Efficient charge generation via exciton dissociation in organic bulk heterojunctions necessitates donor–acceptor interfaces, e.g., between a conjugated polymer and a fullerene derivative. Furthermore, aggregation and corresponding structural order of polymer and fullerene domains result in energetic relaxations of molecular energy levels toward smaller energy gaps as compared to the situation for amorphous phases existing in homogeneously intermixed polymer:fullerene blends. Here it is shown that these molecular energy level shifts are reflected in interfacial charge transfer (CT) transitions and depending on the existence of disordered or ordered interfacial domains. It can be done so by systematically controlling the order at the donor–acceptor interface via ternary blending of semicrystalline and amorphous model polymers with a fullerene acceptor. These variations in interfacial domain order are probed with luminescence spectroscopy, yielding various transition energies due to activation of different recombination channels at the interface. Finally, it is shown that via this analysis the energy landscape at the organic heterojunction interface can be obtained. The semiconductor exhibiting higher molecular energy levels—specifically the lowest unoccupied molecular orbital (LUMO) but also the highest occupied molecular orbital (HOMO)—is then called the (electron) donor, whereas the other is called the (electron) acceptor. This so‐called type II heterojunction enables efficient electron transfer from the donor to the acceptor, which may be accompanied by the formation of charge transfer (CT) states, where the electron is located on the acceptor LUMO and the hole on the donor HOMO. In order to maximize the charge generation yield in organic solar cells considering limited exciton diffusion lengths in both materials, the so‐called bulk heterojunction concept has been introduced, which enables charge generation throughout the bulk of the blend film by intimately mixing of donor and acceptor phases at the nanoscale.Since then intense research efforts were conducted to investigate, modify, and ultimately control the phase separation between the commonly used polymer donors and fullerene‐based acceptors. Since too intimate mixtures do not only promote charge generation but also increase charge recombination rates, balancing the domain sizes constituting the bulk heterojunction is part of the optimization process for maximizing photovoltaic device efficiencies.In general it has been demonstrated that the aggregation of both materials may improve charge generation, separation, and transport.Besides the above mentioned two‐step charge generation process, constituted of an excitation of the donor material followed by a charge transfer to the acceptor or vice versa, lately a single step charge generation process via direct excitation of the CT‐state located at the donor–acceptor interface has been discovered and discussed with growing intensity.FigureWe hypothesize that the energetic position of the CT transition of donor–acceptor bulk heterojunctions depends directly on the interfacial order of the two phase domains and can thus be probed by a combination of photoluminescence (PL)ab (anthracene‐containing poly(p‐phenylene‐ethynylene)‐alt‐poly(p‐phenylene‐vinylene) (PPE‐PPV) copolymers (AnE‐PV), equipped with octyloxy side‐chains at the PPE‐part and 2‐ethylhexyloxy side‐chains at the PPV‐part) and amorphous AnE‐PVba (AnE‐PV equipped with 2‐ethylhexyloxy side‐chains at the PPE‐part and octyloxy side‐chains at the PPV‐part)—both structures may be found in the Supporting Information—in various ratios were applied to create those different interfaces as mentioned above.One option to modify the photovoltaic parameters and the morphology of bulk heterojunction solar cells distinctly, is ternary blending of three components, either two polymers and one fullerene or one polymer and two fullerenes.2Figure−2 (left) and normalized to the CT‐emission peaks (right). From the left graph in Figure CTαEL), a second transition at around 942 nm (CTβEL) and a third transition at around 1007 nm (CTγEL). Even though already three CT transitions are clearly visible, this observation seems to be in contradiction to our expectation of four different recombination channels, as depicted in Figure In order to address the potential recombination channels depicted in Figure According to the literature, CT absorption bands can usually be fitted by Gaussian functions.Intriguingly, yet working for some of the compositions with lower contents of the amorphous polymer fraction, a set of three different Voigt profiles did not yield satisfactory results for all blends investigated. By applying one more Voigt profile, i.e., allowing an additional emission peak, to the fitting procedure, however, good fits could be simultaneously obtained for all blend ratios between the semicrystalline and the amorphous polymer. For detailed information about the individual fit results the reader is referred to the Supporting Information. Due to possible reorganization, the electronic CT‐state energy is often given as an intersection between the electroluminescence and the external quantum efficiency spectra of a given solar cell.AEL ≈ 1.51 eV, CTBEL ≈ 1.39 eV, CTCEL ≈ 1.31 eV, and CTDEL ≈ 1.23 eV. These peak positions are depicted in FigureFigureRoughly, the from electroluminescence obtained CT peak positions consistent for all blend compositions are around CTELA corresponds to the amorphous/amorphous or disorder–disorder heterojunction CT1EL  relates to the semicrystalline/semicrystalline or order–order heterojunction (CT4EL). In order to assign the peaks with intermediate energies (CTBEL and CTCEL), their occurrence with respect to the relative amount of semicrystalline polymer or PCBM domains is crucial. For that the directly from the fitting procedure resulting integrated normalized relative electroluminescence contributions of the CT transitions were analyzed, as shown in Figure 2EL transition is only probable under presence of semicrystalline polymer,CEL peak, significantly present in blend ratios containing less than 50% of the amorphous polymer (AnE‐PVba), corresponds to it. Thus the remaining CTBEL transition can be assigned to the transition between aggregated PCBM and amorphous polymer (AnE‐PVba), i.e., CT3EL. It is interesting to mention that the CTAEL = CT1EL transition, taking place for intimately mixed PCBM molecules with amorphous polymer in the expected range of high amount of amorphous polymer  with PCBM.1PL to a transition between the amorphous PCBM and the amorphous polymer molecularly intermixed with PCBM ,3EL transition corresponds to a transition from aggregated PCBM to the polymer in the intercalated phase. We conclude that the dominance of the CT3EL peak correlates well with the fact that most of the volume inside the donor–acceptor mixture consists of amorphous polymer intercalated by PCBM adjacent to PCBM aggregates. Note that in GiWAXS spectra, obtained on the same blends earlier, indeed the occurrence of a broad peak was detected for higher amorphous polymers fractions, which had been assigned to a specific molecularly intermixed, intercalated polymer:fullerene phase.The difference in transition energies between CTTo further reason that in electroluminescence the recombination takes place between the neat amorphous polymer phase and either isolated or aggregated PCBM, we argue that charge injection from the electrodes as well as charge transport will be much more efficient within the neat instead the intercalated amorphous polymer. Furthermore, due to the energy offset, it will be highly improbable that holes transported within the neat amorphous polymer will overcome the energy barrier to access the intercalated amorphous polymer, whereas holes transported within the intercalated phase most probably will relax to the neat amorphous polymer phase. It has been recently shown by Gelinas et al. that charge generation can be promoted over relatively large separations, i.e., upon charge transfer, electron and hole can be separated by a few nanometers directly and with no measureable time delay into delocalized charge‐separated (CS) states within crystalline domains.4 transition involving the two semicrystalline phases of polymer and fullerene, which could be separated in space due to the inevitable existence of an amorphous polymer halo around any polymer crystallite,4 transition energy and the energetic separation of free and delocalized charge carriers in this case. In other words, we consider the CT4 transition to occur directly via tunneling or resonant coupling between the delocalized CS states in the semicrystalline regions. As a consequence, the CT4 transition should not include lowering of the energy separation between electron and hole due to Coulomb relaxation.As a consequence, specifically for the CTE is to be added to any CT‐transition energy.4 transition. For example, the PCBM energy level split between the amorphous and the crystalline phase may be derived by subtraction of CT1PL/EL − CT3PL/EL yielding on average about 134 meV. In the same way the energy split between neat amorphous polymer and PCBM intercalated amorphous polymer with respect to the crystalline polymer phase yield 205 and 274 meV. These values serve as a lower limit, as any additional effects due to reorganization energy or Coulomb binding energy with respect to free charge carriers within the molecular energy levels are being neglected so far. Since both, the reorganization energy as well as the Coulomb binding energy should yield larger effects, the higher the disorder, we may indeed take the differences from CT‐transition energies as lower limit for the energy split due to disorder effects.In principle, the remaining three CT‐transition energies are not identical but correlated with the transport levels of free charge carriers. In order to yield the CS energy, in general an individual energy offset of ΔEa‐c between the amorphous HOMO/LUMO and the crystalline HOMO/LUMO levels are summarized in TableThe resulting lower limit for energy level offsets ΔFigureThis reconstruction of the interfacial energy diagram of the organic multiheterojunction is depicted in αβ) fraction. Furthermore, even though it is not very probable to observe a direct interface between crystalline donor and acceptor in case of polymer‐based organic bulk heterojunctions, a charge transfer may possibly traverse (tunnel through) a thin interfacial layer of amorphously intermixed materials. Hence, the revisited model for the due to order varied interface between donor polymer and acceptor fullerene has to include in total six different CT‐transition energies, arising from contacts between all possible domains that can be formed within a bulk heterojunction. The corresponding sketch of involved domains and interfaces is depicted in FigureIn fact, it has been shown that the initially presented hypothesis is insufficient to describe the full complexity of the organic multiheterojunction. Indeed it is required to differentiate between a neat amorphous polymer (α) and a fullerene intercalated or intimately mixed amorphous polymer  and disorder (amorphous) molecular energy levels located at the heterojunction, could be directly probed spectroscopically. In fact, several other methods  are currently applied to shed light into the question on how to quantify the energetic changes that occur due to changes in phase order within bulk heterojunctions.3In summary, we have demonstrated the existence of several CT transitions within one single organic semiconductor bulk heterojunction. By systematic variation of the order within the conjugated polymer phase through application of mixtures of semicrystalline and amorphous representatives of one and the same polymer backbone within ternary blends with the fullerene derivative PCBM, the domain order at the heterojunction interface could be tuned from ordered–ordered, over ordered–disordered, up to disordered–disordered. In direct relation with the respective interfacial area, the strength of the corresponding CT transition varied systematically and could be therefore unambiguously assigned via electroluminescence characterization. Via photoluminescence also fullerene‐intercalated amorphous polymer phases could be detected. Thus it could be shown that the CT transition can indeed be used to probe the interfacial order at the heterojunction, allowing deep insights into the internal structure of the bulk heterojunction by simple means.Finally, the information of the different CT transition peaks could be transferred into knowledge about lower limits for disorder‐induced molecular energy level changes, such as the HOMO and LUMO: due to the unambiguous assignment of certain CT transitions to specific interfaces, lower limits for the energy relaxation due to order, or in other words, a lower limit for the difference in HOMO and LUMO levels between certain amorphous and semicrystalline phase domains could be derived.In conclusion, sensitive luminescence spectroscopy on a properly designed ternary bulk heterojunction, bearing polymers with high luminescence quantum yields and tunable order, revealed both: the existence of six different CT energy levels depending on the domain order at the heterojunction as well as quantitative information about energy relaxation within organic semiconductors due to the effect of ordering.4ab and amorphous AnE‐PVba with concentrations running from 0% to 100% AnE‐PVba in steps of 10%. The global polymer:PCBM weight ratio was held constant at 2:3. The synthesis of the polymers is described elsewhere.2. Thin film steady‐state PL spectra and solar cell EL spectra were recorded with an Avantes AvaSpec ULS‐2048 fiber spectrometer. PL excitation was applied with a laser diode emitting at 405 nm. EL was conducted at an injection current of 100 mA applied with a Keithley 2601 Source Measure Unit. For PL normalization and evaluation of optical band‐gap, thin film transmission, and reflection spectra were recorded with a Varian Cary 5000 spectrophotometer under VW conditionTernary polymer:polymer:PCBM blends were prepared from mixtures of semicrystalline AnE‐PVAs a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


HIV‐positive individuals have led to the identification of scores, if not hundreds, of antibodies that are both broadly reactive and potently neutralizing. This development has provided renewed impetus toward an HIV vaccine and led directly to the development of novel immunogens. Advances in identification of donors with the most potent and broad anti‐HIV serum neutralizing responses were crucial in this effort. Equally, development of methods for the rapid generation of human antibodies from these donors was pivotal. Primarily these methods comprise single B‐cell culture coupled to high‐throughput neutralization screening and flow cytometry‐based sorting of single B cells using HIV envelope protein baits. In this review, the advantages and disadvantages of these methodologies are discussed in the context of the specificities targeted by individual antibodies and the need for further improvements to evaluate HIV vaccine candidates.Beginning in 2009, studies of the humoral responses of  Immunological Reviews.This article is part of a series of reviews covering B cells and Immunity to HIV appearing in Volume 275 of 1Natural immunity to many viral diseases relies upon circulating neutralizing antibodies from long‐lived plasma cells in the bone marrow or the production of neutralizing antibodies from memory B cells after re‐activation by the infecting pathogen, frequently years after the original exposure. Successful vaccines such as that for smallpox present a non‐pathogenic form of the infectious agent and induce a similar natural immunity. For HIV, however, natural immunity appears ineffective. Thus, for example, superinfection occurs unhindered by HIV envelope protein (Env)‐specific antibodies,The first HIV bnAbs were isolated by our laboratory using phage displayA second major factor in generating new bnAbs was the development of single B‐cell approaches for the isolation of human antibodies250 value of more than 1:300 across a minimum of four different clades.An important step in the identification of HIV bnAbs was the ability to study large cohorts and identify those with potent and broad serum neutralizing activity. This was first achieved by defining criteria to rank 1800 HIV‐positive serum samples from the IAVI Protocol G cohort for broad and potent activity against relatively neutralization‐resistant isolates to represent circulating viruses.In the first experiment, we screened more than 30 000 individual B cells from one donor for the ability to neutralize two HIV strains and also bind to recombinant gp120 and gp41 protein subunitsThis approach of high‐throughput neutralization screening of single B‐cell cultures was re‐employed to isolate bnAbs from multiple donors Table . These bHowever, simultaneous advances in our ability to discern the specificities that mediate elite neutralizationMany of the bnAbs identified by screening B‐cell cultures are trimer‐preferring or ‐specific, such as PG9 and PGT151, and as such, they may not be identified by antigen selection using molecules such as monomeric gp120  data generated from total RNA from PGT121/4 donor lymphocytes revealed an extensive family tree of possible PGT121/4 heavy‐ and light‐chain combinations with as little as 6% amino acid mutation but still notable neutralization breadth.3The ability to identify bnAbs over the last decade has dramatically increased our knowledge of the specificities underlying broad and potent neutralization of HIV.Thus, while the serum neutralizing specificity of new donors can often be identified, there may be cases where serum profiles appear similar to previously studied donors, but the nuances of a particular individual's bnAb response differ. In turn, this may mean that a typical isolation strategy could risk missing novel bnAbs that target known epitopes in different ways. This idea is suggested by the complexity with which existing bnAb families target their shared epitopes in subtly different ways. These differences are outlined below for the major classes identified to date, namely, those targeting the trimer apex, high‐mannose patch, CD4‐binding site, gp120‐gp41 interface, and MPER.The first class of bnAbs targeting a shared site, but with subtle differences in the exact epitope bound, is the trimer apex‐binding Abs Figure . The pioThe second class of bnAbs to consider is made up of those binding the high‐mannose patch on the gp120 subunit of the Env trimer (Figure The third class of bnAbs target the CD4‐binding site Figure  and haveThe fourth class of bnAb is a highly divergent set, derived from multiple donors by a variety of methods, but all members of the class target the gp120‐gp41 interface (Figure The fifth class of bnAbs comprise those which target the MPER Figure ; namely 4Given the great progress over the past decade in isolating bnAbs and studying their modes of action, an obvious question is do we still need more bnAbs? The more recent discovery and characterization of gp120‐gp41 interface bnAbs suggest it is still worthwhile to search for new bnAbs because they could reveal novel sites of vulnerability on Env. Furthermore, even if new bnAbs are only subtly different to those currently identified, defining these differences can substantially improve our mechanistic understanding of each bnAb class. This in turn will help us to understand how to induce such bnAbs by immunization and how to evaluate if any similar responses or precursors are stimulated by current immunogens.The advances in Ab isolation methods over the last decade have made a huge contribution to the discovery of such a large number of HIV bnAbs in a relatively short‐time period. Therefore, another important question is what is the best way to improve this technology in order to seek out new bnAbs and possibly novel epitopes? There are considerable advantages, in terms of cost and time, to reducing the number of B cells that are screened. Thus far, this has been achieved by pre‐enriching for Env‐specific B cells by single‐cell FACS. The development of native‐like stabilized trimers has greatly improved our ability to use this method to select for B cells that bind the functional Env trimers as compared with non‐functional forms of Env. However, even with stabilized near‐native trimers as FACS baits, non‐neutralizing binders and strain‐specific nAbs are captured.The further development of Ab isolation methods is not only important to help identify novel bnAbs but also to evaluate Abs induced by candidate immunogens. Until recently, very few immunization studies had induced even Tier‐2 autologous neutralization, let alone a broad response.Following the identification of PG9 and PG16 in 2009 and the many other bnAbs that came after, it has become possible to design immunogens based on our knowledge of these extraordinary antibodies. The hope is that by tailoring immunogens to elicit bnAbs rather than strain‐specific and non‐neutralizing Abs, it will be possible to elicit broad and potent immune responses.


HCV) elimination include a 90% reduction in new infections by 2030. Our objective is to review the modelling evidence and cost data surrounding feasibility of HCV elimination among people living with HIV (PLWH), and identify likely components for elimination. We also discuss the real‐world experience of HCV direct acting antiviral (DAA) scale‐up and elimination efforts in the Netherlands.The World Health Organization targets for hepatitis C virus (WHO HCV elimination targets among HIV‐infected (HIV+) people who inject drugs (PWID) and men who have sex with men (MSM), review cost‐effectiveness of HCV therapy among PLWH and discuss economic implications of elimination. We additionally use the real‐world experience of DAA scale‐up in the Netherlands to illustrate the promise and potential challenges of HCV elimination strategies in MSM. Finally, we summarize key components of the HCV elimination response among PWLH.We review modelling evidence of what intervention scale‐up is required to achieve HCV elimination among HIV+ MSM and PWID is potentially achievable but requires combination treatment and either harm reduction or behavioural risk reductions. Preliminary modelling indicates elimination among HIV+ PWID will require elimination efforts among PWID more broadly. Treatment for PLWH and high‐risk populations (PWID and MSM) is cost‐effective in high‐income countries, but costs of DAAs remain a barrier to scale‐up worldwide despite the potential low production price ($50 per 12 week course). In the Netherlands, universal DAA availability led to rapid uptake among HIV+ MSM in 2015/16, and a 50% reduction in acute HCV incidence among HIV+ MSM from 2014 to 2016 was observed. In addition to HCV treatment, elimination among PLWH globally also likely requires regular HCV testing, development of low‐cost accurate HCV diagnostics, reduced costs of DAA therapy, broad treatment access without restrictions, close monitoring for HCV reinfection and retreatment, and harm reduction and/or behavioural interventions.Modelling indicates WHO HCV Elimination targets is potentially achievable among HIV‐infected populations. Among HIV+ PWID, it likely requires HCV treatment scale‐up combined with harm reduction for both HIV+ and HIV‐ populations. Among HIV+ MSM, elimination likely requires both HCV treatment and behaviour risk reduction among the HIV+ MSM population, the latter of which to date has not been observed. Lower HCV diagnostic and treatment costs will be key to ensuring scale‐up of HCV testing and treatment without restriction, enabling elimination.Achieving  The advent of highly effective HCV direct acting antiviral (DAA) therapy, with sustained viral response (SVR) exceeding 90% in both HCV monoinfected and HIV/HCV coinfected populations Hence, to achieve the WHO incidence elimination targets, efforts must focus on both prevention of The WHO strategy does not include discussion relating to elimination of viral hepatitis among HIV‐infected populations specifically. Yet, because of the shared transmission routes many people living with HIV (PLWH) are coinfected with viral hepatitis. Globally, approximately 6.2% (3.4% to 11.9%) of PLWH are coinfected with HCV, equating to approximately 2.28 million (IQR 1.27 to 4.42) HIV/HCV coinfected individuals As such, to achieve the WHO HCV elimination incidence reduction target among PWLH it is crucial to tackle transmission among HIV‐infected MSM and PWID. However, although the prevention interventions required to eliminate HCV globally will be equally applicable to coinfected populations, the intervention level required and targeting may be different based on specific epidemic characteristics.The objective of this paper is to review the modelling and cost evidence surrounding feasibility of HCV elimination among HIV‐infected (HIV+) key populations such as MSM and PWID and identify the likely components required for HCV elimination among PLWH. We use the real world experience of HCV DAA scale‐up in the Netherlands through the Dutch Acute HCV in HIV Studies to illustrate the promise and potential challenges of HCV elimination strategies in a key population (MSM).2This analysis is comprised of four parts: (i) A review of the theoretical mathematical modelling literature examining what prevention and treatment scale‐up is required for HCV elimination among HIV‐infected PWID and HIV‐infected MSM populations. (ii) A review of the cost‐effectiveness of HCV treatment for HIV‐infected populations and discussion of cost considerations for elimination. (iii) A discussion of the real‐world experience of HCV DAA scale‐up among HIV+ MSM in the Netherlands (iv) A summary of probable and possible components of the HCV elimination response among PWLH.33.13.1.1Numerous burden of disease models have shown that existing or modestly increased levels of treatment targeted at individuals with more advanced liver disease can achieve the WHO HCV mortality target (65% reduction by 2030) in a variety of global settings. For example, a regional European Union model showed that HCV treatment only need to increase from 150,000 patients in 2015 to 187,000 patients in 2025 to achieve the mortality elimination target A wide body of literature since 2011 has utilized epidemic modelling to explore what level of prevention scale‐up could result in control and elimination among PWID, and whether HCV treatment could be used for prevention. Several initial modelling studies in the UK and general PWID populations of varying prevalences have indicated that harm reduction alone is unlikely to achieve HCV elimination among PWID populations In addition, modelling studies have pointed to several key additional elements which are required for achieving elimination among PWID: One Australian study highlighted the need for enhanced HCV screening among PWID in order to sustain HCV treatment rates required for elimination, a situation which is likely applicable to many global settings Despite several studies modelling HIV and HCV coinfection transmission among PWID 3.1.2An HCV epidemic among HIV‐infected MSM has been observed in the United States, Western Europe, Australia, Taiwan, Hong Kong and Japan, with HCV incidence and prevalence among HIV‐infected MSM substantially higher than the HIV‐uninfected MSM population As with PWID, modelling indicates the level of intervention required among HIV+ MSM to achieve the WHO elimination targets varies by epidemiological setting, particularly given substantial variation in incidence trends. For example, among HIV+ MSM incidence of HCV over the past decade has remained relatively stable in the UK and the Netherlands. In the UK, a modelling study indicated that scaled‐up rates of DAA therapy (from 46% to 80% treated within a year of diagnosis and from 7%/year to 20%/year thereafter) could reduce incidence among HIV+ MSM over 60% by 2030, but could not meet elimination targets In contrast, HCV incidence among HIV+ MSM has steadily increased over the past decade in Switzerland and Germany. In Switzerland, this has occurred alongside an increase in self‐reported risk behaviour . A recent modelling study in Switzerland projected that if these trends continue, elimination (or even reductions in HCV incidence) could not be achieved through HCV treatment alone, and requires additional reduction in high‐risk behaviour  Finally, preliminary modelling in the Netherlands indicated immediate treatment of all diagnosed HIV+ MSM with DAAs could only result in moderate reductions in HCV incidence among HIV+ MSM (approximately 30% within 15 years), but not reach WHO elimination targets 3.2There is a wide body of evidence that HCV treatment is cost‐effective for HIV‐infected populations, including HIV+ MSM with a risk of reinfection, Despite the evidence HCV treatment is cost‐effective, the high costs of HCV treatment Unfortunately, the high costs of HCV treatment have so far resulted in prioritization of HCV therapy (or restrictions on insurance reimbursement) even in developed countries However, although HCV therapy is likely cost‐effective, the high costs per treatment results in a substantial budgetary impact in countries with a large HCV infected populations. This has resulted in HCV treatment restrictions even in resource rich countries in the U.S. and Europe Promisingly, HCV DAA therapies could be produced as generics at a fraction of the current costs In addition, even the costs of HCV diagnosis and monitoring remain prohibitive in many developing countries Finally, in many settings additional financing will be required to build the capacity of health services for diagnosis and treatment of HCV. However, the economic implications of this health systems strengthening  has not been estimated. It is possible that integration of HCV testing and treatment within HIV services will prove to be an effective and cost‐effective approach 3.3In the Netherlands, surveillance data indicate that among PLWH, the vast majority of acute HCV infection diagnoses occur among MSM HIV and HCV care is well‐organized in the Netherlands. All patients diagnosed with HIV are cared for by a team of infectious diseases physicians and specially trained HIV nurses in 26 treatment centres spread across the country. Screening for chronic HCV is universal at entry into HIV care and more than 99% of the HIV‐infected patients in care in the Netherlands have been tested for HCV at least once In the Netherlands, DAAs were available from September 2014 for HCV‐infected patients with significant liver fibrosis or cirrhosis, and October 2015 regardless of fibrosis stage. This led to a very rapid uptake of HCV therapy among HIV+ MSM in the ATHENA cohort, with 79% attaining SVR just 14 months after restrictions were lifted prevalence of HCV can be achieved in a well‐organized healthcare system of a resource rich country. However, to achieve HCV elimination according to the WHO targets, the incidence of new HCV infections needs to decrease by 90% by 2030 The data described above demonstrate that a very rapid decline in the While the substantial reduction in HCV incidence among HIV+ MSM observed after widespread scale‐up of HCV treatment in the Netherlands is reason for optimism, an observational study cannot prove a causal relationship. Further modelling work will be required to disentangle the estimated impact of HCV treatment as prevention initiatives among HIV+ MSM in the Netherlands, and coverage required for elimination. We consider it unlikely that HCV treatment as prevention alone will result in a 90% reduction in the incidence of HCV (the WHO target) among HIV+ MSM in the Netherlands, and discuss additional steps likely required in the following section. Indeed, no further decline in the HCV incidence has been observed in 2017 so far 3.4probable and possible barriers exist which could hamper HCV elimination efforts among PLWH (Table probable barriers):Regular HCV testing of high‐risk populations, both HIV+ and HIV‐: Among HIV+ PWID, modelling indicates that elimination likely requires elimination efforts among the broader PWID population Development of low cost, simple, reliable and accurate HCV diagnostics. Even in low‐income countries with low cost DAAs, the price of HCV diagnostics remains a barrier Reduced costs for DAA treatment. Despite the availability of generic and low‐cost DAAs in some resource limited settings, costs of DAA therapy in the vast majority of countries remains a barrier to widespread scale‐up of HCV treatment Broad access to HCV treatment without restrictions: Modelling studies indicate that restricting treatment for those with more advanced fibrosis and/or by drug use status, as is occurring in many settings Close monitoring for reinfection and retreatment of reinfections: Treating those at risk of transmission, the target group for HCV treatment as prevention efforts, will result in reinfections. Among PWID, lower rates of reinfection compared to primary infection were reported in the IFN‐era Harm reduction and other behavioural interventions to prevent infection/reinfection: Despite the importance of harm reduction such as OST and needle and syringe programs for preventing HCV infection among PWID In summary, despite the promise of HCV elimination using HCV treatment as prevention from both theoretical modelling studies and real‐world observations in the Netherlands, numerous WH Table . As suchpossible barriers to elimination and therefore may be required for elimination among PWLH. We note that further evidence is required to support whether these items are necessary components of an elimination response:Testing among HIV‐negative MSM receiving HIV pre‐exposure prophylaxis (PrEP): There is growing evidence of HCV infection among HIV‐negative MSM receiving PrEP, a group which could contribute to HCV transmission to HIV+ MSM. In Amsterdam, the prevalence of chronic HCV among HIV‐negative MSM in a PrEP implementation programme was 4% (15/375), comparable to the prevalence of 4.2% in HIV+ MSM Coordinated multi‐country prevention and treatment effort: Among HIV+ PWID, the contribution of cross‐border transmission and import/export of infections to the epidemic is unclear. Among HIV+ MSM, the highly connected nature of the HCV epidemic among HIV+ MSM in Western Europe and frequent travel of MSM between European cities has been documented Licensing for treatment in the acute stage: Currently none of the approved DAA regimens are licensed for the treatment of acute HCV. Depending on the regional legislations, this may result in a compulsory “wait for documented chronicity” policy and thus increase the duration of time for onwards HCV transmissions to occur. HIV modelling studies have highlighted the importance of the acute HIV stage on the HIV epidemic Monitor if transmission of HCV clones with acquired DAA resistance occurs: It was recently shown that the HCV Q80K polymorphism, associated with DAA resistance, is frequently detected and transmitted among HIV+ MSM in the Netherlands In addition, the following items would address 4Both theoretical modelling studies and emerging real world evidence from the Netherlands indicate that HCV elimination among HIV‐infected key populations such as MSM and PWID is potentially achievable. Due to a number of factors, elimination of HCV among HIV+ MSM may be complex; and require both high coverage HCV treatment and behavioural interventions. Elimination among HIV+ PWID will likely require elimination efforts among PWID more broadly. In addition to HCV treatment as prevention initiatives, elimination among PLWH also likely requires regular HCV testing, development of low‐cost accurate HCV diagnostics, reduced costs of DAA therapy, broad treatment access without restrictions, close monitoring for HCV reinfection and retreatment, and harm reduction and/or behavioural interventions.Finally, we note that the vast majority of existing research is limited to high‐income settings, and more research is required relating to HCV elimination among PLWH in resource limited settings. For example, more modelling work is needed to assess what scale‐up is required for HCV elimination among PLWH populations in resource‐ limited settings where transmission routes may vary and the HCV epidemics among HIV‐infected populations may differ. Many developing countries have high burdens of injecting drug use as well as high HCV among HIV‐infected PWID


Eukaryotic gene expression is extensively controlled at the level of mRNA stability and the mechanisms underlying this regulation are markedly different from their archaeal and bacterial counterparts. We propose that two such mechanisms, nonsense‐mediated decay (NMD) and motif‐specific transcript destabilization by CCCH‐type zinc finger RNA‐binding proteins, originated as a part of cellular defense against RNA pathogens. These branches of the mRNA turnover pathway might have been used by primeval eukaryotes alongside RNA interference to distinguish their own messages from those of RNA viruses and retrotransposable elements. We further hypothesize that the subsequent advent of “professional” innate and adaptive immunity systems allowed NMD and the motif‐triggered mechanisms to be efficiently repurposed for regulation of endogenous cellular transcripts. This scenario explains the rapid emergence of archetypical mRNA destabilization pathways in eukaryotes and argues that other aspects of post‐transcriptional gene regulation in this lineage might have been derived through a similar exaptation route. A compelling example of this divergence is provided by mRNA destabilization mechanisms triggered by specific nucleotide and structural elements, base‐pairing between RNA molecules and unusual patterns of mRNA translation When discussing mRNA decay it is important to distinguish between the core machinery responsible for RNA degradation catalysis and controlled mechanisms that target specific subsets of mRNAs. In eukaryotes, the core machinery contains the Xrn family 5′–3′ exoribonucleases and the RNA exosome, a molecular complex combining 3′–5′ exoribonuclease and endoribonuclease activities Prokaryotes degrade their mRNAs using a distinct set of endo‐ and 5′–3′ exoriboucleases The difference between controlled mechanisms of mRNA decay in the two groups is arguably even more striking. In eukaryotes, the regulation is provided by small interfering (si), micro (mi) and Piwi‐interacting (pi) RNAs, nonsense‐mediated decay (NMD), and RNA‐binding protein‐based mechanisms targeting mRNAs with characteristic sequence or secondary structure elements for degradation Prokaryotic logic of controlled mRNA decay is fundamentally different Assuming that eukaryotes originated through symbiosis between archaea and bacteria and inherited a prokaryotic version of mRNA metabolism What could have triggered rapid rewiring of the mRNA metabolism in the newly established eukaryotic domain? Here we propose that at least some of the mechanisms regulating eukaryotic mRNA stability might have emerged as a part of cellular defense against RNA pathogens. According to this scenario, subsequent appearance of specialized innate and adaptive immunity systems allowed the host cells to repurpose, or “exapt” Viruses are exceptionally successful pathogens using their cellular hosts as a source of translation machinery, nucleotides and, occasionally, structural molecules and enzymes http://www.ictvonline.org/virusTaxonomy.asp; 2014 Release) lists 523 prokaryotic and 2666 eukaryotic viruses. Although not an exhaustive catalog of all known species, this sizeable sample suggests that RNA viruses, retroid viruses, and viroids account for ∼62% of eukaryotic viral species, whereas the corresponding figure for prokaryotes is only ∼1%. This striking over‐representation of eukaryotic RNA pathogens does not take into account eukaryotic retroelements, which appear more diverse than such elements in bacteria Although RNA‐dependent viruses have been isolated from both eukaryotic and prokaryotic hosts, an overwhelming majority of these pathogens infects eukaryotes Leviviridae and CystoviridaeEarlier phylogenetic analyses suggested that positive‐strand RNA viruses sharing picorna‐like genome architecture emerged through a “Big‐Bang” event preceding radiation of the major eukaryotic supergroups Eukaryotic retroid viruses and retrotransposable elements likely descended from prokaryotic retroelements Regardless of the exact evolutionary trajectories followed by specific groups of viruses and retroelements, it is fair to assume that eukaryotes have been exposed to an increasingly wide range of RNA‐based pathogens since their early days. If so, long‐term survival of the newly established eukaryotic lineage would have been impossible without adequate defense mechanisms.One such mechanism present in most extant eukaryotes and likely used by the LECA is RNA interference (RNAi) Metazoans use several additional levels of antiviral defense including the Piwi/piRNA system targeting retrotransposons, innate immunity based on recognition of pathogen‐associated molecular patterns (PAMPs) by host‐encoded pattern recognition receptors (PRRs), and immunoglobulin‐ and T‐cell receptor‐dependent adaptive immunity RNAi itself has important functions unrelated to immunity and might completely forego its antiviral responsibilities in many mammalian cells The animal and plant miRNA pathways appear to have evolved independently following radiation of the corresponding supergroups, Opisthokonta and Archaeplastida, from a common ancestor Below, we discuss the possibility that, similar to RNAi and the miRNA pathway, two well‐characterized eukaryotic systems regulating stability of cell‐encoded mRNAs, emerged as intrinsic defense mechanisms against RNA pathogens.NMD is a conserved eukaryotic mechanism destabilizing mRNAs with unusually positioned translation stop codons As discussed in several recent reviews Saccharomyces cerevisiae.mRNAs are typically recognized as NMD targets in the cytoplasm during initial rounds of translation. The presence of exon‐exon junctions >50–55 nt downstream of the termination codon, upstream ORFs in the 5′UTR, or an excessively long 3′UTR increases the likelihood of an mRNA to undergo NMD. Mechanistic details of this pathway differ depending on the species, and a single organism may utilize more than one distinct branch of NMD Fig. . For exastramenopiles, alveolates, Rhizaria) Notwithstanding this functional diversity, genes encoding NMD and EJC components are conserved across metazoans, fungi, plants, and members of the unicellular supergroups: Amebozoa, Excavata, and SAR  inhibited replication of +RNA viruses from the families of Alphaflexiviridae and Tombusviridae. Similar to Togaviridae, these viruses use a subgenomic strategy to express a full complement of their proteins. This gives rise to unusually long 3′UTRs in some of the virus‐encoded RNAs. The authors showed that reducing the length of the 3′UTR in the corresponding RNA species rescued them from the inhibitory effect of NMD. Moreover, a +RNA virus from the Potiviridae family containing a single ORF and a relatively short 3′UTR escaped NMD restriction Antiviral effects of NMD have been also shown in plants Smg7 and the Upf1 genes lead to retarded development and seedling death as a result of elevated expression of antibacterial defense genes A study by Gloggnitzer et al. points at a wider role of NMD in the innate immunity in plants Upf1 has been additionally identified as a cell‐encoded protein interacting with bicistronic RNA of the human non‐LTR retroelement LINE‐1 Of note, RBPs from the Staufen family can induce an NMD‐related process that destabilizes a subset of mammalian transcripts in a Upf1‐ and translation‐dependent manner Retroviridae family, human T‐lymphotropic virus type 1, inhibits the NMD machinery in part through interaction between Upf1 and the virus‐encoded Tax protein Viruses often evolve mechanisms allowing them to evade or disrupt host defenses and this trend is certainly apparent in the case of NMD. For example, Rous sarcoma retrovirus containing several ORFs in its genomic RNA contains a specialized stability element downstream of the first ORF encoding Gag and Pol proteins Flaviviridae family member, interferes with NMD by sequestering an EJC‐associated factor, PYM1/WIBG Similarly, coat protein of hepatitis C virus (HCV), a Another characteristic form of controlled mRNA decay in eukaryotes relies on recognition of specific sequence motifs and structural elements present in a subset of cellular transcripts. RBPs containing CCCH‐type zinc fingers (ZFs) have been widely implicated in this regulation One of the most extensively studied members of this protein group is mammalian Zfp36 also known as tristetraprolin (TTP) TTP has been shown to regulate the stability of several mammalian mRNAs including those encoding proto‐oncogenes, growth factors, and cytokines (including some interferons) Conversely, basal levels of TTP can transiently increase in response to toll‐like receptor (TLR) and cytokine signaling Drosophila homolog of TTP called dTIS11 has been shown to destabilize ARE‐containing targets including mRNA of the antimicrobial peptide cecropin A1 ArabidopsisProteins containing the TTP‐like TZF domain have been identified in metazoa, fungi, plants, Amebozoa, and Excavata In line with this model, a large fraction of human ARE sequences is associated with Alu elements, an abundant SINE group Liang et al. provide an important insight into the CCCH ZF protein evolution The Zc3h12 ZFs are especially closely related to the TTP ZFs see Fig. . The besZc3h12a/Regnase or Rc3h1/Roquin1 genes are somewhat evocative of the effects brought about by the Zfp36/TTP knockout Regnase recognizes secondary structure elements containing a stem and a trinucleotide loop and its PIN domain participates in RNA binding along with the ZF Flaviviridae , Picornaviridae  and Togaviridae (Sindbis virus). It may also interfere with replication of some −RNA viruses  and reverse‐transcribing lentiviruses  Flaviviridae, the ZF domain was also shown to stimulate binding of Regnase to viral RNAs Importantly, several recent studies suggest that, in addition to its role in cellular mRNA metabolism, Regnase can restrict replication of +RNA viruses from the families of Although cis‐elements mediating antiviral effects of Regnase are presently unknown, efficient replication of RNA viruses often requires conserved RNA structures that might, at least in theory, function as a Regnase “specificity code.” Interestingly, repression of viral replication by Regnase depends on the ability of this protein to form oligomers Iridoviridae family and +RNA viruses from the order of Nidovirales (mentioned in the previous chapter as viruses with a Upf1‐like RNA helicase) encode TZF‐domain proteins of unknown function , combining it with other RNA interaction domains (as in Regnase and Roquins) or forming quaternary protein units (as in Regnase and ZAP) might have extended versatility of this system allowing it to recognize a wide range of linear and folded RNA epitopes. Interestingly, no prokaryotic proteins containing more than one CCCH ZF have been identified so far  should be addressed to the authors.Table S1. Viral TZF‐containing proteins showing detectable sequence similarity to human TTP/ZFP36 Click here for additional data file.


NIPD) for sickle‐cell disorder (SCD) is moving closer to implementation and studies considering stakeholder preferences are required to underpin strategies for offering NIPD in clinical practice.Non‐invasive prenatal diagnosis  were recruited from haemoglobinopathy clinics at two maternity units. Health professionals, predominately midwives, who offer antenatal care (N=62) were recruited from one maternity unit. No miscarriage risk was a key driver of decision making for both service users and providers. Service providers placed greater emphasis on accuracy than service users. Current uptake of invasive tests was 63%, whilst predicted uptake of NIPD was 93.8%. Many service users (55.4%) and providers (52.5%) think pressure to have prenatal testing will increase when NIPD for SCD becomes available.Adults who were carriers of NIPD is welcomed by parents and uptake is likely to be high. To promote informed choice, pretest counselling should be balanced and not exclusively focused on test safety. Counselling strategies that are sensitive to feelings of pressure to test will be essential.There are clear differences between service users and health professionals’ preferences for prenatal tests for sickle‐cell disorder. The safety of  Recent improvements in life expectancy for people affected with SCD have been attributed to earlier detection and improvements in comprehensive management.SCD is caused by mutations in the β‐globin gene. Inheritance is autosomal recessive, so both parents must be carriers of a mutation for the baby to be at risk of SCD. In the UK, there is a universal neonatal screening programme for SCD and carrier screening is offered through an antenatal haemoglobinopathy screening programme that aims to offer screening by 10 weeks in pregnancy.Service users and health professionals will differ in their preferences for the three attributes of prenatal tests to be examined: safety, accuracy and time of testing;Service users will value the safety afforded by NIPD and hypothetical uptake of the test will be high.It is important that research exploring views of NIPD is carried out alongside the technical development of these new tests to ensure that social and ethical issues are addressed and stakeholder needs are met. Previous research exploring views of NIPD for single gene disorders, including SCD, has been undertaken using qualitative interviews and focus groups with carriers of single gene disorders and health professionals.22.1Ethical approval was obtained from a National Research Ethics Service Committee (10/H0714/3).2.2Two groups of participants were recruited: (i) service users: adults who were either carriers of SCD or affected with the condition, aged 18 or over and attending haemoglobinopathy antenatal clinics at either St Thomas's Hospital or University College London Hospital, and (ii) service providers: health professionals who deliver antenatal care and see women with pregnancies at risk of SCD at St Thomas’ Hospital.Convenience sampling was used to participants to the study. For the service users, group potential participants were invited to anonymously complete the questionnaire whilst waiting for their clinical appointment. For the service provider group, potential participants were approached in person in their workplace and invited to anonymously complete a hard copy of the questionnaire.2.3The questionnaire had three components: (i) DCE choice sets, (ii) structured questions about prenatal testing and NIPD and (iii) demographic questions. Questionnaire design has been described previously, as the questionnaire used here was a modified version of the questionnaire used in our previous study looking at prenatal testing for cystic fibrosis.2.4DCE analysis followed published guidance.33.1Questionnaires were completed by 78 service users and 62 health professionals. Questionnaires were excluded if the internal consistency question was incorrect  or if the choice set was incomplete . Ultimately, 67 service user and 62 health professional questionnaires were included in the analysis. The service user group included carriers of SCD (88.2%) and people affected with the condition (18.8%). The majority were female (89.4%). A small proportion had a child with SCD (16.4%). Health professionals were predominately midwives (88.1%) and female (91.8%). Demographic information for service users and providers is summarized in Tables 3.2Service users and providers prefer a test with greater accuracy, early testing and no risk of miscarriage Table . The dir3.3The MRS demonstrates the strong preference held by both service users and health professionals for a test with no risk of miscarriage. Notably, service users were prepared to wait longer and accept lower accuracy compared to service providers for a test that had no risk of miscarriage Table .3.4Participants ranked the importance of five attributes of prenatal tests: safety, early testing, accuracy, financial cost and comprehensive information . Safety 3.5When service users were asked whether they would have invasive testing for SCD, 63.1% said they have had or are likely to have invasive testing, with the most common reasons for testing being to “help make a decision about whether or not to continue the pregnancy” (47.4%) and “to plan and prepare for the possible birth of a baby with SCD” (36.8%) Table . More thAll participants were asked their views on pressure to have prenatal testing for SCD and whether they felt that offering NIPD would increase feelings of pressure to have a diagnostic test Table . ApproxiForty‐three (64.2%) service users responded to the question on what they saw as the benefits of NIPD. These primarily fell into three categories: no miscarriage risk (n=21); decision making, knowledge and preparation (n=17); and simpler and less stressful testing (n=6). Fifty‐nine (95.2%) health professionals described potential benefits of NIPD, with the majority mentioning decision making, knowledge and preparation (n=46); no miscarriage risk (n=15); and early diagnosis (n=11). Forty‐five service users reported that they had no concerns (67.2%) and five said they had concerns but did not elaborate. Twenty‐nine (46.8%) health professionals reported that they had no concerns. Twenty‐six (41.9%) health professionals described concerns, with the most common being increased pressure to test and terminate (n=11), availability of appropriate counselling (n=7), accuracy (n=6) and costs (n=4).To address the question of whether health professionals thought NIPD should only be offered if it would change pregnancy management, a question was included asking whether NIPD should only be offered to women considering termination of pregnancy. Only four (6.6%) strongly agreed or agreed compared to 57 (93.4%) who strongly disagreed or disagreed.4The aim of this study was to determine the preferences held by potential service users and providers for prenatal tests for SCD. Test safety was an important driver of decision making for both groups. Differences were evident in the emphasis placed on test accuracy, with service providers placing greater emphasis on this attribute than service users. It was clear that service users were prepared to wait longer and accept lower accuracy than service providers for a test with no risk of miscarriage. In addition, over half of the service users chose tests based only on test safety and did not consider the other attributes. Differences between health professionals and potential service users have been seen in other DCE studies comparing non‐invasive and invasive tests for cystic fibrosisPrevious research looking at NIPD for single gene disorders found that both potential service users and providers thought that the ease and safety of NIPD may put couples under increased pressure to have prenatal testing.We found that willingness to have NIPD was high, with over 90% of service users saying they would have NIPD for SCD if it was available compared to 63% who said they would currently choose invasive testing. In addition, approximately 50% reported that risk of miscarriage was a barrier to invasive prenatal testing. These findings are similar to those of other studies exploring the potential uptake of NIPD for single gene disorders.The changes to the population accepting the offer of prenatal testing need to be considered in our approaches to counselling for NIPD for SCD. Most notably, many couples who would not previously have had prenatal testing may take up NIPD, and as a result may find themselves faced with a decision about termination of pregnancy. This issue is particularly important in the setting of prenatal testing for SCD where in the UK and other countries a prenatal carrier screening programme is in place and there are existing concerns about informed consent processesThe cost of NIPD for SCD will need to be considered in strategies to implement this test in the NHS. The potential cost of NIPD relative to the current invasive testing pathway has been explored and NIPD for SCD using current approaches was estimated to cost £1210, which was £190 more than invasive testing. Moreover, the anticipated high uptake of NIPD we found in this study (approximately 95% compared to 65% for invasive testing) would result in an incremental cost of NIPD over invasive testing of £48,635 per 100 pregnancies at risk of SCD. As the increased uptake will include parents who would want to have NIPD for information only and would not consider termination of pregnancy, there is a need to address the issue of whether it is appropriate to direct resources to test when pregnancy management would not change. Consideration of this question must include the benefits of the information for early reassurance or for planning and preparation if the baby is found to be affected by SCD.4.1Several limitations of our study may mean that our findings are not widely generalizable. Recruitment was only conducted at two centres for service users and only one centre for service providers and both centres were located in central London. As this was not a random sample and convenience sampling was used, it is possible that sampling bias will limit the generalizability of our results. In addition, participants were self‐selected and there may be responder bias towards people with strong pre‐existing views on NIPD. Another limitation of the study was that the numbers of participants recruited were not sufficient to allow subgroup comparisons. In an equivalent study looking at NIPD for cystic fibrosis, we found there were differences in preferences between people affected with the condition and those who were carriers.5When making decisions about prenatal testing for SCD, potential service users and providers do not place the same emphasis on the test attributes. It is likely that the safety of NIPD will be welcomed by parents and uptake will be high. It is therefore important that pretest counselling is balanced and not predominately focused on the safety of NIPD. Care must also be taken to minimize feelings of pressure to have NIPD. Considerations for implementation need to include current carrier screening and prenatal diagnosis pathways. Offering NIPD as a next step in the current pathway could create pressure to test at a time when news of carrier status is still being processed and decisions need to be made quickly about next steps. Thorough pre‐ and post‐test counselling will be essential and NIPD should be offered by health professionals specifically trained in counselling for prenatal testing for SCD.The authors declare no conflict of interest. Click here for additional data file.


Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:1573–1588, 2017.Systems orientated research offers the possibility of identifying novel therapeutic targets and relevant diagnostic markers for complex diseases such as osteoarthritis. This review demonstrates that the osteoarthritis research community has been slow to incorporate systems orientated approaches into research studies, although a number of key studies reveal novel insights into the regulatory mechanisms that contribute both to joint tissue homeostasis and its dysfunction. The review introduces both top‐down and bottom‐up approaches employed in the study of osteoarthritis. A holistic and multiscale approach, where clinical measurements may predict dysregulation and progression of joint degeneration, should be a key objective in future research. The review concludes with suggestions for further research and emerging trends not least of which is the coupled development of diagnostic tests and therapeutics as part of a concerted effort by the osteoarthritis research community to meet clinical needs. © 2017 The Authors.  Osteoarthritis (OA) has been recognized in the earliest forms of man, and throughout animal species, located wherever there is a diarthrodial articular surface. Yet, as we approach 275 years since William Hunter's description of ulcerated cartilage as “a very troublesome disease”A biological system is a set of elements  with multiple and diverse functions; these elements interact in a specific and non‐linear manner to produce coherent behaviors over time. Evolution has defined specialized interactions creating functional systems and sub‐systems at the cell, tissue, organ, organismal, and population/ecological levels.Fundamental definitions and frameworks for a systems approach have been well‐describedpre‐osteoarthritis as the homeostatic mechanism becomes dysregulated. The early consideration of the abnormal characteristics of these components, and inclusion of known prior risk factors promoting a propensity to OA, would be useful in determining at risk groups. When considered in this way it is clear that the historical focus on the end‐stage OA phenotype has distracted from recognising the relationship between all factors. Despite understanding that the inciting factors are likely to be heterogeneous we still recognize similar disease phenotypes; this suggests that at least some common elements of the system are likely to be dysregulated at some stage.OA is the most prevalent chronic joint disease and the most common co‐morbidity of the ageing population. Incidence increases with age and is also associated with other predisposing factors such as obesity and joint trauma.In the interest of brevity notable systems orientated studies in OA will be considered generally as “top‐down”  and “bottom‐up” . The studies are chosen as examples of the research objectives associated with a systems orientated approach to OA.Tissue engineering and regenerative therapies are a major focus of attempts to modify the progression of OA.Interaction networks may be generated from the elements of a biological system; abstractions of these networks can facilitate an understanding of the architecture, activity, and key players in that system. Much like a spider's web a perturbation in one part of a network will be propagated throughout. Network medicine postulates a “disease module” hypothesis, where disease‐associated genes or proteins are likely share the same topological neighborhood in a network. Defining communities of network elements  is a useful way to identify elements that have a close relationship, shared functionality, or disease association. A systems biology approach to comprehending OA is founded on the hypothesis that OA is a multi‐system disorder resulting from the dysfunction of a number of networks that, together, alter the homeostatic balance of the joint. Therefore, comprehensive and multisystem approaches are necessary to understand the complexity of OA and direct the development of innovative treatment strategies. To date most studies pertaining to using a systems approach in OA research are principally based on interrogation of a single ‘omics survey in a single tissue at a single time point. A reference set for transcriptomic and proteomic studies is provided in Table Network‐based approaches make use of known or inferred functional and physical interactions between the elements of a system or can be developed from statistical associations  a priori giving a high‐level understanding of the organization of the system.Wnt‐signaling, apoptosis, matrix organization, and mitotic cell cycle.Protein–protein interactions represent compound data arising from many cell types and biological contexts so may not be indicative of the biological system under investigation and so generic networks without biological specificity may arise. Soul et al.Other network approaches have included the use of Boolean dynamics to consider the coupled sequential reactions  between elements of a pathway to define a mechanistic network that was predictive of the response of primary chondrocytes to different ligands, including those associated with OA pathophysiology.Sox9) or hypertrophic (Runx2) phenotype. There is currently a dearth of validated dynamic, mechanistic models and a critical need to link these “bottom up” studies to the network models generated by “top‐down” approaches.The limitation of many network approaches is that they require mapping of expression data onto pre‐existing protein interactions and so rely heavily on prior knowledge of signalling and metabolic pathways. Furthermore, statistical associations are made with respect to end‐stage disease phenotypes, rather than having pre‐osteoarthritis as the focus of investigations. Critically, network approaches cannot capture spatiotemporal, dynamic changes in the system. Generally, network approaches in OA have been useful in identifying novel targets and sub‐networks, however, further mechanistic evaluation, perturbation, simulation, or validation of the proposed sub‐networks are performed infrequently. Complex disease phenotypes change with time and are subject to biochemical and biophysical fluxes. Often, the signals of interest may be spatially constrained, or example cell‐matrix interface. Network models cannot capture this and so require to be coupled to dynamic models to provide a description of how a system progresses both in space and time. Only a few studies have considered this for chondrocytes or with respect to OA. Using observed immunohistochemical changes in cartilage from ageing mice and candidate proteins associated with cartilage destruction and ageing Hui et al., developed an integrated computational model accounting for progressive collagen loss and increased MMP13 production.metabolic flux and flux‐balance analysis (FBA). There are few examples in the literature relating to constraint‐based approaches to modelling metabolic fluxes in joint tissues and no large‐scale FBA simulations, including gene‐knockout simulations, have been carried out for OA associated tissues. In Salinas et al.,An understanding of the metabolic derangements associated with the joint tissues contributing to OA, and their molecular context, would be invaluable to defining pathogenic pathways especially given the evidence of whole‐body metabolism effects on OA risk.Unlike constraint‐based metabolic flux analyses of micro‐organisms a metabolic understanding of OA requires the construction and coupling of metabolic networks for multiple tissues from the same organism. Common interactions may be defined by metabolites that are secreted or consumed between tissues, but defining these elements, the post‐transcriptional modifications that govern tissue‐specific metabolic activity profiles,18F‐fluoride PET (positive‐emission tomography) uptake with stress distributions in the subchondral bone of coxo‐femoral joints from patients. These few examples suggest that there is still considerable work required to link clinical or functional measurements with in silico models for a number of OA‐associated tissues. There are efforts to develop standardized, open‐source finite element joint models,Physiological models derived from advanced imaging techniques (computed tomography (CT), micro‐CT, magnetic resonance imaging (MRI), and in vitro techniques, e.g., quantitative microscopy) may be used to simulate musculoskeletal systems and are useful approaches to developing a systems understanding of OA. The data are derived directly from the applicable study group, physiological conditions may be applied in a repeatable manner, and temporal changes may be simulated. Predictions of the material properties of the constituent tissues may be made that could not otherwise be easily measured experimentally; multiple tissues, or specific tissue elements, may be considered in their physiologically relevant setting. The approach is non‐destructive and tissue‐failure conditions may be estimated in a non‐invasive manner. The temporal impact of pathology or treatment can be simulated in the model. Overall, these approaches are cheaper, faster, and knowledge‐driven compared to in vivo models. The integration of high‐resolution geometry available from advanced imaging techniques and constraints defined by biomechanical data may be used to develop finite element simulations of the joint tissues. Although these imaging techniques have been more widely applied to muscle and bone this modelling approach is uncommon for cartilage and sub‐chondral bone in the context of OA although some reports are found in the literature. For example, using high‐resolution micro‐CT of the mouse tibia it has been possible to estimate the mechanical characteristics of the femoro‐tibial joint DMM surgery using finite element analysis (FEA).The purpose of multiscale models is to develop early patient‐orientated intervention packages based upon a realization of trauma risk, the predicted performance of an intervention, and the prognostic capacity of biomarkers or clinical measurements as proxies for cell‐level responses.Mechanotransduction is the transfer of biomechanical forces into intracellular chemical or electrical signals and many diseases are associated with dysregulation of this activity.2+ responsive osmomechano‐TRP channel TRPV4 was found to be critical to transduction of mechanical and osmotic signalsTrpv4 revealed a reduction in age‐associated OA at 12 months, but not in a DMM model.Trpv4 knockout. Defining differential mechanotransduction pathways for age‐ and trauma‐associated OA could establish therapeutic targets. Modifications to a known small molecule TRPV4‐antagonist has shown analgesic and anti‐inflammatory properties that could have potential in a number of conditions including osteoarthritis.Work by Guilak et al. has considered numerous modelling strategies, including FEA, to deduce mechanical responses in chondrocytes and associated peri‐cellular matrix, which helped define the complex mechanical environment consisting of changes in tension, fluid pressure/volume, shear, etc.Caenorhabditis elegans) to reduce the complexity of the systems under investigation. This has not been possible with OA given the particular complexity of the mammalian skeleton. However, recently some advances have been made in developing the zebrafish as a model of cartilage dysregulation.As discussed earlier there are limits to the application of in vitro models of OA tissue derangement. Physiology‐based models allow perturbations to be integrated into a physiological environment so it is relevant to the scope of this review. Animal models of complex disease can facilitate a deeper understanding of the natural history of the pathology by providing controlled representations of subsets of human disease, however, there is no single standardized in vivo model and models that better represent the dynamics of human OA are required.Using developmental stages is often useful in systems orientated studies as they are conceptually simpler and easier to visualize. Depending on the model, spatiotemporal changes in expression profiles can be followed and contribute parameters to dynamic in silico simulations. Chondrogenesis, endochondral ossification, and OA pathways share regulatorsWe have highlighted the inherent complexity that researchers face in trying to answer the many unresolved questions in OA pathogenesis; this complexity it also demonstrable at the clinical level, not least given the multiple co‐morbidities that may be present in clinical presentations of OA. There are systems orientated approaches that may be applied to integrate mixed predictors  of risk. Decision trees are one form of machine‐learning (ML) classification tools that may be applied to systems biology problems including clinical decision‐making for complex conditions.Runx1 mRNA,Runx1‐mediated chondroprotective properties, including kartogeninStandard treatments in OA have broadly consisted of physical interventions and behavioral modifications , pharmacological, and surgical interventions. The limitations of traditional pharmacological approaches to the symptomatic treatment of OA arise from their equivocal efficacy and/or unacceptable side‐effects. A number of next‐generation therapeutics are in clinical trial, though few have been developed to a point where regulatory approval has been granted.pre‐osteoarthritis.Arhgdib was found to discriminate between healthy and diseased cartilage derived from the RAAK dataset.High‐throughput screening has become possible with ‘omics technologies to define prognostic markers for OA (reviewed hereSystems biology requires considerable resources and high returns are expected. Critical appraisal of the capacity of systems biology to meet its aims in the context of OA research is required. In systems approaches where many thousands of predictions are left unverifiedpre‐osteoarthritis, is critical. Clinical measurements need to be coupled to predictive models of cellular response to help direct rational intervention programmes for patients at high risk. The advent of mobile health and wearable technologies, and an understanding of social network trends on health, will facilitate collection of clinical and mechanical meta‐data to incorporate into patient‐specific models. Systems pharmacology approaches recognize that single therapeutic interventions for complex diseases are unlikely to be efficacious and insufficiently tailored to patients. RNA therapeutics will emerge as an important tool in network medicine and have the potential to promote personalized interventions in osteoarthritis. Ultimately, there is still much that is unclear about the mechanisms regulating the homeostatic system that still requires resolution before relevant multiscale models may be employed.In the course of the review we describe a number of approaches used by colleagues to gain a systems understanding of basic biology and OA development, but the functional output and clinical impact—changing research and clinical practice, reliable diagnostics, and disease‐modifying therapeutics—arising from these studies is not apparent at this time. The promise of systems approaches has been heralded for the last two decades as a source of new therapeutics and robust diagnostics.AJM, MJP, CP, and PDC all revised and contributed to the article. All authors approved the final submitted manuscript.


Geoengineering has been proposed to stabilize global temperature, but its impacts on crop production and stability are not fully understood. A few case studies suggest that certain crops are likely to benefit from solar dimming geoengineering, yet we show that geoengineering is projected to have detrimental effects for groundnut. Using an ensemble of crop‐climate model simulations, we illustrate that groundnut yields in India undergo a statistically significant decrease of up to 20% as a result of solar dimming geoengineering relative to RCP4.5. It is somewhat reassuring, however, to find that after a sustained period of 50 years of geoengineering crop yields return to the nongeoengineered values within a few years once the intervention is ceased. We simulated a negative impact on groundnut yields under geoengineered climate compared to a future global warming climateCrop yields are sensitive to water stress across whole Indian subcontinent, more sensitive to temperature stress across western partA sustained 50 year geoengineering intervention is shown to have no lasting effects on groundnut yields once the intervention is ceased This is likely to cause regional variations in temperature as well as impact on the biosphere [Parmesan and Yohe, Challinor et al., Bala et al., Pongratz et al., Tilmes et al., One of the geoengineering approaches that has received significant attention is the injection of sulphur dioxide into the lower stratosphere. The intention is that it would increase scattering of sunlight back to space and consequently reduce global temperature [Mueller et al., United Nations, Foley et al., Lobell et al., Bala et al., As with climate change scenarios, one of the main concerns with geoengineering is the possible impact on agriculture, as crop and livestock production systems are strongly dependent on weather and climate. A primary concern would be any potential effects on large human populations that depend on local crops or livestock for food or income, especially in rainfed systems, which is often the case for developing countries [Pongratz et al. [Xia et al. [Pongratz et al. [Parkes et al. [Our current understanding of geoengineering effects on crop production is far from comprehensive. To the knowledge of the authors, only three studies have investigated geoengineering impacts on crop productivity. z et al.  assesseda et al.  analyzedz et al. , they res et al. , which a6 ha and the second largest production, approximately 7 × 106 metric ton annually [Madhusudhana, Food and Agriculture Organization (FAO), Birthal et al., We investigate the effects of solar dimming geoengineering for the groundnut crop for the Indian subcontinent and assess its impact on crop yields. India is one of the major groundnut producing countries in the world. It has the largest groundnut planting area of approximately 7.5 × 1022.1Kravitz et al., Iles and Hegerl, Talawar, Challinor et al., FAO, Ramirez‐Villegas et al., Talawar, Talawar, We focus on India because of its large population that is dependent on locally grown rainfed crops. The consequences of geoengineering will be important if significant changes in weather patterns result, especially if the onset or intensity of the Indian monsoon is affected [2.2Andrews et al., Taylor et al., Kravitz et al., Sabeerali et al., Mitchell and Jones, Andrews et al., 2 level. The future projection runs, RCP4.5 and G3, are initialized using this HIS run, starting in 2006, with a simulated increasing CO2 concentration. In the simulations, the CO2 concentration increases from 380 ppm in 2006 to 538 ppm by 2100. We chose RCP4.5 since it is a moderate climate change scenario compared to RCP 8.5 of CMIP5 business as usual trajectory that we are currently on [Taylor et al., To study how geoengineering may influence groundnut yields we use output from climate model simulations from CMIP5 [2 or sulphate aerosol into the lower stratosphere has begun at 2020 and shut off at 2069, and the simulation terminates at 2089 [Kravitz et al., Kravitz et al., In the GeoMIP simulations, the geoengineering‐based injection of SO2.3Challinor et al., Ramirez‐Villegas and Challinor, 2 acts in GLAM to enhance water use efficiency and increase assimilation [Challinor and Wheeler, Challinor et al., Ramirez‐Villegas et al., Parkes et al., Challinor et al. [Challinor and Wheeler [To estimate groundnut yields for geoengineered (G3) and nongeoengineered (RCP 4.5) scenarios, we used a regional‐scale process‐based crop model  [r et al.  and Chal Wheeler .Challinor et al., Koehler et al., Parkes et al., Ramirez‐Villegas and Challinor, Ramirez‐Villegas and Challinor, 2 response parameterizations) for each of the five groundnut growing regions  at the original resolution of the BNU‐ESM climate model . This choice of resolution follows the definition of GLAM as a regional‐scale crop model and is consistent with previous GLAM studies, whereby GLAM is run at scales commensurate with those of climate models [ions see , were ust test and two‐sided Kolmogorov‐Smirnov tests for the modeled yield data for the scenarios.Using the calibrated parameter ensemble, we first performed all scenario model runs using the standard version of GLAM. Then, in order to understand the driving processes in the future scenarios, we also ran simulations with versions in which heat stress at the time of flowering was switched off (NOHTS), water stress was switched off (IRR), and both heat and water stress were switched off (IRR NOHTS). We then chose to focus on two future time periods both of 20 years' duration with the earlier period when geoengineering was active, from 2050 to 2069, and the second period when geoengineering was switched off, from 2080 to 2099. By studying these two time periods, we could then evaluate the effect of geoengineering on crops yields and determine if effects persisted after geoengineering was no longer active. The significance of changes in meteorology and yields were quantified using a statistical analysis approach including both the student 3We use BNU‐ESM climate model projections from the CMIP5 framework to project the future climate and also to run the off‐line GLAM crop model. We use the RCP 4.5 which is a reference scenario for G3. The RCP4.5 is also a conservative assumption that is consistent with relatively ambitious emission reductions. We chose this value rather than the higher RCP 8.5 to build into the results scope for unforeseen future offsets by mitigation and adaptation. The BNU‐ESM for the RCP 4.5 pathway shows an increase in maximum temperature across all India throughout our simulation period Figure  which isFor both the RCP 4.5 and G3 future scenarios, precipitation changes  are more geographically variable with negative changes in central and part of western India and increases elsewhere Figures b and 1c.The simulated future scenarios were used as inputs into GLAM, and the results are shown in Figures TO = 28°C) and so the projected temperature decreases and results in a yield reduction mediated through accelerated development (and hence shorter stages of development). The projected reductions in solar flux also acts to reduce yields through reduced light interception by the plants . This is also seen by the large yield gains from increased water supply , the precipitation is slightly decreased for G3  resulting in reduced yields of up to 20% relative to the RCP4.5 scenario. The results for the west and central regions were shown to be statistically significant, whereas the yields for other regions of India were not. Groundnut yields were shown to be sensitive to regional variations in meteorological variables, and our process simulations showed significant importance of water stress for all India, whereas heat stress was found to be important only for the west. Although we have shown that geoengineering can have significant negative effects on yields, we have also shown that once geoengineering is switched off, even after a continuous 50 year period, the yields tended toward the values of the nongeoengineered climate change scenario (RCP) with no statistical difference. This is important as it statistically shows that a sustained period of geoengineering intervention is not likely to set us on a vastly different climatic path and that the lower groundnut crop yields for geoengineered climate would rebound once geoengineering was ceased.Supporting Information S1Click here for additional data file.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.


Homarus gammarus) in a designated lobster reserve and nearby fished area in southern Norway. In the area open to fishing, the fishery is regulated by a closed season, a minimum legal size and a ban on the harvest of egg‐bearing females. Due to the differences in size and sex‐specific fishing mortality between the two areas, males and females are of approximately equal average size in the fished area, whereas males tend to be larger in the reserve. Our results show that females would mate with males larger than their own body size, but the relative size difference was significantly larger in the reserve. Sexual selection acted positively on both body size and claw size in males in the reserve, while it was nonsignificant in fished areas. This strongly suggests that size truncation of males by fishing reduces the variability of traits that sexual selection acts upon. If fisheries continue to target large individuals  with higher relative reproductive success, the weakening of sexual selection will likely accelerate fisheries‐induced evolution towards smaller body size.Removing individuals from a wild population can affect the availability of prospective mates and the outcome of competitive interactions, with subsequent effects on mating patterns and sexual selection. Consequently, the rate of harvest‐induced evolution is predicted to be strongly dependent on the strength and dynamics of sexual selection, yet there is limited empirical knowledge on the interplay between selective harvesting and the mating systems of exploited species. In this study, we used genetic parentage assignment to compare mating patterns of the highly valued and overexploited European lobster ( Disentangling how harvesting might affect the stability of a mating system is no trivial task, especially in many marine species which are not easily observed in their natural environment. Most studies of sexual selection and mate choice have been limited to controlled environments and model species  by comparing paternity data from a lobster reserve and an adjacent area open to fishing across multiple years. The clawed lobsters, consisting of European lobster and the American lobster (Homarus americanus), are long‐lived iconic species with high commercial value and therefore subject to intense fishing pressure  phenotypes.22.1The study was conducted in an area open to fishing and in a designated lobster reserve established in September of 2006, located at the Skagerrak coast in south‐eastern Norway Figure . Tempora2.2Captured egg‐bearing females were also measured and individually tagged with T‐bar tags. In addition, tissue samples were collected along with samples of offspring, where one egg was randomly sampled at the top of the egg mass near each of the ten pleopods and stored in separate vials with ethanol . Sampling fertilized eggs of each female allowed us to deduce the genotype of the father of each offspring based on the known mother–offspring genotype combination, which should help increase the likelihood of finding the actual fathers when running parentage assignment with the sampled males.2.3nreserve = 331, nfished = 319), and a total of 1,150 offspring from the 115 females. All individuals were genotyped with ten microsatellite loci developed for European lobster (see André and Knutsen ™ (Bio‐Rad) using fluorescent‐dyed forward primers (Life Technologies). The loci were pooled into one triplex , three duplexes  and one simplex (locus hgc120). Fragment analysis of PCR products was carried out on capillary sequencers CEQ™8000 (Beckman Coulter) and ABI ™ 3130xl (Applied Biosystems) and manually scored using genemapper v3.7 (Applied Biosystems) and ceq™ 8000 genetic analysis system v 8.0, respectively. As the length of the alleles slightly differed between the instruments, msatallele  was tested using fstat v.2.9.3.2 , all samples were pooled in subsequent analysis. The fixation index  was measured at each locus with fstat. One sample t test was used to assess whether FIS estimates differed significantly from zero. Pairwise linkage disequilibrium for each pair of loci was tested with a likelihood ratio statistic using the Markov chain algorithm of Raymond and Rousset . All critical significance levels for multiple testing were adjusted with R‐package fdrtool, after Benjamini and Hochberg (NA) and the theoretical exclusion probability given one parent is known for each locus and combined (EXC) were estimated with gerud 2  and false allele (ε2), were estimated with a combination of methods  and 967 eggs were used (and pooled) in the final parentage analysis . Inferred multiple paternities were only accepted as true cases of multiple paternity if offspring differed from the first male at five or more loci, did not show sign of scoring error (miss‐matching mothers genotype) and if the loci in question had not been calculated by colony due to missing alleles. The input files in colony were set up with two replicate runs and analysed with the highest precision settings with full‐likelihood, and with very long runtime on a PowerEdge M820, Linux CentOS 6.7 machine. For more details on the settings used, see Appendix Genotypes from seven males were excluded due to missing data (≥5 missing loci), and eighteen females had eggs from which DNA yield was insufficient to allow successful genotyping of the batches. Altogether, a total of 612 males, 97 females (prDM) were quantified using the software prDM  and probability of moulting, respectively, as predicted from CLmeasured using linear and logistic regression. The model predictions showed that almost all males below 90 mm CL  moulted annually and that the probability of moulting decreases to below 0.75 in larger sizes classes (>113 mm CL). The overall probability that males of all sizes would moult once every year was >0.5  and tested whether such patterns differed between areas, comparing models with Area x female size – interaction Equation  against We excluded six putative matings between mates sampled in different areas. Males were duplicated in the data file if the same male has mated with multiple females.2.7Standardized selection differentials on male body size and claw size were calculated, subtracting the mean trait value of potential fathers from the mean of successful fathers in each area (Arnold and Wade 1984). The size of maturity for males is not known in this population, but to reduce the probability for including immature males among potential fathers, only males with 80 mm or larger CL were included in the selection differential calculations. Size at maturity for European lobster in Scotland has been estimated to 80 mm CL for males and 79 CL for females  as the response variable (0 or 1). Claw width and body size were strongly correlated traits (r = .90) and could therefore not be included in the same model due to high collinearity , as covariate together with CL. We also fitted a model only including relative claw size (CWres). To evaluate whether trait‐fitness relationships differed between areas, we also included models testing for an interaction effect with area for each of the traits  using the following model structures: avggrad) for each trait with the Janzen‐Stern logistic regression approach  would evidently determine the prospects of obtaining mating success in the different years. Although we maintain that sexual selection is likely to be the primary mechanism underlying these selection differentials, we also conducted a more specific analysis of sexual selection, where we included only males sampled in 2010 because they represent the population at the time of reproduction and also had a sufficient number of assigned paternities (in following year) to warrant further analyses . We estimated standardized selection gradients, which capture the sensitivity in the fitness function when trait values change, and therefore better represent the strength and shape of selection than selection differentials alone . colony inferred genotypes from 41 unsampled males that sired offspring with 42 females. There was little exchange of individuals across area boundaries, although five females  had mated with males from the opposite area. Two of these interarea pairs involved a large male from the reserve, estimated to have been ~140 mm CL at the time of mating.A total of 511 52.8%) offspring were assigned a known father  of the broods showed evidence of being sired by a second male and therefore concluded to be multiply mated females. The paternal contribution among the multiply mated females was highly skewed in favour of a primary male (9:1 ratio) in both these cases  had mated with more than one female, of which five had mated with two females and three had mated with three females. Polygamous males were not significantly larger than males with only one recorded paternity . On average, the level of polygamy was higher for males, with females mating with 1.01 males and males (known and unknown) mating with 1.16 females.Of the inferred males with known identity, eight , but not in the reserve . Interarea pairs (n = 5) were removed prior to analysing the area‐specific size‐assortative mating pattern . Females in the fished area also paired with males of larger sizes, as all but three of the 15 pairs had a male larger than the female, with the average size difference smaller (6.4%) and marginally statistically significant . There was a strong positive size‐assortative mating pattern , with females mating with larger males relative to their own size in the reserve compared to the fished area .Across all sampling years, females were larger than males in the fished area  and claw size (CW) were significantly positive in the reserve, while they were more variable and nonsignificant in the fished area Table . Corresprt Table . A simplrt Table . Using t4We investigated the mating system of the exploited European lobster in its natural environment inside and outside a coastal marine reserve to establish whether harvesting can affect mating patterns and sexual selection. Our genetic parentage assignment clearly demonstrates a positive size‐assortative mating pattern, where females have a strong disposition to mate with comparatively larger males. Moreover, we show that this within‐pair size difference was larger in the reserve than in the fished area. We also documented that male size (body and claws) strongly influenced their mating success inside the reserve, while selection differentials on these traits were weaker and not significant in the fished area. Sexual selection was stronger on relative claw size, rather than on absolute claw and body size. Overall, our findings suggest that fishing can greatly affect mating patterns, with potential consequences for reproductive output and the rate and trajectory of fisheries‐induced evolution.4.1Having been afforded protection from fishing for almost a decade, lobsters in the reserve might display a good depiction of what can be considered more “natural” mating behaviour, given that females have access to a wider diversity of male phenotypes. Therefore, the increased scope for sexual selection on male traits is the likely explanation for the higher positive selection differentials in the marine reserve relative to the fished area. Although females in the fished area tended to mate with males larger than themselves, the average difference in body size between sexes was much smaller  than in the reserve . These results are in line with those for wild‐mated female American lobsters obtained by Gosselin, Sainte‐Marie, and Bernatchez , who fouHigh fishing mortality could also explain the lower assignment probability for males in the fished area in 2010, as a male should have lower chances of surviving and successfully mate the following year, relative to a similar sized male in the reserve. This implies that the selection differentials may not be purely due to sexual selection but may also reflect an unidentified component of fisheries selection against large individuals. Arguably, results from this work show that selective harvesting is indeed affecting mating patterns, but we only compare the trait distributions of males with and without paternity on the broods  sampled. The consequences for true fitness and selection can therefore only be inferred. However, we find no reason to assume different trait distributions between the observed and unobserved fathers.Contrasting no‐take reserves and complementary monitored control areas where harvesting continues as usual may be one of the best options available to study the effects of harvesting regimes in situ. To test the generality of the findings, and indisputably attribute spatial variability in ecology to that in fishing pressure, the approach should be tested using multiple pairs of reserves and fished areas. Moreover, temporal replicates, tracking several selection episodes and ideally also including natural and fisheries selection, could be used to estimate life time fitness and to test individual consistency and temporal stability in sexual selection.4.2Female choice appears to play an important role in driving the positive assortative mating pattern; some of the largest males had mated with small females, while the largest females never mated with small males. Females can have a direct benefit from choosing larger males. First, female clawed lobsters usually moult in a males’ shelter, where she will mate soon after and cohabitate for some time, a strategy believed to increase successful pre‐ and postcopulatory guarding of the soft‐shelled female  on both claw (3.04) and body size (3.56) were relatively high, both being well above the median (1.93) calculated from 140 published estimates . In contrast, a recent study in a region of the United Kingdom found no incidence of multiple paternity in the European lobster , a reduction in mean and variability in these traits due to fishing is expected to lead to faster harvest‐induced evolution than under the assumption of random mating , a maximum size limit of 320 mm total length (~116 mm CL) was implemented for lobster caught along the Skagerrak coast. As for the benefit of spatial management, a handful of small reserves established along the coastline are unlikely to have any strong effects on the evolutionary trajectory. If, however, the number and size of reserves are increased, with sexual selection recovering within, the potential for increased reproductive output from large females  and spill‐over of larger, more “attractive” males from the reserves could possibly strengthen sexual selection and buffer fisheries‐induced evolution in fished areas (see also: Baskett and Barnett In conclusion, our paper presents novel empirical support for how fishing affects mating behaviour in wild European lobster. Selective fishing reduces the phenotypic variability for sexual selection to act upon, but at the same time, the strength of sexual selection may be relaxed through lowered density and biased sex ratio. Sexual selection is an integral part of evolution and should therefore be mandatory to consider in evolutionary enlightened management.None Declared.https://doi.org/10.5061/dryad.1b1f023.Data available from the Dryad Digital Repository:  Click here for additional data file. Click here for additional data file. Click here for additional data file.