EMBO Molecular Medicine by Demminger and colleagues  is timeliness to exemplify the steps needed to develop effective vaccines.Undoubtedly, vaccination is one of the health interventions showing major impact on humankind. Vaccines remain one of the most effective and safest ways to tackle infections. The current coronavirus pandemic is not an exception, and we all hope that ongoing international efforts will succeed in developing a vaccine soon. In this scenario, the present work published in this edition of   EMBO Molecular Medicine.When vaccination against a virus uses another virus or the tale of AAV‐mediated vaccines against influenza infections, in this issue of  Spanish flu, highlighting the devastating impact of these viral infections.This study focuses on influenza, a seasonal pandemic with major annual impact on the health systems worldwide. This infection is associated with high mortality and morbidity in very young and old individuals. Stories in the recent news have highlighted the unusual deadly influenza pandemic lasting from 1918 to 1920, the so‐called The current influenza prophylaxis is based on immunization with trivalent or quadrivalent influenza vaccines containing antigens from influenza A and B viruses. However, this vaccine is mostly strain specific and the process to produce it is time‐consuming. In addition, evidence indicates it does not provide protection against emerging zoonotic influenza A virus strains. Overall, the need to develop a novel vaccine conferring protection as broad as possible is widely acknowledged.via interference with viral replication or via Fc‐receptor‐mediated cellular cytoxicity  acting Demminger and colleagues developed AAV vectors expressing wild‐type HA, the surface exposed neuraminidase (NA), or chimeric HA (cHA) containing head regions from influenza A virus subtypes, or HA headless constructs. Constructs were based on proteins encoded by the pandemic influenza virus A/California/7/2009 (H1N1) pdm . Control assays confirmed the robust expression of all antigens.Next, they assessed the immunogenicity in mice challenged intranasally testing side‐by‐side empty AAV vector (expressing GFP) and whole‐inactivated virus (WIV) as controls. As expected, all AAV constructs expressing influenza antigens did elicit reactive antibodies that correlated with the total serum IgG titers. Interestingly, AAV vectors induced antibodies against a panel of different influenza viruses. Somewhat unexpectedly, WIV vaccination also elicited broadly reactive antibodies. Interestingly, neutralizing antibodies were found only in the sera of mice challenged with AAV containing HA, or cHA, and these antibodies were specific for the virus used for prime immunization. Of significant therapeutic relevance, authors demonstrated that AAV vector vaccination induced strong Fc‐activating antibody response against Cal/7/9 but also against the mouse‐adapted heterologous influenza virus PR8.These positive results encouraged authors to test whether AAV vectors do protect mice from homologous  and heterologous (PR8) challenges. Although the mouse model has its limitations in terms of translational potential, it offers the opportunity to obtain first proof‐of‐concept information. As read‐outs of infection, Demminger and co‐workers used weight loss and survival of mice over a 14‐day period. Mice vaccinated with the AAV construct were protected, and preliminary data suggest that three doses of the vaccine were better than two doses. Although these findings are sound, it is important to note that determination of viral titers in the tissues over time should be determined to solidify the read‐outs based on animal behavior, clinical parameters, and/or survival. Some control experiments revealed a reduction in viral titers in vaccinated animals.In a remarkable tour de force, authors went on to assess the protective efficacy of the AAV vectors in ferrets. This animal model reproduces very closely the course of human influenza. In these experiments, authors also introduce a group immunized with a commercial quadrivalent influenza vaccine of season 2017/18. Animals were vaccinated intranasally three times in 4‐week interval whereas the control group immunized with the commercial vaccine were given two doses by the intramuscular route. It can be argued that the different experimental regime of the groups is not suitable for a comparison between the different vaccine formulations. However, the aim of this work was to demonstrate whether the AAV vectors do confer protection and not whether they are better than the commercial vaccine. In a 3‐day infection protocol, ferrets vaccinated with the AAV vectors showed clear signs of recovery assessing serous nasal exudate, congestion, frequent sneezing, wheezing, and depression. Notably, only in those ferrets vaccinated with the AAV‐HA construct and the commercial vaccine was there a reduction in the load of virus although no sterile immunity was induced. Virus clearance was associated with a significant reduction in pathophysiology and induction of neutralizing antibodies.This elegant study follows a comprehensive design from in vitro characterization of the AAV vectors to animal pre‐clinical studies probing two different research models, one of them of significant translational potential Fig . The useThis work also reinforces the utility of AAV vectors as platform to develop vaccines. AAV vectors are already approved by the EMA and FDA for its use in humans and have been tested in pre‐clinical immunization studies against infectious diseases. Demminger and colleagues demonstrate that they should be considered as a valid platform for a broadly protective influenza vaccine. I anticipate that this comprehensive work will lead to significant advances in our quest to develop effective influenza vaccines and may result in similar research to tackle other infections.EMBO Molecular Medicine (Demminger et al, I will encourage the vaccinology field to consider and set clear parameters that should be determined in any research like the one presented in this issue of


New HIV diagnoses have fallen in the past decade due to increased HIV testing, earlier diagnosis, earlier antiretroviral treatment, improved linkage to care and engagement in care, and the recent increased uptake of pre‐exposure prophylaxis (PrEP). We propose a novel method to compute the rate of new HIV diagnoses at the Metropolitan Statistical Area (MSA) level in the US to support the evaluation of comprehensive treatment and prevention efforts over time.The number of new HIV diagnoses, number of individuals with a PrEP indication and aggregated person‐time exposed to PrEP during the years 2012 to 2017 were used to compute a new HIV diagnosis rate for people at risk of HIV excluding those already on PrEP for the 105 MSAs in the US with published HIV surveillance data. In our calculation of person‐time with a PrEP indication, time‐at‐risk excluded time on PrEP and time after an HIV diagnosis. We used a multivariate Poisson regression model to estimate HIV diagnosis rates by year and location.From 2012 to 2017, the aggregate HIV diagnoses rate among high‐risk individuals with an indication for PrEP in the 105 MSAs decreased from 4.14 per 100 person‐years (PY) (95% CI 4.10 to 4.19) to 3.26 per 100 PY (95% CI 3.22 to 3.30). For the 25 US MSAs that overlapped with an ongoing large randomized clinical trial of PrEP in men who have sex with men (MSM), the HIV diagnosis rate from 2012 to 2017 decreased from 4.86 per 100 PY (95% CI 4.80 to 4.93) to 3.61 per 100 PY (95% CI 3.56 to 3.66), a decline that was more rapid than in non‐study MSAs .We propose a model to estimate the background HIV diagnosis rate in people at risk for HIV and with a PrEP indication in US MSAs (excluding those on PrEP) using publically available surveillance data which can evaluate trends over time. Data generated using this methodology could be used by policy makers and local HIV prevention specialists to evaluate and monitor their prevention efforts for the population at risk in their communities. The challenge stems partially from the need to understand the rate of new HIV infections specifically in the subgroup of high‐risk individuals with an indication for PrEP . Since new HIV infection diagnosis rates vary substantially by community, per‐community or aggregate estimates of PrEP effectiveness require knowledge of new diagnosis rates within specific communities. Such community‐level data are important to guide local prevention efforts tailored to each community's specific needs. It is equally important to understand local HIV diagnosis rates in PrEP‐eligible individuals when designing and interpreting clinical trials involving PrEP and novel HIV prevention strategies, especially when placebo arms must be omitted for ethical reasons Here we present a computational approach that utilizes publicly available MSA‐level HIV surveillance data collected annually by the CDC in order to calculate the rate of new HIV diagnosis among PrEP‐eligible individuals. By doing this, we sought to quantify differences in new HIV diagnosis rates by community in PrEP‐eligible individuals, and to establish a background diagnosis rate, analogous to a placebo rate. Using this approach, we have generated an estimate of the rate of new HIV diagnoses among PrEP‐eligible individuals on an MSA level. As an example of this method's utility, we demonstrate how this rate varies over time in MSAs participating in an ongoing PrEP clinical trial compared to control MSAs that are not participating in the trial. This methodology for estimating a background rate of new HIV diagnoses among PrEP‐eligible individuals could be useful for public health and policy officials assessing the impact of HIV prevention efforts on a community scale, and for creating a hypothetical placebo arm in active comparator PrEP clinical trials.22.1Data were obtained from publically available sources with the exception of patient‐level PrEP usage data, which was obtained from a pharmacy claims database which provided continuous or discontinuous TVD exposure periods . The number of new HIV diagnoses in 136 MSAs from 2012 to 2017 were obtained from CDC HIV Surveillance Reports 2.2The proportion of adults with a PrEP indication by MSA is computed using the published data by state and US Census data. The temporal changes by calendar year are computed using the natural rate of change in each MSA overall population as per the census estimates.The rate of new HIV diagnoses per 100 PY is obtained as follows: The numerator corresponds to the count of new adult HIV diagnoses by MSA on a given year as reported by CDC, which is adjusted so that those without a PrEP indication are removed from the calculation by counting only those individuals with a transmission category  who are included in the denominator. These excluded individuals (non‐PrEP risk categories) represented 0.19% of all new diagnoses. The denominator corresponds to person‐time for individuals who are (1) HIV seronegative, (2) have an indication for PrEP, and (3) are not taking PrEP. The denominator is calculated using a survival analysis interval censoring approach, by starting with the number of adults with a PrEP indication alive at the beginning of the period multiplied by one year of follow up, minus the number of adult PrEP users prevalent during the same period multiplied by the average person time not at risk, minus the number of adult new HIV cases multiplied by the average person time at risk, assuming that the count of cases follows a Poisson distribution and that the time between events follows an exponential distribution. This approach censors the individuals at risk in the denominator who acquired HIV at the midpoint of the interval, with those dying during the interval being also censored, and both not contributing time in the next interval. Additionally, this approach includes in the denominator only person‐time that individuals were at risk and not taking PrEP during a given year excluding the small number who become HIV positive during the period N = number of HIV diagnosesn = Number of HIV diagnoses without PrEP indicationsPT = Total person time for those with PrEP indicationsPrEP = Total person time of subjects on PrEP during exposure to TVDPTHIV = Total person time of subjects who became HIV positivePTMore specifically, letPrEP = cumulative periods of time exposed to TVD minus the time after and if they become HIV positive (1 per 100 person years of TVD exposure). PTHIV = amount of time during the observation period after subjects become HIV positive during the interval.Moreover PT = number of subjects with a PrEP indication alive at the start of the observation period times one year. PTWe computed an aggregate rate of new HIV diagnoses among PrEP‐eligible individuals by performing the aforementioned calculation using merged data from 105 MSAs that had complete PrEP indication information available. We also performed identical calculations with MSAs stratified by overlap with the DISCOVER trial, an ongoing double blind active controlled study of F/TDF versus emtricitabine plus tenofovir alafenamide (F/TAF) for PrEP being conducted in 2,500 men who have sex with men (MSM) and transwomen We performed a sensitivity analysis using self‐reported testing information at the MSA level and explored the potential bias due to differential proportions of HIV testing over time and place. In addition, we conducted a sensitivity analysis using precise MSM HIV incidence rate estimates from San Francisco (which has a robust HIV diagnosis and reporting programme). Statistical analyses were carried out using STATA 15.2.3All data used in this manuscript were obtained from publically available databases and publications with the exception of some patient‐level PrEP utilization data which were obtained in an anonymized form from a commercial source .3Using merged data from 105 MSAs that had complete PrEP indication information available, we calculated the estimated rate of new HIV diagnoses among PrEP‐eligible individuals between 2012 and 2017. Over that period, we observed a decrease in the rate of new HIV diagnoses, from 4.14 per 100 PY (95% CI 4.10 to 4.19) to 3.25 per 100 PY (95% CI 3.22 to 3.30), a mean decrease of 6.0% per year (95% CI 5.3 to 6.4) , we utilized MSA‐level HIV testing data from the CDC Behavioural Risk Factor Surveillance System 4The methodology we present here uses publically available HIV surveillance data to calculate estimated HIV diagnosis rates among PrEP‐eligible individuals while excluding those on PrEP. Having the background rate of HIV diagnosis in those who are not on PrEP allows for a comparison between the observed rate of new HIV diagnoses post intervention with the expected rate over time and may be useful for evaluating the impact of HIV prevention efforts, such as the proposed federal government effort to improve PrEP coverage in certain counties and states. Furthermore, computation of an incidence rate for certain risk factors is possible using the same data sources. The overall rate in 2017 among MSM who represent (69.7%) of all cases was 3.42 per 100 PY, 95% CI 3.37 to 3.47. Using the same method and the availability of race both for numerator and denominator, 2017 rates can be computed for Whites , Hispanics  and African Americans .In addition, we used this methodology to calculate an estimated background rate of HIV diagnosis, analogous to a placebo rate, in the MSAs where a large PrEP trial (DISCOVER) is being conducted. The methodology revealed several notable findings. First, the HIV diagnosis rates in the DISCOVER MSAs was higher overall than in the non‐DISCOVER MSAs, likely reflecting the efforts to conduct the trial in geographies with high historic HIV prevalence and current higher incidence. Second, we demonstrate that the temporal declines in new HIV diagnosis rates differed between DISCOVER and non‐DISCOVER MSAs, likely reflecting the aggressive prevention efforts taking place in these higher risk MSAs. And lastly, we observed that the DISCOVER MSAs had a continued decline in the HIV diagnosis rate after the implementation of the DISCOVER trial (2015 to 2017), while rates in non‐MSAs remained steady. While our methodology cannot determine causality and regression toward the mean may be operating, the findings demonstrate how our methodology could be useful in tracking the impact of PrEP and other prevention strategies on an MSA level over time.This approach may also be useful in the design and interpretation of active comparator noninferiority trials for HIV prevention. Since a true placebo arm is often excluded in modern PrEP clinical trials We note several limitations in the approach reported here. Because CD4 count data stratified by PrEP indication are not available, it was not possible to use the well‐characterized CD4 depletion model to estimate incidence rates Additionally, because PrEP indication data are available on a state scale but not an MSA scale, our model relies on the assumption that PrEP indication frequencies within MSAs are equivalent to those in their parent state. While this assumption is imperfect, our MSA PrEP indication estimates are likely underestimates, since the proportion of individuals in the entire state is probably lower than metropolitan areas. Despite these limitations, our findings suggest that a computational HIV diagnosis rate derived using this methodology can provide a reliable estimation of HIV incidence among high‐risk individuals.Publically available HIV surveillance data can be used to compute an estimated HIV diagnosis rate among high‐risk, PrEP‐eligible individuals. This methodology may be useful for local policy makers and prevention specialists to monitor ongoing prevention efforts. Additionally, data generated using this methodology may be useful for investigators conducting HIV prevention studies, especially when the lack of a placebo arm makes the determination of efficacy challenging.5The model we propose allows for the estimation of the background HIV diagnosis rate in people at risk for HIV and with a PrEP indication in US MSAs (excluding those on PrEP) using publicly available surveillance data which can evaluate trends over time. Data generated using this methodology could be used by policy makers and local HIV prevention specialists to evaluate and monitor their prevention efforts for the population at risk in their communities.RM, JA, CC, MD and SM are employees of Gilead Sciences and hold stock interest in the company.RM designed the model and computational framework and analysed the data. JA performed the sensitivity analysis. CC wrote the manuscript with input from all authors. JB, MD and SM contributed to study conception, manuscript writing, and were in charge of overall direction and planning. SS provided data for sensitivity analysis and guidance on study design. All authors discussed the results and provided critical review of the manuscript.


The World Health Organization (WHO) recently completed the first phase of a RSV surveillance pilot study in fourteen countries (two to three in each WHO region) building on the Global Influenza Surveillance and Response System (GISRS). This active surveillance strategy had several objectives including understanding RSV‐related health burden in a variety of settings. A range of approaches can be used to estimate disease burden; most approaches could not be applied by participating countries in the WHO surveillance pilot. This article provides the recommendations made by WHO for strengthening and expanding the scope of the RSV surveillance in the next phase to enable burden estimation. Shi et al have estimated that globally RSV‐associated lower respiratory tract infection (LRTI) accounted for between 94 600 and 149 400 deaths annually. In addition, 33 million RSV‐associated LRTI resulted in 3.2 million hospital admissions.Recently, significant progress has been made in the development of a range of RSV vaccine candidates, in particular for the protection of infants via maternal immunization. In addition, new longer acting monoclonal antibodies that can protect children for several months are in late‐stage clinical development. These developments have ignited interest in this area. Accurate estimates of RSV‐related health burden including costs are needed to undertake economic evaluations comparing the cost‐effectiveness of alternative interventions.Ahead of the potential availability of new RSV vaccines for pregnant women and young children and new generation monoclonal antibodies and to improve awareness of its importance among policy‐makers, health staff and the public, the World Health Organization (WHO) established an RSV surveillance pilot study in 2017 in fourteen countries (two to three in each WHO region) building on the Global Influenza Surveillance and Response System (GISRS), hereafter referred to as the pilot. The pilot had several objectives including contributing to understanding of the health burden of RSV‐related infection in a range of settings. This article reviews the potential approaches that can be used to estimate disease burden and explores their applicability to the WHO RSV surveillance based on the influenza platform.2The WHO RSV surveillance pilot (1) took place in fourteen countries ranging from high to low income and from tropical to temperate settings. The main aim was to assess whether global RSV surveillance could fit into the GISRS platform. A secondary aim was to assess the feasibility of collecting data points needed for burden estimates in participating countries. The focus of the pilot was to collect data to estimate the proportion of respiratory hospitalizations associated with RSV. In some settings, this was extended to outpatient or primary care settings. Many countries were successful in implementing the clinical and laboratory aspects of the surveillance including collection of the required sample of clinical specimens.3The burden of disease can be characterized in several different ways. Burden on the health service is often quantified in terms of numbers of outpatient/primary healthcare consultations; emergency department attendances and hospital admissions. Burden on the population may also be described by the numbers of infections including short‐term morbidity and any longer‐term chronic consequences of infection plus deaths associated with infection.Burden of disease estimates may also include the cost of diseases . For example, a study in Bangladesh demonstrated a median direct cost of USD 10 million of RSV‐associated hospitalizations among children <5 years of age in 2010 and an indirect cost of USD 3 million.Obtaining an accurate estimate of the disease‐specific healthcare burden can be challenging, as often the investigation, laboratory testing and reporting of infectious diseases such as RSV are incomplete or absent. RSV‐associated infection is known to be very common as shown with ad hoc age‐stratified serological studies, for example a study in Kenya indicated that 100% of the population are likely to be infected by 3 years of age.4Method 1: Regression modelling using routine data sources. This ecological approach uses national or large‐scale routine laboratory surveillance and administrative health service data that have been collected consistently over several years. It has been utilized in some countries to measure the burden of a wide range of infections including influenza and RSV.Method 2: Multiplicative modelling. This approach has been applied to estimate influenza‐related mortality for the 2009 pandemic by multiplying age‐specific symptomatic infection attack rate and symptomatic case fatality rate from a range of income settings. With this method, a respiratory mortality multiplier was defined to consider differences in outcome according to setting. Simulation models with these parameters were then applied across all countries to derive global estimates of influenza‐related mortality.Method 3: Data linkage. Linkage of routine hospital administrative data to laboratory records has been used in developed settings such as Australia to better describe RSV epidemiology.Method 4: Enhanced surveillance/prospective cohorts. The establishment of prospective population cohorts, with the comprehensive systematic collection of epidemiological information and biological samples from participants, allows RSV epidemiology and burden to be studied more directly—both in the community and in primary and secondary care settings. The Influenza and RSV in Infants Study (IRIS) is a multi‐country prospective study in four low‐income settings and was set up to assess the frequency of influenza and RSV in children <1 year of age who have been hospitalized and to ascertain predictors of more severe disease.Method 5: Simple, rapid assessment approach. This methodology has been used to estimate the national burden of influenza using surveillance data from sentinel sites and by applying several adjustments to obtain a national estimate.A range of approaches, many deployed for influenza, can be used to estimate local‐, country‐ and international‐level RSV‐related burden in terms of numbers of cases and disease incidence either using established disease surveillance systems and vital statistics or through special studies. Their potential application in the context of the WHO surveillance pilot (1) is explored:5The results of the pilot were reviewed at a face‐to‐face meeting  organized by WHO. Some countries were only able to implement a convenience sampling strategy and relied on attending physicians to collect samples and baseline clinical information. Most of the participating pilot countries lacked data from routine hospital admissions, catchment populations and administrative registers to estimate the burden of RSV‐associated hospitalization stratified in different age bands and disaggregated by week or month. This meant none of the burden estimation methods outlined above could be applied at this first stage of pilot—including method 5.6Building on the learning from the pilot, an extension phase is planned and coordinated by WHO. Going forward in the next phase of the WHO RSV surveillance, countries agreed at a further face‐to‐face meeting  on the importance of prioritizing hospitalization burden associated with RSV in young children. The importance of defining a minimum data set was identified and that additional efforts would be required to collect harmonized burden‐related data based on surveillance. Most countries proposed the need to set up systems to collect healthcare attendance data more systematically as part of surveillance that could inform policy‐making in circumstances where no data from any of the recognized approaches outlined earlier are available in that country. In the absence of resources for special disease burden studies, a tiered approach towards disease burden estimation using surveillance data was proposed for the second phase of the WHO surveillance. Table The next phase of the WHO Global RSV surveillance programme will assist countries to better describe RSV health burden by collecting the necessary data needed. The critical data still to be collected in phase 2 of the WHO RSV surveillance are as follows: (a) documentation of total hospital admissions, respiratory admissions and patients screened but not enrolled into the surveillance programme and (b) the sampling fractions for cases in different time periods during the year. If countries opt for more sophisticated estimates of RSV disease burden, information will be additionally required on (a) what the catchment population is from which cases are drawn at each site (ie how many people would go to the surveillance facility if they developed symptoms of an RSV‐like illness), (b) the number of in‐referrals from outside the catchment area, (c) catchment population denominators and (d) Healthcare Utilization Surveys (to estimate the proportion of catchment population who would present at the sentinel facility for an acute respiratory illness).The WHO RSV surveillance is making important progress to estimate RSV‐associated hospital‐related burden in a range of settings through the inclusion of additional data required to refine these estimates. The WHO candidate case definitions for severe and very severe RSV‐associated lower respiratory tract infections (LRTIs) require additional information on spO2, pulse oximetry and IMCI danger signs to be collected, as proxy indicators for disease severity.7Accurate in‐country disease burden estimates are important for local policy‐makers to make decisions on the introduction of prevention strategies and to employ cost‐effectiveness models when new RSV vaccines or monoclonal antibodies become available. Even simple estimates of proportion of hospitalizations associated with RSV are important to raise awareness of RSV disease burden among policy‐makers and providers. The WHO Global RSV surveillance programme aims to provide countries with a platform based on established approaches to collect these data. The next phase of the programme will assist countries with tools for collection of burden data, closely monitor sampling strategies for patient selection and testing, and strengthen the surveillance to provide simple, robust burden estimates for informed immunization policy decisions.


Estrogen receptor signaling plays an important role not only in breast cancer but also in other cancers including ovarian cancer, prostate cancer and acute myeloid leukemia. This series of articles includes new findings of estrogen receptor co-regulators in breast and ovarian cancer with potential as novel targets for therapy. The major isoforms of estrogen receptor—namely estrogen receptor alpha and estrogen receptor beta—can have opposing functionality and these differing roles are described in reviews of estrogen signaling in prostate and ovarian cancers and acute myeloid leukemia. In breast cancer, mutated forms of the estrogen receptor can be selected for on endocrine treatment and frequently lead to treatment resistance while novel imaging techniques are being evaluated to monitor diagnosis and response to treatment. These developing research fields are overviewed. The series of seven articles  in this Special Issue address estrogen receptor (ER) signaling pathways in cancer and is presented by international experts engaged in research across a variety of cancer types. While estrogen signaling has long been known to have a major role in breast cancer, there is emerging evidence that it can regulate function in other cancer types also. These include ovarian cancer, prostate cancers and acute myeloid leukemia (AML) and these are reviewed here alongside studies of breast cancer.ESR1 (the ERα gene). The inhibition of both DOT1L and ER showed additive effects on cell growth and their dual inhibition is suggested as a means to enhance the current effects of endocrine therapy [The two original articles both describe studies with ER-coregulators, which control the transcriptional activity of ER ,2. A stuThere is increasing recognition that estrogen signaling may be important in other cancer types.Furthermore, the major isoforms—ERα and estrogen receptor beta (ERβ)—have been shown to possess different functionalities. Di Zazzo et al. review data supporting a role for estrogen signaling in the epithelial to mesenchymal transition (EMT) in prostate cancer . In thisParallel findings for these isoforms and splice variants are also observed in ovarian cancer . While EIn AML, these opposing actions of ERα and ERβ continue to be the dominant view with ERα activation increasing the effects of chemotherapy while ERβ inhibits both leukemogenesis and leukemia cell proliferation and this area is reviewed by Roma and Spagnuolo . The ratESR1, are increasingly being recognised as a mechanism of resistance to endocrine therapy in luminal disease and this area is reviewed by De Santo et al. [In breast cancer, mutations in the ligand binding domain of the ERα gene, o et al. . These mo et al. . The theo et al. . The useo et al. . These ao et al. .


Resection of the seizure generating tissue can be highly beneficial in patients with drug‐resistant epilepsy. However, only about half of all patients undergoing surgery get permanently and completely seizure free. Investigating the dependences between intracranial EEG signals adds a multivariate perspective largely unavailable to visual EEG analysis, which is the current clinical practice. We examined linear and nonlinear interrelations between intracranial EEG signals regarding their spatial distribution and network characteristics. The analyzed signals were recorded immediately before clinical seizure onset in epilepsy patients who received a standardized electrode implantation targeting the mesiotemporal structures. The linear interrelation networks were predominantly locally connected and highly reproducible between patients. In contrast, the nonlinear networks had a clearly centralized structure, which was specific for the individual pathology. The nonlinear interrelations were overrepresented in the focal hemisphere and in patients with no or only rare seizures after surgery specifically in the resected tissue. Connections to the outside were predominantly nonlinear. In all patients without worthwhile improvement after resective treatment, tissue producing strong nonlinear interrelations was left untouched by surgery. Our findings indicate that linear and nonlinear interrelations play fundamentally different roles in preictal intracranial EEG. Moreover, they suggest nonlinear signal interrelations to be a marker of epileptogenic tissue and not a characteristic of the mesiotemporal structures. Our results corroborate the network‐based nature of epilepsy and suggest the application of network analysis to support the planning of resective epilepsy surgery. To render patients seizure free and minimize adverse effects, localization of brain tissue necessary and sufficient to generate seizures  is not conclusive or the localized region is contiguous to eloquent cortex , intracranial EEG  is the gold standard to obtain information with superior spatial resolution and signal‐to‐noise ratio. Despite being labor‐intensive and prone to interrater variability, visual analysis by trained neurophysiologists/epileptologists is still the method predominantly used in clinical routine to analyze these signals. The signals showing the first continuous epileptiform activity constitute the seizure onset zone (SOZ), which is then used as a proxy for the EZ. However, the considerable portion of suboptimal outcomes suggests that classic approaches treating signals mostly as if they were isolated from each other could be too simplistic to capture the essential information in all cases. Instead, novel quantitative approaches incorporating interrelations among different parts of the epileptic brain might be necessary. Besides, automated analyses could abbreviate the labor‐intensive process of visual analysis and introduce more objectivity into it.In the past, there has been research on numerous quantitative approaches to analyze iEEG signals to assess their potential to indicate epileptogenicity of the underlying tissue and thus be able to predict resection targets. A signal characteristic considered over the last two decades as a potential marker of the EZ were high frequency oscillations (HFOs) but meta‐analyses could not confirm an indicative role in epilepsy surgery decision‐making  of the interaction between any pair of nodes. Different measures capture different signal characteristics and thus associated results must always be considered specific to the chosen dependence measure . Incorporating multiple complementary types of interrelations in parallel might help to get a more complete view of the ongoing interactions and thus also contribute to improve on the outcomes in epilepsy surgery.Recently, Rummel et al.  used fouPreceding work has demonstrated a clear difference in linear and nonlinear connections after surrogate correction  using assortativity, a graph theory measure quantifying similarity of connected nodes. We then analyze both types of interrelations based on their range, that is, if they act predominantly within or between hemispheres. We complement the characterization of the linear and nonlinear interrelations by calculating their strengths in dependence on their location and on their relation to the resected brain tissue. The entirety of our findings suggests a conception of seizure related activity where linear and nonlinear interrelations possess fundamentally different roles.22.1In this study, we included 20 consecutive patients with drug‐resistant epilepsy who received implantation of depth electrodes targeting the mesiotemporal structures during evaluation for epilepsy surgery at the Inselspital Bern . More detailed patient data are given in Table This study was approved by the Ethics Committee of the Kanton of Bern (project no. 2017‐00697). All decisions regarding the actual treatment of the patients  were made solely on clinical grounds prior to this study and all patients gave written and informed consent that EEG and imaging data may be used for research purposes.Of each patient, we analyzed recordings containing the first two artifact‐free seizures occurring during iEEG monitoring and in order to maximize homogeneity we concentrated our analysis on those signals recorded from the depth electrodes implanted into the mesial temporal lobes even though some patients had individualized additional electrodes implanted. In addition, permanently artifact corrupted channels were excluded (<5% of channels). Decisions on artifacts and seizure onset and termination were made by experienced epileptologists/electroencephalographers (K.S. and H.G.) based on visual inspection.Since the iterative generation of surrogate time series (see below) is computationally expensive, continuous analysis of interictal EEG with the chosen methods is prohibitive. Rather we needed to restrict our analysis to segments of few minutes duration. Kuhnert, Elger, and Lehnertz  have fouIn summary, we included two recordings of 20 patients yielding 40 preictal epochs in total. For 24 epochs, we know the channels recording from tissue that was later resected causing a favorable outcome  and, for 18 epochs, these channels were located in the mesiotemporal structures (Patients 1–9).2.2To contrast different regions regarding the strength of connectivity, we separated interrelations in various ways.In all patients, we separated interrelations according to if they occurred between channels inside the hemisphere of seizure onset , between channels inside the contralateral hemisphere , or between channels of different hemispheres. Moreover, in the subgroup of patients who underwent resective surgery targeting the mesial temporal areas with favorable seizure control , we separated interrelations according to if they occurred between channels recording from the subsequently resected brain tissue, between channels recording from the brain tissue ipsilateral to surgery but left untouched by surgery, or between these channels recording from opposite regions.Likewise, we grouped the node strengths in all patients according to whether the channels were recording from the hemisphere ipsilateral or contralateral to seizure onset and in Patients 1–9 also according to if they were recording from the subsequently resected brain tissue or from the untouched brain tissue ipsilateral to surgery.2.3In this study, we analyzed undirected surrogate‐corrected linear and nonlinear interrelation matrices, calculated in a sliding windowed procedure from iEEG recordings as described in detail in the study by Rummel et al. . In brieThe nonlinear interrelation matrices were determined by mutual information, a measure that quantifies the amount of information one variable provides about the other. Since mutual information is sensitive to both linear and nonlinear dependences alike, we used multivariate IAAFT surrogate time series with conserved Pearson correlation matrix  . We analyzed the node strengths groupwise with respect to the hemispheres and where applicable with respect to the resected area.2.5N = 40 and tests including only patients which had surgery in the mesiotemporal structures with a favorable outcome have a sample size of N = 18.We calculated the graph measures from surrogate‐corrected interrelation matrices derived from windows of 8 s duration displaced consecutively by 1 s over an epoch of 180 s directly preceding the visually determined seizure onset. These values were then averaged epoch‐wise for subsequent tests. For the PCA analysis and the identification of core nodes, we used the average of all matrices of an epoch. Accordingly, tests including all patients have a sample size of Due to exclusion of channels permanently corrupted by artifacts, the number of nodes in different patients varied. In addition, the absolute values of linear and nonlinear interrelations differ between patients and epochs. To enable comparisons across multiple epochs and patients, we normalized all node‐related measures to the relative amount an entity (node/region) contributed in an epoch. That is, the actual interrelation strength in each entity was divided by the value that would be expected if the total amount of interrelation strength was uniformly distributed. Hence, the resulting normalized values were factors indicating the entities' relative strengths with values <1 if an entity was underrepresented and >1 if it was overrepresented.α = 0.01. In situations where the median of more than two groups was of interest, we first applied a Kruskal–Wallis test. If the null hypothesis that all groups stemmed from distributions with the same median was rejected, we applied pair‐wise post hoc Mann–Whitney U‐tests to determine which medians differed. Bonferroni correction was used in these cases to account for multiple testing. If the central position of only two groups needed to be compared, we used Mann–Whitney U‐tests directly. To determine whether the median of a group deviated from an expected value, we used sign tests of the difference. In all boxplots, the central line indicates the median of the distribution, the first (q1) and third quartile (q3) are indicated by the bottom resp. top edges of the box and the whiskers comprise all data points in the range q1–1.5 * (q3 − q1) to q3 + 1.5 * (q3 − q1). Values beyond this range are displayed as dots.We used nonparametric testing throughout this study since test sizes were rather small and distributions potentially skewed. For all statistics, we used a significance level 33.1p < 10−4 in N = 40 tests). Accordingly, results from subsequent analyses based on the linear resp. the shift surrogate‐corrected nonlinear interrelation matrices were very similar. In contrast, both were dissimilar to the mutual information matrices corrected with multivariate IAAFT surrogates . Unlike the shift surrogate‐corrected nonlinear interrelations, the multivariate IAAFT surrogate‐corrected nonlinear interrelations provide additional information over the linear interrelations. Due to these findings, we restrict the presentation of our results to the interrelation matrices corrected by IAAFT surrogates in the sequel, which were also used in the study by Rummel et al. . In all epochs, the nonlinear excess networks had on average a negative assortativity, while the linear networks typically had positive assortativity on average. The median was in both groups significantly different from zero .The distribution of epoch‐wise averaged p < 10−32, Kruskal–Wallis test). In a post hoc analysis of pair‐wise tests we found (a) linear interrelations within both hemispheres were significantly stronger than between them ; (b) within the nonfocal hemisphere there were significantly stronger linear than nonlinear excess interrelations (p < 10−11), while there was no such difference within the focal hemisphere; (c) nonlinear excess interrelations were significantly stronger within the focal hemisphere than within the nonfocal hemisphere (p < 10−10); (d) nonlinear excess interrelations were significantly stronger between hemispheres than within the nonfocal hemisphere (p < 10−12); and (e) nonlinear excess interrelations between hemispheres were significantly stronger than the linear ones (p < 10−12). The number of multiple comparisons was 18: three types of interrelations (results from the shift surrogate‐corrected nonlinear interrelations are not shown) contrasted within three subdivided regions (nine tests) and each type of interrelation contrasted across the three regions (nine tests). The Bonferroni‐corrected significance level was thus 5.5 * 10−4.To examine the interrelations depending on their spatial range, we distinguished them with respect to the hemispheres see . For linp = .177, Kruskal–Wallis test) and no deviation of any of the groups from the expectance under a uniform distribution of all linear interrelations . The post hoc analysis found: the NSs of nonlinear excess interrelations were (a) significantly stronger in the focal than in the contralateral hemisphere  and (b) significantly stronger in the resected nodes than in the ipsilateral nonresected nodes (p < 10−05). Moreover, the NSs of nonlinear excess interrelations in the focal hemisphere and in the resected tissue were significantly larger than would be expected under a uniform distribution of all nonlinear excess interrelations  whereas in the nonfocal hemisphere they were significantly weaker (p < 10−07). Within the resected brain tissue the median overrepresentation of nonlinear excess NS was 1.97 (range 1.07–3.45), whereas in the remaining ipsilateral channels, it was 0.84‐fold underrepresented (range 0.40–1.36). The number of multiple comparisons was 6: nonlinear excess interrelations contrasted according to two regional subdivisions (two tests) and all subdivisions contrasted against the expected value (four tests). The Bonferroni‐corrected significance level was thus 0.0017.We grouped the NSs  with respect to the hemisphere and where applicable with respect to the resected area see . The ress Figure a. There s Figure b, the prp < 10−15, Kruskal–Wallis test). The post hoc analysis found (a) significantly weaker linear interrelations bridging the resected and the nonresected area than acting inside or outside the resected area ; (b) significantly stronger linear interrelations inside the resected area than outside (p < 10−05); (c) significantly stronger nonlinear excess interrelations acting inside the resected area and bridging outwards than among channels outside (both p < 10−06); (d) significantly stronger nonlinear excess than linear bridging interrelations (p < 10−06); (e) significantly stronger linear than nonlinear excess outside interrelations (p < 10−06). Analogous to the analysis by hemisphere (see above) the number of multiple comparisons was 18 and the corresponding Bonferroni‐corrected significance level was 5.5× 10−4.In the 11 patients who underwent resective surgery targeting the mesiotemporal areas the mean percentage of channels recording from the resected area was 0.22 (range: 0.07–0.44). Nine of these patients had favorable outcome (Engel Class I or II), whereas two had unfavorable outcome (Engel IV). We applied group statistics only to the favorable outcome group. Individual results of the Class IV patients are presented below. Linear and nonlinear excess interrelations separated with respect to the resected area see  are show3.3The epoch‐wise averaged linear interrelation matrices are displayed in The epoch‐wise averaged nonlinear excess interrelation matrices are displayed in Without showing the results, we remark that we have repeated this analysis, overlaying the left and right hemispheres rather than the focal and nonfocal ones. The findings were similar and the differences between reproducible linear and specific nonlinear excess matrices were even more pronounced.3.4Three of the included patients had Engel outcome Class IV after surgery. Because clinical characteristics in these patients were heterogeneous and patients with unfavorable outcome are most valuable for future improvement of computer‐assisted presurgical evaluation, we discuss the results separately instead of attempting group statistics. Where applicable and necessary, we repeated the analysis with the full channel set, including extra‐temporal locations.Patient 13 is a 19‐year‐old female patient experiencing about one dyscognitive seizure a day despite prior surgery  and medication with several seizure‐suppressing drugs. Epilepsy duration was 7 years. The MRI showed an asymmetry of the posterior parts of the hippocampi , a discrete malformation of the right amygdala as well as malformation of the right collateral sulcus and parahippocampal gyrus.The semiology of seizures observed in phase I EEG monitoring was consistent with seizure onset in right mesiotemporal structures, which led to a direct first resection without phase II monitoring. Since the seizures did not reduce satisfactorily after surgery, eight depth electrodes were implanted into the hippocampus, amygdala, insula, temporo‐anterior and temporo‐polar regions on the right as well as into the left hippocampus . During the four recorded seizures, a seizure onset in the remains of the right uncus and a stereotypical rapid propagation pattern to ipsilateral temporo‐anterior and temporo‐polar electrodes was observed. Propagation to the contralateral hippocampus was delayed and completely missing in seizure 3. Based on these findings, a second resection of the remains of the right amygdala, hippocampus, temporal pole and parahippocampal gyrus was performed . Altogether 14 seizures were recorded, 13 of which started in the right hippocampus according to visual EEG analysis. None of them propagated to the left hemisphere and neither were they noticed by the patient nor were clinical signs observable in the video monitoring which classifies them as “subclinical.” However, the SOZ of the 11th seizure was identified in the left hippocampus and amygdala. Semiologically, smacking and cramping of the right hand were observed. Interictal spikes were observed on both hemispheres. Because most of the seizure onsets were localized in the right hippocampus, which importantly also showed MR findings typical for sclerosis, a right‐sided selective amygdala‐hippocampectomy was performed “palliatively,” that is, expecting a significant reduction of seizure rate .Results of our quantitative analysis of preictal EEG prior to seizure 1 and 2 were very similar, see Figure We also analyzed the seizure originating in the left mesiotemporal structures. Prior to seizure 11, the linear interrelation pattern was very similar to the situation found before seizures 1 and 2, see Figure Patient 15 is a 31‐year‐old male patient experiencing focal seizures with and without secondary generalization. Epilepsy duration was 27 years. The MRI showed infratentorial atrophy, whereas potentially epileptogenic lesions were not detected. Before surgery, the patient experienced several seizures per day despite medication with several seizure‐suppressing drugs. The seizure semiology and phase I EEG monitoring with scalp electrodes suggested seizure origin in the left supplementary sensor‐motor area (SMA). To better localize the SOZ and to monitor seizure propagation pathways, an extended phase II EEG monitoring with additional electrodes outside the left SMA was performed. Altogether 10 depth electrodes were implanted symmetrically . Four depth electrodes targeted the mesiotemporal structures of both hemispheres. Several clinical and subclinical seizures with stereotypical EEG pattern starting in the left SMA and rapid propagation to the contralateral hemisphere were recorded per hour. Seizure onset was localized to the left superior frontal gyrus. To reduce the seizure rate, surgery was performed in the left SMA, initially leading to almost seizure freedom. Six months after surgery, the seizure rate had increased again and 24 months after surgery was back to the presurgical state.Our quantitative analysis of preictal EEG revealed that linear signal interrelations were stronger and more extended in the right than in the left hemisphere, see Figure 4We examined surrogate‐corrected linear and nonlinear interrelations in the immediate preictal phase of patients with focal epilepsies to test the hypothesis that nonlinear excess interrelations between iEEG signals are different compared to linear ones and provide additional important information to identify epileptogenic brain tissue. The linear and nonlinear dependence measures were chosen based on preceding work, which demonstrated a clear difference in linear and nonlinear connections after surrogate correction  or to nodes with a very different degree . Our finding of consistently negative assortativity of the nonlinear excess networks  was left intact by surgery despite removal of a considerable fraction of the nonlinear excess interrelations and the right hippocampus. In the context of our findings in the good outcome group, we hypothesize that a slightly more aggressive surgery might have led to improved outcome in terms of postsurgical seizure control.The observations made in Epochs 1 and 2 of Patient 14 were in full agreement with our findings in the good outcome group. A considerable part of the right mesiotemporal channels exhibiting nonlinear excess EEG interrelations prior to these seizures was removed. Based on the radiological suspicion of mesiotemporal sclerosis on the right and our findings in these two epochs alone, one could have expected a much better postsurgical outcome. However, before seizure 11, the only seizure with onset in the left mesiotemporal structures, nonlinear excess interrelations were widely distributed over all channels and almost equally strong on the left hemisphere Figure f. We hypIn Patient 15, we observed that only a small fraction of the channels showing strong nonlinear excess interrelations was removed. Specifically, before onset of the first seizure, these were expressed much more strongly in the signals recording from the left amygdala than in the actually resected left SMA Figure c. The noIn all cases, our analysis suggests the resection of additional brain areas. If resection beyond the clinical gold standard is feasible is highly dependent on the specific case and must be assessed by physicians and patients under consideration of the expected benefits and impairments. In some cases, this kind of additional information could lead to larger or different, more promising resections whereas in others it could help to preclude unhelpful surgery. Both would be highly beneficial for patients if available early on.4.3In general, network results based on signal interrelations are always specific to the applied dependence measure and rather complement than invalidate each other. It has repeatedly been stated that linear and nonlinear measures for signal interrelation yield qualitatively similar results when applied to simulated signals or real iEEG (Ansari‐Asl, Senhadji, Bellanger, & Wendling, Regarding network analysis of iEEG a large amount of studies have been undertaken in the past decade (see, e.g., Lehnertz, Geier, Rings, & Stahn, Ortega, de la Prida, Sola, and Pastor  studied Several EEG‐based studies found indications for a functional decoupling of the focal zone around seizure onset (Kramer et al., 5In this study, we found fundamentally different properties of linear and nonlinear iEEG interrelation networks when corrected by appropriate surrogates. Linear interrelation patterns are organized locally in block‐like manner and highly reproducible between patients. In contrast, nonlinear excess networks have star‐like structure with interactions overrepresented in the focal brain regions and are specific to the individual pathology. This makes them a candidate mechanism to efficiently transmit information from the focal zone to distant brain areas or alternatively to act as compensatory mechanism toward the focal zone, an important research question that needs further investigation. Another subject to address in future studies is about the mechanisms underlying these long‐range nonlinear excess interactions. Two potential candidates might be neuronal burst firing (Cain & Snutch, Regarding epilepsy surgery, our results corroborate the potential of network analysis to support identification of the epileptogenic zone using either linear interrelations to measure its decoupling or the excessive presence of nonlinear excess interrelations. Combination of these two approaches to enhance the overall accuracy is planned to be investigated in upcoming projects.None of the authors has any conflict of interest to disclose.Appendix S1: Supporting informationClick here for additional data file.


Birmingham Women’s and Children’s NHS Foundation Trust was formed in February 2017 following an acquisition. The Library and Knowledge Services (LKS) merged while operating across two hospital sites. A priority for the merged Library and Knowledge Service was to integrate e‐collections. A literature review identified six papers reporting health libraries that had merged and integrated e‐collections.A priority for the merged Library and Knowledge Service was to integrate e‐collections.To ensure equitable and cost‐effective access to an extended collection, an audit of pre‐existing e‐collections was conducted. Electronic licence agreements enabling cross‐site access were negotiated. A new OpenAthens ID was created.The integration of e‐collections enabled Trust staff access to a greater number of e‐journals and additional e‐content, and an overall cost‐saving was achieved.This case study supports existing literature stating that integrating collections increases the number of e‐journals. It further identifies cost‐difference in acquiring cross‐site access to e‐journals compared to databases providing full‐text e‐journals and additional e‐content.Integrating e‐collections enables equity of access and value. A national co‐ordinated approach to procurement of e‐collections will further support equity and best value throughout NHS LKS. To facilitate equity of access and best value, merged health libraries should integrate e‐collections.A journal audit is a valuable tool to identify information need and to integrate e‐collections.Health library managers should involve all team members in the integration process to ensure understanding, engagement and shared responsibilities.Cost and usage data analysis are important collection development decision aids and can be used for ongoing assessments of value.An integrating collections policy should prioritise information need, equity of access and value.Birmingham Women’s Hospital delivers care to more than 50 000 women, men and their families every year providing gynaecology, fertility, neonatal, maternity and foetal medicine services. It also accommodates a regional genetic laboratory. Birmingham Children’s Hospital is a specialist paediatric centre caring for children and young people up to 16 years of age, and also provides child and adolescent mental health services. Birmingham Women’s and Children’s (BWC) NHS Foundation Trust was formed in February 2017 following the acquisition of Birmingham Women’s Hospital by Birmingham Children’s Hospital. The merger of the two hospitals enabled more seamless care for women, children and families in Birmingham and the wider region.The Trust employs approximately 6000 staff with almost 3500 at Birmingham Children’s Hospital, 2000 at Birmingham Women’s and 500 in mental health services, and receives more than 641 000 patient visits per year. The two hospitals are teaching centres, and international centres for research and development working closely with the University of Birmingham, other NHS Hospitals and charitable organisations. In November 2019, the Care Quality Commission awarded the Trust an overall inspection rating of Good.The Library and Knowledge Services (LKS) also merged in February 2017 while continuing to operate across two hospital sites. Prior to the merger, each LKS had a Library Manager, an Information Specialist and a Senior Library Assistant. Around the time of merger, one Library Manager accepted redundancy and one Specialist left the organisation. Subsequently, two new posts were created: a Senior Library Assistant and a Library Apprentice. The revised cross‐site LKS structure consists of one Library Manager, one Information Specialist and a team of four operational staff. All team members work cross‐site, and since the merger staff training and team building has been a priority.The merger of the LKS provided impetus to develop a collections policy prioritising information need, equity of access and value while also providing strategic direction and organisational relevance. The integration of e‐collections of the two libraries was organised in line with subscription renewals of January 2018 with preparation starting 4 months earlier. Identifying specialist collections within departments was a challenging part of the process that highlighted the need for a policy statement guiding actions around departmental collections. The integration of e‐collections was a staged process involving all LKS staff to ensure understanding, engagement and shared responsibilities. Regular communication was an important part of the process. The regional OpenAthens Administrator provided advice and support.This case study starts with a literature review identifying merged health libraries that had integrated e‐collections; six relevant papers were identified. The focus of individual papers varied although each acknowledged that integrating collections and access management were important aspects. This case study also describes methods used to integrate e‐collections at BWC NHS Foundation Trust including an audit of journal holdings and an attempt to identify departmental subscriptions. A single collection was established based on the findings of the audit. This paper outlines processes completed to enable cross‐site access including negotiation of electronic licence agreements and OpenAthens for access management. The paper aims to support NHS LKS undergoing mergers and seeking to integrate e‐journal collections to enable equity of access and best value.embase, emerald, health business elite, health management information consortium, the kings fund, lisa, medline and google scholar. These databases were searched from inception to November/December 2018. Search terms used included health libraries and merger and e‐journals. Synonyms, acronyms and variations of these words were used. In addition, the reference lists of included papers were assessed for additional relevant studies. The search retrieved 95 articles with six meeting the inclusion criteria reporting merged health libraries that had integrated e‐collections. Three of these papers reported on merged health libraries in the United States, and the other three in Canada. No papers were located that reported on merged health libraries in the United Kingdom. The focus of individual papers varies from general impact of mergers to consolidating resources and change management. However, all papers acknowledge to some extent that integrating collections and access management are important aspects of library mergers.A search for literature reporting on merged health libraries that had integrated their e‐collections was conducted. Databases searched included Wos and Oddan  providesThe publication of McGowan’s article  and RegeAmong the long term benefits to the consolidation of the libraries are not only cost reduction but higher productivity and greater availability of combined resources to both campuses (2003: 51).Barnes  providesDavis, Shorr, Campbell, & McGowan  uses qua.… since electronic resource delivery is highly valued it makes sense to devote more time and funds to maintaining current electronic resources and teaching users how to utilise these for evidence based practice. Combining collections with one interface can result in increased electronic resources for all users The most recent publication is Kenefick and DeVito  that detKenefick and DeVito  also sugIn summary, Wos and Oddan  providesThis case study supports the findings of the literature review that mergers and integrating collections result in an increased number of journals. It also expands existing literature as it outlines the methods used to integrate collections, and provides details of obtaining electronic licence agreements and organising access management as well as detailing the cost implications of integration.Verify journal holdings of individual library sites reporting cost, supplier and usage dataIdentify departmental subscriptionsIdentify duplicate titles between library sites and departmentsReview usage data to identify titles providing good, fair and low value to inform re‐subscription, further promotion or cancellation of titleApprove a single collection of e‐journals.An audit of journal holdings was completed in order to integrate e‐journal collections. There were five stages to the audit:The first stage of the audit verified journal holdings of individual library sites. The scope was e‐journals including single titles and subject‐specific titles available through bibliographic databases. Print journals were outside of the scope of the audit although review of remaining print holdings is an ongoing project.Individual library sites held an inventory of titles in an Excel spread sheet. The inventories were combined to create a single list of e‐journals that included cost, supplier and usage data. Access to the titles was through an A–Z journal list accessible via the Intranet.The second stage of the audit identified departmental subscriptions. Communications Department sent a global email requesting Trust staff contact the library to discuss locally held subscriptions. Titles identified were added to the inventory.The third stage identified duplicate titles between holdings at Birmingham Women’s LKS, Birmingham Children’s, and individual departments.The fourth stage of the audit investigated usage data to identify titles providing value. A value threshold was decided based on cost per download compared to document supply cost that included LKS staff time; cost per download being less than document supply indicated good/fair value, whereas cost per download being more indicated low value. Good value permitted re‐subscription while low value provoked discussion between LKS staff and end‐user and informed promotion or cancellation of title.During the final stage of the audit, a single collection of e‐journals was established with reference to findings of stages 1–4 of the audit.Once the audit was completed, e‐licence and access management were arranged to enable Trust staff at both hospital sites authorised access the newly integrated collection.Journal suppliers were informed of the Trust merger and aim to integrate e‐journal collections, and e‐licence agreements recognising both sites as authorised users were obtained. The e‐licences provided use terms and conditions for access, downloading, document supply and storing. Purchase orders were completed quoting the NICE Electronic and Print Content Framework Agreement and new OpenAthens Organisation Identifier (org ID) recognising the newly merged Trust and certified cross‐site access.Existing access management of e‐journals at both sites was through OpenAthens with two separate org IDs in place. A new single org ID was created to identify the newly merged Trust. Prior to migrating users, a number of OpenAthens housekeeping tasks were completed including identifying and deleting duplicate and expired accounts. Following migration, journal holdings in the Link Resolver were combined.See Table The cost of enabling cross‐site access to a single database providing access to 600 + e‐journals, healthcare knowledge and additional e‐content was £11.5K; although this was considered to be a substantial increase, it was a negotiated cost that achieved a preferential outcome for the LKS. There was no additional cost to allow cross‐site access to the remaining 33 e‐journal titles. Overall, following the merger and integration of e‐resources, more Trust staff had access to more e‐journals, and a total cost‐saving of £3.8K was achieved.The audit was a valuable tool to identify and ensure holdings were being managed, and to integrate e‐journal collections. It also identified a departmental collection, and furthermore that departmental staff were unaware of cross‐site holdings. The LKS acquired management of the collection organising procurement and cross‐site access, and continues to work with the department to promote and seek good value. The audit came close to identifying a single departmental title but this was lost to follow‐up with no response to emails.Additional departmental subscriptions and/or small specialist collections may still exist but have proved challenging to identify. Therefore, LKS teams may benefit from working with finance and procurement departments to identify subscriptions through budget codes and purchase order systems. It is very worthwhile trying to identify departmental holdings as there are a number of advantages of library staff managing all holdings including widening and managing access, monitoring usage to ensure continued value, and supporting compliance with licences and copyright legislation. It is also in the interest of cost‐savings for individual departments and the Trust as a whole.Cost of cross‐site access to a database was £11.5K; indeed without the cost‐savings through cancellation of titles the LKS budget would have fallen short of meeting the increased cost. However, prior to purchase of a database providing access to health care knowledge including full‐text e‐journals and additional e‐content, an appraisal and cost‐effectiveness study including collection of accurate and meaningful usage data needs to be conducted, and value aligned to judgement of the subject needs of end‐user. The appraisal should include appropriateness of health care knowledge for UK practice, and its evidence base. In addition, the increasing availability of free articles via the Internet and social media, and open access should be considered together with document supply services and cost.The results of this case study as indicated in Table The integration of e‐collections following a hospital merger was a staged process that included an audit of e‐resources, and involved all LKS staff to ensure understanding, engagement and shared responsibilities. The integration resulted in Trust staff having cross‐site access to a greater number of e‐journals, health care knowledge and additional e‐content, and a cost‐saving was achieved. Promotion and evaluation is an ongoing project for the LKS at BWC to ensure continued value. The variance between cost of acquiring cross‐site access to e‐journals and databases needs consideration and options investigated following a pragmatic appraisal of need and value. Equally, progress with national procurement of e‐collections will enable LKS to further support equity and value.All authors have no conflict of interest.


In mammals, odorants induce various behavioral responses that are critical to the survival of the individual and species. Binding signals of odorants to odorant receptors (ORs) expressed in the olfactory epithelia are converted to an odor map, a pattern of activated glomeruli, in the olfactory bulb (OB). This topographic map is used to identify odorants for memory‐based learned decisions. In the embryo, a coarse olfactory map is generated in the OB by a combination of dorsal‐ventral and anterior‐posterior targeting of olfactory sensory neurons (OSNs), using specific sets of axon‐guidance molecules. During the process of OSN projection, odor signals are sorted into distinct odor qualities in separate functional domains in the OB. Odor information is then conveyed by the projection neurons, mitral/tufted cells, to various regions in the olfactory cortex, particularly to the amygdala for innate olfactory decisions. Although the basic architecture of hard‐wired circuits is generated by a genetic program, innate olfactory responses are modified by neonatal odor experience in an activity‐dependent manner. Stimulus‐driven OR activity promotes post‐synaptic events and dendrite selection in the responding glomeruli making them larger. As a result, enhanced odor inputs in neonates establish imprinted olfactory memory that induces attractive responses in adults, even when the odor quality is innately aversive. In this paper, I will provide an overview of the recent progress made in the olfactory circuit formation in mice. Binding signals of odorants detected in the olfactory epithelium (OE) are transmitted to the olfactory bulb (OB) and converted to a topographic map of activated glomeruli. Odor information is then conveyed by projection neurons to the olfactory cortex (OC) for making decisions of the odor quality to mediate behavioral outputs. Decisions are made via two distinct neural circuits; one is innate and the other is learned pathway. Partial rescue is also observed with the ca mutant of protein kinase PKA. Thus, PKA‐mediated transcriptional regulation appears to be involved in A‐P projection, using Gs without involving Golf and CNG channels  OR, whereas dominant‐negative PKA results in an anterior shift  is transcriptionally regulated by cAMP and found in an anteriorlow/Sema3Ahigh axons are sorted to the central compartment of the bundle, whereas Nrp1high/Sema3Alow axons are confined to the outer‐lateral compartment. OSN‐specific KO of Nrp1 or Sema3A not only perturbs axon sorting within the bundle but also causes shifts of glomeruli along the A‐P axis. These results demonstrate that pre‐target axon sorting within bundles contributes to establish olfactory map topography.In contrast to the Sperry model, OSN axons are able to converge and form loci even in the absence of the target OB  navigate to the D domain of the OB through the repulsive interactions with its ligand Slit1 expressed in the ventral (V) domain of the OB , including ORs, are known to possess two conformational states, active and inactive  have proven useful. The β2‐AR is a GPCR with the highest sequence homology to ORs. When expressed in OSNs with the 2‐AR genes, the activity‐low and activity‐high β2‐AR mutants generate glomeruli anterior and posterior to those of the WT, respectively  for M/T‐cell projection. Recently, it was found that Nrp2 can be used for the study of migration and projection of mitral cells (MCs) in mice  is co‐transfected, GFP+ MCs are confined to the V‐region MCL. Furthermore, GFP signals are detected in the anterior MeA in the hNrp2 transfected mice. Activation of the single axon‐guidance gene hNrp2 Nrp2 appears to be sufficient to induce not only the migration of MCs to the V‐region MCL but also their axonal projection to the anterior MeA. In this electroporation experiment, the dorsal MC marker OCAM can be detected in the anterior MeA, indicating that ectopic expression of hNrp2 alone can induce MC projection to the anterior MeA even in the dorsal‐lineage MCs that normally send their axons to the CoA.For the study of Nrp2‐mediated MC targeting, Inokuchi et al.  also per3.4II domain of the posterodorsal OB. In contrast, the glomeruli responsive to attractive social cues, for example, methylthio‐methanethiol (MTMT), are confined to the posteroventral OB , but not avoidance behavior, indicating that this particular OR, Olfr1019, is specialized for freezing. In the KO of Olfr1019, immobility responses to TMT are reduced, but aversion and stress reactions are not affected. Consistent with this observation, the posteromedial CoA and AmPir that are known to induce aversive responses, are not activated by photo‐illumination of the Olfr1019 glomeruli. These observations demonstrate that TMT‐induced fear may be separated into two different components, immobility and avoidance. Furthermore, the TMT‐induced immobility appears not to be the consequence of stress reactions mediated by a stress hormone, adrenocorticotropic hormone (ACTH).Optogenetic experiments demonstrate that activation of single glomerular species can elicit specific innate odor responses Figure . For exaIt is generally thought that an activation pattern of a specific set of glomeruli, an odor map, is transmitted to the OC to discriminate and identify the odorant .Neonatal odor exposure promotes Sema7A expression in the responding OSNs, resulting in glomerular enlargement .We then studied the effect of neonatal odor exposure on odor perception in adults. The mice conditioned to VNL at the early neonatal stage demonstrate increased responsiveness and sensitivity to VNL. In the preference test, the VNL‐conditioned mice spend significantly longer times in the room where a VNL‐spotted filter paper is placed. These imprinting effects cannot be seen in the mice where Sema7A signaling is blocked in neonates.It is notable that attractive responses to imprinted odors are observed even for aversive odorants, for example, 4‐methyl‐thiazole (4MT), a derivative of fox odor TMT . Stress‐reducing effects of imprinted odor memory can be seen in the odor conditioned mice. Mice usually demonstrate strong stress responses in an unfamiliar environment. When they are transferred to a new cage, the rectal temperature rises and remains elevated for approximately 20 min. However, in the mice conditioned to 4MT in neonates, stress is immediately eased by the imprinted odor as adults and a plasma concentration of the stress hormone ACTH is lowered. These stress‐reducing effects are not seen if the mice are conditioned to 4MT after the critical period.4.4Male mice normally demonstrate strong curiosity for the mouse scents of both male and female. However, the M/T‐specific cKO of PlxnC1, where Sema7A signaling is blocked, shows aversive responses by attempting to avoid interactions with unfamiliar mice . Abnormal social behavior is also seen in the naris‐occluded mice, when the occlusion is performed during the entire critical period. Thus, Sema7A signaling in early neonates appears to be involved in modulating the hard‐wired circuit for smooth social interactions.How does the imprinted odor memory induce the stress‐reducing reactions? To study this, we have analyzed various OC regions in the 4MT‐imprinted mice for their activation by the 4MT memory. Sharp contrasts are seen in the anterior MeA and AmPir between the 4MT‐conditioned and unconditioned mice . By the imprinted 4MT odor, the anterior MeA is activated and the AmPir is suppressed. The anterior MeA is known to mediate attractive social responses . Attractive responses can be induced even to the imprinted 4MT whose odor quality is innately aversive. Furthermore, the 4MT memory rapidly eases stress reactions by reducing the plasma concentrations of the stress hormone ACTH. Such imprinting effects can be seen only for the odor experience during the first week after birth. In the OC, imprinted 4MT memory activates the anterior MeA to mediate attractive responses and suppresses the AmPir to block the ACTH‐mediated stress reactions . It is important to determine in the future how the two conflicting decisions, innate aversion and memory‐based attraction, are balanced for the imprinted 4MT.It has been shown that early exposure to environmental odors affects social responses later in life. Male mice normally demonstrate strong curiosity toward unfamiliar mice. However, when Sema7A signaling is blocked in neonates, the mice avoid social interactions with strangers. It is possible that mice innately avoid stressful interactions with unfamiliar mice. However, Sema7A‐mediated imprinting may help pups adapt to their community for smooth social interactions with fellow mice. It will be interesting to examine whether similar imprinting described in the mouse olfactory system can be found in humans. It is also important to determine in the human infants when the critical period starts and how long it lasts. These imprinting studies will give new insights into our understanding of neurodevelopmental disorders, such as autism spectrum disorders and attachment disorders, that may be caused by improper sensory inputs during the neonatal period. The mouse olfactory system will continue to serve as a useful tool for the developmental studies of neural circuit formation in the mammalian brain.


Prlo‐DRB*4, Prlo‐DRB*16 and Prlo‐DRB*102, were the most common alleles in the European populations, suggesting directional selection is acting on this locus. Our research shows empirical support for the importance of functional immune diversity for adaptation and survival in novel environments.The adaptive potential of invasive species is related to the genetic diversity of the invader, which is influenced by genetic drift and natural selection. Typically, the genetic diversity of invaders is studied with neutral genetic markers; however, the expectation of reduced diversity has not been consistently supported by empirical studies. Here, we describe and interpret genetic diversity at both neutral microsatellite loci and the immune‐related MHC‐DRB locus of native and invasive populations of raccoon to better understand of how drift and selection impact patterns of genetic diversity during the invasion process. We found that despite the loss of many MHC (major histocompatibility complex) alleles in comparison with native populations, functional MHC supertypes are preserved in the invasive region. In the native raccoon population, the number of supertypes within individuals was higher than expected under a neutral model. The high level of individual functional divergence may facilitate the adaptation to local conditions in the invasive range. In the invasive populations, we also detected increased population structure at microsatellites compared to the MHC locus, further suggesting that balancing selection is acting on adaptively important regions of the raccoon genome. Finally, we found that alleles known to exhibit resistance to rabies in the native range,  These samples were obtained from local pest control programmes  and introduced (Europe) range of raccoons. Raccoons from the native range n = 2 were sam2.2For samples collected from the native range, DNA was extracted using Qiagen DNeasy Blood and Tissue Kit or a Serapure Bead  fragment of exon 2 that contains the functionally important protein binding region (PBR), using DRB 5c and DRB 3c primers  previously used for MHC characterization in a native raccoon population by Castillo et al. . The seqThe concentration of PCR products was estimated by eye from agarose gels, and products from each row were pooled into approximately equimolar quantities. The pools were then purified using MinElute PCR Purification Kit (QIAGEN). Next, each pool was diluted between 10 and 100×, depending on the electrophoresis band intensity. The pooled amplicons were amplified in multiplexed PCR with primers including Illumina P5/P7 sequences, resulting in Illumina‐compatible paired‐end sequencing templates .To investigate the significance of functional MHC class I diversity, we clustered alleles into supertypes. MHC alleles of the same supertype encode biochemically similar amino acids at antigen‐binding sites, and thus, the molecules bind similar antigens, whereas molecules encoded by alleles from different supertypes recognize repertoires of different antigens. Therefore, alleles of different supertypes should have different functional values , mean number of alleles per individual, the number of private alleles (Apriv), the number of segregating sites (S) and nucleotide diversity (π) for whole sequences as well as for synonymous and nonsynonymous sites separately. We used SPAGeDi , expressed as the expected number of alleles assuming the smallest sample size . To evaluate whether the individual number of functional MHC‐DRB variants (represented by MHC supertypes) in native and invasive populations differs from that expected under neutrality, we compared the actual distribution of supertypes observed in individuals to a null distribution obtained from randomly generated genotypes. Because different MHC alleles may be linked within individual genotypes either by disassortative mating or due to spatial proximity of different alleles within the genome , we tested for possible associations between alleles using pair.ia function from poppr R package  was used to select the optimal number of principal components to be retained. Next, we used DAPC to generate assignment probabilities to actual populations (sampled sites). Due to the fact that we detected hierarchically arranged levels of genetic structure in invasive raccoon populations in our previous study  implemented in the R package adegenet  in synonymous and nonsynonymous sites were similar for native and invasive populations. Clustering analyses revealed 10 unique supertypes among all available MHC‐DRB alleles and nine supertypes among individuals sampled in the native range (Table AR) ranged from the highest at the D2 sampling site (9.00) to the lowest in PL2 (5.72). The raccoons from D1, considered to be the site of the initial raccoon introduction, displayed the highest estimates of both synonymous and nonsynonymous nucleotide diversity per site, despite a low number of alleles. Within different European locations, the number of supertypes ranged from 5 to 7 , while five pairs showed significant association within the PL and D populations: Prlo‐DRB*04/Prlo‐DRB*14 , Prlo‐DRB*16/Prlo‐DRB*62 , Prlo‐DRB*19/Prlo‐DRB*102 , Prlo‐DRB*02/Prlo‐DRB*25  and Prlo‐DRB*01/Prlo‐DRB*257 . Although no significant associations between alleles were found in population sample from Florida, we are aware that the lack of associations between alleles can be a result of much smaller sample size.A comparison of the number of supertypes in observed and simulated data sets was performed on three separate raccoon groups. We treated separately (a) individuals from native population (Florida); (b) individuals from Czechia (site CZ), because genetic structure at the MHC‐DRB locus  and numerous lines of evidence , but lower than simulated when complete association is assumed . No significant differences were observed in the CZ population . Because no associations were found in the FL population, the only possible comparison was between observed (2.81) and simulated unlinked alleles (2.00). Here, the difference was statistically significant (p = 3 × 10–7).Comparisons of the number of supertypes between observed and simulated data sets showed that in German and Polish populations, the observed mean number of supertypes is higher than simulated without assuming association between alleles . The only exception was D1 where, despite relatively low sample size, allele frequencies were evenly distributed, with no pronounced dominance of Prlo‐DRB*04 and Prlo‐DRB*14 alleles, and only one allele exhibited a frequency lower than 10% , but not for the MHC‐DRB locus . When the correlation between MHC‐DRB and geographic pairwise distances was controlled for neutral diversity, the pattern was also not significant . We also did not find an association between MHC‐DRB and microsatellite interpopulation diversity while controlling for geographic distance .Global among‐site differentiation expressed as Rho/(1 − Rho) was 0.190 and 0.240 for MHC‐DRB and microsatellite loci, respectively. Despite the absence of private alleles among European sites , the MHC‐DRB population structure was high and significant for most comparisons. However, no differentiation was found between D2 and D3, and PL1 sites  based on nine microsatellite loci separated native populations from Florida as well as the European CZ site along the first  axis. The differentiation between the remaining sites in the invasive range (D1–D4 and PL1–PL2) was visibly weaker and distributed along the second  axis Figure a. When t44.1Newly established invasive populations often exhibit lower levels of genetic diversity in comparison with native populations resulting from the loss of alleles during introduction events  and at least one representative of each divergent lineage would be kept in each locus. In such a case, it is not parsimonious to invoke balancing selection.In the invasive population extending north‐east from the invasion source in Germany to western Poland (sites PL and D), the simulated genotypes represented lower mean individual supertype number when random segregation of alleles was assumed, but higher supertype number with the assumption of allele association, according to the pattern present in the studied populations. This finding suggests that the maintenance of divergent alleles and high number of supertypes is a result of linkage disequilibrium between alleles. All pairs of linked alleles found in this study are placed far apart on allele network Figure  and reprPrlo‐DRB*4, Prlo‐DRB*16 and Prlo‐DRB*102) were present and frequent in Polish and German populations, while Prlo‐DRB*4 and Prlo‐DRB*16 were found in Czech population  to reduce the number of variables, it does not rely directly on allele numbers and frequencies, which makes it more appropriate to analyse and compare two different types of genetic markers. Regardless of the marker type, the raccoons from CZ were clearly distinct from the other sites as shown by DAPC. This is in line with the findings presented in Biedrzycka et al.  and supp4.3Our data suggest that raccoon MHC genes are highly diverse and may represent multiple invasions. Additionally, known and unknown introduction events  as such events potentially add more genetic variation and can introduce novel adaptive alleles into invasive populations. Moreover, wildlife managers should pay attention to reducing the possibility of mixing raccoons belonging to different genetic clusters. To do so, the efforts should be concentrated on preventing the further spread of raccoons forming populations at the invasion front. Furthermore, to reduce MHC diversity, a management plan should focus on reducing raccoons at source habitats where the racoon numbers and genetic diversity are the highest. One important question that still needs to be answered is: What is the role that urban populations play in adding functional genetic diversity and zoonotic disease risk to native wildlife and humans?5Our research showed that functional genetic diversity is maintained in invasive raccoon population, despite a strong reduction of MHC‐DRB allele number. We found that invasive raccoons have high number of functionally important MHC supertypes and individual allele divergence compared to the native populations. Additionally, low population structuring displayed at the MHC‐DRB locus than in microsatellites implies the role of balancing selection. Many studies have found that despite experiencing genetic bottlenecks, successful invasive populations retain a higher level of genetic diversity than expected, probably due to multiple introduction events (Uller & Leimu, Here, we showed that immune genetic diversity might be retained despite a strong reduction in the number of MHC alleles. Because MHC loci capture an important fraction of the genetic variation underpinning resistance to pathogens, we predict that raccoon invasion in Europe will proceed based on observed levels of functional genetic diversity.None declared. Click here for additional data file. Click here for additional data file. Click here for additional data file.


We report a rare case of peripheral pulmonary hamartoma that caused haemoptysis from the non‐adjacent bronchus. A 54‐year‐old man was admitted to our hospital because of haemoptysis. Contrast‐enhanced chest computed tomography revealed a tumour measuring 25 × 24 mm in the left lower lobe. Fluid was observed around the pulmonary artery in addition to the consolidation of the lingular segment of the left lung. Bronchoscopy confirmed continuous bleeding from the lingular bronchus, and surgery was planned. The patient was promptly diagnosed with pulmonary hamartoma intraoperatively, and tumour enucleation was performed. The haemoptysis resolved after surgery, and the patient was discharged on post‐operative day 10. As peripheral hamartoma does not generally cause haemoptysis, we suspected that the haemoptysis occurred due to the unusual condition in which the bleeding from the tumour reached the non‐adjacent lingular branch along the pulmonary artery sheath. Here, we report a case involving a middle‐aged man who developed a peripheral pulmonary hamartoma with haemoptysis from the non‐adjacent bronchus and exhibited resolution of the haemoptysis after tumour enucleation. Pulmonary hamartoma is the most common benign lung tumour. It is usually asymptomatic and is often found incidentally. However, endobronchial hamartoma can occasionally cause haemoptysis. On the other hand, extrabronchial pulmonary hamartoma  rarely causes haemoptysis, and haemorrhage from the bronchus non‐adjacent to the tumour is even rarer. Here, we report a case involving a middle‐aged man who developed a peripheral pulmonary hamartoma with haemoptysis from the non‐adjacent bronchus and exhibited resolution of the haemoptysis after tumour enucleation.A 54‐year‐old man was referred to our hospital with a chief complaint of haemoptysis. Chest X‐ray showed consolidation in the left lower lung field. Contrast‐enhanced chest computed tomography (CT) revealed a tumour measuring 25 × 24 mm in the left lower lobe Fig. A, C. FluDespite the existence of differences between reports, pulmonary hamartoma is a benign tumour that occurs in the bronchi in 3–20% of patients and at the periphery in the rest The treatment of haemoptysis caused by tumours is divided into palliative treatment and radical modalities. Several studies have reported that BAE is effective as a palliative treatment. Fujita et al. reported that 96% of patients with non‐small cell lung carcinoma were successfully treated with BAE Preoperative chest CT showed consolidation around the lingular segment, and bronchoscopy revealed bleeding from the lingular bronchus. The tumour was not adjacent to the lingular bronchus, and the causal relationship between the tumour and bleeding was unclear. However, chest CT revealed fluid accumulation, which was thought to be caused by the hamartoma that extended from around A6 to the lingular bronchus, and haemoptysis stopped after tumour enucleation. On the basis of the results mentioned above, this case exhibited an unusual condition in which the haemorrhage from the pulmonary hamartoma reached the non‐adjacent lingular bronchus along the pulmonary artery sheath, resulting in haemoptysis. To the best of our knowledge, this is the first report of a peripheral pulmonary hamartoma with haemoptysis from the non‐adjacent bronchus that resolved after enucleation of the tumour.Appropriate written informed consent was obtained for publication of this case report and accompanying images.