In England, demand for primary care services is increasing and GP shortages are widespread. Recently introduced primary care networks (PCNs) aim to expand the use of additional practice-based roles such as physician associates (PAs), pharmacists, paramedics, and others through financial incentives for recruitment of these roles. Inequalities in general practice, including additional roles, have not been examined in recent years, which is a meaningful gap in the literature. Previous research has found that workforce inequalities are associated with health outcome inequalities.To examine recent trends in general practice workforce inequalities.A longitudinal study using quarterly General Practice Workforce datasets from 2015–2020 in England.The slope indices of inequality (SIIs) for GPs, nurses, total direct patient care (DPC) staff, PAs, pharmacists, and paramedics per 10 000 patients were calculated quarterly, and plotted over time, with and without adjustment for patient need.Fewer GPs, total DPC staff, and paramedics per 10 000 patients were employed in more deprived areas. Conversely, more PAs and pharmacists per 10 000 patients were employed in more deprived areas. With the exception of total DPC staff, these observed inequalities widened over time. The unadjusted analysis showed more nurses per 10 000 patients employed in more deprived areas. These values were not significant after adjustment but approached a more equal or pro-poor distribution over time.Significant workforce inequalities exist and are even increasing for several key general practice roles, with workforce shortages disproportionately affecting more deprived areas. Policy solutions are urgently needed to ensure an equitably distributed workforce and reduce health inequities. Pre-existing health inequalities, which are widening owing to COVID-19, may be exacerbated by an inequitably distributed health workforce. In addition, the composition of the general practice workforce is changing with the introduction of PCNs. Here, recent longitudinal trends are explored in the distribution of practice-based staff, including GPs, nurses, and allied health professionals. It is found that significant workforce inequalities exist and are even increasing for several key general practice roles, with workforce shortages disproportionately affecting more deprived areas. Policy solutions are urgently needed to expand access and reduce health inequities. The number of consultations in general practice has increased significantly in recent years, but staff numbers have not kept up with this demand. The percentage of GPs in the NHS workforce has been steadily decreasing, and the GP workforce is ageing; doctors are increasingly working part-time, which foreshadows future worsening shortages. In 2015, then-Secretary of State for Health Jeremy Hunt promised an additional 5000 GPs for the NHS by 2020, but this was not achieved; instead, it is predicted that there will be a shortage of 7000 GPs by 2024. England’s increasingly depleted primary care workforce will likely be a 'make-or-break' issue for the NHS in coming years. Evidence suggests that these workforce shortages are not equally distributed across England; areas with higher levels of deprivation tend to have fewer GPs relative to the patient population despite a greater burden of chronic disease. Moreover, GP supply has been linked to health-outcome measures, including mortality rates, self-reported health, and life expectancy, even after controlling for sociodemographic measures.Health workforce shortages, especially in primary care, have long plagued healthcare systems globally, and the gap between the growing need for services and sufficient staff has been widening.The NHS Long Term Plan, published in 2019, emphasised the importance of general practice services and established PCNs, or groups of neighbouring practices that enable economies of scale and integrated care. PCNs were to boost the primary care workforce through the Additional Roles Reimbursement Scheme (ARRS), which guaranteed financial resources to recruit specified additional roles such as pharmacists, PAs, and paramedics into general practices. The expanded presence of allied health professionals in primary care is intended to achieve a dual aim of shoring up general practice by reducing pressure on existing clinical staff, as well as delivering outcomes specified in the PCN network contract. and through an exclusive focus on GPs.While it is still too early to evaluate recruitment under the ARRS, this research aims to quantify recent trends in the distribution of the general practice workforce, including nurses and additional roles, to uncover any existing workforce inequalities since 2015. It will also examine whether and how the skill mix within practices has changed. This analysis fills a gap in the previous literature on workforce inequalities, which is limited by age , Practices were matched with their respective PCNs using the NHS Digital GP and GP practice-related data page.Quarterly data from September 2015–December 2020 (20 quarters) were obtained from the NHS Digital General Practice Workforce collection. and SPLWs and pharmacy technicians were introduced in June 2019. Atypical practices, that is practices with <750 total patients or <500 patients per GP FTE, were excluded, following previous analyses. The same method as that described in the General Practice Workforce Data Quality Statement was employed to account for missing workforce data: weighted average estimates were substituted for missing values for GPs, nurses, and total DPC staff in each quarter.Full-time equivalent (FTE) data were collected on the following staff: GPs, nurses, total DPC staff , pharmacists, physiotherapists, PAs, occupational therapists, podiatrists, paramedics, social prescribing link workers (SPLWs), and pharmacy technicians. In December 2016 and June 2017, only data on GPs were available. Paramedics were introduced into the dataset in September 2016, IMD scores for 2016, 2017, 2018, and 2020 were calculated based on a weighted mean of IMD 2015 and IMD 2019  and divided into deciles on a scale of 0–1. Deprivation increases with IMD decile; 0.1 corresponds to the least deprived decile, and 1 corresponds to the most deprived decile. Missing IMD data were imputed with IMD scores from the adjacent year, where available.Practice-level Index of Multiple Deprivation (IMD) 2015 and 2019 scores were obtained from the Fingertips National General Practice Profiles. While not an exact measure of patient need, this formula has been used similarly in previous research, and is one of the best summary measures available to capture patient need in GP practice populations. Practice-level Carr-Hill data were obtained from the NHS Payments to General Practice collection on NHS Digital, 2015–2016 through 2019–2020, five datasets in total. Higher Carr-Hill values indicate higher patient need. Missing Carr-Hill data were imputed with values from the adjacent year, where available.The Carr-Hill formula was used to adjust for patient need. This resource allocation formula considers patient demographics and health status, list turnover, market forces, and rurality to calculate a weighted practice population. The SII is a measure of the difference in FTE staff per 10 000 patients between the highest and lowest IMD decile. A negative SII value corresponds to pro-rich inequality, that is, fewer staff in more deprived deciles; a positive value corresponds to pro-poor inequality, that is, more staff in more deprived deciles. Confidence intervals that overlap zero indicate no significant distributional inequality. These unadjusted and adjusted SIIs were plotted for the quarters with available data from September 2015–December 2020. The SII analysis was also done from March 2019–December 2020 with workforce variables and IMD deciles aggregated to the PCN level for GPs and nurses only, weighted by patient population. Additional roles recruited and employed by PCNs may have been recorded in the newly introduced Primary Care Network Workforce datasets instead of the General Practice Workforce datasets beginning in March 2020; as a result, PCN-level SII analyses of total DPC staff and each additional role from March 2020 onwards may not be entirely accurate or complete, and were, therefore, excluded from this analysis.The practice-level number of FTE staff per 10 000 patients was calculated for GPs, nurses, total DPC staff, pharmacists, physiotherapists, PAs, occupational therapists, podiatrists, paramedics, SPLWs, and pharmacy technicians. Socioeconomic gradient bar charts depicting the mean FTE staff per 10 000 patients by IMD decile for each workforce variable at each quarter were plotted and visually assessed for linearity. Next, a linear regression analysis of FTE staff per 10 000 patients by IMD decile was conducted at each quarter, with and without Carr-Hill adjustment. The coefficient of this regression is the slope index of inequality (SII).A sensitivity analysis using head count (HC) instead of FTE staff was conducted and compared with the practice-level SII graphs. All analyses were done using Stata (version 16.1).Practice populations ranged from 750–89 786 patients. The descriptive statistics, including the percentage of missing data for each workforce variable (FTE staff per 10 000 patients) across the quarters with available data, are shown in Supplementary Table S1.Occupational therapists, podiatrists, and physiotherapists were excluded owing to insufficient data to detect distributional trends. SPLWs and pharmacy technicians also had low overall numbers but were included in the first step of the analysis because their employment sharply increased in the quarters studied.The socioeconomic gradient graphs of mean FTE staff per 10 000 patients by IMD decile are included in Supplementary Figure S1. SPLWs and pharmacy technicians were excluded from the regression analyses because they did not show any consistent socioeconomic gradient.Across all quarters, there were significantly fewer FTE GPs per 10 000 patients in practices in higher IMD deciles, that is, higher levels of deprivation. This inequality has widened slightly over time, as the SIIs have moved further from the null. While the PCN-level indices for GPs were less negative than the practice-level indices, the SII trends over time appeared similar.P<0.05) in 2019.For nurses, the longitudinal trend appears to be moving in the opposite direction, towards a more equal or pro-poor distribution over time. Before adjustment, the positive SII values for nurses suggest that there were more nurses per 10 000 patients employed in more deprived practices ; after aThe distribution of total DPC staff mirrors that of GPs, in that there were fewer total DPC staff employed in more deprived areas relative to the patient populations . SimilarFor both pharmacists and PAs, a trend of increasing pro-poor inequality was observed at the practice-level, both before and after Carr-Hill adjustment . StartinFor paramedics, widening pro-rich inequality was observed in both the adjusted and unadjusted practice-level SII estimates . This inInequalities were observed in the distributions of GPs, paramedics, and total DPC staff, with fewer staff employed in more deprived deciles . The opp on GP inequalities from 2004–2005 to 2013–2014 and updates it to include nurses and additional roles as well as PCN-level indices for GPs and nurses. In addition to its novelty, another strength of this analysis is its exhaustive use of available data. Instead of merging all 20 quarters of data, analyses were run separately at each quarter, minimising the number of excluded practices.This analysis extends a previous analysis As a result, the adjusted SIIs reported may underestimate the true adjusted inequality indices, that is, there may be higher levels of pro-rich inequality than this analysis reports. Furthermore, while SIIs have been used and justified in previous health and workforce inequality analyses, the reliance on a crude regression analysis masks variation across and within IMD deciles. The distributions of each workforce variable were assessed visually, and variables that did not show a linear gradient were excluded from further analysis. However, slight non-linear trends are still present in the remaining variables, which can be seen in Supplementary Figure S1. Finally, the publicly available general practice workforce datasets are limited, especially for newly introduced additional roles. Although substitution was employed for missing values where possible, as described in the Method section, the potential for inaccurate or incomplete workforce data as reported by practices remains a limitation of the analysis.However, this research has several limitations. First, the Carr-Hill formula, which has not been substantively updated since it was first implemented in 2004, has been criticised for not adequately considering the extra pressures that practices in areas of high socioeconomic deprivation face. Moreover, the present findings are comparable with a recent longitudinal report by The Health Foundation, which plotted general practice workforce supply over time by IMD quintiles; while this report did not include significance testing, the observed trends align with the results presented in this article. The most recent longitudinal SII analysis of workforce inequalities reported a slight pro-poor, although not statistically significant, SII estimate in 2013–2014. However, the SIIs in this analysis had been approaching the null since 2011–2012. It is, therefore, likely that GP workforce inequality has been increasing since 2011–2012.The finding that GP workforce shortages disproportionately affect practices in areas of higher deprivation, an inequality that has widened since 2015, is consistent with recent cross-sectional analyses of workforce inequalities. Previous reports have shown that practice closures have been increasing since 2013. Likewise, during this study period, far more practices were dropped from the datasets than added, which may have driven the widening inequality observed.This previous longitudinal analysis also found that the primary driver of GP inequality was the opening and closing of practices in more deprived areas, rather than recruitment into existing practices.et al as a disproportionate care law (DCL), or an increase in healthcare delivery with social disadvantage, but not relative to population need. The finding of an ICL operating for GP supply in England is especially striking given that this form of inequality has primarily been documented in countries with proportionately less or more fragmented public investment in health care; a DCL is more commonly observed in countries with publicly funded health systems that provide universal coverage such as the NHS.This research demonstrates the existence of an inverse care law (ICL), or a decrease in healthcare delivery with social disadvantage, within general practice in recent years. This is contrasted with previous analyses examining study periods before 2015, which had primarily found what is described by Cookson NHS Long Term Plan. Health needs are often greatest in areas of high socioeconomic deprivation, but we observe a relative undersupply of some key clinical staff in primary care in these areas. One effect of this is the persistence of the ICL in general practice. Addressing this is even more critical in the context of the COVID-19 pandemic, which has disproportionately affected under-resourced and underserved communities, and has widened existing health inequities patterned by class, race, and geography.Reducing health inequalities is a core government commitment and was specified as a primary goal within the  In addition, the impact of substituting nurses and allied health professionals for GPs in under-doctored practices on quality of care and health outcomes is not entirely known. Further research is needed to better understand the effects of skill-mix redesign in the health workforce.In England, PCNs are to be tasked with reducing health inequalities. To do this, practices and PCNs in the poorest areas will need relatively more GPs and allied health professionals to respond to the demand for primary care services. The finding — that in general there remains a pro-rich inequality in recruitment of staff in primary care — should be of significant concern to policymakers. They will need to consider why practices and networks in deprived areas are relatively understaffed, and how this can be reversed. Given the significant scale of the proposed expansion of the primary care workforce, policymakers must urgently consider what evidence-based mechanisms can be used to encourage equitable recruitment of additional roles in primary care. This might include but not be limited to incentivisation of DPC posts in understaffed areas, enhanced training offers for these roles, and offering practices and networks in understaffed areas additional recruitment support.Significant workforce inequalities exist, and are even increasing, for many general practice roles, with some workforce shortages disproportionately affecting more deprived areas. Expanded use of additional roles under the ARRS may partially alleviate GP workload in overstretched practices, but there is a risk that additional workforce will gravitate to more affluent areas, further perpetuating inequity in primary care staffing. These findings underscore the need for more intentional and directed policies addressing workforce inequalities in order to expand access and reduce health inequities.


We quantified intra-population genetic variance and predicted individual genetic performance along a climatic gradient. This approach can be adopted in most breeding and conservation programs, boosting the speed of adaptation under climate change.In forest tree breeding, assisted migration has been proposed to accelerate the adaptive response to climate change. Response functions are currently fitted across multiple populations and environments, enabling selections of the most appropriate seed sources for a specific reforestation site. So far, the approach has been limited to capturing adaptive variation among populations, neglecting tree-to-tree variation residing within a population. Here, we combined the response function methodology with the  Additive genetic variance  from 21 test sites in Austria ranging from Alpine to lowland regions. We utilized height and wood density measured directly in forest stands on individual mature trees with reconstructed pedigree. Using the response function methodology combined with mixed-model genetic evaluation, we quantified the intra-population As outlined above, response functions have been proposed to capture adaptive variation at the population (provenance) level, unlike the conventional breeding focusing mainly on intra-population The European larch data used in this study were previously sampled and genetically analyzed by Lstibůrek et al. . We willWe extracted climatic variables for the test sites from the WORDCLIM dataset ; Z is the genetic relationship matrix; a is the vector of additive genetic (breeding) values, where A is the average numerator relationship matrix, σa1a2 is the additive genetic covariance between traits 1 and 2, and ⊗ is the Kronecker product operator. The random residual error effect was modeled using an unstructured covariance matrix structure aswhere e1e2 is the residual covariance between the two traits. Random effects were assumed to be independent.where σWe utilized the above predictions of the fixed site effects and calculated the respective all-pairwise differences. Next, we trimmed the dataset so that each half-sib family was represented in at least six sites to achieve even representation of families while maximizing their distribution across multiples sites  to describe the within-population genetic variation following major climatic gradients. We tested the linear, quadratic, and Gaussian models as they have been predominantly used in previous studies  and wood density , respectively. We observed negligible additive genetic correlation between the respective traits . Our data did not show a significant genotype by environment interaction (GxE), as shown earlier by Lstibůrek et al. . 87% of all pairwise differences between sites were statistically significant (p < 0.05). Details are provided in Experimental site effects (considered fixed) were found statistically significant  varies from 35 to 120. This uneven number is explained because it is a product of natural crosses among parents; hence the families' sizes were only revealed at the pedigree reconstruction stage. In Nb and the mean model's p-values of the RFs curve fitting were significant (p < 0.05), except one (genotype L2).In  of 0.27 . All p-vIn Phenotypic data were regressed onto random genetic and fixed site factors using the mixed linear animal genetic model. The genetic variation observed in this study resembles typical values for height and wood density in conifers  take place in operational afforestation sites with a designated seed source. Further, uneven gametic contributions within and among the respective sites are optimally accounted for within the combined genetic evaluation protocol, i.e., multi-site animal genetic model. Unlike the traditional breeding programs relying on transfer within and among fixed-seed zones, the current approach is flexible. Seed transfer delineation is dynamic in line with particular CC development.There are possible pitfalls of this proposed strategy that should be addressed here. Although used in many studies, the quadratic model fitting is a simplistic representation of the trait response to the environment. It assumes a physiological response that increases to a maximum value, then drops immediately  identification of a common seed source representing a specific population, i.e., a provenance, (2) phenotypic evaluation followed by pedigree reconstruction  can be found at: ML and SS conceived the project. VP and DC conducted statistical analyses. VP and ML wrote the manuscript. SS, DC, JS, and HK contributed to discussions. All authors contributed to the article and approved the submitted version.VP was financed by the Internal Grant Agency of the Faculty of Forestry and Wood Sciences, Czech University of Life Sciences Prague. DC, HK, and SS were supported by the Austrian Research Promotion Agency (FFG) and the Cooperation Platform Forst Holz Papier (FHP) and LIECO nurseries and the Austrian Federal Forests (ÖBf). ML was supported by grant “EXTEMIT – K”, no. CZ.02.1.01/0.0/0.0/15_003/0000433 financed by OP RDE. ML and JS were supported by the Ministry of Education, Youth, and Sports program INTER-EXCELLENCE, subprogram INTER-ACTION [grant number LTAUSA19113].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Furthermore, NiOx can be fabricated via scalable and controlled physical depositionmethods such as RF sputtering to facilitate the quest for scalable,solvent-free, vacuum-deposited HaP-based solar cells (PSCs). However,the interface between NiOx and HaPs isstill not well-controlled, which leads at times to a lack of stabilityand Voc losses. Here, we use RF sputteringto fabricate NiOx and then cover it witha NiyN layer without breaking vacuum.The NiyN layer protects NiOx doubly during PSC production. Firstly, the NiyN layer protects NiOx from Ni3+ species being reduced to Ni2+ by Ar plasma, thus maintaining NiOx conductivity.Secondly, it passivates the interface between NiOx and the HaPs, retaining PSC stability over time. This doubleeffect improves PSC efficiency from an average of 16.5% with a 17.4%record cell to a 19% average with a 19.8% record cell and increasesthe device stability.The interfaces between inorganic selective contacts andhalideperovskites (HaPs) are possibly the greatest challenge for makingstable and reproducible solar cells with these materials. NiO One of the main factorsfor the high PCE in PSCs composed of polycrystalline thin films isthe intrinsically low defect concentration in the bulk of halide perovskites(HaPs).x) is a sturdy andefficient hole-transport material (HTM), which has been reported toimprove the stability of PSCs over that achieved with organic HTMs.9 Radio-frequency (RF) sputtering is a scalable fabrication methodfor metal oxides with a highly controlled oxygen partial pressure.Wang et al. have shown that sputtered NiOx at low temperatures has controlled transparency and conductivitythat affect the efficiency of the PSC.10 Moreover, sputtered NiOx is preferredover wet-chemically processed NiOx becauseit leaves no residues of solvents or precursors that can damage thestability and efficiency of the final device.11 However, sputtered NiOx hole-transportlayers (HTLs) with no further treatment have parasitic resistancethat leads to PSCs with moderate fill factors (FFs) and efficiencies.14Nickel oxide , a very small bandgapsemiconductor, as an ultra-thin ∼2 nm layer on NiOx, essentially an in-situ modification of the NiOx surface without breaking vacuum. This NiyN interlayer becomes a buffer between theoxide and the HaP film. We then investigate its effects on PSC performanceand stability.In this work, we use RF magnetron sputtering for an alternativein-situ route to passivate the HaP/NiOyNprotects NiOx from the reduction of Ni3+ cationsto Ni2+ during the Ar plasma cleaning step, typically usedto improve the wettability of the oxide, thus maintaining NiOx conductivity. The NiyN layer also passivates the interface between NiOx and MAPbI3 and inhibits the reactionbetween MAPbI3 and Ni3+. Although NiyN is conductive and could be expected to introducetrap states that damage PV performance, we show that thin enough NiyN can passivate the interface between NiOx and HaP, thereby improving device efficiencyand stability.We find that NiFluorine-doped tin oxide -coated glass substrates  werecleaned in a sonication bath with soap (Decon 90) and deionized waterand then rinsed in deionized water followed by dry ethanol.x and NiyN were deposited using RF sputtering  from2-inch NiO and Ni targets, respectively . NiOx deposition was done at room temperaturewith an Ar gas flow of 30 sccm and a total chamber pressure of 3 mTorr.NiyN deposition was done at room temperatureand a total chamber pressure of 3 mTorr, with Ar and N2 gas flows of 15 and 45 sccm, respectively. In the first stage, theNiOx target was set to 80 W for 1 h, thenthe chamber was purged for 10 min, and finally, the Ni target wasset to 60 W for 3 min. Ar plasma cleaning was performed by applyinga power of 30 W on the substrate with an Ar flow of 30 sccm.NiO3 was synthesized from MAI (Greatcell Solar) andPbI2  precursors in a 1:1 ratioat a concentration of 1.5 M. The precursors were dissolved overnightat 60 °C in γ-butyrolactone  anddimethyl sulfoxide  at a 7:3 ratio.The solution was spin-coated at 4000 rpm for 35 s with 800 μLof a toluene  anti-solvent drip after 30s. For device fabrication, 20 mg of PCBM was dissolved in 1 mL ofchlorobenzene overnight, spin-coated at 2000 rpm for 30 s, and annealedfor 10 min. After 15 min of cooling, a solution of 3 mg of bathocuproine in 6 mL of isopropanol wasspin-coated at 4000 rpm for 30 s. Finally, thermal evaporation wasperformed to deposit round Ag contacts of 3 mm diameter. A schemeof the device structure is given in the Supporting Information (SI).MAPbI–10 Torr) with He I photons (21.22 eV)generated by a He discharge lamp, with a pass energy of 5 eV and a0.02 eV step size.Ultraviolet photoemission spectroscopy(UPS) was used to probe the vacuum level and the position of the Fermilevel with respect to the valence band edge, leading to the determinationof the work function (WF) and ionization energy (IE) of the measuredmaterials at a resolution of 0.15 eV. UPS was performed in an ultrahighvacuum  withan Al Kα anode (1486.6 eV) was used to probe the Ni 2p, N 1s,and C 1s core levels at a resolution of 0.8 eV. Scans were taken witha pass energy of 25 eV and a 0.05 eV step size at a low base pressureof 10–8 Pa (∼5× 10–10 Torr). The binding energy (BE) wascalibrated vs carbon (C 1s = 285 eV). Survey scans were collectedwith a pass energy of 200 eV and a 1.0 eV step size, followed by high-resolutionscans with a pass energy of 50 eV and a step size of 0.1 eV. The sampleswere exposed for ∼1 min to air during the sample transfer.XPS in Bar-Ilan University (BIU) wasperformed using a Thermo ScientificNexsa spectrometer XPS system with an Al Kα anode (1486.6 eV)at a base pressure of ∼7 × 102 atmosphere.Optical transmission measurements were performed using an opticalfiber-based custom-made system that consists of a CCD array spectrometer and two integrating spheres. The measurementswere done over a 400–1000 nm spectral range with a circulardiameter of 3 mm in a NTwo-probe measurementswere done with a Keithley 2400 source measurementunit (SMU) in a potential range of −0.5 to 0.5 V at a 50 mV/pointrate.J–V characteristicsweremeasured with the same Keithley source at a potential range of −1.2to 0.2 V at a 20 mV/point rate in ascending and descending scan directions.The device was illuminated through an optical fiber using a laser-drivenlight source  xenon lamp calibrated to theAM1.5G solar spectrum.2 atmosphere, using an air photoemission system , illuminated by a deuterium (D2) UV source, coupledwith a motorized grating monochromator.Photoemission yield spectroscopy (PEYS)was used to measure theIE  ofperovskite surfaces. The measurements were done under a NTransient PL decay measurementson the HaP thin films depositedon NiO/FTO substrates with and without a NiN interlayer were measuredin ambient conditions. The HaP thin films were excited with a 450nm picosecond pulsed laser diode source, and PL decay characteristicsin the 760–765 nm emission wavelength range were recorded witha photomultiplier tube.x layer from a NiO target on an FTO-coatedglass substrate. To modifythe NiOx surface, we used reactive sputteringtreatment of the Ni target with a plasma composition of 25% Ar and75% N2.We used RF sputtering to deposita NiOx surface, we used XPS on NiOx samples modified and unmodified by reactivesputtering. TheXPS spectra from both NiOx samples showa broad and shallow nitrogen signal around a binding energy of 399eV, which typically fits the signal of nitrogen in organic matrices position in the material. The O 1s feature is a superposition oftwo core level peaks corresponding to oxygen bound to Ni3+ and Ni2+. The BE of the O 1s peak is found to increaseby 0.6 eV upon Ar+ etching, from 528.6 to 529.2 eV, forNiOx  positions obtained by linear extrapolationof the leading edge of the filled states are shown in x valence band shows a 0.39 eV shift away from EF upon Ar+ etching, whereas the NiyN-modified NiOx valence band remains at the same position (negligible shift of 0.03eV). Comparing the valence bands of the NiOx reference and NiyN-modified NiOx after 5 s of Ar+ etching, wefind the VBM to be 0.30 eV closer to the Fermi level for NiyN-modified NiOx (1.00eV below EF) than for the unmodified NiOx (1.30 eV below EF) .To understand theeffect of Arelow EF) 2. Thistx (left) and NiyN-modifiedNiOx (right) before and after 5 s Ar+ etching,plotted relative to the vacuum level (Evac), are shown in Ef), as they were measured,are shown in the SI . The WFfor unmodified NiOx decreases from 4.75to 3.94 eV after 5 s Ar+ etching and from 4.52 to 4.10eV for NiyN-modified NiOx. Together with the linearly extrapolated VBM values, the IEvalues are found to be 5.24 eV for Ar+-etched, unmodifiedNiOx and 5.10 eV for NiyN-modified NiOx  and the p-type character of the film, as shown by the positionof the Fermi level in the lower part of the gap (x we synthesized is O-rich as reported inthe literature.28 The charge carrier (hole) densityin the NiOx is the result of nickel vacancies(VNi″) and/or oxygen interstitials (Oi″).29 Ar+ etching presumably creates more oxygen vacancies (VO··), becauseoxygen is lighter than nickel and, thus, more easily ejected fromthe etched surface. Oxygen vacancies can reduce the charge carrierconcentration as x films. NiyN-modified NiOx is more resistantto such a process, as seen by the XPS and UPS results, demonstratingthe passivating effects of NiyN on theNiOx surface. This passivating effectmakes the NiyN-modified NiOx film less prone to electronic structure changesduring subsequent processing steps, which is further corroboratedby the unchanged energies of the valence states with respect to theFermi level in NiO of Ni3+ S2 and th of Ni3+ S2 and th+ etching of both surfaces (Evac) shift, which agrees withthe 0.42 eV change in the NiyN-modifiedNiOx WF measured by UPS  above EF upon Ar+ etching  betweenthe FTO electrode and the NiOx surfacewith and without a NiyN layer, both beforeand after Ar plasma treatment. The Ar plasma treatment dramaticallyincreased the unmodified NiOx resistance∼40×, from 1.9 MΩ before to 80 MΩ after. TheNiyN layer reduced the NiOx resistance from 1.9 to 0.09 MΩ before plasmatreatment, but even more critically, after Ar plasma treatment, theresistance increased about three times to 0.3 MΩ, lower thanthat of the unmodified NiOx . The VBM shift toward EF as NiyN gets thicker mayexplain the minor resistance increase after Ar plasma, as part ofthe NiyN layer is etched away.The NiOied NiOx 3b. The dx with and without NiyN, beforeand after plasma etching, we marked NiyN-modified NiOx after plasma treatmentas the best candidate for PSCs. Thus, we deposited MAPbI3 on top of NiOx with and without a NiyN layer after plasma treatment and then performedtime-resolved photoluminescence (TRPL) to measure differences in chargeextraction after NiyN deposition and plasmatreatment. A fit of the PL decay to a double exponential curve typicallyindicates two kinds of decay mechanisms, which are common when HaPis deposited on a selective contact.34 A short lifetime τ1 correspondsto charge transfer and detrapping of charge due to light exposure,and a longer lifetime τ2 corresponds to recombinationsat the interface and within the absorber. Because τ1 and τ2 were hard to distinguish, we chose to calculateonly τ2 according to a monoexponential fit at a decaytime > 60 ns.35 Similar lifetimes, 31and29 ns for MAPbI3 on NiOx andNiyN-modified NiOx, respectively (yN modificationdid not cause more (or less) recombination at the interface. Furthermore,PEYS, AFM, SEM, and XRD measurements  do not differ between MAPbI3 layers deposited on NiyN-modified or unmodified NiOx.Aftermeasuring the transparency and resistance of NiOectively 4a, indicyN modification does not show any differencein morphology .AFM imaging of the surfaces before and after NiFigures S6 and S7) measurementsshow no significant difference in MAPbI3 morphology andcrystallinity . Both XRD analysisand SEM micrographs did not show any signs of PbI2 beyondthe peak-to-noise ratio level.SEM andXRD , but only the descendingdirection is shown for simplicity . The similarity in Voc between these two samples is consistent withthe similar results obtained by TRPL. At the same time, the FF improvesdramatically from a 65% average with unmodified NiOx to 75% with the NiyN layer, regardlessof its thickness or the etching time  for these samples andis explained by the decrease in transparency, as seen in yN modificationis to have as thin a NiyN layer on NiOx as possible, which can passivate the HTLwith a minimum loss in Voc and transparency.The minor decrease in r plasma 5d. A sliing time 5e is coning time S9 for thyN layer has on the complete solar cell FF, we performeddark I–V measurements todetermine the shunt resistance (Rsh) andthe series resistance (Rs) of the SC , we ascribe this to aVBM shift toward the vacuum level that increases the driving forcefor hole extraction from the HaP film but at the expense of the Voc (To further investigate the huge effect that the Nisurement S3, we asyN modificationon cell stability, 18 cells were measured over 4 days at room temperaturein a N2-filled chamber with a controlled environment andrelative humidity that did not surpass 5%. The cells were held inthe dark between the measurements and were exposed to light for 4h before and naturally during measurements. For both types of cells,the PCE decreased over the 4 days. However, on average, the referencecell PCE values were reduced by almost 50%, from 15.9 to 9%, whilethose of the NiyN-modified cells werereduced only by 15%, from 16.5 to 14% (To test the effect of the Ni5 to 14% 7a.yN thanthose without it. The Voc and Jsc of both types of cells show inverse changesover the 4 days of testing. The Jsc ofthe reference cells decreased by 40% to, on average, 14 mA/cm2, whereas the Jsc of the NiyN-modified cells remained almost constantat 20 mA/cm2 (Voc of the referencecells increased by 3% up to 1.1 V, and the Voc of the NiyN-modified cells decreasedby 8% down to 0.95 V (yN modified-NiOx; see yN-modifiedcells remained constant for 3 days, and only on the fourth day didthe series resistance increase by 30% (Jsc reductionand the Rs increase in untreated PSCsare prominent evidence of a reaction at the NiOx–MAPbI3 interface inhibited by the NiyN treatment. The NiyN layer plays a dual role, as it helps maintain the Ni3+ concentration, passivates the NiOx–MAPbI3 interface, and presumably preventsa reaction between the more active Ni3+ and MAPbI3. These two features of the NiyN layerimprove the solar device FF and, most importantly, cell stability.Further investigation of the SC statistics revealsa differentaging mechanism for cells with Ni0 mA/cm2 7e. The Vo 0.95 V 7c. The Fe by 30% 7d,f, stiI–V characteristics over 4 days,but this time we repeated the I–V measurements for 100 cycles every day (yN-modified cells do not. Although in theinitial response, the shunt resistance of the reference cell is higherthan that of the NiyN-modified SC, itsseries resistance is higher by 150%, which leads to the two typesof cells having equal FFs. After 4 days of cycling, the series resistanceof the reference cell increased by 70%. Similar to what Khenkin etal.36 showed, the initial Voc of the reference PSC started every day at 1.1 V butdecreased as the number of cycles progressed, which is not the casefor the NiyN-modified cells. We interpretthis finding as an acceleration of the reaction at the interface bythe constant change in the electric field applied to the SC underworking conditions. The Jsc and Rs reductions suggest that an insulating interfaciallayer formed between NiOx and MAPbI3. In contrast, in the cells prepared with NiyN-treated NiOx, Jsc only undergoes a 3 mA/cm2 loss over the100 cycles. The constant Rs and Rsh over time indicate no change in charge transportand therefore no change of the material under working conditions.These results altogether support that NiyN modification inhibits reaction at the NiOx–MAPbI3 interface and prevents the formationof a blocking layer that deteriorates the SC performance.To further investigate the current reduction source, we measured very day 8a,b. Thex HTLs with NiyN inorganiclayers, which is the first time that such a modification was everdone. We showed that NiyN plays a dualrole, improving the PSC’s efficiency and stabilizing the NiOx–HaP interface. The NiyN layer formed on NiOx retains the Ni3+ species within the nickel oxide bulk,preserving its conductivity and thereby improving the overall cellefficiency. Furthermore, the same NiyNlayer protects HaP from the reactive Ni3+ species, necessaryfor nickel oxide conductivity, and prevents degradation of HaP, givingdevices superior stability over those built with untreated referenceNiO films. Although, with time, some of the NiyN might be oxidized by the NiOx layer, in a well-encapsulated device, the interfacial reactivitycan be kept below the level where charged defects will start to changethe overall defect density of the transport and absorber layers. Theseinteractions should be investigated further beyond the scope of thisstudy. We also showed that when NiyN isthin enough, its small energy gap, which can block sunlight and introducetraps for charge carriers at the interface, has a negligible effecton the overall SC efficiency. Our first reported solid-state, inorganic,in situ passivation route via RF sputtering deposition presents amajor step toward solvent-free fabrication of reproducible and stablePSCs.We used a physical vapor deposition methodto improve NiO


The results support the assumption of an urban–rural divide in terms of perspectives and values. The discourse indicates that rural actors who are most affected by the wolves’ vicinity have more negative attitudes towards them. At the same time, they feel abandoned and dominated by urban perspectives and politics. In addition, linkages to right-wing populist positions and conspiracy narratives that can be interpreted as a consequence of political alienation are found.Given that wolves have been extinct in Germany for approximately 150 years, their return and growing population over the last decade has caused intense discussion and conflict. To develop a widely accepted and just coexistence between humans and wolves, a comprehensive understanding of the conflict is needed. There are indications that the conflict goes beyond dealing with the wolf population and marks a spatial–cultural divide between urban and rural areas. Nevertheless, the social dimensions of the human–wolf conflict in Germany have been little studied. The aim of this paper is to narrow this gap by means of a media discourse analysis including reader comments in order to provide insights into the constituent elements of this conflict. We conducted a qualitative content analysis. The sample comprises articles ( Worldwide, the recolonization of large carnivores such as wolves and bears often results in human–wildlife conflicts  attention?Against this background, the aim of this study is to narrow this knowledge gap and gain a better understanding of the social conflict over wolves in Germany by means of a media discourse analysis. We follow the working thesis that conflicts surrounding the recolonization of wolves may reveal deeper underlying social tensions, such as the rural‒urban divide , nor would it be useful with regard to the qualitative research approach, we reduced the number of articles for analysis. Qualitative approaches aim to show the range of different qualitative manifestations of a research subject and to theorize possible interrelationships and patterns. Determining the sample size and assessing its appropriateness therefore followed the frequently applied criteria in qualitative research studies of “theoretical saturation,” “informational power,” and “informational redundancy”  had to be a central topic of the article. Only mentioning it was not sufficient.ii.The article must have focused on the normative dimension of the human‒wolf relationship in that meaning was attributed to this relationship. It did not matter whether the normativity came directly from the author or, for example, in the context of an interview or quotation.iii.High numbers of reader comments attributed to an article were regarded as indicating high resonance by either favouring this perspective or holding antagonistic views. It can therefore be argued that these articles constitute good representations of discursive struggles, and they were preferentially included in our sample.The selection of these articles was guided by the following criteria:Data processing was performed using MAXQDA software. All the articles and reader comments were evaluated and interpreted following the seven-step guide to qualitative content analysis by Kuckartz  on the bThe results show varying discourse themes around wolves and variations in the frequency with which the different media cover wolves  actor groups and their mutual relations or perspectives/perceptions, including conflict over interpretative sovereignty; (2) an instrumental utility discourse about benefits versus costs, risks and goal conflicts connected with wolf resettlement; (3) an urban–rural divide and political alienation; 4) links to right-wing populist discourses, narratives of conspiracy and forms of escalation; and (5) issues in wolf management. Table  links toi.agriculture versus nature conservation andii.rural people versus the “urban elite”.Actors with opposing values, perspectives and interests are a central part and constituting category of the “human–wolf” conflict in Germany, which largely indicates a social conflict over wolves. The results show that views in the conflict about wolf resettlement in Germany are characterized by two strongly opposed dichotomies:In addition, there are distinct actor groups, such as politicians, scientists and experts, hunters and the media, forming or supporting the parties in the conflict. In the content studied here, these positionings resulted from attribution of criticism and devaluation of opposing positions. It was apparent that the conflict over the reintroduction of wolves is also a conflict over interpretative sovereignty. Frequently, strategies were used to devalue opposing arguments and strengthen the actor’s own position. Credibility, expertise and knowledge became contested attributes denied to the other side through degrading attributions. These devaluation mechanisms were used by wolf supporters and wolf opponents alike but with different attributions and levels of intensity. Thus, references to other social groups of actors came primarily from opponents of the wolf-recolonization efforts. From the perspective of many wolf-resettlement opponents, nature conservationists and urban people were “wolf romanticists” and “wolf freaks” with “eco-social romantic fairy tale ideas” who are “far removed from reality”. Further attributions included that they are “ignorant and [have a] know-it-all attitude”, they enforce “double standards” and tell “lies”, they “lack expertise”, and they engage in “unobjective/ideological/emotional discussions”.Opponents of wolf recolonization considered politicians to lack a firm “grip on reality” and “expertise” and to be “idle/actionless” while practising politics only for the benefit of the “urban elite”. Some wolf-recolonization opponents accused the media of being biased and “pro-wolf”. However, the analysis of the positioning of the articles studied contradicted this accusation (see 3.1), as it showed that media coverage of the wolf population is varied and not one-sided.In contrast, some proponents of wolf resettlement accused those opposed of “scaremongering”, having “irrational fears”, exhibiting “hysteria”, and engaging in an “emotional debate”. Farmers were considered “complainers” who destroy nature with their agricultural practices.Another main theme throughout the media articles and reader comments was a benefit-versus-cost discourse. The “damage” caused by wolves was frequently used as an argument against wolf resettlement. Often mentioned, damage included “livestock killed by the wolves”, potential “risks to people and especially to children”, “fear of wolf attacks” and a resulting “diminished quality of life” due to the perceived restricted range of motion for humans and playground areas for children. In addition, the cost of prevention measures funded by taxpayer money was criticized. Target conflicts in nature conservation were cited, particularly regarding the high nature value of extensive pastureland, which is regarded as endangered by wolf recolonization. A further ethical conflict was seen regarding animal welfare when wolves kill grazing animals in fenced pastureland.Q2: “But here, where every German citizen has only 2000 square metres of usable space to ‘survive’, one should differentiate a little as to who is allowed to sit at the table. The wolf does not fit here.” (Reader_TopAgrar)In addition to the discourse about damage, the general benefits of having wolves were questioned by wolf-recolonization opponents . In particular, wolf recolonization in Germany’s densely populated “cultural landscape” was repeatedly denounced (Q2–4). Here, the statements documented the perception of or fear of competition with wolves and the narrowing of humans’ interests and scope for action:Strikingly, the wording of comments frequently implied active settlement by wolves instead of a natural dispersal process (Q3).Q6:“If a tiger is shot in India, the German is the first to raise a finger. But we don’t care about our own fauna.” (Reader_Spiegel)Reader comments also included arguments for the protection of species of larger predators in distant regions of the world but not in their own immediate vicinity (Q4). This form of colonization of other countries to further species conservation was considered critical by some wolf-recolonization advocates, who noted that the conservation of large carnivores and mammals in countries in the Global South is more precarious (Q5-6).Q12: “As you often hear, the fears you describe exist, whether justifiably or not is irrelevant. I ask myself why we want to do this to ourselves, or to those who are afraid, just to prove a few opinionated wolf romantics right. Fear is not a nice thing, and one should not make fun of it.” (Reader_NOZ)While some saw the wolf as an unnecessary “luxury” that causes great financial damage, which must be remedied by adequate compensation, a main argument against wolf dispersal was the perceived fear and risk of humans being attacked by wolves (Q7-8), especially the danger that wolves present to children, which was a recurring theme in the discourse (Q9). This fear, particularly of the danger posed to children by wolves, was a motif that ran throughout the reader comments (Q10-11). In this context, the feeling of not being taken seriously was also found.In contrast, the views and arguments expressed by supporters of and experts in wolf resettlement revealed that compared with other everyday risks, they viewed the risk as very low or acceptable. Compared with wolf-recolonization opponents, who focused on the uncertainty and potential risk posed by future developments, wolf-recolonization supporters and experts highlighted that there have been no fatal wolf attacks on humans thus far (Q13). Other arguments emphasized that humans constantly live with certain risks (Q14). In addition, proponents of wolf recolonization argued for the potential benefits of wolves as a regulating force against game browsing and damage to crops. They considered the wolf to play an important role in “reestablishing the ecological balance”. In the Lusatian region, a main habitation area for wolves in Germany, tourism experts even believe that the wolf population has the potential to become a tourist attraction (Q15).Beyond a pure cost–benefit argumentation that considers the wolf merely from an instrumental utility perspective, there were also reader opinions that attributed an intrinsic value to the wolf. These actors criticized the colonial dominance of humans and recognized the right of the wolf as a species to exist for its own sake. However, this last argument was rarely put forward.The reader comments documented contrasting perceptions of the realities of life in the city and the country, coupled with processes of political alienation. The discourse painted a picture of disparity between city and country, between an “urban elite” and the rural population (Q16). There was a perceived political “dominance by urban people” and their opinions over the rural population. At the same time, the rural population was portrayed as an oppressed minority (Q17–18).Q19: “The people in the areas where wolves live are already suffering from massive restrictions on their quality of life. […] It’s unfair for nature-minded urban people to be in favour of the wolf, which they will never encounter in their lives!” (Reader_FAZ)Q20: “We are bombarded with wolf propaganda by the nature conservation authorities. But when concerned graziers show the cruel reality, the state authorities intervene. What a complete dismissal of the rural population!” (Reader_TopAgrar)In addition, the difference in the degree to which urban and rural populations are affected by wolf recolonization was emphasized by some readers’ comments. The support for wolf resettlement found among urban people, who are rarely affected by the presence of the wolves, was perceived as arrogance towards the rural population (Q19–22).Here, a motif of perceived spatial injustice emerges, which was also associated/contextualized with other developments and conflicts in rural areas, such as the energy transition and the conflictual designation of areas for wind turbines (Q23) or the growing demand for organic food and sustainable agricultural practices, which increasingly pressures farmers to change (Q24). Urban people were accused of lacking an understanding of nature, being out of touch with reality, and lacking judgement (Q25–27). The city of Berlin recurred not only as a symbol of urban living and as an urban space but also, alongside the city of Brussels, as a political centre of power where decisions about rural areas are made removed from the context in which the issues occur (“in distant Berlin”) (Q28–29). Mixed into this was also a demonstrable loss of trust in politics and the state, feelings of political alienation and sympathy with populist positions . Politicians were frequently accused of inaction, but the opposite was found in the media coverage. Despite the many other challenges brought by development in rural regions, such as demographic change, the wolf population received special attention and played a comparatively large role in both the media and politics (Q33). Thus, the wolf recolonization conflict was discussed at the highest political levels during the study period, including by the Chancellery, the Prime Minister of Saxony, and the Ministers of the Environment and Agriculture.The political alienation that was evident in the analysed material was regularly linked to sympathy for right-wing populists and antidemocratic positions (Q30–32). In the discourse on the wolf population, vocabulary typically used by right-wing populist groups appeared conspicuously often. The use was documented exclusively in reader comments by those opposed to wolf recolonization in Germany. In addition to the motif of the “lying press” (Q34-35), there were recurring allusions to “conspiracies” by “elites” and rejecting the European Union, and references to the 2015/2016 refugee crisis in Germany and Europe were made using the term “Willkommenskultur” (Q36), which was influential at that time.It was not just the reader comments that took up this issue; the articles themselves examined it as well. The articles revealed that defensiveness about wolf recolonization is being made an election campaign issue by the right-wing populist party AFD in Germany (Q37–38). In fact, the Federal Minister of Agriculture considered the wolf an “AFD acceleration programme”, as anti-wolf positions were strongly represented by and related to the AFD party.In addition, various conspiracy narratives were found in the reader comments. One common narrative was that the wolves are hybrids that were deliberately brought to Germany and released. Another narrative asserted that important information was being kept secret (Q39). It alleged a conspiracy among scientists, politicians and actors from NGOs: scientists were purportedly falsifying results in their expert reports, and NGOs would earn money from the wolves.The media discourse on wolves is divisive and polarizing, and there are signs of increasing escalation and intensity in the conflict, which manifest in hostility and threats. Not only emotional statements and mutual accusations but also insults were observed in exchanges between the parties in the conflict, and these pointed to hardened positions (Q40). In addition, various forms of protest, such as vigils, demonstrations and social media campaigns, as well as calls for “illegal wolf killing”, were mentioned. This call for criminal action was justified as a form of self-defence (Q41–43).One of the most frequently discussed topics were wolf management issues. These included “stock regulation/wolf population control”, subsequent demands for “legislative changes”, aspects of “herd protection” and “compensation payments”, demands for “conflict management” and “counselling and information services”, such as training and education to increase public knowledge about wolves.Overall, in concrete discussions of suitable wolf management measures, the discourse notably took on a more objective tone. Emotional or sarcastic expressions were less commonly observed. The positions expressed seemed more conciliatory and solution-oriented, and the conflict appeared less intense in such discussions.Regarding population regulation, there were very few voices in favour of extirpating the wolf completely, but many favoured population regulations. Opinions ranged from allowing “strict population regulation/sharp hunting” (Q44–45), setting an “upper limit” on moderate regulation and removing wolves “in case of danger” or “problem wolves”. The latter is also seen and advocated as a measure to promote acceptance of the wolf population (Q46). The demands for wolf population regulation went hand in hand with demands for legislative changes that allow the removal of wolves as well as the designation of wolf-free or protected grazing areas. In addition, herd protection measures and the adaptation of pasture management, including installing fences, stable security, using guard dogs and scaring away wolves, were discussed. Commenters who had already had good experiences with these measures also spoke up here. There were several indications that damage from wolves mainly occurs where there is no or minimal herd protection.There were many reader comments that demanded comprehensive “compensation payments” for the herd protection measures enacted and the damage caused by wolves. However, this appears to be somewhat irritating, as 100% of these costs are already being reimbursed. In this context, it seems natural that the idea of “conflict management” was often alluded to in the articles (Q47) in reference to learning processes, training and information services that could increase acceptance.The aim of this article was to analyse the human‒wolf conflict in German media discourse, determining how the conflict is constituted and documented, whether it is a symbol of an urban‒rural divide, and why the topic is covered so intensively in the media. In the following, we will discuss our findings in relation to results from other geographical contexts (5.1) and justify our argument that the human‒wolf conflict is indicative of broader societal conflicts, such as the urban‒rural divide (5.2).Our findings about the human–wolf conflict in Germany have much in common with the results of studies in other regions of Europe and North America. The return for wolves in regions where they have been previously extinct creates conflicts (e.g., Ericsson and Heberlein Dressel et al.  have shoOur analysis further reveals a prevailingly utilitarian discourse in the German media that questions the wolf against the background of a cost–benefit discourse. The wolf is seen as “useless” and harmful by farmers, and its presence thus deepens the already existing conflict between stakeholders in agriculture and in nature conservation (see also Friedrich et al. The observation by Figari and Skogen  that theAll the media investigating the wolf topic continually reported on it. The issue received significant attention, and the debate can be regarded as emotionalized. A similar level of attention was also observed in Northern European countries after wolf populations began to spread again (Ericsson and Heberlein Consistent with Wilson’s  observatMoreover, these results show that the wolf symbolizes the political alienation of the rural population, who feel dominated by urban worldviews influencing policies and the will of supposed majorities. Eriksson  showed tAdditionally, some scientists, especially ecologists normatively involved in conservation, seem dismissive of these concerns. As an example, Chapron and López‐Bao  criticizNevertheless, the geographical and spatial disparities between urban and rural areas have led to increasing social distortions in recent years, which are also reflected in the successes of right-wing populist parties (e.g., Kallert et al. However, further research is needed to elaborate whether the conflict line is the rural‒urban divide or whether other conflict lines are congruent with this line; thus, other sociocultural aspects may be relevant, as described in recent theoretical and empirical sociological research in Germany (e.g., Lux et al. In Europe, wolf recolonization is a result of, and supported by, international nature conservation law, such as the Washington and Berne Convention and the EU Habitats Directive. However, this is a top-down approach that insufficiently considers local specificities and region-specific development opportunities in rural areas. The wolf and its coexistence with humans thus seem politically imposed, which inhibits successful coexistence (König et al. To date, human‒wolf conflict in Germany remained understudied. In particular, qualitative analyses that allow for an integrative understanding are lacking. The present media analysis contributes towards filling this gap. Overall, it shows that media analyses achieve considerable density in the qualitative description of human‒wildlife conflicts. However, this description takes place on a superordinate aggregated societal level, which can differ greatly from local specific and varying manifestations. In addition, media filter information according to newsworthiness and also attention or entertainment value. However, even the reader comments analysed here do not provide a representative depiction of the human‒wolf conflict but rather indicate the different dimensions and facets that play a role. For a deeper understanding of the conditions for successful coexistence, therefore, further analyses are needed that look at different local characteristics and also at the interactions of different social factors with varying management instruments and communication approaches.The recolonization of wolves in densely populated cultural landscapes causes major social conflicts reflected by a high amount of media attention. There is a divide in the perspectives of urban and rural actors, which is supported by the narrative of “pro-wolf” urbanites and “anti-wolf” ruralites and is also visible in the media coverage. The unequal distribution of the nonmaterial costs and benefits of the recolonization of the wolf population results in “coexistence inequalities”, which in turn may lead to political alienation and a sense of imposition. However, the human–wolf conflict symbolizes a larger conflict based on spatial disparities and inequalities between urban and rural areas. Damage prevention and compensation measures alone will not be sufficient to resolve the conflict. Going forward, it will be important to consider regionally specific development options, strengthen local decision-making competences and facilitate honest participation and dialogue so that different actors with different opinions are valued in decision-making. Only in this way can a just and sustainable coexistence between humans and wolves be established.Supplementary Information


C. elegans that express reporter proteins that are sensitive to changes in the protein folding environment and respond by misfolding and displaying readily scorable phenotypes, such as localized YFP fluorescence or paralysis. We found that nPM exacerbated protein aggregation in body wall muscle cells, increasing the number of large visible protein aggregates, the amount of high molecular weight protein species, and proteotoxicity. Taken together, the data point to nPM negatively impacting proteostasis. Therefore, it seems plausible that nPM exposure may exacerbate symptoms of AD and age-related dementia in a manner that is at least partially dependent on proteostasis decline.The proteostasis network comprises the biochemical pathways that together maintain and regulate proper protein synthesis, transport, folding, and degradation. Many progressive neurodegenerative diseases, such as Huntington’s disease (HD) and Alzheimer’s disease (AD), are characterized by an age-dependent failure of the proteostasis network to sustain the health of the proteome, resulting in protein misfolding, aggregation, and, often, neurotoxicity. Although important advances have been made in recent years to identify genetic risk factors for neurodegenerative diseases, we still know relatively little about environmental risk factors such as air pollution. Exposure to nano-sized particulate air pollution, referred to herein as nanoparticulate matter (nPM), has been shown to trigger the accumulation of misfolded and oligomerized amyloid beta (Aβ) in mice. Likewise, air pollution is known to exacerbate symptoms of AD in people. We asked whether nPM contributes to the misfolded protein load, thereby overwhelming the proteostasis network and triggering proteostasis decline. To address this, we utilized  C. elegans engineered to express misfolded Huntington’s disease (HD)-associated protein Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Does the manuscript adhere to the experimental procedures and analyses described in the Registered Report Protocol?  If the manuscript reports any deviations from the planned experimental procedures and analyses, those must be reasonable and adequately justified.Reviewer #1: YesReviewer #2: Yes**********  2. If the manuscript reports exploratory analyses or experimental procedures not outlined in the original Registered Report Protocol, are these reasonable, justified and methodologically sound?  A Registered Report may include valid exploratory analyses not previously outlined in the Registered Report Protocol, as long as they are described as such.Reviewer #1: YesReviewer #2: Yes**********  3. Are the conclusions supported by the data and do they address the research question presented in the Registered Report Protocol?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. The conclusions must be drawn appropriately based on the research question(s) outlined in the Registered Report Protocol and on the data presented.Reviewer #1: YesReviewer #2: Yes**********  4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.  Reviewer #1: YesReviewer #2: Yes**********  6. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. Reviewer #1: In the manuscript entitled “Nanoparticulate air pollution disrupts proteostasis in Caenorhabditis elegans”, Manriquez et al. evaluate the impact of environmental risk factors such as air pollution on proteostasis. Using well established C. elegans protein conformation disease (PCD) models, the authors examine protein aggregation using visual and biochemical assays and monitor proteotoxicity using behavioral readouts for movement. Based on this analysis, the authors conclude that nanoparticulate matter (nPM) exacerbates protein aggregation and proteotoxicity in the PCD models and has no impact on stress responses in wild type animals. Taken together, the authors suggest that exposure to environmental air pollution may overwhelm the proteostasis network and worsen symptoms associated with Alzheimer’s disease and dementia. Overall, the manuscript is well organized and well communicated. This reviewer concludes that the manuscript is appropriate for publication in PLOS ONE after addressing the issues outlined below.To support the authors’ conclusions, the following additions to the experimental data are recommended.1. To better view the visual aggregates that were quantified in Figure 1B, include an inset with higher magnification that depicts the type of aggregate that was scored. Specifically, magnify the area at the end of the arrowheads.2. It is unclear what the reader should observe in Figure 1C. Addition of arrows to denote what the authors refer to as AB puncta and arrowheads to highlight what the authors refer to as larger aggregates is needed.3. Supplemental Figure 2 shows the quantification of relative protein levels in each polyQ model. To support the conclusion that protein levels do not change, a representative gel image of the area quantified would be helpful. Specifically, polyglutamine tracts can lead to accumulation of proteins in the stacking gel of SDS PAGE gels. It would be important to note if protein accumulated in the stacking gel and if this was included in the final determination of protein levels. An image of a representative gel/blot to resolve this would be informative.4. In Supplemental Figure 2, the authors state that there is no statistical difference  were statistically significant.8. In Figure 3, it is unclear if the high molecular species are normalized to total protein in each lane or to control. Or are the values determined only by comparing intensity of the HMW species vs monomers for each strain. This reviewer recommends calculating relative amounts of HMW in total for each strain.9. In Supplemental Figure 4, the figure legend states that protein was collected for the Q40 strain immediately instead of after 72 hours, which was the procedure for aggregate counting. Is there a rationale for why the treatment with nPM was not similar to the procedure for aggregate counting?10. Supplemental Figure 4 shows the quantification of high molecular weight species in the Q40 and Q44 models. A representative native gel image highlighting the area quantified would be beneficial.The recommendations above ask for additional data and further analysis of the data to strengthen the authors conclusions. This reviewer offers additional suggestions to address within the text to further strengthen the manuscripts conclusion.1. Have the authors examined the impact of nPM on stress responses in PCD models? The authors demonstrate no impact on N2 animals but the extra challenge of expressing a misfolded disease protein along with the nPM may lead to activation of a stress response.2. In Supplemental Figure 1A, nPM leads to an unexpected decrease in Q40 aggregates. To explain this surprising result, the authors state that Q35 is a “better sensor” than Q40. The authors should also consider that the treatments are done at different times in the developmental cycle of the worm, which may affect proteostasis demands.3. In the conclusions section, it would be helpful if the authors offer their insight into why nPM that exacerbates protein misfolding in body wall muscles can suggest deficiencies in protein folding of AB species in neurons leading to dementia.Reviewer #2: The paper by Garcia Manriquez et al is a well written report that describes a study in C.elegans models to assess the proteotoxicity of nanoparticulates associated with air pollution. The study is technically sound. The methods used appear appropriate and appropriate statistical methods were used to assess the data. One short-coming of the study is uncertainty regarding the potential exposure in vulnerable neurons of human brain relative to the dose of nanoparticulates that the worms were exposed to.The only technical comment I have is that in Fig 1C, the authors should indicate the number of replicates that were examined to produce the representative images.**********  7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: NoReviewer #2: No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. 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SOD2) plays a crucial role in cells’ protection against mitochondrial oxidative damage. A genetic polymorphism in the mitochondrial targeting sequence of the SOD2 gene has been implicated in various diseases, including prostate cancer. Paller et al. have shown an increase in prostate-specific antigen (PSA) doubling time in patients with the Ala/Ala (wildtype) genotype when treated with pomegranate/grape extract antioxidants. We developed and validated a pyrosequencing assay that detects the common germline SOD2 SNP (rs_4880) with the aim of identifying men with castrate-resistant prostate cancer eligible for an antioxidant therapy clinical trial. We first selected 37 samples from the 1000 genomes study with known genotypes determined using Illumina-based sequencing and confirmed them by Sanger sequencing. In a blinded design, we then performed the new pyrosequencing assay on these samples and assigned genotypes. Genotypes for all 37 samples  were all concordant by pyrosequencing. The pyrosequencing assay has been live since May 2018 and has proven to be robust and accurate. Manganese superoxide dismutase-2 ( Biochemically recurrent (BCR) patients experiencing rising prostate-specific antigen (PSA) following local therapy  make up the largest subset of prostate cancer patients undergoing monitoring and treatment in the United States. The standard of care for these patients ranges from watchful waiting to androgen-deprivation treatment (ADT), but many of them seek treatment alternatives to ADT because of its significant impact on quality of life, including potential obesity, erectile dysfunction, hot flashes, decreased muscle mass, and osteoporosis. Consequently, the use of complementary medical products is widespread among prostate cancer patients, with 39% of recently diagnosed patients and 51% of patients overall reporting that they have used such products as herbal, vitamin, and mineral dietary supplements (primarily antioxidants) to try to boost their immune system and prevent disease progression ,3,4,5,6.SOD genes that have evolved in mammals: manganese superoxide dismutase (MnSOD) in mitochondria, copper-zinc superoxide dismutase (Cu-Zn SOD) in the cytosol, and extracellular superoxide dismutase (EC-SOD) in the extracellular compartments [SOD2 gene, has a scavenging role during oxidative phosphorylation, where it converts superoxide anion into oxygen and hydrogen peroxide [Sod2 gene was incompatible with life [Superoxide dismutases (SODs) are antioxidant enzymes and there are three artments . MnSOD, peroxide ,8,9. Theith life ,11.SOD2 gene that encodes for valine  instead of alanine (Ala) at codon 16 has been shown to be less efficiently imported into mitochondria [Signal sequences at the N-terminus target proteins to their respective compartments within the cell ,13,14. Achondria ,18,19. Achondria , althougchondria ,21,22.SOD2 biomarker is supported by three studies. Muscadine grape skin and pomegranate trials showed a 6–12 month lengthening of PSA doubling time (PSADT) in the BCR AA-SOD2 population but not in the control group or in VV or VA patients [SOD2 genotype randomly assigned to β-carotene (antioxidant) treatment (versus placebo) had a relative risk of 0.6  for fatal prostate cancer, but found no significant association in men with the VV/VA genotypes [Identifying biomarkers that will predict response to antioxidant therapies could potentially allow some patients to improve their quality of life and help reduce morbidity. The rationale to further explore this patients . Similarenotypes .In order to test the hypothesis that identifying biomarkers will predict response to antioxidant therapies, a SOD2 test was validated in a Clinical Laboratory Improvement Amendments (CLIA)-certified molecular laboratory and was developed for a phase III trial to determine whether 48 weeks of antioxidant treatment compared with placebo significantly lengthens PSADT in AA-SOD2 BCR men. The trial, which is called Muscadine Plus (MPX) in Men with Prostate Cancer (NCT03535675), is an 80-patient, phase III, randomized, double-blind clinical trial was being performed at 15 sites in the United States and using muscadine grape skin extract. Unfortunately, the trial closed for futility 1 July 2022, as it would not meet its primary endpoint. An analysis is underway to see if any of the biomarker endpoints were significant. The CLIA test is also being used in two other diet-based trials, one weight loss intervention in breast cancer (NCT04499950) and another plant-based diet in prostate cancer (NCT05471414), to see if genotype correlates with outcomes.SOD2 gene using pyrosequencing. After the pyrosequencing is performed, a given patient’s result is pattern-matched to the three pyrograms representing each of the genotypes. We selected 13 homozygous wildtype (WT), 12 heterozygous, and 12 homozygous variant samples with established genotypes from the 1000 genomes project, confirmed their genotypes using Sanger sequencing, and used them for orthogonal validation of the pyrosequencing assay. The assay has been live since May 2018, and approximately 120 runs were completed to select 80 men with castrate-resistant metastatic prostate cancer with the Ala/Ala genotype to be eligible for the clinical trial.We report the analytic validation of the codon 16 polymorphism in the mitochondrial leader sequence of the We obtained 37 DNA samples from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research  under a research agreement with the 1000 Genomes Consortium See . SamplesSOD2 SNP analysis was restricted to a small region around codon 16 within exon 2 (NM_ NM_000636.4(SOD2):c.47T>C ). Five different forward primers and two different reverse primers were designed using Primer3 . All primers were ordered from IDT Technologies  with M13 forward and M13 reverse universal primers. The best-performing primers  were as follows: forward primer 5′-tgtaaaacgacggccagt-ACTGACCGGGCTGTGCTT-3′  and reverse primer 5′-biotin-caggaaacagctatgacc-GCGTTGATGTGAGGTTCCAG-3′, , where M13 forward and reverse universal primers are indicated in lower case letters. Samples were PCR amplified using 100 ng genomic DNA. Cycling conditions were as follows: 96 °C for 1 min; 35 cycles of 95 °C for 30 s; 62 °C for 30 s; 72 °C for 1 min, and a final extension of 72 °C for 10 min. The expected size of the full-length PCR product was 174 bp, including the M13 primers. After amplification, PCR products were visualized by gel electrophoresis  and Sanger sequenced to confirm the previously determined genotypes. Based on previous findings, 2, 0.2 mmol/L of each dNTP, 5 pmol of forward primer (listed above), 5 pmol of reverse primer (biotinylated), 0.8 units of HotStar TaqDNA polymerase (Qiagen), 10 ng of template DNA, and dH2O to a 25 ul final volume. Pyrosequencing was done on the PyroMark Q24 (Qiagen) instrument, as previously reported, in a reaction containing 0.3 μmol/L sequencing primer (5′- AGCAGGCAGCTGGCTCCG -3′) and annealing buffer. The nucleotide triphosphate dispensation order for codon 16 was G,A,T,A,C,T,A,G,A,T,A,T.PCR-amplified products were generated using the same primers optimized for Sanger sequencing above, except that the reverse primer was biotinylated at the 5′ end to capture the bottom (anti-sense) strand for pyrosequencing. Each reaction contained 1X PCR buffer, 1.5 mmol/L MgClA total of 3 mL of peripheral blood from each patient was submitted to the Molecular Diagnostics Laboratory at The Johns Hopkins Hospital. DNA was isolated from 350 ul of peripheral blood using the Qiagen EZ1 Advanced XL instrument per the manufacturer’s protocol . Isolated DNA was quantified using Qubit 2.0 . We identified EBV-transformed lymphoblastoid cell lines to use as controls for each of the three genotypes . GM12878 is a commonly used reference sample whose use is advocated by the National Institute of Standards and Technology and the Centers for Disease Control (CDC). DNA samples were PCR amplified and pyrosequenced as described above. Analysis was done using Pyromark Q24 software, and pyrograms were pattern-matched for the genotype. The full protocol is available upon request.SOD2 gene using the 1000 genomes data sequenced on the Illumina platform. We then tested all possible primer combinations  were included on the same pyrosequencing run. Results were concordant and reproducible .We included four different controls in this assay . As such, we expected the heterozygous control to be present between 40% and 60%. This range is easily achievable by pyrosequencing technology, which has a limit of detection between 3% and 5%.We calculated the percent of C:T alleles in all of the samples that were pyrosequenced. The background noise for the homozygous (C/C) wildtype was ≤5% T, and for the homozygous (T/T) variant, it was ≤5% C. The heterozygous (C/T) control was consistently between 45–55% C:T. Non-template controls consistently demonstrated no signal.SOD2 polymorphism status were pyrosequenced. Pyrosequencing results showed 100% concordance compared to the consensus genotype , 204 Ala/Val, and 105 Val/Val (49.9%). Controls never failed on any of the runs, and NTC was never contaminated/amplified.SOD2. In contrast, samples that showed the Ala/Val genotype were reported as heterozygous, and those determined to have the Val/Val genotype were reported as homozygous variants for SOD2. Reports cited that the Val/Val genotype has been associated with a modestly increased risk of cardiomyopathy in the Japanese population [Samples determined to have an Ala/Ala genotype were reported as homozygous wildtype for pulation  and diabpulation  in generSOD2 p.A16V SNP. Analytical validation was performed on 37 samples used in the 1000 genomes project. The assay achieved 100% accuracy. Genotypes determined by the pyrosequencing assay demonstrated complete concordance with orthogonal assays (Sanger and Illumina sequencing) run on the same samples.We developed an assay for an ongoing clinical trial in a CLIA-certified laboratory that is routinely able to genotype samples for the The validated assay has been used in a phase III, randomized, double-blind clinical trial (NCT03535675) and two ongoing trials in breast and prostate cancer . All samples are being received and processed in a timely manner. The outcomes of these trials will tell us more about the prognostic potential of this biomarker.SOD2 gene (rs_4880) have a higher risk of aggressive prostate cancer in the presence of low antioxidant concentration8. Previous studies have demonstrated that this subgroup of men may benefit from compounds that reduce oxidative stress [SOD2 genotyping assay with outstanding analytical sensitivity, specificity, accuracy, and precision.Men with Ala/Ala genotype in the gene rs_480 have ae stress ,31. In tSOD2 genotype has been implicated in various diseases, including prostate cancer [SOD2 genotype, one might question why AA-SOD2 BCR prostate cancer patients would benefit from MPX treatment. The answer lies in the Physicians’ Health Study finding of a 10-fold increase in the risk of aggressive prostate cancer from high to low quartiles of antioxidant status in men with the AA genotype (but not in men with the VA or VV genotypes) [SOD2 genotype who had low consumption of dietary antioxidants [SOD2 men will show a significant decrease in PSA slope when treated with MPX compared to placebo. If the trial shows that the AA-SOD2 BCR subpopulation will benefit from ellagic acid supplementation, the results will encourage physicians to check patients’ SOD2 genotypes to make recommendations about antioxidant supplementation. Negative results should put to rest the claim that MPX and ellagic acid as single oral agents are active in a clinically meaningful way in this patient population. Finally, the development of this assay will allow for further exploration of the predictive capacity of AA-SOD2 to predict the response to antioxidants in other cancers, such as early-stage breast cancer.Further, this e cancer ,33,34,35notypes) . In addinotypes) ,36. Furtoxidants ,38. This