event Scale (IES), the Cancer Worry Scale‐Revised (CWS‐R), and the Memorial Anxiety Scale for Prostate Cancer (MAX‐PC). The HADS contains two sub‐scales of seven items that measure the presence and severity of general anxiety and depression The IES is a 15‐item scale measuring PCa‐specific distress through the frequency of intrusive or avoidant thoughts about PCa The CWS‐R is a six‐item scale that measures worry about the risk of developing cancer and the frequency and impact of that worry on mood and daily functioning The MAX‐PC includes three scales assessing PCa anxiety, PSA anxiety, and fear of recurrence. In the present study, we used the PCa anxiety (11 items) and PSA anxiety (3 items) scales We assessed HRQoL using the 36‐item short‐form health survey (SF‐36) version 2.0 Men were asked to rate their perceived risk of PCa compared with the average man's risk: lower; the same; slightly increased; moderately increased; or strongly increased BRCA1/BRCA2, the effect of having an altered gene, and risk of PCa. Knowledge scores were created by taking the sum of the correct responses to the nine items.We developed a ‘knowledge’ questionnaire based on a measure developed by Lerman et al. The internal consistency reliability, as assessed by Cronbach's coefficient α, was high for all measures used, ranging from 0.79 for the SF‐36 General Health scale to 0.96 for the SF‐36 Role Physical scale. Fourteen of the 15 scales had an α coefficient >0.80.The dataset contained a small amount of missing data. For all scales, except the SF‐36, if ≥75% of a subscale was complete then a total score  was calculated. If <75% was completed, data were excluded. For the SF‐36 score, scales were excluded when there was <50% of a sub‐scale completed, as per the recommendation of the scale's authors spss 22.0 statistical computer package  was used to manage and analyse the data. Scores for each questionnaire were calculated in accordance with each scale's scoring system. Descriptive statistics, including means and standard deviations, were used to summarize the sample characteristics and questionnaire data.The P value of <0.01 was regarded as statistically significant.All psychometric scales  were skewed towards better scores. Neither log nor square‐root transformations of these scales produced normal distributions, but given the large sample size within each genetic cohort, parametric tests were used. To minimize the potential effect of multiple testing on the Type I error rate, a BRCA1 mutation carriers, BRCA2 mutation carriers and controls on the dependent variables risk perception, HRQoL (SF‐36), the psychological measures  or knowledge. As UK participants made up the largest proportion of participants, a UK dataset was used as a normative comparator for HRQoL, by randomly selecting individuals matched to our sample on age. Means were then compared using a paired Student's t‐test Univariate analysis was used to examine if there were any measurable differences at baseline between The impact of other variables on psychosocial outcomes was also explored. Independent variables included demographics , prior PSA screening, family history of PCa, time since genetic testing, and comorbidities coded from clinical interview into a Charlson Comorbidity Index score anova, Student's t‐tests, chi‐squared tests and Pearson's correlations, as appropriate. For categorical independent variables, strength of association was calculated with Cohen's d for any significant relationship. Subsequently, multivariate linear regression analyses were performed employing all independent variables found to be associated significantly at the univariate level with a psychosocial outcome.The associations were investigated initially with n = 35), as were nine men who were untested for their familial mutation, remaining at 50% risk. The data presented are therefore from 432 men, 351 of whom were recruited in the UK, 50 from the USA and 31 from Spain. No significant differences in responses were observed between nationalities.Of the 65 centres participating in the IMPACT study, 23 agreed to take part in the HRQoL sub‐study, including all 19 UK centres, two in Spain and two in the USA. The main reasons for electing not to participate as a centre were financial; there was no specific funding to support this sub‐study at collaborating sites outside of the UK. A total of 780 men enrolled in the HRQoL study, of whom 476 enrolled prior to their first screening visit . This corresponds to 26% of the participants in the IMPACT study taking part in this sub‐study. Those who returned their questionnaire >1 month after their initial screening visit or had not returned the study consent form were excluded (Uptake into the HRQoL sub‐study was 85–100% at participating sites. There was no significant difference in the participants’ sociodemographics (employment status or education) between the men in this sub‐study and those in the parent IMPACT study.BRCA1 gene, 160 (37.0%) carried a mutation in the BRCA2 gene and 174 (40.3%) were controls. The median time from undergoing genetic testing to joining the IMPACT study was 7.2 months (range 0 months–15.4 years); 47.4% of men joined within 6 months of testing, and 39.6% of men had had at least one PSA measurement before they joined the IMPACT study.In all, 98 men (22.7%) carried a mutation in the The sociodemographic characteristics and family cancer history of the cohort are shown in Table P < 0.001; Table BRCA2 mutation carriers were more likely to rate their risk of PCa as moderately or strongly increased compared with the general population than the control group.Participants’ perception of their lifetime risk of PCa was influenced significantly by their carrier status . The overall mental functioning SF‐36 score was significantly better in the study cohort compared with the normative sample, but the effect size was small and both mean values were close to the standardized mean of 50 . Means also did not differ significantly across genetic groups.Overall physical functioning SF‐36 scores did not differ significantly from the normative sample .The overall mean anxiety and depression scores for the HADS were 4.9 and 2.8, respectively, which were not higher than previously reported general population norms P = 0.02 and P = 0.03, respectively; Table None of the independent variables showed a significant association with either the anxiety or depression scores. Those with higher risk perception had slightly higher scores on the anxiety and depression scales , therefore, a multivariate analysis was not undertaken.Scores for PCa anxiety (MAX‐PC) were only associated with risk perception  or poor HRQoL. A small subset of participants had higher levels of distress, but perception of risk contributed more to explaining the variance in distress level than did genetic status. General population screening studies in the UK and European series have reported similar findings: that PCa screening does not have a detrimental effect on measures of HRQoL and psychological health BRCA2 mutation carriers had the highest perceived risk of PCa, most frequently classifying risk as ‘slightly’ or ‘moderately’ increased, and controls most frequently classifying risk as the ‘same’ as the general population.It was reassuring that participants’ perceptions of PCa risk were influenced by carrier status, largely reflecting what would have been communicated during genetic counselling BRCA1/BRCA2 mutations and cancer risk. The knowledge questionnaire was designed specifically for this study, but was adapted from that used in other studies BRCA1/BRCA2 testing. The high levels of knowledge reported in our cohort could reflect that they have recently revisited their risk status in making a decision to undergo screening in the IMPACT study; however, men were asked to complete these questionnaires prior to their first screening appointment and so may not have had a detailed discussion about risk of PCa since being informed about their genetic status.Knowledge levels were high across the cohorts, irrespective of genetic status, education level and time since testing, showing that men retained accurate information about inheritance of The sociodemographic characteristics of the cohort indicate that employment and education levels are similar to those observed in the UK general population The HRQoL assessments did not detect any clinically relevant differences in either physical or mental health when compared with general population samples, both matched and unmatched by age BRCA mutation carriers and controls for both the IES and CWS; however the differences were small and mean scores remained below clinically relevant levels for the IES. Importantly, at the multivariate level, risk perception was found to have a stronger association with distress levels than genetic status itself.In terms of general distress, scores were within previously reported population norms No significant association was observed between anxiety and having a family history of PCa, supporting previous reports A number of studies have reported that anxiety about cancer screening affects a small number of people who are predisposed to anxiety, and that this anxiety continues throughout participation in cancer screening We did not observe a significant association between distress and age. While this supports several earlier studies It is important to consider whether we would have observed different results if all men in the IMPACT study had been included in this psychosocial sub‐study; however we found no difference in sociodemographic characteristics between the men in the sub‐study and those in the IMPACT study as a whole. It could be that those more predisposed to anxiety may be inclined not to join the psychosocial sub‐study; however, no evidence of this has been found by others We obtained a very high uptake level for the psychosocial sub‐study, with at least 85% opting in at participating sites. Uptake was also found to be high in the European Randomized study of Screening for Prostate Cancer Swedish cohort, with 84–94% of men with abnormal PSA levels completing a questionnaire measuring anxiety levels A strength of the present study is the use of a number of different, standardized psychological measures that offer extensive insight into the psychosocial profile of the participants and that allow comparison of the results with a number of other PCa screening studies that have used the same or similar measures.It should be noted that our sample was restricted to men who have previously engaged with health services by undergoing genetic testing and who responded positively to an invitation to take part in a research study. In addition, there was limited variability in ethnicity, which may limit the generalizablity of the findings to other populations.The data presented represent a snapshot of men's psychosocial profiles when they joined the IMPACT study. Follow‐up data will inform whether the PCa screening process has an impact on HRQoL or distress over time.BRCA1/BRCA2 mutations taking part in a PCa screening study. Uptake into the study was very high, and participants had very high levels of knowledge about genetics and PCa. As a whole, the cohort did not demonstrate any clinically concerning levels of general or cancer‐specific distress or poor HRQoL. A small subset of participants reported higher levels of distress, but perception of risk was more strongly associated with distress levels than was genetic status. It is important for healthcare professionals who are providing PCa screening to be aware of these predictors of distress so that men with potential for heightened distress can be identified and adequate counselling and support can be offered. Follow‐up data will determine whether these factors have an impact on adherence to screening and whether men experiencing abnormal PSA results experience more distress.To the best of our knowledge, this is the first study to report the psychosocial and HRQoL profile of men with Prof. Rosalind Eeles: Janssen Pharmaceutica, provided medical education support to GU ASCO February 2013, and received an honorarium and expenses for attending and speaking at the UK Cancer Convention, October 2013 from Succinct Communications. The remaining authors have no other conflict of interest to declare.CWS‐RCancer Worry Scale‐RevisedHADSHospital Anxiety and Depression ScaleHRQoLhealth‐related quality of lifeIESImpact of Event ScaleMAX‐PCMemorial Anxiety Scale for Prostate CancerPCaprostate cancerSF‐3636‐item short‐form health surveyAppendix S1. The IMPACT Collaborators.Click here for additional data file.


This fMRI study investigated brain activity while soccer players were imagining creative moves in real soccer decision‐making situations. After presenting brief video clips of a soccer scene, participants had to imagine themselves as the acting player and think either of a creative or obvious move that might lead to a goal. Findings revealed stronger activation during trials in which the generation of obvious moves was required, relative to trials requiring creative moves. The reversed contrast (creative > obvious) showed no significant effects. Activations were mainly left‐lateralized, primarily involving the cuneus, middle temporal gyrus, and the rolandic operculum, which are known to support the processing of multimodal input from different sensory, motor and perceptual sources. Interestingly, more creative solutions in the soccer task were associated with smaller contrast values for the activation difference between obvious and creative trials, or even with more activation in the latter. Furthermore, higher trait creative potential  was associated with stronger activation differences between both conditions. These findings suggest that with increasing soccer‐specific creative task performance, the processing of the manifold information provided by the soccer scenario becomes increasingly important, while in individuals with higher trait creative potential these processes were recruited to a minor degree. This study showed that soccer‐specific creativity tasks modulate activation levels in a network of regions supporting various cognitive functions such as semantic information processing, visual and motor imagery, and the processing and integration of sensorimotor and somatosensory information. In makiSeveral empirical research reports demonstrate that creativity is a key factor for success in soccer performance. Kempe and Memmert , for exaFrom a more general view, the domain of sports might be considered as promising field to investigate creative performance in a more ecologically valid way  or an obvious/conventional move (control condition) that might lead to a goal. The general experimental test design has been validated extensively in different sport settings  participated in this study. As important inclusion criteria, participants were required to have been actively playing soccer for at least 10 years (at least once per week). On average, participants have been actively playing soccer for approximately 17 years , and they indicated to play soccer for about 7 hrs per week . All participants were right‐handed , in which participants were required to mentally generate moves with the intention to score a goal in a given soccer decision‐making situation. The objectivity, reliability and validity of the SVT‐S has been established in previous studies Memmert, . The sti2.3The 30 videos administered during fMRI assessment were taken from a pool of videos of previous studies of our laboratory  using a 32‐channel head coil. Structural images were acquired using a MPRAGE T1‐weighted sequence . BOLD‐sensitive T2*‐weighted functional images were acquired using a single shot gradient‐echo EPI pulse sequence . Head motion was restricted using firm padding that surrounded the head. To record the verbal responses of the participants, an MR compatible microphone was used . Stimuli were presented using the Software Presentation .2.6t test treating subject as a random effect. A voxel‐wise FWE correction (at p < .05) was used for corrections for multiple comparisons. Only activation clusters exceeding a spatial extent threshold of 10 voxels (3 × 3 × 3 mm) are reported.Functional MRI data analysis was performed using SPM 12 software , which ran in a MATLAB 2015b environment . The Data Processing Assistant for Resting‐State fMRI  , as compared to imagining creative/original moves in soccer decision‐making situations, resulted in stronger activation in a mainly left‐lateralized network including the cuneus, middle temporal gyrus, the rolandic operculum, and smaller clusters involving the left angular and postcentral gyrus, and the left pallidum (see Table 3.2p < .05) for the left rolandic operculum and the left postcentral gyrus, and trends toward significance (p < .10) for the left cuneus and the right parietal cortex  in the soccer task and the identified patterns of brain activity during performance of this task, contrast estimates were computed for the significant activation clusters in the control > creative contrast (see Table p < .05) for the left middle temporal gyrus and the left postcentral gyrus—very close to those regions for which significant negative correlations with soccer‐specific originality were found  was clearly different from the domain‐specific creativity measure obtained during performance of the soccer task. Creative potential showed mostly positive associations with the contrast estimates (control > creative), which were significant . The majority of participants indicated that they were actively playing in soccer clubs, the highest soccer league the participants indicated they had ever played ranged up to the third‐highest national league; only four participants were not actively playing in soccer clubs.To assess some facets of participants' expertise in soccer, participants were asked to indicate the current amount of soccer training per week and the highest soccer league they have ever been playing in their career. The amount of training per week ranged from 0 to 12 hr  than the control condition  = 8.48, p < .001). The mean reaction time in the control condition  was significantly shorter than in the creative condition  = 6.59, p < .001).The creative condition of the soccer task resulted in more original responses , and none of the task performance scores was significantly associated with trait creative potential . Likewise, there were no significant correlations of soccer‐specific expertise with soccer task performance in the control and creative condition and with creative potential (r ranging between −.11 and .12).There was no significant correlation between the originality scores in the control and in the creative condition . This finding adds further evidence to the common view of domain‐specificity of creativity and nicely complements the result pattern found in the related EEG study by Fink et al. . This stWhat still remains somewhat puzzling in the overall pattern of findings is the fact that the results of the task contrast seem to be at odds with the findings obtained in the correlational analyses. On the one hand, the creative condition of the soccer task generally  yielded less brain activation than the obvious condition in a brain network supporting visual and motor imagery, and the processing of sensorimotor and somatosensory information Figure . HoweverThis pattern of findings is strikingly similar to that observed in the accompanying EEG study  revealed no single brain region that was more activated in the creative versus control condition (even at the uncorrected level). Rather, they revealed stronger activation in the control condition in brain regions similar to the findings reported here, though, however, the significance of these analyses is limited given the considerably lower number of time segments and trials available for analysis (resulting in lower reliability). A further limitation of the self‐paced response mode could be also seen in the fact that there might be various individual differences in the strategies participants are pursuing in generating a creative move. Hence, an analysis of brain activity in shorter and fixed time intervals, which also considers different stages of the creative ideation process, and which may resemble real‐play conditions more realistically , would be extremely valuable here. Also, in this study participants were explicitly instructed to think either of an obvious or a creative move. It would be also interesting to see the “typical” behavior of participants in such real‐life soccer decision‐making situations and how this is reflected in brain activity. Notwithstanding these restrictions we conclude that the applied experimental paradigm facilitates the investigation of creative behavior in a novel, ecologically valid way. This study revealed that finding creative soccer moves is a complex cognitive process involving the processing of multimodal input from different sensory, motor and perceptual sources, along with domain‐specific mechanisms such as visual or motor related mental imagination.


To determine the influence of propofol or methohexital, with and without doxapram, on the examination of laryngeal function in dogs.Experimental study.Forty healthy dogs randomly assigned to 4 groups: propofol with saline (n = 10), propofol with doxapram (n = 10), methohexital with saline (n = 10), or methohexital with doxapram (n = 10).Propofol and methohexital were administered to effect. Investigators examined laryngeal function  simultaneously with video laryngoscopy. Doxapram or saline was administered, and laryngeal function was reevaluated (second). Laryngeal motion, quality of laryngeal exposure, and the degree of swallowing, laryngospasm, and jaw tone were scored at each evaluation. Adverse events were recorded. Initial and second videos were evaluated by a masked observer, and still images obtained from both evaluations were evaluated for change in rima glottidis size by 2 masked observers.P = .001). Laryngeal function did not differ between dogs receiving propofol or methohexital, irrespective of doxapram administration. Doxapram improved breathing scores in both groups (P < .001). Jaw tone increased with propofol during the second evaluation (P = .049). Swallowing was more prevalent at initial examination (P = .020). Methohexital resulted in an increased heart rate (P < .001) compared with propofol. Twenty‐five percent of dogs receiving methohexital developed seizure‐like activity (n = 5/20).Administration of doxapram and saline was delayed with propofol (Evaluation of laryngeal function did not differ between healthy dogs anesthetized with propofol or methohexital. Methohexital provided shorter examination times with less jaw tone but was associated with adverse events.This study provides evidence to recommend propofol over methohexital as an induction agent for laryngeal function examination. The diagnosis is confirmed with direct laryngeal examination, oral laryngoscopy, transnasal laryngoscopy, or echolaryngography.Anesthetic protocols that have been evaluated for laryngeal evaluation include propofol, thiopental, ketamine, alfaxalone, and various combinations of these agents.Identifying a new protocol for laryngeal examination may improve the quality of evaluation and accuracy of diagnosis of laryngeal paralysis. Methohexital is a rapidly acting barbiturate anesthetic agent, similar to thiopental.The objective of this study was to determine the influence of propofol or methohexital with or without doxapram on the examination of laryngeal function in healthy dogs. Our primary hypothesis was that no difference in evaluation of laryngeal function in healthy dogs would be found between methohexital and propofol, as assessed by a prior published scale for laryngeal function assessment.22.1Forty healthy young adult dogs were recruited among dogs presented to the shelter program at the Oklahoma State University Center for Veterinary Health Sciences from local shelters for routine spay or neuter between September and December 2016. The experimental protocol was approved by the university's institutional animal care and use committee, and each participating shelter signed an informed consent for enrollment of their dogs in the study. Inclusion criteria included the absence of respiratory abnormalities, normal physical examination findings, and a body weight greater than 5.0 kg.2.2Each dog was randomly assigned to 1 of 2 groups, a control group (group P) and an experimental group (group M), by using a computerized randomization protocol . Group P consisted of 20 dogs in which layngeal evaluation was performed under propofol‐induced anesthesia. Half of these dogs (n = 10) were randomized to receive doxapram for respiratory stimulation while half (n = 10) were given a saline placebo. Group M consisted of 20 dogs in which laryngeal evaluation was performed under methohexital‐induced anesthesia. Dogs in this group were similarly randomly assigned to receive doxapram (n = 10) to stimulate respiratory function or saline placebo (n = 10).Prior to induction, a complete physical examination was performed on each dog by the same individual (MBB). An intravenous catheter was placed in a cephalic vein. The induction protocol excluded any premedications from being administered. Dogs in group P were scheduled to receive up to a 6.0‐mg/kginitial, was performed at administration of propofol or methohexital, and the second examination, referred to as second, was performed after administration of doxapram or saline.Appropriate plane of anesthesia was confirmed by loss of jaw tone allowing for adequate visualization of the larynx. An experienced diplomate of the American College of Veterinary Internal Medicine  performed video laryngoscopy using a 30 °, 2.7‐mm rigid cystoscope . Direct visual observation of the larynx was also performed concurrently by an experienced diplomate of the American College of Veterinary Surgeons (DRD) and a surgical intern (MBB). A tertiary investigator noted whether the dog was undergoing inspiration or expiration along with depth of respiration to allow documentation of any paradoxical arytenoid motion. Two video and visual examinations were obtained for each dog. The first examination, referred to as Data were recorded for each dog for initial and second laryngeal evaluations by using a modification of a previously published scale and included breathing score (0‐3); laryngeal motion score (0‐2); quality of visualization/laryngeal exposure ; and the presence or absence of swallowing (0‐2), laryngospasm (0‐2), and jaw tone , who is a diplomate of the ACVIM. Each video was evaluated for degree of laryngeal motion and presence or absence of laryngospasm and swallowing by the same scores noted above.http://imagej.nih.gov/ij). The ratio of each value at maximal abduction and adduction was calculated. The 3 ratio measurements were then averaged to allow for normalization of data. The difference in the ratio at maximal adduction prior to doxapram or saline administration and the ratio at maximal abduction postadministration was calculated. The difference in ratio of maximal adduction compared with maximal abduction was also calculated before and after doxapram or saline administration to account for the effects of doxapram and time.An objective evaluation of the change in rima glottidis between inspiration and expiration was performed individually by 2 investigators (DRD and SDL) using still images from the obtained videos. The height and width of the rima glottidis was measured on 6 images, 3 at maximal abduction and 3 at maximal adduction, in ImageJ 1.50i  with still image variables of maximal abduction and adduction of the larynx, yielding the ability to detect a standard deviation difference with 88% power. The variables of interest were evaluated for normality by using Kolmogorov‐Smirnoff tests, with no compelling evidence to suspect nonnormality. Analysis of variance assuming a factorial arrangement in a completely randomized design was used to evaluate the initial and second differences between induction agent administered, observer, and doxapram administration. Simple effects of each factor given the other factors  were evaluated by using planned comparisons within the ANOVA model. Contingency tables and χt test was performed on adverse events that occurred with administration of the induction agents. Fisher's exact test was used to evaluate for the rate of seizure activity between induction agent groups. Statistical significance was set at P < .05.All statistical analyses were conducted in SAS version 9.4 . A power analysis was performed in R software . There were 27 males and 13 females with no known history or physical examination findings consistent with respiratory disease or laryngeal paralysis. The body weight of the dogs ranged from 5.0 to 29.5 kg, with a mean body weight of 15.4 kg.P = .001). Time from end of first injection to administration of second injection of saline for group P (120.111 ± 25.6 seconds) was not different from group M . Time from end of first injection to administration of second injection of doxapram for group P (71.7 ± 10.2 seconds) was not different from group M .Half of the calculated total dose of methohexital was administered as the initial steady bolus over a mean of 22 seconds . The total volume administered was then titrated to achieve the appropriate anesthetic plane for examination. Propofol was administered slowly and to effect over a mean of 208 seconds . Mean doses administered for methohexital were 7.4 mg/kg  and for propofol were 6.8 mg/kg . Time from beginning of first injection to administration of the second injection  was longer in group P (263.2 ± 67.9 seconds) compared with group M  or after administration of doxapram .There was no difference between observers (DRD and MBB) performing visual laryngoscopy in determination of laryngeal function, either before  or after (second) administration of doxapram , laryngeal exposure (P = .30), and the presence or absence of swallowing in the second phase (P = .0545), laryngeal spasm , or jaw tone  in evaluation of breathing score  between methohexital and propofol; however, there was a difference observed in second breathing scores (P < .001). Post hoc analysis revealed improved breathing scores in group M dogs that received methohexital and doxapram vs methohexital and saline, in group P dogs that received propofol and doxapram vs propofol and saline, and in group P dogs that received propofol and doxapram vs group M dogs that received methohexital and saline (P < .001). These improved breathing scores did not result in paradoxical movement of the arytenoid cartilages. There was no difference in jaw tone between groups during initial evaluation (P = .22). There were more patients in group P with increased jaw tone during the second evaluation compared with group M (P = .049). There was no difference in laryngeal exposure between groups P and M (P = .377). There were more patients in group P with swallowing present during the initial examination (P = .020), although a difference was not noted for the second examination (P = .534). There was no difference in the presence of laryngospasm between groups P and M .There was no difference in initial breathing scores (3.2P < .001) and M (P = .0065) without doxapram administration. In this evaluation, 1 observer (DRD) measured a smaller difference in the change from initial narrow to second wide  compared with the other observer . There was also a difference in laryngeal function, identified by the change in rima glottidis size from initial narrow to second wide, seen by 1 observer (DRD) in group P between dogs receiving doxapram  and those receiving saline . No further difference in laryngeal function was seen with administration of doxapram or between agents administered.There was a difference in ratio measurements between observers (DRD and SDL) in the evaluation of the difference between initial narrow  and second wide  of both groups P , laryngospasm , and laryngeal function postdoxapram or saline administration  for groups P and M combined compared with the findings of the direct observers (DRD and MBB). No difference between blinded and nonblinded observers was seen with initial evaluation of laryngeal function (P = .118).Evaluation of video laryngoscopy by the masked observer (SDL) revealed a difference in the presence of swallowing . Seizure‐like activity was seen in 25% of the methohexital cases (n = 5/20); 2 episodes occurred concurrently with vomiting (n = 1) and regurgitation (n = 1). There was a difference between seizure‐like activity in group M vs group P (P = .047). All seizure‐like episodes occurred as the dogs were returning to a lighter plane of anesthesia at completion of the laryngeal function examination; 1 dog began having seizure‐like activity directly after intubation. After identification of seizure‐like activity in 3 of the first 4 dogs receiving methohexital, the anesthetic protocol was modified to include an additional bolus of methohexital if dogs were returning to a light plane of anesthesia and required intubation for their elective gonadectomy procedure after laryngeal function examination. After altering the protocol, 2 of the remaining 16 dogs still developed seizurelike signs. No adverse events were seen in dogs in group P.Adverse events identified in group M were tachycardia, seizure‐like activity, vomiting and regurgitation. Mean heart rate of dogs in group M prior to administration of methohexital was 132 beats/min , and mean heart rate after administration of methohexital was 229 beats/min . Dogs receiving propofol (group P) had a lower mean heart rate after administration  compared with dogs receiving methohexital  compared with direct observers (DRD and MBB). This difference may reflect the use of videos for evaluation by the masked observer, or potential bias by the direct observers, aware of the agents used. Video evaluation removes knowledge of respiratory status (presence of apnea), depth of respiration, and phase of respiration, which clearly could inhibit the ability of the masked observer correctly to identify adequate laryngeal function without paradoxical motion. It is also possible that the masked observer could pause the video and evaluate at leisure, which may improve the identification of occasional swallow or laryngospasm events. The difference in laryngeal function is more difficult to explain. The masked observer (SDL) appreciated more instances of “no function” in the second examinations compared with the direct observers. Laryngospasm or flutter may have been observed by the direct observers and qualified as brief function, whereas the masked observer had more time to evaluate the video and the opportunity to rewind to confirm weak or brief function. Some dogs who received doxapram maintained extremely exaggerated laryngeal function in which the larynx remained maximally abducted. The direct observers could identify these episodes because they correlated with exaggerated breathing, whereas the masked observer would not be able to recognize this change.The use of video laryngoscopy compared with direct observation of laryngeal function warrants additional study. The authors believe that the direct observers’ evaluation should serve as gold standard when masked observers are unable to detect laryngeal function in dogs identified as functional through direct assessment. Indeed, direct observers can identify subtle changes in respiratory rate, identify the depth of respiration, know the pattern of laryngeal motion as it correlates to phase of respiration, and identify the plane of anesthesia. Each of these components of an examination is important to evaluate a dog successfully for laryngeal paralysis.Doxapram did not influence laryngeal function in this study, irrespective of induction agent used, prompting us to accept our secondary hypothesis. However, administration of doxapram increased breathing scores. Such increase can enhance the confidence of an experienced observer assessing laryngeal function, but breathing can also result in paradoxical motion of the larynx. This effect could have contributed to the variability of laryngeal function scores in the current study.Examination consistently took longer when dogs received propofol rather than methohexital. Achieving an adequate plane of induction was delayed with propofol, which pushed back initial evaluation times as well as the time to administration of the second injection . After adequate anesthetic plane was reached, no difference was detected between agents in times between the end of the first injection and the start of the second injection. Lack of adequate anesthetic plane also required administration of a greater volume of propofol than anticipated in 5 dogs. Likewise, dogs receiving propofol consistently had more jaw tone and swallowing after initial drug administration, resulting in a longer time required to achieve the appropriate plane of anesthesia. This finding was expected because propofol administration can result in apnea if administered too fast. Therefore, slow administration of propofol is preferred to achieve an adequate plane of anesthesia to evaluate laryngeal function with the dog sedate enough to allow for adequate visualization.Evaluation of still images and the difference in rima glottidis size between the initial and second phases supported the absence of difference in methohexital and propofol for functional laryngeal evaluation. However, minor differences were identified between the 2 observers (DRD and SDL). One observer (SDL) identified a larger difference in the ratios obtained for the narrowest rima glottidis at the initial phase and the widest rima glottidis at the second phase compared with the other observer (DRD). This difference could reflect the difficulty in identifying landmarks in some images. Portions of the ventral floor of the larynx were sometimes cut off during recording of videos, complicating height measurements and associated mean ratio differences. These subtle differences in still image measurements highlight the value of visual examination to identify absolute change in rima glottidis size.Adverse events were more severe in dogs receiving methohexital compared with those receiving propofol. Seizure‐like activity occurred in 25% of dogs as they approached a lighter plane of methohexital anesthesia. The majority of these complications occurred early in the study (3 of the first 4 dogs). The seizure‐like activity ceased with additional administration of methohexital. After noticing the consistent timing of seizure‐like activity in the first 3 events, the study protocol was modified with a second bolus of methohexital administered before the animal reached a light plane of anesthesia. This altered protocol suppressed the onset of seizure‐like signs in all but 2 of the remaining study animals. This side effect of methohexital is important to note because some dogs concurrently vomited. Vomiting and regurgitation can result in aspiration pneumonia, for which laryngeal paralysis dogs are already at risk.Limitations of the current study include a small sample, lack of a standardized scale measurement within the video laryngoscopy, and lack of masking direct observers to the anesthetic induction agent used. A larger sample may further isolate specific adverse events associated with methohexital; however, a power analysis conducted did yield the ability to detect a standard deviation difference with 88% power. The effects of doxapram may have been more apparent with a larger sample as well. Use of a scale within the field of view on laryngoscopy would have allowed direct measurements of the area of the rima glottidis rather than calculating ratios. The authors used a height:width ratio to standardize measurements, as previously described for objective evaluation of the change in the rima glottidis.In conclusion, no difference was detected in evaluation of laryngeal function of dogs anesthetized with methohexital or propofol at a similar depth of anesthesia. Therefore, methohexital was not found to be a better induction agent to evaluate laryngeal function. Methohexital provided shorter examination times and decreased jaw tone compared with propofol but resulted in more adverse events. Administration of doxapram did not influence the evaluation of laryngeal function, irrespective of the induction agent. The results of this study provide evidence to support continued use of propofol as the preferred induction agent for laryngeal function examination.The authors declare no conflicts of interest related to this report.


Using these five topological measures, we constructed a linear model of biotic integrity, here a composite of taxonomic and functional diversity known as the California Stream Condition Index, of groups of BMI communities within a watershed. This model can account for 66% of among‐watershed variation in the mean biotic integrity of communities. These observations imply a role for co‐occurrence networks in assessing the current status of biotic integrity for BMI communities, as well as their potential use in assessing other ecological communities.Ecological monitoring of streams has often focused on assessing the biotic integrity of individual benthic macroinvertebrate (BMI) communities through local measures of diversity, such as taxonomic or functional richness. However, as individual BMI communities are frequently linked by a variety of ecological processes at a regional scale, there is a need to assess biotic integrity of groups of communities at the scale of watersheds. Using 4,619 sampled communities of streambed BMIs, we investigate this question using co‐occurrence networks generated from groups of communities selected within California watersheds under different levels of stress due to upstream land use. Building on a number of arguments in theoretical ecology and network theory, we propose a framework for the assessment of the biotic integrity of watershed‐scale groupings of BMI communities using measures of their co‐occurrence network topology. We found significant correlations between stress, as described by a mean measure of upstream land use within a watershed, and topological measures of co‐occurrence networks such as network size ( Using 4,619 sampled communities of streambed benthic macroinvertebrates, found in 67 watersheds covering the state of California, we found patterns of co‐occurrence were associated with the percent of land in a watershed dedicated to human activity. Using five topological measures of the co‐occurrence networks generated in our study, we were able to construct a linear model of watershed health, which could describe 66% of the variation in the health of watershed, as assessed by the California Stream Condition Index. We propose further investigation into the potential for the assessment of the health of ecological communities using topological measures of co‐occurrence networks. With ecological networks, connectance has been found to be negatively correlated with the skewness of their degree distributions  .The type and geographic extent of land use in the upstream vicinity of each sampling site data is derived from the National Land Cover Data set (NLCD)  and the mean percent developed upstream land use (land use), and for mean geographic separation distance in meters between samples (distance), we used the 2.6netassoc    Butts,  in R . This reflects a general decline in both the number of genera found in an individual sampling site  and the number of functional feeding groups , in relation to land use.The size of our co‐occurrence networks declined significantly with a rise in land use , we still found that connectance tended to be larger in co‐occurrence networks constructed from groups of stressed communities with a rise in land use . This positive association between stress and connectance appears to reflect a greater relative decline in the number of nodes relative to land use  than with the number of edges .While network size was found to have a strong negative correlation with land use, along with the mean number of edges per node  tended to decline with land use, while those of generalist functional feeding groups  tended to increase with land use Table .r = .25, p < 10–4). We also found evidence of a negative relationship between both mean co‐occurrence strength and the number of co‐occurrences , and connectance . This potentially indicates a preferential loss of weak co‐occurrences in networks assembled from communities under high levels of land use.In addition to a rise in connectance, networks assembled from communities with higher land use were on average found to have stronger co‐occurrences (p < 10–4). However, despite evidence of significant network modularity there was only a relatively weak positive correlation with its value and land use .Weaker trends were observed with regard to variables, modularity and degree heterogeneity, which describe structural arrangements of co‐occurrences. The mean modularity of our networks (0.35) was found to be both greater than the common modularity threshold of 0.3 . However, we did find support for our hypothesis that a decline in degree heterogeneity would be driven, at least in part, by a rise in connectance . Similar to our results with modularity, we found evidence, using a Wilcoxon signed‐rank test, for significantly higher values for degree heterogeneity in our co‐occurrence networks than their randomized null counterparts (p < 10–4). The higher mean degree heterogeneity of our co‐occurrence networks (1.82), as compared to that of the null networks (1.14), indicates our networks are skewed more toward a relatively small number of highly connected nodes than what would be expected by chance.Across our watersheds, we observe a trend where land use is associated with a slight decline in degree heterogeneity (3.2N), connectance (C), mean co‐occurrence strength (S), modularity (M), and degree heterogeneity (ζ), a linear model was constructed to best predict the mean value of the CSCI score for a set of samples , and the mean strength of their co‐occurrences (H3). Evidence supporting our hypothesis regarding a negative correlation between network size and land use (H1) reflects a well‐established link between environmental stress and both the loss of biodiversity and measures of biotic integrity  and degree heterogeneity (H5), were less clear. This may reflect limitations in our use of co‐occurrence rather than co‐abundance networks. However, prior evidence from assessments of biotic integrity for stream communities of BMIs has shown a strong correlation between results generated using community data sets described through the abundance or presence/absence (Beentjes, Speksnijder, Schilthuizen, Schaub, & Hoorn, Analyses of co‐occurrence networks have been used to identify candidate keystone taxa (Berry & Widder, Even with these limitations we found, a simple linear model composed of topological measures of co‐occurrence networks could describe a significant portion of the observed variation in the biotic integrity of our BMI communities Table . AnalysiVariations in our models appeared to be driven more by both land use and the standard deviation on land use than either altitude or geographic separation distance Table . This reThese trends may reflect co‐occurrence patterns unique to stream communities of BMIs. However, the framework we have used to test these hypotheses is not dependent upon the particular identities of taxa present in communities and may have the potential to be applied to other systems. Assessments of the biotic integrity of freshwater ecosystems have been carried out at a variety of spatial scales (Booth et al., 4.1It is increasingly becoming feasible to characterize entire ecological communities, from prokaryotes through metazoa, through metagenomic approaches (Baird & Hajibabaei, No conflict of interest exists in this manuscript.A.L.S. both constructed the co‐occurrence networks used in this study and performed the analyses involved with measures of their topology. R.M. and S.T. were involved in developing the California Stream Condition Index. A.L.S. wrote the first draft of the manuscript, with all authors contributing to subsequent revisions.