What makes a protein a good immune regulator? We can make some intuitive guesses based on the characteristics of an immune response. Because immunity is not only dangerous to the pathogen but also potentially harmful to self, its regulators should only be active when the threat of infection is imminent, and their activities should be spatially and temporally appropriate to the infection pressure. In-depth studies of the plant immune regulator non-expressor of pathogenesis-related (PR) genes 1 (NPR1) have revealed the molecular mechanisms contributing to these characteristics and also discovered some surprising connections that the immune system has to normal plant physiological processes to minimize the fitness costs. Because NPR1 transcription is not dramatically affected by pathogen challenge . Bo. Bo55/SeS55/59A mutant in which sumoylation is no longer inhibited by phosphorylation at this site, defense response is constitutively elevated . Th. Th35]. 3a/cul3b . TherefoBecause NPR1 is not only a positive regulator of SAR but also an inhibitor of ETI, it is not difficult to imagine that NPR1 degradation plays a role in ETI induction. Because this PCD-associated immune response is tightly restricted to the site of infection, there is likely a threshold at which NPR1 is degraded to relieve its repression on cell death. Fu et al. proposed that this is achieved by the SA concentration gradient radiating from the site of pathogen challenge , combine-elements in the PR-1 promoter and their activities [Posttranslational modifications of the master immune regulator, NPR1, play a major role in the signaling decision for cell death or cell survival during pathogen challenge. However, there are still some very basic questions that remain to be answered: How is the transcription cofactor activity of NPR1 orchestrated, and what genes does it control during specific immune responses? Modification of NPR1 affects its ability to interact with TFs which may affect their associations with both positive and negative cistivities . Anothertivities –41. SincAlthough phosphorylation has been shown to regulate multiple NPR1 functions, the specific kinases responsible for some key phosphorylations remain elusive. To date, two members of the SnRK family of kinases have been shown to interact with and phosphorylate NPR1 ,42, both521 and Cys529) in the C-terminus and that this activity is crucial for the SA-induced transactivation activity of NPR1 [Several methods have been used to demonstrate that NPR1 can directly bind SA ,44. One


Diabetic macular edema is a serious visual complication of diabetic retinopathy. This article reviews the history of previous and current therapies, including laser therapy, anti-vascular endothelial growth factor agents, and corticosteroids, that have been used to treat this condition. In addition, it proposes new ways to use them in combination in order to decrease treatment burden and potentially address other causes besides vascular endothelial growth factor for diabetic macular edema. Macular edema occurs when high blood glucose damages the small capillaries supplying blood to the retina. This breakdown of blood vessels leads to the extravasation of blood and its solutes from the capillaries to the extracellular space under the macula, the central area of the retina, and causes it to thicken and swell (edema). The macula holds tightly packed cones that provide sharp central vision, enabling the person to see vivid detail and color. In this review, we briefly discuss the treatment options currently available for the treatment of macular edema and we review the rationale for emerging agents, many of which are currently being evaluated in clinical trials.Diabetic macular edema (DME) is a common complication of diabetes and the leading cause of vision loss in adultsLaser photocoagulation has been the gold standard for the treatment of DME for the past two decades. Laser treatment involves placing tiny laser burns within thickened areas of the retina in both direct  treatment of microaneurysms and scattered spots in other areas of edema (grid). Laser photocoagulation involves the application of a precise and directed high-energy laser to the retina, and the heat generated as it is absorbed into the tissue causes clotting of the blood vessels and leads to the localized destruction of the tissue. These burns in the light-sensitive membrane in the back of the eye serve to destroy the diseased areas of the tissue and to seal off the damaging blood vessels that threaten vision.2.This treatment for macular edema is very prevalent since it is a quick and cost-effective procedure that is usually completed after one session without the risk of endophthalmitis that an intravitreal injection poses. However, laser photocoagulation focuses on the symptoms of edema instead of addressing the retinopathy. The benefits to visual acuity are unremarkable, as there is only a 50% reduction in vision loss and vision already lost cannot be regained using laser photocoagulation. Lesions on the retina left by the destructive laser have been observed to expand over time. At 2 years, laser sears increased 50% per year and 4.6% a year afterwards, and 11 out of 203 patients experienced foveal encroachment. Still, laser photocoagulation is a highly effective treatment for macular edema and is still a feasible option for those unresponsive to anti-vascular endothelial growth factor (anti-VEGF) treatments3. In addition, the RISE and RIDE studies demonstrated that 39.2% of patients had 15-letter gains in visual acuity and a mean improvement of 12.4 letters vs. sham over 24 monthly injections and were the pivotal studies that allowed approval of ranibizumab by the US Food and Drug Administration4.The VEGF family is the most critical with regard to the pathogenesis of diabetic retinopathy owing to its signaling the induction of angiogenesis as well as increasing vascular permeability. Because of VEGF’s central role in the pathogenesis of DME, VEGF antagonists are a logical choice for therapy. The first anti-VEGF agent used for ophthalmology was pegaptanib  but was replaced by the development of ranibizumab . In the randomized clinical trial Protocol I, researchers observed that ranibizumab triples the mean change in visual acuity compared to the corticosteroid triamcinolone, and in 1 year the same steroid showed a visual acuity loss three times higher than that produced by the VEGF antagonist5.Bevacizumab  is another VEGF antagonist that has come into widespread clinical use in the treatment of retinal disease. The BOLT study compared bevacizumab vs. macular laser in patients with DME. The bevacizumab arm gained a median of 9 ETDRS letters vs. 2.5 letters of laser treatment (P=0.005), with a mean gain of 8.6 letters for bevacizumab vs. a mean loss of 0.5 letters for laser. Forty-nine percent of patients gained 10 or more letters (P=0.001) and 32% gained at least 15 letters (P=0.004) for bevacizumab vs. 7% and 4% for MLT. The percentage who lost fewer than 15 letters in the laser arm was 86% vs. 100% for bevacizumab (P=0.03)6. These visual acuity results indicate that a large portion of patients with DME may be effectively treated with dosing every 8 weeks compared to the monthly injections of other anti-VEGF agents.Aflibercept is the most recent anti-VEGF medication approved to treat DME. In the randomized clinical studies VIVID and VISTA, researchers compared intravitreal aflibercept injections, which have recently demonstrated clinically equivalent efficacy to monthly ranibizumab in neovascular age-related macular degeneration, to laser monotherapy for the treatment of DME. The results of the trials demonstrated that aflibercept, given either every 4 weeks or every 8 weeks , is superior to laser and results in 10.7–12.4 letters gained at 1 year. In addition, 32–41% of patients gained 15 letters at 1 year as wellHowever, it is widely accepted that the systemic use of anti-VEGF agents results in an increased risk for arterial thromboembolic events, obstructions of a blood vessel caused by a blood clot that has become dislodged from another point in the circulation, which can show a higher incidence of stroke among patients receiving this therapy. However, it is noteworthy that no significant increased rate of death, stroke, or myocardial infarction was seen in the RISE and RIDE or VIVID and VISTA groups.8. This analysis looked at the two arms of patients who received ranibizumab and looked at vision at 12 months and 3 years. The study showed that patients could be divided into three groups: those who were good responders with 10 or more letters of improvement, those who were fair responders with 5 to 9 letters of improvement, and those who were suboptimal responders with 5 or fewer letters of improvement. The study showed that the best-corrected visual acuity (BCVA) response after three anti-VEGF injections (12 weeks) is a strong predictor of long-term BCVA response at 12 months and 3 years. This study demonstrated that physicians can recognize suboptimal DME responders much earlier in the treatment cycle and should consider different therapies in patients who are suboptimal responders.About 50% of patients with DME experience only a moderate reduction of edema and improvement in vision from VEGF antagonists alone. However, for most clinicians, it takes many months or years to determine the need for a switch in therapy. The EARLY analysis was a post hoc analysis of Protocol I of the diabetic retinopathy clinical research group9.Mounting evidence exists to show that inflammation is a significant aspect of the pathogenesis of DME. Leukocytes in the blood release a variety of cytokines and chemokines that significantly increase vascular permeability, leading to more fluid build-up under the retina. These cytokines also carry VEGFs, which can aggravate and worsen macular edema by promoting angiogenesis10. Steroids have been shown to lower the central subfield thickness (CST) and improve visual acuity for suboptimal responders to anti-VEGF and pseudophakics. Corticosteroids also appear to be effective for both chronic and treatment-naive macular edema, while anti-VEGF therapy is seen to be a less effective treatment for chronic DME.Corticosteroids have shown the ability to lower inflammatory mediators and VEGF, while anti-VEGF therapy treats only the VEGF portion. Anti-VEGF treatment does not work for all patients; 50% of patients respond significantly and quickly, 25% of patients have an intermediate response, and 25% of patients do not respond to anti-VEGF treatment11.There are currently two approved corticosteroid therapies for DME: dexamethasone  and fluocinolone . The MEAD study showed that dexamethasone intravitreal implant was able to improve vision in patients over a 3-year period. Patient vision improved overall by 4 letters and pseudophakic patients improved by 6 letters over a year period. In the MEAD study, there was a nearly 60% frequency of cataract surgery, and 40% of patients on steroids were later prescribed medication for intraocular pressure (IOP). However, patients’ increase in IOP usually peaked at 6–8 weeks and then returned to baseline by the end of 4 months, and one patient required IOP-lowering surgery12.The advantage of corticosteroid delayed delivery systems, such as dexamethasone intravitreal implants, is that patients require fewer treatments compared to the monthly injections required by most anti-VEGF agents. The newest steroid to be approved is the fluocinolone implant. The FAME study showed that the fluocinolone implant caused an improvement of ≥15 letters in 28.7% of patients in the study group vs. 18.9% in sham eyes and a 6-letter improvement in vision at 24 months. IOP medications were required in 42% of patients with seven patients requiring IOP-lowering surgery13. VEGF is a logical drug target that treats DME well, but it requires monthly retreatment to maintain efficacy and it does not address additional inflammatory cytokines upregulated in DME. It is in these patients that supplementing with therapies that work using a different mode of action may be of value. Diabetic maculopathy is a combination of both VEGF-mediated factors as well as inflammatory mediators. Corticosteroids decrease inflammatory cytokines and have a modest anti-VEGF effect, while anti-VEGF agents have a modest anti-inflammatory effect9. Using a corticosteroid in combination with an anti-VEGF agent allows the patient to benefit with increased efficacy as well as increased duration of effect. As the category of sustained release steroids increases, the physician’s arsenal of managing these “hard to treat” patients increases as well.Although anti-VEGF medication remains the mainstay of therapy for DME, there are many cases for which anti-VEGF therapy alone is not adequate enough to control the macular edema. This should not be unexpected, as the clinical trials were able to significantly improve vision in fewer than 50% of patients14. After 12 months, it was observed that while ultimately there was no difference in vision, there were differences in vision at different monthly time points and the optical coherence tomography (OCT) CST was significantly better in the combination group, with a “sawtooth” effect14. Subgroup analysis suggested that the greatest benefit of dexamethasone implant was in the group with the most bevacizumab injections prior to enrollment in the study14. In conclusion, although visual acuity changes are not superior to continued bevacizumab monotherapy, dexamethasone combined with bevacizumab significantly improves visual acuity and significantly improves macular morphology in eyes with refractive chronic DME.A 12-month randomized study of eyes with persistent DME assessed the efficacy of a corticosteroid (dexamethasone) delivery system as an adjunct to the VEGF antagonist bevacizumab compared with continued bevacizumab monotherapy15. The study covered 12 patients who received a single injection of fluocinolone acetonide and were followed for 6 months. Out of the 11 patients who completed the study, nine maintained or improved their best-corrected visual acuity from baseline, and the 11 patients experienced an average decrease in CST of 300.6 microns from baseline. However, there are no good data on combination therapy with the implant, but it may be appropriate for patients with suboptimal edema reduction with anti-VEGF monotherapy15.In another study, researchers explored the effect and safety of fluocinolone acetonide in chronic DME patients considered insufficiently responsive to available therapies with or without intravitreal corticosteroid therapyCombination therapy is a rational approach to battling DME, as it targets VEGF-mediated angiogenesis while combating the multiple factors in the inflammatory cascade. Combination therapy may be an alternative for patients who are unresponsive or have a suboptimal response to VEGF antagonists by providing a sustained duration of action, which translates into increased efficacy and convenience. However, there are potential adverse effects, such as cataracts and elevated IOP, that need to be considered.BCVA, best-corrected visual acuity; CST, central subfield thickness; DME, diabetic macular edema; IOP, intraocular pressure; OCT, optical coherence tomography; VEGF, vascular endothelial growth factor.


WUSCHEL independently of photosynthesis and that this likely involves inter-regional cytokinin signaling. Metabolic signals, on the other hand, are transduced to the meristem through activation of the TARGET OF RAPAMYCIN (TOR) kinase. Surprisingly, TOR is also required for light signal dependent stem cell activation. Thus, the TOR kinase acts as a central integrator of light and metabolic signals and a key regulator of stem cell activation at the shoot apex.A major feature of embryogenesis is the specification of stem cell systems, but in contrast to the situation in most animals, plant stem cells remain quiescent until the postembryonic phase of development. Here, we dissect how light and metabolic signals are integrated to overcome stem cell dormancy at the shoot apical meristem. We show on the one hand that light is able to activate expression of the stem cell inducer DOI:http://dx.doi.org/10.7554/eLife.17023.001 Plants are able to grow and develop throughout their lives thanks to groups of stem cells at the tips of their shoots and roots, which can constantly divide to produce new cells. Energy captured from sunlight during a process called photosynthesis is the main source of energy for most plants. Therefore, the amount and quality of light in the environment has a big influence on how plants grow and develop. An enzyme called TOR kinase can sense energy levels in animal cells and regulate many processes including growth and cell division. Plants also have a TOR kinase, but it is less clear if it plays the same role in plants, and whether it can respond to light.Arabidopsis. The experiments show that light is able to activate the production of WUSCHEL independently of photosynthesis via a signal pathway that depends on TOR kinase. The stem cells do not directly sense light; instead other cells detect the light and relay the information to the stem cells with the help of a hormone called cytokinin.Plant stem cells only start to divide after the seed germinates. In shoots, a protein called WUSCHEL is required to maintain stem cells in an active state. Here, Pfeiffer et al. studied how shoot stem cells are activated in response to environmental signals in a plant known as Further experiments show that information about energy levels in cells is relayed via another signal pathway that also involves the TOR kinase. Therefore, Pfeiffer et al.’s findings suggest that the activation of TOR by light allows plant cells to anticipate how much energy will be available and efficiently tune their growth and development to cope with the environmental conditions. Future challenges are to understand how TOR kinase is regulated by light signals and how this enzyme is able to act on WUSCHEL to trigger stem cell division.DOI:http://dx.doi.org/10.7554/eLife.17023.002 Light is the sole energy source of plants and therefore one of the most important environmental factors influencing their development and physiology. Consequently, several of the core developmental decisions during the lifecycle of a plant from germination to seedling development and flowering are strongly influenced by light conditions. After germination, higher plants undergo two distinct developmental programs depending on the availability of light, termed skotomorphogenesis and photomorphogenesis. Skotomorphogenesis, the dark adaptation program, is characterized by an etiolated phenotype, including an elongated hypocotyl, closed cotyledons, the formation of an apical hook and etioplast development. Importantly, stem cells at the shoot and root tip remain dormant and thus growth in etiolated seedlings is mainly dependent on cell elongation rather than cell division. In contrast, photomorphogenesis, the developmental program triggered in light, leads to seedlings with short hypocotyls, unfolded cotyledons and development of chloroplasts. In the light, shoot and root meristems are activated, leading to root growth and development of the first leaves by cell division and expansion   . Howeverignaling . Recentlignaling .Arabidopsis thaliana is based on the activity of the homeodomain transcription factor WUSCHEL (WUS), which is expressed in the organizing centre and necessary and sufficient to non-cell- autonomously induce stem cell fate by protein movement. Stem cells in turn express CLAVATA3 (CLV3), a short secreted peptide, that acts via the CLV / CORYNE receptor system to limit the expression of WUS in the organizing center  as well as cells of the underlying organizing center (pWUS:3xVENUS-NLS). With the help of these tools, we were able to genetically dissect the individual contribution of light signaling and photosynthesis-driven nutrient sensing on the stem cell system of the SAM. We show that both pathways ultimately converge at the level of TOR kinase activation, revealing a role for TOR as a central regulator of stem cell activation in response to environmental cues.Here, we analyzed the role of light and nutrients for post-germination stem cell activation in the shoot apical meristem (SAM) of young seedlings. Stem cell control in the SAM of g center . The useArabidopsis seedlings remains dormant during skotomorphogenesis and therefore, plants are unable to advance to the organogenic stage in the absence of light. However, since light acts as signal and energy source alike, we first asked which of the two roles is dominant for SAM development. While supplementation of sugar to wild-type seedlings grown in the dark is known to be inefficient to trigger development  and niche cells (pWUS:3xVENUS-NLS) we found that stem cell identity was actively maintained independently of growth conditions and was even observed in the dormant state mediated by germination in the dark . Again,nditions  confirmiWUS reporter (pWUS:3xVENUS-NLS) was below detection level in dark-grown seedlings. In contrast, GFP signals were readily detectable in light-grown plants from three days after germination onwards with the signal steadily increasing over time  to analyze the effect of light signaling with minimal influence of photosynthesis-derived metabolites. Even at low intensities, blue, as well as red light were sufficient to robustly induce WUS expression  caused strong cop1-like phenotypes and a substantial activation of the WUS promoter in the SAM showing that transgenic activation of light signaling is sufficient to trigger stem cell activation in darkness  and auxin , which codes for one of seven homologous cytokinin dehydrogenases involved in CK catabolism in Arabidopsis met all criteria  and is highly expressed in etiolated seedlings as well as under shade conditions . In contrast to either single ckx mutant, ckx5/ckx6 double mutants showed basal WUS reporter activity in darkness, similar to its behavior under CK treatment. Furthermore, we observed a dramatic enhancement of the effect of sucrose on WUS activity and also light dependent reporter induction was increased two-fold over either single mutant, which was also detectable by qRT-PCR  of different sugars with diverse energy content for four days in darkness  kinase and photosynthesis-mediated activation of the root meristem has recently been described to be under the regulation of TOR . CharactWUS expression in a dose dependent manner  or 15 mM glucose for 24 hr, we detected a substantial increase in phosphorylation of S6K compared to seedlings that were kept in darkness  and energy availability caused by glucose supplementation (PC2) as the two major components in our sample set, with light treated samples being influenced by both components. Interestingly, glucose treatment only had a mild effect on seedling metabolism. We detected slight increases of glucose, fructose and sucrose as well as a clear increase of mannose levels in glucose treated seedlings, while raffinose levels were reduced  that were all previously described to be reduced in amiR-tor seedlings  and shade conditions (CKX6), respectively  also plays an important role in light, sugar and CK crosstalk at the shoot apex. Meristems of weaker stip mutants arrest at seedling stage but can be rescued by addition of sucrose to the medium  construct was obtained by LR reactions of the pDONR221-mCHERRY-NLS plasmid (pFK143) with pFK317, a pGreen-IIS (CLV3 1.4 kb promoter and 1.2 kb terminator sequences and kanamycin resistance cassette. The pWUS:3xVENUS-NLS construct (pTS81) was generated accordingly, using a pGREEN-IIS based binary vector with a 4.4 kb genomic WUS promoter upstream of the ATG and 2.8 kb WUS terminator downstream of the stop codon (pFK398) and a Basta resistance cassette. In both constructs the N7 NLS  carrying the pGREEN-IIS and GreenGate based plasmids. The Agrobacteria strain GV3101 was used for the transformation of plants with the plasmid pAP101.All used plant lines were in the Col-0 background. oral dip  using thTo select for the presence of the corresponding resistance markers, plants were grown on plates supplemented with 5 mM D-Ala, 20 µg/ml Hygromycin B or 50 µg/ml Kanamycin or grown on soil and sprayed with the herbicides Inspire  (7.6 µl/l)  or BastapCLV3:mCHERRY-NLS and pWUS:3xVENUS-NLS, respectively. The double reporter line is comprised of a cross of both lines. The WUS-GFP rescue line  was alscop1-4 (pPIN1:PIN1-GFP (cop1-4 (phyA-211 (phyB-9 (cry1-304/cry2-1 (hy5 (SALK_096651C), ckx5-1 (SALK_064309) and ckx6-2 (SALK_070071)  . All of , cop1-6  and cop1 and A05466 (AGCCAACTTAAAGGCTATGCAG) and the PCR product was digested with Eco81I to screen for homozygous ckx6 mutants at the locus of the first gRNA. The genomic region of T1 plants that produced undigested PCR fragments was amplified with the oligos A05465 and A05468 (ACTTGAGGGTCTCATGCAAAAT), and sequenced after subcloning the PCR product into pGEM-T Easy ) to confirm the mutation of the CKX6 locus. T2 plants of homozygous T1 mutants were used in this manuscript.A homozygous double reporter−2*s−1 of white light for 6 hr. Afterwards plants were either kept in white light, transferred to darkness or to LED cabinets equipped with red (673 nm), far-red (745 nm) and blue (471 nm) LEDs . Unless otherwise noted, constant red, far-red or blue light was applied with an intensity of 30 µmol*m−2*s−1. All white light treatments were carried out at 150 µmol*m−2*s−1 of fluorescent white light with a 16-h-light/8-h-dark cycle. Starting with the light induction of germination, plants were kept at 21–22°C. 0.5x MS plates were supplemented with 1% (30 mM) sucrose or 15 mM glucose only if mentioned explicitly. Growth in a CO2-deficient environment was accomplished by growing unsealed vented petri dishes in a sealed plastic bag with 5 g NaOH and 5 g CaO , 0.8% Phytoagar in vented petri dishes that were sealed with micropore tape . Germination was induced by 150 µmol*m 5 g CaO .−2*s−1 of blue and red light each. Both treatments were started 24 hr before and continued until the harvest of the material. To exclude ungerminated seeds and empty seed shells form the metabolite measurements, only above root tissue was harvested. All seedlings were rinsed with distilled water prior to harvest.Four day old seedlings that were harvested for protein extracts and metabolite measurements were grown vertically on top of 100 µm nylon meshes . For the glucose treatment in these experiments seedlings were transferred with the mesh to plates containing 15 mM glucose. Light treatments entailed irradiation with 60 µmol*mAbout 30–40 seeds, that were imbibed as described above, were sown in 3 ml 0.5x MS in petri dishes of 35 mm diameter. Plants were kept in darkness for three days after the induction of germination by 6 hr light treatment. The medium of three day old etiolated seedlings was supplemented with 15 mM glucose and/or 0.5–2 µM AZD-8055 . Stock solutions of 1000x concentrated AZD-8055 were diluted in DMSO, therefore control plants were mock treated with the same volume of DMSO.A detailed protocol of the in situ hybridization procedure was provided previously .Arabidopsis seedlings with the Plant RNA Purification Reagent  according to the instructions of the manufacturer, digested with TURBO DNAse  and purified with RNeasy Mini Kit . Equal amounts of RNA were used for oligo dT primed cDNA synthesis with the RevertAid First Strand cDNA Synthesis Kit . The qPCR reaction was set up using the SG qPCR Master Mix  and run on a Chromo4 Real-Time PCR System  with technical duplicates each. The relative expression levels were calculated using the ddCt method with PP2A expression as a reference. Results shown are the means of 2 independent biological replicates. The following oligos were used: PP2A: A01067: TAA CGT GGC CAA AAT GAT GC and A01068: GTT CTC CAC AAC CGC TTG GT; WUS: A00317: TTA TGA TGG CGG CTA ACG AT and A00318: TTC AGT ACC TGA GCT TGC ATG; PHYB total: A05986: AGC AAA TGG CTG ATG GAT TC and A05987: GCT TGT CCA CCT GCT GCT AT; PHYB 3'UTR: A05984: GCG ACC ATT GTC AAC TGC TA and A05985: CTC CGA CGT CGT TAG ACA CA.Total RNA was extracted from 100 mg seven day old 3Fe(III)(CN)6, 0.5 mM K4Fe(II)(CN)6, 1 mM EDTA, 1% Triton X-100, 1 mg/ml X-gluc) at 22°C over night. After staining, the seedlings were incubated with 60% and subsequently in 95% ethanol to remove chlorophyll.Four day old seedlings were harvested in 90% acetone and incubated at −20°C for at least 1 hr. Seedlings were washed with PBS and incubated in substrate buffer (1x PBS (pH 7.0), 1 mM KWUS and 41 µm (80 pixles) diameter for CLV3. Quantifications in each figure were normalized to the median of the fluorescence levels of wild-type plants grown in red light for four days. Only exception: in cop1 mutant plants (second box in each box plot). For all these experiments plants of one experimental set were always grown and analyzed in parallel to the untreated (dark-grown) and the corresponding reference sample.To image the fluorescent reporter activities in the SAM, seedlings were split in half by pulling one cotyledon away from the SAM with forceps. The exposed meristem was imaged with a Zeiss Imager M1, the Plan-APOCHROMAT 20x/0.8 objective  and YFP- and mCHERRY-specific filter sets. For the quantification of VENUS and mCHERRY signal intensities the settings for the intensity of the fluorescent lamp and exposure times were unchanged for each channel. 16-bit B/W pictures of at least 20 SAMs per sample were analyzed by FIJI , using tIn situ sections were analyzed with the same microscope and a 40x/0.95 Plan-APOCHROMAT objective . Equipment and settings used for confocal microscopy was described earlier .Proteins were extracted from 50 mg materials in 250 µl 2x Laemmli buffer  supplemented with 1.5% phosphatase inhibitor cocktail 2 . After adding extraction buffer, samples were briefly mixed and heated at 95°C for 10 min. Cellular debris was removed by two centrifugation steps . 20 µg protein were separated on a 10% SDS gel and transferred to PVDF membrane. Phospho-p70 S6 kinase (Thr(P)-389) polyclonal antibody  was used to detect S6K phosphorylation. S6K1/2 antibody  was used to detect total S6K1 and S6K2.Three biological replicates were harvested for and analyzed as described  by the M In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Ian Baldwin (Senior editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.Thank you for submitting your article "Integration of light and metabolic signals for stem cell activation at the shoot apical meristem" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.All reviewers agree that your work is of general interest and in principle support publication. However, they also agree that a few issues have to be addressed before the work can be accepted. In your revision, please pay special attention to the following points:2-free atmosphere.1) The reviewers are worried about the validity of some of the pharmacological experiments because of possible indirect or side effects of the drugs (in particular of norfluorazon and lincomycin). Therefore, they believe that your paper would be substantially strengthened if you could repeat the basic introductory experiments  and 2 incop1 allele used. They ask that some of the basic experiments are verified with a cop1 null allele.2) The reviewers also have some concerns regarding the plant material, especially with regard to the CLV3/WUS activity, but also wonder whether some of the basic observations could be confirmed by qPCR analysis of the endogenous genes?3) The reviewers acknowledge the use of reporters as an output for Please find the reviewer comments attached below for more details, and please respond to them to the best of your capacity.Reviewer #1:Arabidopsis. Overall, the data appear solid and are presented in a transparent manner, and the conclusions are largely justified from my point of view. However, the authors' conclusions strongly hinge on the quantification of two reporters under different conditions or in different genetic backgrounds, and I believe some improvements are required to make the manuscript acceptable for publication.This manuscript from the Lohmann lab and colleagues presents a complex set of data, which indicate that SAM activation is both light- and energy-status dependent in cop1 alleles in their work, the cop1-4 mutant. However, within the cop1 allelic series, this allele has always been the most mysterious. Unlike true null alleles, cop1-4 produces a truncated protein that comprises both the RING finger and coiled coil domains, yet alleles that retain these domains as well but are truncated further C-terminal or carry certain point mutations in the WD40 repeat domain also have a strong phenotype.1) I have one major concern with respect to the genetic material. Several analyses are based on experiments with a mutant in COP1, a central negative regulator of photomorphogenesis. Understandably, the authors use one of the viable cop1-4 are representative. I understand that it would be too much to ask to repeat reporter experiments in another cop1 background, but I think the authors could check the principal physiological responses in true cop1 null alleles, which should be easily available. In particular, the authors could repeat the experiment of cop1 on WUS expression by qPCR instead of a reporter, the difference appears strong enough although it is restricted to few cells  Another genetic concern: I believe the authors are formally right to conclude that HY5 has no role in light-dependent stem cell activation, however it is important to remember that unlike cop1 shoot formation on +/- suc, are those responses fully penetrant? If so, this should be explicitly stated, if not, proportions clearly indicated.3) With respect to responses illustrated by images only, like phyB mutant was not tagged? Maybe the authors can present evidence from additional lines and/or correlation with expression levels. As is, their statement regarding ML1 is misleading, because one out of two lines shows the other phenotype, so it's a 50:50 choice.4) One of the two ML1 transgenic lines deviates from the highlighted phenotype . Why is 5) With regard to the cytokinin experiments, and the sometimes inconclusive contribution, I wonder whether the actual cytokinin used is of importance? Given the variety of cytokinins and their reported shoot- and root-specific activities, the authors might find a "better" cytokinin, which might have a more clear effect on WUS expression.6) Because of the importance of the fluorescence measurements, I would like the authors to confirm that samples analyzed for one graph were grown in parallel and analyzed with identical settings.Reviewer #2:cop1 seedlings is activated by adding sugar thereby leading to the formation of leaves (even flowers) in darkness. This suggests that both light signals (constitutively on in cop1) and metabolic signals cooperate to enable SAM activation. The authors explore the molecular mechanisms underlying this activation. The authors show that the CLV3 expression domain is rather constant across conditions while WUS expression is induced by light. A number of experiments indicate that this light activation comprises both light and metabolic signals. TOR is implicated in the metabolic regulation of WUS expression while the phytohormone CK is involved in light regulation of WUS expression. This paper should be of broad interested for developmental biologists. Below I list a few issues that the authors should clarify.This is a very interesting manuscript exploring how light and metabolic signals activate the SAM. The SAM is dormant in etiolated seedlings and gets activated upon light perception. As shown previously the SAM of etiolated cop1 +/- sucrose and WT +/- light and the use of NF and Linc . This would not lead to the rather drastic effects of these drugs on chloroplasts. In principle the effect of CO2-free air could be complemented by adding sucrose. Haydon et al., 2013 also complemented the NF effect by adding sucrose which to some extent rules out non-specific effects of NF.1) Based on WUS expression in and Linc , the autWUS expression by transmitting the light signal . In WUS expression. However, on WUS . It therefore rather appears that these genes are involved in basal regulation of WUS expression and not in light regulation. It would actually be interesting to test this by adding sucrose to the double mutant and look for signs of leaf development in darkness rather than hypocotyl elongation as shown in supplementary 2) The role of CK is potentially quite complex and I'm not sure that the current data allows drawing a clear conclusion. The authors propose that CK promote , on WUS  given th3) A potential difficulty with the role of CK in this process and the use of drugs like NF is that CK biosynthesis requires plastids and chloroplast development is promoted by CK. Not sure how this can be untangled but it should be discussed.4) All figure legends should be more explicit and provide detailed information about the exact light conditions , presence of sucrose and numbers of seedlings used per experiment. For example, I don't see which data correspond to + sucrose (hatched box) in 2A.Reviewer #3:WUSCHEL (WUS) and of it repressor CLAVATA3 (Clv3) by metabolic, hormonal and light signals using fluorescent markers driven by the promoters of these genes. This manuscript covers a lot of ground and makes a thorough analysis of the factors regulating the expression of these reporter genes. Specifically the role of cytokinins as well as sucrose and sugars in inducing WUS expression is shown and the obtained data suggest that the Target Of Rapamycin (TOR) kinase plays a central role in connecting metabolic signals to WUS induction. This manuscript is well written on the overall, although some Results parts could be shortened, and given the central and conserved roles of both TOR and WUS/Clv3 in promoting organ formation in plants, it is likely to be of interest for a large audience.This interesting manuscript investigates the regulation of the stem cell inducer Nevertheless, I have a few points that, to my opinion, should be addressed by the authors:WUS 5'UTR is included in the chimaeric construct used, which should be more clearly defined? This could be important given the translation regulations of WUS expression . Thus I am not sure that a transcriptional fusion with a reporter gene completely recapitulates WUS or Clv3 regulations.1) It is not clear from the Methods if the 2) Therefore I think that the data obtained with the fluorescent markers should be backed up, for the most important experiments, with another method like QPCR or even better immunolocalization. In situ hybridization was used but only for the first experiment.WUS induction by sugars or light is quite clear and interesting. However, this effect could be indirect and linked to the pleiotropic effect of TOR inhibition on development and metabolism. This part of the paper is mainly descriptive and the authors could try to find more mechanistic evidence by mining the available data on the impact of TOR inhibition on transcriptome or metabolome. The western blot used to measure S6K phosphorylation, a classical readout for TOR activity, could be improved. The molecular mass of both phosphorylated and total S6K bands should be indicated together with a picture of the whole membrane. Much less proteins seem to have been loaded for dark-grown plants? Also a control experiment with AZD should be performed to check for lower S6K phosphorylation.3) The effect of TOR inhibition on 4) Some differences in metabolite abundance are quite small and should be confirmed by statistical analyses to avoid over-interpretation of the results. All reviewers agree that your work is of general interest and in principle support publication. However, they also agree that a few issues have to be addressed before the work can be accepted. In your revision, please pay special attention to the following points:2-free atmosphere.1) The reviewers are worried about the validity of some of the pharmacological experiments because of possible indirect or side effects of the drugs (in particular of norfluorazon and lincomycin). Therefore, they believe that your paper would be substantially strengthened if you could repeat the basic introductory experiments  and 2 in2-deficient environment as proposed by reviewer #2 and described earlier in Kircher and Schopfer, 2012. The new experiments lead to the same conclusion as our previous experiments with norflurazon. The growth of plants in a CO2-deficient environment also inhibited development in light and could be rescued by the addition of sucrose to the medium  The reviewers also have some concerns regarding the plant material, especially with regard to the cop1 allele used. They ask that some of the basic experiments are verified with a cop1 null allele.cop1 mutants in several of our experiments. Since cop1 null mutants arrest at early seedling stage , we used the weak allele cop1-4. The cop1-4 mutant expresses a truncated COP1 protein that lacks the WD repeats. A recent publication shows that the comparatively weak phenotype of the cop1-4 mutant relies on the SPA proteins that can partially substitute the lack of the WD repeats that are missing in cop1-41. In absence of the SPA proteins the COP1-4 protein has no activity.We used cop1 mutants in darkness is not restricted to the cop1-4 allele. It has already been described in previous publications for the alleles cop1-1, cop1-6 and cop1-8 .Addressing the concerns of Reviewer #1, we want to point out that the development of cop1 mutant alleles  and cop1-19  and all confirmed the results obtained with cop1-4. We show that in all alleles tested WUS expression is induced in darkness  The reviewers acknowledge the use of reporters as an output for CLV3/WUS activity, but also wonder whether some of the basic observations could be confirmed by qPCR analysis of the endogenous genes?WUS reporter line because it is by far the most sensitive and robust way to quantify WUS promoter activity. We have characterized these reporters in great detail and found them to faithfully recapitulate in many different settings, including other transgenes, suggesting that positional effects have minor influence on their output (see Author response image 1pWUS:mCHERRY-GUS reporter in 4 day old seedlings as an example).Many conclusions of our manuscript are based on the analysis of the WUS expression by qRT-PCR to independently verify our results obtained with the reporter line. We were able to confirm our findings, however the lack of spatial resolution and the small number of expressing cells translated to low signal to noise ratio in the qRT assays. Therefore, we only detected mild expression changes of WUS with some variation in 4 day old seedlings grown in darkness, red light or in presence of sucrose . We believe it to be essential to analyze WUS expression with cellular resolution and thus view the qRT-PCR results as a qualitative rather than a quantitative confirmation of our reporter gene analysis.We want to point out that quantitative analysis of Please find the reviewer comments attached below for more details, and please respond to them to the best of your capacity.Reviewer #1:This manuscript from the Lohmann lab and colleagues presents a complex set of data, which indicate that SAM activation is both light- and energy-status dependent in Arabidopsis. Overall, the data appear solid and are presented in a transparent manner, and the conclusions are largely justified from my point of view. However, the authors' conclusions strongly hinge on the quantification of two reporters under different conditions or in different genetic backgrounds, and I believe some improvements are required to make the manuscript acceptable for publication.1) I have one major concern with respect to the genetic material. Several analyses are based on experiments with a mutant in COP1, a central negative regulator of photomorphogenesis. Understandably, the authors use one of the viable cop1 alleles in their work, the cop1-4 mutant. However, within the cop1 allelic series, this allele has always been the most mysterious. Unlike true null alleles, cop1-4 produces a truncated protein that comprises both the RING finger and coiled coil domains, yet alleles that retain these domains as well but are truncated further C-terminal or carry certain point mutations in the WD40 repeat domain also have a strong phenotype.Therefore, I believe it is important to check whether the results obtained with cop1-4 are representative. I understand that it would be too much to ask to repeat reporter experiments in another cop1 background, but I think the authors could check the principal physiological responses in true cop1 null alleles, which should be easily available. In particular, the authors could repeat the experiment of Done – Please see response to Reviewing Editor point 2 above.2) Another genetic concern: I believe the authors are formally right to conclude that HY5 has no role in light-dependent stem cell activation, however it is important to remember that unlike cop1, none of the COP1-target mutants has a phenotype that is as absolute skotomorphogenic as cop1 is photomorphogenic. If their conclusions with respect to cop1 are right, multiple mutants combining various COP1 targets should eventually show the pertinent defect. Among others, the hy5 hyh double mutant comes to mind. In the shoot, HYH is stronger expressed than HY5, and hy5 hyh double mutants have been reported to have meristematic defects, like "delayed" organ formation. I believe the authors should check light-dependent SAM activation at least in hyh single and hy5 hyh double mutants, and maybe also pertinent triple mutants with additional factors.The hy5 mutation did not have any effect WUS expression in our reporter line. However, Reviewer #1 is right in pointing out that other COP1 targets might be also involved in downstream signaling and therefore the effects of the hy5 mutation might be masked by redundancy. Since we cannot exclude that HY5 is involved in regulation of meristem activity, we now state in the text:"However, hy5 mutants showed no defect in activation of WUS expression , suggestand in the Discussion:hy5 on WUS expression . Since hy5/hyh mutants showed deficiencies in development of the first leaved future experiments analyzing such a double mutant might give further insights.""However, we cannot exclude that the HY5 homolog HYH with partially overlapping function but higher expression level in the shoot might mask any effect of hy5 double reporter background to test this hypothesis. However, these experiments will take six months to carefully execute and go well beyond the scope of this manuscript.We currently prepare CRISPR-CAS9 mutants of HYH in the 3) With respect to responses illustrated by images only, like cop1 shoot formation on +/- suc, are those responses fully penetrant? If so, this should be explicitly stated, if not, proportions clearly indicated.cop1-4 mutants developing leaves on sucrose. In contrast, only approximately one third of WT plants develop leaves on norflurazon CO2-deficient environment in light when supplemented with sucrose. In the absence of sucrose we never see development of the cop1 mutants in darkness or WT on norflurazon or CO2-deficient environment in light.We now clarify the percentage of plants with shoot formation in the text. We see that 100% of 4) One of the two ML1 transgenic lines deviates from the highlighted phenotype . Why is pML1:PHYB Y276H expressing lines in darkness, some showed a cop-like phenotype, while others resembled WT seedlings. We now show that this correlates with the expression level of PHYB by analyzing the total and endogenous PHYB expression by qRT PCR. Line AP88.2.1 (cop phenotype in darkness) shows 15x higher level of PHYB than line AP88.1.3 (WT phenotype)  . Additio5) With regard to the cytokinin experiments, and the sometimes inconclusive contribution, I wonder whether the actual cytokinin used is of importance? Given the variety of cytokinins and their reported shoot- and root-specific activities, the authors might find a "better" cytokinin, which might have a more clear effect on WUS expression.WUS expression. However, we did not see striking differences in the activation of WUS expression (see To elucidate the role of different cytokinin molecules we now assayed the effect of Kinetin and Zeatin on 6) Because of the importance of the fluorescence measurements, I would like the authors to confirm that samples analyzed for one graph were grown in parallel and analyzed with identical settings.All datasets were grown and analyzed in parallel with the reference sample (which is either the red light treated sample or the glucose treated sample) as well as an untreated (dark grown) sample and we now state so explicitly in the Materials and methods section:"For all these experiments plants of one experimental set were always grown and analyzed in parallel to the untreated (dark-grown) and the corresponding reference sample."not done at the same time. The plot shows WUS expression in dark-grown WT, red-light-grown WT and red-light-grown phyB mutants grown and analyzed days apart (box 1-3 and box 4-6).We already mentioned in the Methods that microscope settings were identical during all measurements. To illustrate that the measurements were highly reproducible we present an example of two experiments, which were Reviewer #2:2 free environment (see for example Kircher and Schopfer 2012). This would not lead to the rather drastic effects of these drugs on chloroplasts. In principle the effect of CO2-free air could be complemented by adding sucrose. Haydon et al., 2013 also complemented the NF effect by adding sucrose which to some extent rules out non-specific effects of NF.1) Based on WUS expression in cop1 +/- sucrose and WT +/- light and the use of NF and Linc , the aut2-free environment would be a good alternative for the norflurazon and lincomycin treatments. We now have carried out the suggested experiments and found them to confirm our earlier results. For further details please see response to Reviewing Editor point 1 above.We want to thank Reviewer #2 for pointing out that growth a CO2) The role of CK is potentially quite complex and I'm not sure that the current data allows drawing a clear conclusion. The authors propose that CK promote WUS expression by transmitting the light signal . In We appreciate the reviewers' thought on this complex matter and agree that the proposed role of CK in transmitting light signals likely is only one of many.ckx5/ckx6 mutants grown in darkness for a prolonged time do not develop leaves, even in the presence of sucrose  A potential difficulty with the role of CK in this process and the use of drugs like NF is that CK biosynthesis requires plastids and chloroplast development is promoted by CK. Not sure how this can be untangled but it should be discussed.2-free atmosphere in addition to the pharmacological treatment to show the dependency of CK signaling on energy and this new experiment confirmed our original findings (To disentangle the dependency of CK biosynthesis on chloroplast development we now grew plant in a COfindings .4) All figure legends should be more explicit and provide detailed information about the exact light conditions , presence of sucrose and numbers of seedlings used per experiment. For example, I don't see which data correspond to + sucrose (hatched box) in 2A.We tried to improve the readability of the figures and now add more information about the growth conditions in the figure legends. We also refer to the Materials and methods section, where we explained in detail the quantification of the fluorescence intensities.Reviewer #3:This interesting manuscript investigates the regulation of the stem cell inducer WUSCHEL (WUS) and of it repressor CLAVATA3 (Clv3) by metabolic, hormonal and light signals using fluorescent markers driven by the promoters of these genes. This manuscript covers a lot of ground and makes a thorough analysis of the factors regulating the expression of these reporter genes. Specifically the role of cytokinins as well as sucrose and sugars in inducing WUS expression is shown and the obtained data suggest that the Target Of Rapamycin (TOR) kinase plays a central role in connecting metabolic signals to WUS induction. This manuscript is well written on the overall, although some Results parts could be shortened, and given the central and conserved roles of both TOR and WUS/Clv3 in promoting organ formation in plants, it is likely to be of interest for a large audience.Nevertheless, I have a few points that, to my opinion, should be addressed by the authors:1) It is not clear from the Methods if the WUS 5'UTR is included in the chimaeric construct used, which should be more clearly defined? This could be important given the translation regulations of WUS expression . Thus I am not sure that a transcriptional fusion with a reporter gene completely recapitulates WUS or Clv3 regulations.WUS reporter and the regulatory regions used in the reporter have been shown to fully rescue the meristem defect of wus mutants in the context of a WUS-GFP fusion, which has been published and is also used in our study . To clarify the reporter design, we now state in the text:Reviewer #3 pointed out the importance of the 5'UTR in the regulation of WUS expression. The 5' and 3´ UTRs are part of our pWUS:3xVENUS-NLS construct (pTS81) was generated accordingly, using a pGREEN- IIS based binary vector with a 4.4 kb genomic WUS promoter upstream of the ATG and 2.8 kb WUS terminator downstream of the stop codon (pFK398) and a Basta resistance cassette.""The 2) Therefore I think that the data obtained with the fluorescent markers should be backed up, for the most important experiments, with another method like QPCR or even better immunolocalization. In situ hybridization was used but only for the first experiment.We have now repeated key experiments using qRT-PCR and confirmed our results obtained with the WUS reporter. For a detailed answer, please see response to Reviewing Editor point 3 above.3) The effect of TOR inhibition on WUS induction by sugars or light is quite clear and interesting. However, this effect could be indirect and linked to the pleiotropic effect of TOR inhibition on development and metabolism. This part of the paper is mainly descriptive and the authors could try to find more mechanistic evidence by mining the available data on the impact of TOR inhibition on transcriptome or metabolome. The western blot used to measure S6K phosphorylation, a classical readout for TOR activity, could be improved. The molecular mass of both phosphorylated and total S6K bands should be indicated together with a picture of the whole membrane. Much less proteins seem to have been loaded for dark-grown plants? Also a control experiment with AZD should be performed to check for lower S6K phosphorylation.Normalization of TOR activation assays is challenging in situations of diverging protein synthesis, such as light vs. dark, since TOR kinase has been described as a central regulator of protein synthesis. Therefore, we decided to load equal amounts of fresh weight to the gel instead of equal total protein amounts and to use unphosphorylated S6K1/2 for normalization. In our experience loading equal fresh weight results in rather similar amounts of unphosphorylated S6K1/2 signal, while protein levels correlated strongly with TOR activity, as expected. We now repeated the Western Blot analysis on AZD-8055 treated samples as control to confirm the specificity of the phosphorylation specific antibody . As requAs suggested, we have again screened the literature and databases for candidates that might explain the effect of TOR on SAM development. Obviously, several regulators could be identified, but since we cannot provide any evidence of the contribution of these candidates in meristem regulation we would like to constrain ourselves from speculation at this point.We hope that the manuscript text makes this point rather clear:"Characteristic expression changes that were described in response to glucose-TOR signaling  and E2Fa overexpression , namely affecting genes involved in ribosome biogenesis, protein translation and cell proliferation have also been identified in microarray analyses of shoot apex tissue derived from young seedlings . These genes were rapidly and synchronously induced by photosynthetically active light preceding organ growth, which lead us to hypothesize that stem cell activation in the SAM might also be under control of the TOR kinase."4) Some differences in metabolite abundance are quite small and should be confirmed by statistical analyses to avoid over-interpretation of the results.We now added statistical information to all metabolite measurements and also added a PCA that analyses the overall variability of our data sets and replicates.ReferencesArabidopsis Post-Embryonic Development and Photomorphogenesis. Mol. Plant 8, 479–481 (2015).1) Ordoñez-Herrera, N. et al. A cop1 spa Mutant Deficient in COP1 and SPA Proteins Reveals Partial Co-Action of COP1 and SPA during 2) Demidenko, N. V. & Penin, A. A. Comparative analysis of gene expression level by quantitative real-time PCR has limited application in objects with different morphology. PLoS One 7, 5–10 (2012).


CHRNs) on chromosomes 8 and 15. Here we use 1000 Genomes sequence data from 3 populations  to examine whether natural selection has occurred at these loci. We used Tajima’s D and the integrated haplotype score (iHS) to test for evidence of natural selection. Our results provide evidence for strong selection in the nicotinic receptor gene cluster on chromosome 8, previously found to be significantly associated with both nicotine and cocaine dependence, as well as evidence selection acting on the region containing the CHRNA5 nicotinic receptor gene on chromosome 15, that is genome wide significant for risk for nicotine dependence. To examine the possibility that this selection is related to memory and learning, we utilized genetic data from the Collaborative Studies on the Genetics of Alcoholism (COGA) to test variants within these regions with three tests of memory and learning, the Wechsler Adult Intelligence Scale (WAIS) Block Design, WAIS Digit Symbol and WAIS Information tests. Of the 17 SNPs genotyped in COGA in this region, we find one significantly associated with WAIS digit symbol test results. This test captures aspects of reaction time and memory, suggesting that a phenotype relating to memory and learning may have been the driving force behind selection at these loci. This study could begin to explain why these seemingly deleterious SNPs are present at their current frequencies.Much of the evolution of human behavior remains a mystery, including how certain disadvantageous behaviors are so prevalent. Nicotine addiction is one such phenotype. Several loci have been implicated in nicotine related phenotypes including the nicotinic receptor gene clusters ( Consequences of nicotine dependence are the leading cause of preventable death in the USA and it has been noted that some populations experience higher levels of addiction than others  but the CHRNs).It has been estimated that approximately 10% of the genome has been affected by linkage due to recent selective sweeps . However(CHRNs) are a heterogeneous class of cation (positively charged) channels expressed in the central and peripheral nervous system. There are 11 neuronal CHRN genes, each of which encodes a receptor subunit. The neuronally expressed nicotinic receptors consist of combinations of alpha and beta subunits, encoded in humans by 8 alpha  and 3 beta (β2-β4) genes [Neuronal cholinergic nicotinic receptors (CHRNA5) on chromosome 15 [In the body, the opening of these channels is controlled by the endogenous ligand, acetylcholine, a chemical produced by neurons to activate other nearby neurons. Nicotine, the major psychoactive chemical present in tobacco smoke is a chemical present in the environment that can also stimulate the opening of these nicotinic acetylcholine receptor ion channels . A numbeosome 15 ,10–14. Wosome 15 . Thus, tosome 15 . Althougosome 15 .Here we test the hypothesis that natural selection has acted on these genes. But if the null hypothesis of neutral evolution or demographic processes is rejected, why would nature seemingly select for this trait, especially given the fact that it is believed that nicotine has not been a part of our evolutionary history long enough, and in large enough quantities, for its effects to be visible in our genomes? One hypothesis is that selection acted on another phenotype and the effect on nicotine addiction was secondary and incidental, a genetic phenomenon termed hitchhiking.CHRNA7 knockout mice have impaired reaction times [CHRNA6 knockout mice show that this receptor plays a role in nicotinic modulation of dopaminergic transmission, an important component of learning and memory [Nicotine is known to have an enhancing effect on cognitive performance. It enhances the reorientation of attention in visuospatial tasks  and alteon times  and decron times , while, d memory . Based oCHRNA5-A3-B4 region on chromosome 15q25 and the CHRNB3-A6 region on chromosome 8p11. We provide strong evidence for selection in the CHRNB3-A6 region and moderate evidence for selection in the CHRNA5-A3-B4 region. However, there is only a modest correlation between nicotine dependence and score on the Wechsler Adult Intelligence Scale (WAIS) Digit Symbol test in our dataset. Overall, we suggest that one possible explanation for these results is that SNPs in these regions associated with risk of nicotine dependence are associated with natural selection acting at these loci to improve human memory and learning.To test this hypothesis, we use two summaries of genetic variation that have different statistical power to make inferences, depending on the model of selection and associated population demography. Two different methods were used for detecting natural selection at two loci relevant to nicotine dependence, specifically the LCT, CHRNA5-A3-B4 nicotinic receptor gene region and the CHRNA6-B3 gene region. Within each of these regions, we calculated sliding windows of 10 kb with an increment of 1 kb. We then compared the number of windows within each region with Tajima’s D values above 2 or below -2  to permutations of 10,000 randomly selected regions of the same size across the genome.We calculated Tajima’s D over the regions of the nicotinic receptors on chromosomes 8 and 15, as well as lactase on chromosome 2 as the positive control. As the program utilized does not incorporate ancestral information, all results are based on the folded frequency spectrum, i.e. the distribution of polymorphic sites according to the number of chromosomes that carry a given minor allele rather than the number of chromosomes that carry the derived, non-ancestral allele. Fig 1 shoLCT gene region as a positive control. LCT encodes the protein lactase and mutations in the region give rise to lactase persistence. The lactase persistence phenotype is due to changes in a regulatory region that enhances the expression of LCT located in intron 13 of the neighboring gene, MCM6 [LCT region in AFR, ASN and EUR. As expected, both the Asian and European populations show many windows above 2 but very few exist in the African population. All p-values are derived using permutations of 10,000 regions of the same size from across the genome. For Europeans and Asians, the proportion of windows with extreme values was 8.6% and 6.5%, respectively  and appears inconsistent with the null hypothesis of neutral evolution and demography at the LCT locus especially in the EUR populations. In contrast, the histogram for AFR shows few windows with extreme Tajima’s D values. Indeed, the proportion of windows with extreme values was <1% and was not significantly different from the negative controls, therefore we are unable to reject the null hypothesis of neutral evolution and demography at this locus in AFR.We utilized the ne, MCM6 . Fig 1A ectively . This isCHRNA5-A3-B4 gene cluster on chromosome 15. In this region, we analyzed a 120 kb region that includes the nicotinic receptor gene cluster as well as ~18 kb upstream of CHRNA5. This was done to ensure that we included the large region upstream of CHRNA5 that has been associated with regulation of the level of mRNA expression for CHRNA5 [CHRNA5-A3-B4 region in AFR, ASN and EUR. Overall, we observed increased numbers of sliding windows with extreme Tajima’s D values in each of the three populations, which is not consistent with a neutral evolution and demography in this region  [For the pendence . Fig 1C e (FTND) . The LD e (FTND) . The datLCT, CHRNA5-A3-B4 nicotinic receptor gene region and CHRNA6-B3 nicotinic receptor gene region. Within each of these regions, we calculated the iHS score for all SNPs for which we could unambiguously determine the chimp ancestral allele. We then compared the number of SNPs within each region with iHS values above 2 or below -2, to the 10,000 equally sized permutated regions that were chosen at random.LCT/MCM6 region as a positive control. LCT/MCM6 region for EUR, ASN and AFR populations. As expected, the AFR population shows few extreme values (only 3.2%) and this does not differ significantly from the negative control. In EUR and ASN, the overall average proportion of extreme values for this region is 80.4% and 9.1%, respectively . Using this dataset, we tested the association between genotypes and three of the most relevant phenotypes, namely scores on the Wechsler Adult Intelligence Scale (WAIS) Block Design, WAIS Digit Symbol and WAIS Information tests. These tests were designed to measure aspects of perceptual organization, processing speed and verbal comprehension, respectively .CHRNB3-A6 region and none of the three neurocognitive phenotypes had a significant association with SNPs in the region of CHRNA5-A3-B4 on chromosome 15 (not shown). Of the 17 SNPs in the CHRNB3-A6 region on chromosome 8, one SNP–rs7017612—passed multiple test correction (p≤0.003) for association with the score on the WAIS Digit Symbol test . rs7017612 lies in the intergenic region between CHRNB3 and CHRNA6. This SNP is highly correlated with rs6474413 , a SNP tagging the genome-wide significant bin for decreased risk for nicotine dependence. These data suggest a modest association between genotype at these SNPs one measure of cognitive function.CHRNB3-A6 region–rs6982753- had a nominal p-value with the WAIS Digit Symbol phenotype before multiple test correction and almost passed the multiple test correction (p = 0.009). Interestingly, this SNP has an r2 of 0.91 with rs892413 , a SNP that has previously been associated with increased risk for cocaine dependence [A second SNP in the pendence .CHRNB3-A6 locus. Both the Tajima’s D test and iHS point to an ongoing sweep in humans on chromosome 8. In the case of the CHRNB3-A6 locus, all of the extreme values in the Tajima’s D analysis were positive. High positive Tajima’s D values occur when there is an excess of variants in a region with intermediate allele frequencies. This can occur in either balancing selection or ongoing positive selection. We also found extreme iHS values in the CHRNB3-A6 locus. This region fulfills the criteria for a sweep laid out by Voight and colleagues [Many studies have demonstrated that risk for nicotine addiction has a genetic component. We performed two tests of selection on chromosomal regions containing the genes encoding five nicotinic receptor subunits and each of these analyses indicate that selection likely occurred at the lleagues , i.e. clCHRNB3-A6 locus on chromosome 8 have previously been associated with a decreased risk of nicotine dependence [Several SNPs in the pendence . One of Selective pressures in our ancestral environments were likely not on addiction, but rather on behaviors that were biologically rewarding . Given the role of nicotine in neurological function, it is possible that, in the case of nicotine addiction, the phenotype on which natural selection was working was related to enhancements in memory or cognition. The addiction phenotype would have hitchhiked along because it acts through the same or related mechanisms. The addiction phenotype was likely not selected against in ancestral environments because the availability and opportunity for prolonged use of purified drugs was negligible.CHRNB3-A6 locus with scores on WAIS tests of memory and cognitive function. Our analysis of the individuals in the COGA dataset suggests that one SNP, rs7017612, which lies in the intergenic region between CHRNB3 and CHRNA6, is associated with increased score on the WAIS Digit Symbol test. This test is thought to largely measure processing speed, but also, to some extent, memory. rs7017612 itself has not been reported to be associated with nicotine dependence. However, it is in moderately high LD (r2 = 0.75) with rs6474413, a SNP tagging the genome-wide significant bin for decreased risk for nicotine dependence. Thus, our data are consistent with the possibility that improved performance on this particular cognitive test is modestly associated with a decreased risk for nicotine dependence and that alleles of SNPs in these regions have effects on a subset of cognitive pathways best captured here by the WAIS digit symbol test. It is possible, however, that a function other than addiction or cognition is the true phenotype undergoing natural selection at these loci.To test this possibility, we assessed the association of SNPs in the CHRNA5, increases risk for the development of nicotine dependence, and independently decreases risk for cocaine dependence [Genetic studies of nicotine addiction have identified an inverse relationship between the risk for nicotine addiction and the risk for cocaine addiction. For instance, the minor allele of rs16969968, a missense variant in pendence . One hypAnother alternative hypothesis is that the selective pressure at this locus was on social behavior. Cocaine addiction is characterized by a dampened reward response to social interaction, meaning that it inhibits the positive emotions that accompany social interaction or feelings of belonging. A recent study demonstrated that cocaine users process social gaze (joint attention on an object) differently than controls, resulting in a reduced activation of the reward system during social interactions . Using fCHRNA5-A3-B4 locus. In particular, rs16969968, the SNP that encodes the missense mutation in α5, that is strongly associated with risk of nicotine dependence, lies in a sliding window exhibiting a high Tajima’s D score. The iHS analysis of this locus did not provide evidence for selection. This could indicate that the selective pressure exerted on this locus is older that that seen for the CHRNB3-A6 locus and as such has allowed extended haplotypes to be broken down by recombination. In this scenario, Tajima’s D would still be extreme while iHS scores in the region might be less so. This may be particularly true if the selective pressure being exerted on the CHRNB3-A6 locus is ongoing.There was also moderate evidence for selection at the Here we have used two statistical tests of selection and uncovered evidence of positive selection at the nicotinic receptors on chromosome 8 chromosome 15. Multiple drug-related phenotypes are associated with SNPs in or near these loci, however for several reasons it is unlikely that these phenotypes are the direct targets of this selective pressure. We have proposed two possible explanations 1) phenotypes related to memory and learning and 2) phenotypes related to social behavior. We were only able to discover a modest association with memory-related phenotypes, likely due to the small sample size. We also are as of yet unable to test this second hypothesis because we do not have data in our sample for a phenotype measuring sociality. However this work is the first to explicitly describe signs of natural selection acting on loci underlying substance dependence phenotypes.To determine whether the nicotinic receptor loci are under selection, we used Tajima’s D, and integrated haplotype score (iHS) to examine the landscape of natural selection at three loci previously demonstrated to harbor genetic variants contributing to the risk of nicotine dependence. These tests have different but complementary strengths. Tajima’s D test functions best on recently completed selective sweeps. There are many variables that contribute to how far in the past a sweep can be detected, such as how extreme the sweep was in the first place. Both the mutation rate and the recombination rate affect it as well and vary widely across the genome making generalizations difficult. By contrast, integrated haplotype score iHS functions best for detecting sweeps in progress with alleles at intermediate frequencies, mainly in the range of or after the separation of European, Asian and African populations, during the agricultural phase of human evolution.http://www.1000genomes.org/data), a third party source for population whole genome sequence data. The populations were grouped into EUR , ASN , and AFR . All methods were calculated for the same regions: the CHRNA5-A3-B4 region on chromosome 15q25, the CHRNB3-A6 region on chromosome 8p11, the LCT region as a positive control on chromosome 2q21, and ten intergenic negative control regions where applicable.We utilized 1000 Genomes data for these analyses. Data was obtained from the 1000 genomes website  should be equal. Tajima’s D tests for a skew in the frequency spectrum by comparing two estimates of θ – the number of segregating sites (S), and pairwise nucleotide diversity (π) . ExtremeIn a review, Garrigan & Hammer  have comTajima’s D was calculated using the program Variscan . After aiHS is a measure of whether a SNP is on an unusually long haplotype carrying the ancestral or derived allele. It compares the rate of haplotype decay between haplotypes carrying either the ancestral or derived allele at a given site, referred to as the core SNP. Haplotypes whose core SNP is under selection will be unusually long compared to those evolving neutrally. Long haplotypes with derived alleles are indicated by negative iHS values and those with ancestral alleles are indicated by positive iHS values. Under neutrality, extreme scores are distributed throughout the genome, however under selection, they are clustered across the selected region . iHS is The haplotype decay is calculated until the extended haplotype homozygosity (EHH) reaches 0.05. EHH is defined as “the probability that two randomly chosen chromosomes carrying the core haplotype of interest are identical by descent for the entire interval from the core region to point x”  (p. 833)CHRNB3-A6 where we selected plus or minus 2500 SNPs. We constructed recombination maps using cM maps provided by the SHAPEIT2 program [http://snp.gs.washington.edu/SeattleSeqAnnotation137/). Phased haplotypes were coded as number of copies of the derived allele. All positions in which the derived allele could not be determined unambiguously (i.e. C/G or A/T SNPs) as well as those without known chimp alleles were removed from further analyses. All analyses were run on each population separately. As iHS is greatly influenced by SNP allele frequency, iHS values from WHAMM were standardized using the average and standard deviation of all SNPs on chromosome 15 and 8 binned by allele frequency such that the average iHS value for each bin after standardization was identical. We excluded SNPs with a minor allele frequency less than 5% because low frequency SNPs are difficult to normalize accurately. After removing these SNPs, extracting just the desired gene regions, and removing those with MAF of <0.05, there were ~150–350 SNPs per region, depending on the population. Standardization was done separately for each population using population specific averages and standard deviations. iHS values were then superimposed onto graphs of the regions.First, we extracted the desired regions from the 1000 Genomes dataset. We then selected known SNPs within each region, and extracted a region of plus or minus 2000 SNPs around that SNP, except in the case of  program . AncestrThe haplotype on which a beneficial allele resides tends to be significantly longer than the other haplotypes at the same frequency in the population when adjusted for the recombination background. However, long haplotypes tend to occur in regions with low recombination, and these can be confused with genuine genomic signals of positive selection . This isCandidate regions of positive selection were defined as genomic regions containing an uncharacteristic clustering of SNPs with high iHS statistics. This was quantified as the proportion of SNPs with |iHS| > 2 in the four regions of interest. Candidate regions of positive selection were identified as containing any SNP with an iHS score of |iHS| > 2, as this corresponds to the top ~5% of all scores. The iHS value at a SNP “measures the strength of evidence for selection acting at or near that SNP” however does not provide a formal significance test .COGA recruited subjects in Hartford, Connecticut; Indianapolis, Indiana; Iowa City, Iowa; New York City, New York; San Diego, California; St Louis, Missouri; and Washington, DC. For inclusion in SAGE, cases had to meet lifetime criteria for DSM-IV alcohol dependence, the majority of cases were recruited from alcoholism treatment centers. Control subjects, were both biologically unrelated to cases, and had consumed alcohol but never experienced any significant alcohol or drug-related problems, according to the Semi-Structured Assessment for the Genetics of Alcoholism (SSAGA) . The COGCOGA administered a variety of neuropsychological tests to its subjects including the three used here: Wechsler Adult Intelligence Scale-Revised (WAIS-R) Block Design, WAIS Digit Symbol, and WAIS Information. In total there were 1247 European-Americans with these neuropsychological phenotypes. However, the overlap between this number and those with family GWAS data was 492. Therefore, our analyses were performed on 492 subjects. In WAIS Block Design, the subject replicates models or pictures of two-color designs with blocks. In WAIS Information, the subject answers a series of questions about factual information. In WAIS Digit Symbol, the subject writes down as quickly as possible the symbols that correspond to a series of numbers.SNPs in the region of the nicotinic receptor clusters on chromosomes 8 and 15 were tested for association with the scaled scores of neuropsychological phenotypes in European-Americans from the COGA study using an additive linear mixed effects (LME) model with the lmepack.batch function as implemented in the GWAF package in R using age, sex and FTND score as covariates . NeitherS1 Table2>0.9) with the SNP rs9298626.All SNPs were highly correlated (r(PDF)Click here for additional data file.


The United States Preventive Services Task Force (USPSTF) has recommended against testicular self-examinations (TSE) or clinical examination for testicular cancer screening. However, in this recommendation there was no consideration of the significant fiscal cost of treating advanced disease versus evaluation of benign disease. In this study, a cost-utility validation for TSE was performed. The cost of treatment for an advanced-stage testicular tumor (both seminomatous and nonseminomatous) was compared to the cost of six other scenarios involving the clinical assessment of a testicular mass felt during self-examination . Medicare reimbursements were used as an estimate for a national cost standard. The total treatment cost for an advanced-stage seminoma  or nonseminoma  equaled the cost of 313–330 benign office visits ($156); 180–190 office visits with scrotal ultrasound ($272); 79–83 office visits with serial scrotal ultrasounds and labs ($621); 6–7 office visits resulting in radical inguinal orchiectomy for benign pathology  or 2–3 office visits resulting in treatment and surveillance of an early-stage testicular cancer . A large number of clinical evaluations based on the TSE for benign disease can be made compared to the cost of one missed advanced-stage tumor. An average of 2.4 to 1 cost benefit ratio was demonstrated for early detected testicular cancer versus advanced-stage disease. Testicular cancer remains the most common solid malignancy in men between 15 and 34 years of age [Two-thirds of nonseminomatous germ cell tumors (NSGCTs) and 15% of pure seminomas present with regional or distant metastases. Symptoms associated with metastatic disease occur as the presenting complaint in 10% to 20% of cases –5. DespiIn 2004, the USPSTF made a Grade D recommendation for testicular self-examination (TSE) or clinical evaluation to screen for testicular cancer in asymptomatic males and then reaffirmed this recommendation in 2009 and again in 2011 , 7. GradWe created eight common clinical scenarios involving the evaluation and treatment of a testicular mass identified during self-examination: four benign (scenarios A–D); two localized or regional malignant (scenarios E and F); and two advanced-stage testicular cancer (scenarios G and H) Fig. . We focuCost analysis was based on the Medicare reimbursements. Most of the costs were determined using Current Procedural Terminology (CPT) codes and the par amount from the 2013 Kansas Medicare Physician Fee Database. Laboratory costs were obtained using CPT codes from the 2013 Medicare Clinical Lab Fee Schedule for Eastern Kansas . Chemotherapy drug costs were obtained using the January 2013 Payment Allowance Limits for Medicare Part B Drugs for the appropriate Healthcare Common Procedure Coding System (HCPCS) codes. In the various scenarios involving a testicular malignancy, we estimated the average cost of surveillance for 10 years following initial diagnosis and treatment.The total cost of a stage IA or IB seminoma was broken down into surveillance, chemotherapy and radiation-specific groups, whereas the total cost of a stage IA or IB nonseminoma was sub-divided into surveillance, chemotherapy, and retroperitoneal lymph node dissection (RPLND)-specific groups. The cost of a stage IS, IIA, or IIB seminoma was included in the radiation-specific group cost. The cost of Stage IS, IIA, or IIB nonseminoma was divided into either chemotherapy or RPLND-specific groups. The average cost of the surveillance, chemotherapy, and radiation treatment arms  was reported for scenario E. The surveillance protocol used for scenario E consisted of a clinical examination with tumor markers every 3 months for the first 2 years, every 6 months for the following 2 years and then annually for the next 6 years. Imaging consisted of a CT scan of abdomen/pelvis with chest x-ray every 6 months for the first 3 years and then annually for the following 2 years.The average cost of the surveillance, chemotherapy, and RPLND treatment arms  was reported for scenario F and the length of hospital stay accounted for following RPLND was 3 days (conservative estimate). The surveillance protocol used for scenario F consisted of a clinical examination with tumor markers and a chest x-ray every 2 months for the first 2 years, every 3 months for the following 2 years, every 6 months for the following year, and then annually for the following 5 years. Additional imaging consisted of a CT scan of the abdomen/pelvis every 4 months for the first year, every 6 months for the following 3 years, and then annually for the following 6 years.The total cost for an advanced-stage seminoma (stage IIC or III) was calculated assuming initial intermediate risk, partial response to chemotherapy with residual masses, a limited resection of these masses and then salvage chemotherapy (scenario G). The surveillance protocol used for scenario G consisted of clinical examination, tumor markers, and chest x-ray every 2 months for the first year, every 3 months for the following year, every 6 months for the following 2 years, and then annually for the following 6 years. Additional imaging consisted of CT abdomen/pelvis every 6 months for the first 2 years and then annually for the following 8 years. The total cost for an advanced-stage nonseminoma (Stage IIC or III) was calculated assuming initial intermediate risk, partial response to chemotherapy with residual masses, RPLND, and then salvage chemotherapy (scenario H). The surveillance protocol used for scenario H consisted of clinical examination, tumor markers, and chest x-ray every 2 months for first 2 years, every 3 months for the following 2 years, every 6 months for the following year, and then annually for the following 5 years. Additional imaging consisted of CT abdomen/pelvis every 4 months for the first year, every 6 months for the following 3 years, and then annually for the following 6 years.Breakdown of costs for each step of treatment with scenarios A through H are shown in Figure Based on the type of testicular cancer (NSGCT vs. seminoma), the total treatment cost for an advanced-stage testicular cancer equaled the cost of 313–330 (average 322) benign office visits (scenario A); 180–190 (average 185) office visits with scrotal ultrasound (scenario B); 79–83 (average 81) office visits with tumor marker laboratory testing and serial scrotal ultrasounds (scenario C); or 6–7 office visits resulting in radical inguinal orchiectomy for benign disease (scenario D). In terms of malignant disease; the detection, treatment, and surveillance of 2–3 (average 2.4) early-stage testicular cancers (scenarios E and F) equaled the cost of one advanced disease treatment with subsequent surveillance (scenarios G and H). In comparison, 125–168 (average 147) TSEs with negative clinical evaluations can be performed for the cost of treatment of one localized or regional testicular cancer.Data from SEER indicate that of the 8000 new testicular cancers diagnosed in 2013, ∼12% presented with advanced/distant metastatic disease. This equates to 960 cases per year, the cost of which will follow scenarios G and H as outlined. This suggests that an average of 309,120 negative clinical examinations (322 × 960) following scenario A could be performed in 1 year for the cost of all the distant metastatic disease treated in 2013. If the remaining 7040 cancers are defined as localized or regional, then they will follow the treatment and cost algorithms according to scenarios E or F. This would equate to 1.03 million (7040 × 147) negative clinical evaluations for all the localized/regional cancers treated in 2013. This indicates that a total of 1.344 million false-positive TSEs with a negative clinical examination could be performed for the cost of all new testicular cancer cases diagnosed and subsequently treated in 2013.In terms of cost alone, 960 advanced metastatic cases equated to approximately $48 million for the treatment of testicular cancer in 2013, when we calculated $50,000 spent per case if averaged between seminoma and NSGCT for scenarios G and H. For the remaining 7040 cancers in 2013, the cost could be estimated according to scenarios E and F with an average cost between seminoma and NSGCT of $21,737 per case. This equates to a cost for localized and/or regional treatment of $153 million in 2013. The total cost of testicular cancer treatment in 2013, by Medicare calculation alone, is estimated at $201 million.Testicular cancer remains the most common solid malignancy in young males. Delays in diagnosis can dramatically impact survival with 5-year median relative survivals dropping from 99% for cancer confined to the testis to 74% for metastatic disease . Moul etWith a lack of evidence examining the issue of screening for testicular cancer, the objective of our study was to determine the potential fiscal ramifications of TSE performed correctly, incorrectly or not at all and presenting with localized and advanced testicular cancer. We created practical clinical scenarios that are commonly encountered by any provider performing a scrotal exam. Since most men have testicular cancer only once in their life time it seems appropriate to compare fiscal effectiveness for a clinical evaluation of an abnormal TSE to the lifetime risk, which is 1 in 260 men. Based on the 313–330 false-positive or negative clinical evaluation visits for one advanced seminoma and NSGCT, respectively, it suggests that screening might be cost effective relative to a presentation of cancer as advanced disease. This was not seen with the ratio of 125 and 168 negative office visits to the cost of treatment of one early/localized and potentially early detected cancer.The study accounted for the possibility of a clinician being defensive or unsure/inexperienced with a testicular physical exam finding as presented in scenario B. Bosl et al. showed that the longest period of diagnostic delay was actually due to the physicians in two-thirds of cases . ScrotalWe showed that treatment of one early or regional testicular cancer is half the cost of an advanced case. We also estimated that the total cost of testicular cancer treatment in 2013 was $201 million. Of this, approximately $48 million would be for treatment of advanced metastatic disease and $153 million for localized and/or regional treatment. This is a significant difference or increase from the estimate published in 2000 . The 12%There were limitations of our study. We opted to use Medicare since it is the only national cost standard in the USA. We were aware that most men in the highest incidence age group will not be covered under Medicare, but this was the most practical method to standardize reimbursement. Reimbursement does not correctly represent true cost but we did not have access to this confidential data from our institution which varies between regions based on the contracts with vendors and insurance companies. Due to inconsistencies in hospital master charge lists and real costs, reimbursement by Medicare was considered the best standard. The Healthcare Cost and Utilization Project (HCUP) does not cover the scenarios presented. We worked with our regional Medicare payment scheme but are aware of regional variations and possible higher reimbursement according to the Geographic Practice Cost Index (GPCI). We did not average the cost across the USA, since the difference was not felt to be significant enough to warrant this extra analysis. Medicare reimbursements are often 20–40% below private insurances and as such we may have undervalued our cost analysis. We also did not account for future inflation in costs. Medicare reimbursement is prone to the least degree of variation overtime. We only factored in one new patient visit based on presentation to one provider, whether oncologist, urologist, or primary care provider. Adding additional one or two consults would not have changed overall cost or ratios significantly. We also did not calculate the cost of all possible scenarios with associated subtleties of cost. We only considered the most common presentations. We were unable to factor into our analysis the costs associated with lost work and lost or reduced fertility resulting from any treatment especially for advanced disease, with extensive chemotherapy and inpatient postoperative care after salvage RPLND. Furthermore, given the young age of the population, an analysis of lost quality-adjusted life years would demonstrate a greater cost. Anxiety from over investigation is mentioned as one of the USPSTF's concerns with TSE as a screening tool. We did not factor in the cost of anxiety induced from either management of a false-positive TSE or diagnosis of localized/metastatic cancer. This is a relatively intangible cost, unless related to time off work or mental health visits.Another limitation of the model was that we had to make some assumptions: men actually performed a TSE; they found an abnormality correctly or incorrectly with or without early or late symptoms; and they sought medical evaluation. We had to make an assumption that men will present with an abnormal TSE or constitutional symptoms from metastatic disease and get detected for the cancer, early or late. Although these are assumptions, they are in fact exactly how patients present anyway. We could not directly account for intratesticular masses presenting with metastatic disease, but the regional or advanced diseases scenarios pick up this possibility since this is how they will eventually present.Although cost alone does not equate to screening it does serve as a means to determine how many men can be seen. Testicular cancer is not preventable and may be very treatable as indicated by the USPSTF, but has significant morbidity and mortality despite this if detected late. Care of advanced disease is always going to cost more medically, psychologically, and financially than localized disease and it is important to reduce this cost, morbidity, and mortality through early detection and treatment. How that is achieved is difficult to determine without patient awareness and continued evidence-based research to validate early evaluation and detection, even if screening is not proven yet.