The near complete genome sequences of ten field avian orthoreovirus (ARV) strains collected from young chicken between 2002 and 2011 in Hungary have been determined in order to explore the genetic diversity and evolutionary mechanisms affecting ARVs in this region. Sequence analyses and phylogenetic calculations revealed similar geographic distribution and distinct evolution in case of eight studied strains that were closely related to the recently described Hungarian strain T1781. The remaining two strains showed the highest similarity with the US origin AVS-B. The topology of the phylogenetic trees of certain segments was affected by several potential homologous reassortment events between strains of Hungarian, Chinese and US origin. Analyzing the μB gene a possible heterologous reassortment event was identified in three Hungarian strains. Recombination events were detected in as much as a dozen cases implying that beside point mutations and reassorment this mechanism also plays an important role in the diversification of ARVs. All these mechanisms in concert may explain the reduced effectiveness of immunization using commercial vaccine strains. Reoviridae, subfamily Spinareovirinae, genus Orthoreovirus) are non-enveloped, icosahedral particles with a diameter of 70–80 nmAvian orthoreovirus, while other ARV-like strains, including the Tvärminne avian orthoreovirus (TVAV), the Bulbul orthoreovirus (Pycno-1) and the closely related Steller sea lion reovirus (SSRV), and the Psittacine reovirus strain Ge01, were suggested to be prototype members of further novel species, respectively23Avian orthoreoviruses , medium (M), and small (S)ARV infections are widespread and had been associated with various clinical manifestations in domestic poultry789Although the number of complete genome sequences available in GenBank is still limited, sequence data collected in the last few years suggest that the genetic material of ARV strains circulating in domestic poultry is continuously changing due to the well-known evolutionary mechanisms of reoviruses, resulting in genetically and antigenically heterogeneous strains. As observed in other RNA viruses, point mutation which is the primary mechanism of genetic and antigenic drift is relatively common. Antigenic shift, another evolutionary strategy, occurs when cognate genome segments reassort, and this mechanism is thought to be the driving force of the emergence of novel strains causing disease in poultry1112In this study we determined the near complete genome sequences of ten ARV field strains collected from young chicken with different clinical signs over a 10-year period in Hungary  in orderThe near complete genome sequences of ten, randomly selected Hungarian chicken ARV isolates had been determined by a modified sequence-independent, single-primer amplification method coupled with next-generation sequencing. The genomic organization of these ARV strains was similar and corresponded with that of the previously described reference ARV strains, AVS-B and S1133. With the exception of the S1, all genomic segments were found to encode a single protein and the homologues of the following orthoreoviral proteins were identified by open reading frame prediction and sequence comparison: λA, λB, λC, μA, μB, μNS, σA, σB, σC, σNS, p10 and p17. Where available, the 5′ and 3′ terminal sequences (GCUUUU[U/C] and UCAUC) were conserved among all segments as observed in other ARVs described so far5Sequence comparison of the Hungarian strains revealed 71.1–100/83.0–100% nucleotide sequence identity and aa sequence similarity values with the exception of the μB and σC genes where higher genetic variability was observed, and these values ranged between 62.7–99.8% nt and 71.3–99.7% aa for μB and 52.9–100% nt and 46.2–100% aa for σC, respectively , Table 2Eight out of ten Hungarian strains  showed the highest homology with T1781, another Hungarian ARV strain we characterized recentlyAvian orthoreovirus species . These Hungarian reassortant strains were collected in 2005, 2006 and 2011 in Hungary and strains possessing a closely related gene have never been detected before and after in any other countries. The first seven nt of the 5′ terminal sequences (GCUUUUU) of μB of the three strains showed high level of conservation with ARVs, implicating that this conserved terminal sequence may have been a prerequisite of the acquisition of this gene by reassortment. Apart from this conserved motif the 5′ untranslated region (UTR) sequences showed considerable variability and were 4 nt shorter (25 nt) when compared with the μB encoding genome segment of other ARV strains supporting the theory of the heterologous reassortment. However, alignment of the 5′ UTR containing orthoreoviruses belonging to other genera and unclassified strains revealed the highest similarity with ARV-like viruses, especially with NBV and Pulau virus (data not shown).Interestingly, when analyzing the μB gene, three Hungarian strains, 924-Bi-05, 3457-M-11, and 16821-M-06, formed a monophyletic group distinct from both waterfowl and other gallinaceous ARVs, suggesting that these strains might have acquired this gene through a reassortment event from a different, yet unknown host species  and 3. MThree distinct lineages of orthoreoviruses have been described in previous studies, suggesting a common evolutionary history of each of the following clades: (i) the reptilian orthoreovirus strains with Baboon orthoreovirus and Broome virus, (ii) the classical mammalian orthoreoviruses, (iii) the avian orthoreoviruses along with NBV, TVAV and SSRV. It is possible that reassortment might occur between species belonging to the monophyletic group of the third clade, but the exact genetic breakpoint remains to be discovered. The genetic and phylogenetic distance is considerably smaller between representative members of the different species in the ARV-like clade, than that of the two other clades. Although it is documented that reassortment occurs only between members of a given orthoreovirus speciesTo uncover whether recombination involving cognate genome segments occurs in ARV, particularly the Hungarian isolates, we performed recombination analysis with the studied Hungarian strains and sequences of ARVs (chicken and turkey) available in GenBank. Analysis of the coding region of each genomic segment revealed twelve putative recombination events in the λA (2), λC (3), μA (3), μB (1), μNS (2), and σA (1) genes. Details of the analyses are provided in Our results provide insight into the molecular characteristics of ARV strains randomly selected from a major strain collection. Genome sequencing and bioinformatics analyses of these strains provided new insight into the evolutionary mechanisms of ARV strains detected over a decade in Hungary. We conclude that lineage diversification, recombination involving cognate genome segments, reassortment among homologous strains and even among heterologous strains drive the evolution and genetic diversity of chicken orthoreovirus strains in Hungary. It remains to be studied whether similar mechanisms affect the genetic diversity of ARV strains in other parts of the world. A better understanding of the relative importance of these mechanisms will likely affect current vaccination strategies and the control and prevention of reovirus infections.Samples processed in this study originated from young chicken found dead following a course of disease with different manifestations in commercial poultry flocks between 2002 and 2011 in Hungary and submitted for post mortem evaluation to the Veterinary Diagnostic Directorate, National Food Chain Safety Office, Debrecen. Chicken ARV strains had been isolated from the collected specimens and the tissue culture supernatant of each isolate had been lyophilized for later use . Prior thttp://www.clcbio.com). To determine the missing sequences of the coding regions specific primer sets were designed. Direct sequencing was carried out applying the BigDye Terminator v1.1 Cycle Sequencing Kit (Thermo Fisher Scientific) and run on an automated sequence analyzer .Sequencing was performed using the protocol described in detail elsewhereContigs were aligned with Sanger sequencing reads using MultAlin online softwareThe genome sequences of the Hungarian strains were deposited in GenBank under the following accession numbers: KX398232-KX398331.No animal experiments were done in the study. The routine diagnostic investigations were performed according to the current directives specified in law 1998./XXVIII. and 2008./XLVI., and regulation 41/1997. (V. 28.).How to cite this article: Farkas, S. L. et al. Lineage diversification, homo- and heterologous reassortment and recombination shape the evolution of chicken orthoreoviruses. Sci. Rep. 6, 36960; doi: 10.1038/srep36960 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The extent, productivity and preservation of mangroves are controlled by the interplay of tectonics, global sea level and sedimentation, including tide, wave and fluvial processes. The impact of these processes on mangrove-bearing successions in the Oligo–Miocene of the South China Sea (SCS) is evaluated herein. Palaeogeographic reconstructions, palaeotidal modelling and facies analysis suggest that elevated tidal range and bed shear stress optimized mangrove development along tide-influenced tropical coastlines. Preservation of mangrove organic carbon (OC) was promoted by high tectonic subsidence and fluvial sediment supply. Lithospheric storage of OC in peripheral SCS basins potentially exceeded 4,000 Gt . These results highlight the crucial impact of tectonic and oceanographic processes on mangrove OC sequestration within the global carbon cycle on geological timescales.Modern mangroves are among the most carbon-rich biomes on Earth, but their long-term (≥10 Mangroves are important carbon-rich biomes, but their long-term impact on the global carbon cycle is relatively unknown. Here, the authors show that the Oligo–Miocene mangroves in the South China Sea stored in excess of 4,000 Gt, demonstrating the long-term impact of mangroves on carbon storage. Sedimen erosion 11. The e erosion 11. Sedim3 km) distribution of modern mangroves is controlled by climate , salinity and sea-level fluctuations12712131417The geographical 10–100–103 km Here we integrate palaeogeographic reconstructions, palaeotidal modelling and sedimentary analysis and suggest that higher tidal range and stronger tidal currents caused widespread mangrove development along ancient shorelines in the Oligo–Miocene South China Sea (SCS). Furthermore, this has decreased through time due to tectonic-driven changes in tidal dynamics. We also show that preservation of mangrove OC was optimized along tide-dominated coastlines in the Oligo–Present SCS, which could be a significant component of the global carbon cycle on geological timescales.Southeast Asia comprises a complex mosaic of geological terranes assembled along a system of active and extinct subduction and collisional zones. Critically, the region is also located at the triple junction between three major tectonic plates: the Eurasian, Indo-Australian, and Pacific platesThe Luzon Strait (LS) between Taiwan and the Philippines was wider in the past than the present day . The LS Uncertainty in the palaeogeographic position of Palawan stems from a lack of conclusive data regarding Jurassic–Palaeogene plate reconstructions and the genesis of the Northwest Borneo–Palawan Trough21Tectonics caused significant subsidence in shelf basins in the SCS during the Oligo–Miocene; extension in western and northern basins 16.Tidal modelling (see Methods section) of three representative Oligo–Miocene palaeogeographies and palaeo sea levels  arc  had a siTidal range and bed shear stress model output has been compared with sedimentological and biostratigraphic data from the Oligo–Miocene SCS region. This indicates broad agreement between model results and previous palaeoenvironmental interpretations24In the Nam Con Son Basin, the Late Oligocene–Early Miocene Dua Formation comprises paralic  coals and mudrocks, commonly containing mangrove palynomorphsIn the Pattani Basin, Early–Late Miocene mudstone/coal source rocks with common mangrove pollen are interbedded with medium-grained sandstones —basin 4;24Thalassinoides-like trace fossils, may preserve burrow networks typical of the lobster Thalassina in modern mangroves and seagrasses and intensely bioturbated mudstones, typical of intertidal mud flats seaward of mangrove forestsIn northwest Borneo, the Early Miocene Nyalau Formation of the Balingian Province, Sarawak Basin —basin 7 31The gross depositional environment was a variably tide- and wave-influenced deltaic shoreline, which included mangrove colonization of the intertidal lower coastal-deltaic plain 303436. D3034Total organic carbon (TOC) burial in the Oligo–Miocene SCS, which contains a significant component of mangrove OC2322 since the Middle Miocene (c. 15 Ma)  . This co1 ref. 23; an esti1 ref. 23 suggests per Myr . This re2 per Myr (n=20) measured in the western BDP−1)414, sediments deposited in the BDP were probably exposed to O2 for only a few decades. A maximum average TOC of 1% for sediments deposited in the BDP since 15 Ma may therefore be plausible, especially with an expanded and more productive coastal mangrove biome. An average TOC of 1% would correspond to c. 1,800 Gt OC burial, equivalent to sequestration of 48 p.p.m. atmospheric equivalent CO2 per Myr  of this volume is accounted for by mid-Miocene (c. 15 Ma) to present sedimentation in the BDP. Assuming that the amount of OC buried per unit sediment volume in present-day shelf basins of the SCS was equivalent to that calculated in the BDP , we estimate that between c. 40 and c. 2,000 Gt of OC was buried in SCS shelf basins since 34 Ma 3; see Methods section) is due to OC deposition in a range of coastal–deltaic to deep-marine environments, reworking and decomposition on geological timescales. Assuming a maximum average TOC value of 1% suggests burial of c. 41,000 Gt of OC in SCS shelf basins since the mid-Miocene  increase in accommodation space for mangrove accumulation1832Preservation of mangrove sediment depends on the interplay between the rates of accommodation space creation and sedimentationVertical stacking of tide- and wave-dominated stratigraphic units (facies successions) in the Balingian and Baram Delta provinces  may have32During RSL changes, mangroves must re-adjust to the changing intertidal area to maintain their capacity to store and bury OC1347During both RSL rise and fall, the depth to which preceding mangrove deposits are eroded may be insignificant compared to the thickness of preceding sedimentation. Many peripheral SCS basins have undergone >5 km subsidence since the Oligocene : (1) c. 13Mangroves are one of the most efficient links connecting the atmospheric, biospheric and lithospheric reservoirs of carbon38Highstand palaeogeographic reconstructions for Southeast Asia at seven timeslices during the Late Oligocene–Late Miocene  were gen161821Subsidence curves for 10 peripheral SCS and Borneo basins  from the offshore Balingian Province (Sarawak Basin); (2) outcrops (n=10) of Nyalau Formation in the onshore Balingian Provincen=14) of the Lambir Formation in northeast Sarawak ; and (4) outcrops (n=14) of the Belait Formation in northeast Brunei (BDP). This was supplemented by a regional review of published and unpublished sedimentological and stratigraphic data for additional peripheral SCS and Borneo basins  corFluidity is a finite element hydrodynamic model that uses unstructured, tetrahedral meshes to maximize computational accuracy and efficiency2 and K1 tides in the SCS compare favourably to models that include data assimilation: (1) global tidal models ATLAS TPXO8 and FES2012, which both used structured meshes with c. 20 km (1/6°) and c. 7 km (1/16°) resolution, respectively2 tide, Fluidity underpredicts the amplitude in the Philippine Sea  in the western SCS , are derived by two methods: Method 1—using estimates of total in-place petroleum resources in hydrocarbon fields in Brunei2, in peripheral Borneo and SCS basins throughout periods of the Oligocene–Present, we first assumed an equivalent amount of OC burial per unit sediment volume to that calculated in the BDP since the Mid-Miocene (using Methods 1 and 2), and second calculated various estimates of total sediment volume across the region during the Late Oligocene–Present (−3 and that 82% of sediment mass deposited in the SCS since the Oligocene (c. 34 Ma) was deposited in present-day shelf basinsEstimates of the total amount and rate of OC burial, and equivalent concentration of CO–Present . These i–Present  since thThe rate of OC burial in modern mangroves bordering the SCS  was calculated using estimates of mangrove area in countries bordering the SCSThe proportion of hydrocarbons dominantly sourced by terrestrial- and mangrove-dominated OC  in basins in the SCS region was estimated based on the recoverable hydrocarbon volumes and dominant source rocks in ref. The palaeogeographic and tidal modelling data sets generated during and/or analysed during the current study are not publicly available due to confidentiality restrictions but are available from the corresponding author on reasonable request and with the permission of Getech. The sedimentary and OC data sets are available from the corresponding author on reasonable request.How to cite this article: Collins, D. S. et al. Tidal dynamics and mangrove carbon sequestration during the Oligo–Miocene in the South China Sea. Nat. Commun.8, 15698 doi: 10.1038/ncomms15698 (2017).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables and Supplementary References


The testis as a site for atherosclerotic changes has so far attracted little attention. We used the apolipoprotein E (ApoE)/low density lipoprotein (LDL) receptor deficient mouse model (KO) for atherosclerosis  to investigate whether Leydig cells or the capillary network are responsible for reduced serum testosterone levels previously observed in extreme ages of this model. In KO mice, overall testosterone levels were reduced whereas the adrenal gland-specific corticosterone was increased excluding a general defect of steroid hormone production. In addition to micro-CT investigations for bigger vessels, stereology revealed a reduction of capillary length, volume and surface area suggesting capillary rarefaction as a factor for diminished testosterone. Stereological analyses of interstitial cells demonstrated significantly reduced Leydig cell numbers and size. These structural changes in the testis occurred on an inflammatory background revealed by qPCR. Reduced litter size of the KO mice suggests hypo- or infertility as a consequence of the testicular defects. Our data suggest reduced testosterone levels in this atherosclerosis model might be explained by both, rarefication of the capillary network and reduced Leydig cell number and size. Thus, this study calls for specific treatment of male infertility induced by microvascular damage through hypercholesterolemia and atherosclerosis. Focal tubular sclerosis was also reported in association with atherosclerotic lesions of interstitial testicular vessels2.Decline of androgen blood levels, dysfunctional sperm, and histologic changes, such as focal atrophy of seminiferous tubules and reduction of tubular diameter, are considered signs of male hypogonadism6. In man, early investigations of undescended testes linked reduced blood perfusion to atrophy and hypoplasia of the organ7. Reduction of blood flow due to decreased diameter of microvascular lumen in testes was suggested as a possible cause for degenerative changes of germ cells, oligospermia in humans8 and age-dependent disturbances of spermatogenesis10. A more recent study demonstrated that testicular vasculature might also be important for germ cell defects found in Klinefelter syndrome11.For a given organ, a healthy capillary vascular bed with its regular architecture is the prerequisite for adequate perfusion and function of this organ. The appearance of a regular testicular microvasculature was described previously for different species14. Vice versa, testosterone administration was reported to inhibit atherosclerosis16 implying that lower testosterone levels promote atherosclerosis and coronary artery disease17.Testosterone deficiency was reported together with signs of atherosclerosis in epidemiological studies, however, there is currently no evidence for a causal relationship20.Atherosclerotic plaques develop by retention of lipids in large- and medium-sized arteries as a consequence of shear stress to the vascular wall initiating platelet aggregation and blood coagulation. Plaques decrease downstream blood flow by reduction of the vascular lumen and thus have a negative impact on effective local blood circulation. Although evidence supports local immune processes induced by atherosclerotic plaques, there is considerable data to suggest an additional systemic immune response characterized by proinflammatory cytokines, such as TNFα released by endothelial cells, macrophages, and platelets−/−/LDL receptor−/− mice) displaying enhanced plasma cholesterol levels. In this model a lipoprotein pattern resulting in hypercholesterolemia is caused by targeted mutations of both apolipoprotein E (ApoE) and the low-density lipoprotein (LDL) receptor21. ApoE, mainly synthesized in the liver, transports lipoproteins and cholesterol into the lymphatic system and is a ligand of the LDL receptor family. In the ApoE−/−/LDL receptor−/− model, fatty streaks typical of advanced atherosclerotic lesions were observed in the proximal aorta and renal artery with increasing age22. In general, the degree of atherosclerosis detectable in wild-type (WT) mice is less pronounced compared to men. However, homozygous ApoE−/−/LDL receptor−/− mice develop atherosclerotic alterations more similar to humans23. Disturbances of vasculature in ApoE and ApoE/LDL receptor deficient mice, respectively, were found not only in aortic root but also in lung24, heart25, kidney22 and bladder26.The relationship between vasculature and morphological alterations in different organs was investigated more systematically in a mouse model of atherosclerosis  mice and male C57BL/6 J  mice  as controls were fed standard chow ad libitum throughout the study. They were euthanized with CO2. In both groups, KO and WT mice, animals of 20, 40, 60 and 87 weeks of age were used. Each age group included 10 mice except the oldest group (87 weeks) with three KO mice and six WT mice.This animal study was conducted in accordance to the German Animal Welfare Act and approved by the regional council of Giessen, Germany (GI 20/25A 43/2011). Male ApoEFrom each animal blood was collected from the vena cava caudalis for determination of steroids.27.The left ventricle was penetrated by a cannula and infused with heparinized saline (10 ml of 0.9% sodium chloride with 1000 IU Heparin)27.When the venous effluent was free of blood, the fixation solution  was infused and one testis was removed for histological morphometry, the ventricle was purged again with heparinized saline, and infused with the lead-containing radiopaque polymer  for micro-CT investigations and voxel-based morphometry as described previouslyFor analyses of inflammation-associated parameters by real-time PCR 37-week-old KO and WT mice (n = 10) on standard chow were used.The blood samples from mice of the different age groups were centrifuged and the separated serum was investigated by gas chromatography-mass spectrometry.28, consisting of many sub-steps, includes over-night incubation of the probes, extraction of the steroids by Extrelut® columns , filtration with Sephadex LH-20 columns  and subsequent derivatization with HFBA .This methodThe contrast agent-perfused testes of mice from the different age groups were scanned in a micro-computed tomograph  to investigate testis volume and vessel volume.27.The x-ray system consists of a microfocus tube (20–80 kVp) with a minimum spot size of 8 Im at 8 W. While the sample carrier rotates at 180° at its own axis the sample is irradiated by x-rays in cone-beam geometry to generate projection imagesThese digital images were reconstructed in cross-section data sets by modified Feldkamp-algorithm with isotropic voxel geometry, so 3D images were available for evaluation by analysis software  to investigate testis volume and vessel volume in the agent-perfused testes of WT and KO mice.30. For this, testes of 20, 40, 60 and 87-week-old mice were fixed by vascular perfusion with a mixture of 1.5% glutaraldehyde and 1.5% paraformaldehyde in 0.15 M HEPES buffer. The testis volume was estimated by fluid displacement31 and the testes were cut into cubic tissue pieces (approximately 1 mm³) from which a random selection was osmicated, stained en-bloc with uranyl acetate and finally embedded in epoxy resin . The samples were randomly dropped into gelatine capsules (one sample for each capsule) to ensure random tissue orientation32. After embedding, semi-thin sections (0.75 µm) were cut using an ultramicrotome and stained with methylene blue. This procedure is well known to allow identification of all testicular cell types under physiological and pathological conditions35. In most cases, five sections from different tissue blocks per testis and mouse were investigated at the age of 20, 40, 60 and 87 weeks using design-based stereology18. Specifically, the length, volume and surface area of the capillaries of the testes were estimated. For this purpose, fields of view were sampled by systematic uniform random sampling at a magnification of 400×  = P(cap)/P(testis). The surface density of the luminal capillary endothelium was estimated by counting the number of intersections (l(cap)) of test line segments with the luminal endothelium and relating them to the number of endpoints of the line segments hitting testis (P(testis)). The surface density is given by SV(cap/testis) = 2*l/(l(p)*P(testis)) where l(p) is the length of an individual test line associated with one endpoint. The length density was estimated by counting the number of capillary profiles (Q(cap)) within a counting frame of known area (A(CF)) and the number of corner points of the counting frame hitting testis P(testis). The length density is given by LV(cap/testis) = 2Q/(a/p*P(testis)) where a/p is the area of the counting frame associated with one corner point. The total capillary volume, surface area and length were calculated by multiplying the density with the testis volume36.Stereology was used as a method that allows the quantification of three-dimensional parameters of a given organ from histological sections by superimposing appropriate counting test systems like point grid, line grid and counting frame, on randomly sampled organ sections32 with a disector height of 3 µm (d). After staining, these paired sections were subjected to a systematic uniform random sampling and an unbiased counting frame with a known area (A(CF)) was projected on the fields of view. The number of Leydig cells was estimated by the disector principle, i.e. a cell was counted if the cell was visible in one section but not the other one (Q−). In addition, the number of corner points of the counting frame hitting the interstitium (P(interstitium)) was counted to estimate the reference volume. The number of Leydig cells in relation to the interstitium is given by Nv(LC/interstitium) = Q− /((A(CF)/4)*P(interstitium)*d). Based on this, the number of Leydig cells in relation to the entire testis is given by L = Nv(LC/interstitium)*Vv(interstitium/testis)*V(testis). The disector was not only used for counting of cell number but also for sampling of the cells for volume estimation using the rotator37, i.e. if a cell was counted its volume was estimated by the rotator. From the resulting individual cell volumes, the number-weighted mean volume was calculated as the arithmetic mean of the measured cell volumes.To investigate Leydig cell number and size, epoxy resin blocs were cut in 1 µm semi-thin sections. To exclude duplicate counting of Leydig cells, the first and fourth section of a consecutive row of sections were placed on a glass slide to generate a physical disectorThe number of pups per litter and relative frequency of litter sizes were retrospectively evaluated in KO mice compared to WT mice. For that we analyzed data of 109 litter of KO and 43 litter of WT mice, every litter developed from 1:1-matings of young females and old males.Total RNA from testis and epididymal fat was extracted using RNeasy Mini Kit according to the manufacturer’s protocol. Total RNA (1.0 μg) was used as template for cDNA synthesis by using MMLV RT (Invitrogen). For quantitative comparisons, cDNA samples were analyzed by real-time PCR using the IQ SYBR Green Supermix on the StepOne Plus real-time polymerase chain reaction system (Applied Biosystems). Primer sequences are given in the supplement (Suppl. Table).38. A value of p < 0.05 was considered significant.The statistical analysis was performed using the statistics software R, version 3.0.240.For each parameter  the potential influence of age  and mouse-strain (KO vs. WT) was investigated by a two-factorial ANOVA (with interaction-effect of age groups and mouse strain). When indicated this was complemented by multiple mean comparisons of the two mouse strains for the four age groups and simultaneous confidence intervals for the respective differences in mean valuesThe joint (multivariate) distribution of families of capillary parameters  was analyzed using a MANOVA (with the same two factors as above and their interaction).43. Since detection of relationships between variables is of primary interest, and to simplify the presentation, scale divisions or units are not provided, but only their orientation (by indicating their minimum and maximum).Three-dimensional scatter plots and parallel plots were used as exploratory tools for the presentation of the multivariate distribution of the mentioned parameter families. Parallel plots attempt to display relationships between several metric variables  by connecting their one-dimensional (=univariate) scatter plots. To this end, the measurement scales of the variables were placed parallel to each other, and the values that belong to the same multivariate observation, i.e. individual, were linked by line segments across the univariate scatter plots. This allows us to identify the values that constitute a multivariate observation by tracing the respective connected line segments (“polylines”) through the univariate scatter plotsThe distribution of litter size of the two mouse strains was compared using Fisher´s exact test and the arithmetic means of those distributions were compared with Welch´s two-sided two-sample t-test .n denoting the number of independent experiments unless otherwise indicated. A Welch´s two-sided two-sample t-test was used to determine statistical significance for qRT-PCR results.Data are given as mean values ± SEM, with −/−/LDL receptor−/− mice (Stock No: 012307) and C57BL/6J (Stock No: 000664) are now available from Jackson Laboratory .ApoEThe datasets analysed in this study can be made available from the corresponding author on reasonable request.−/−/LDL receptor−/− mice under additional Western diet27. To investigate whether testosterone serum levels are also reduced independently of ageing and dietary effects we compared WT and KO mice between 20 and 87 weeks on standard chow.Reduced serum testosterone levels had been described in very old ApoEIn KO mice, the overall serum testosterone levels were reduced as opposed to WT mice (p < 0.05) Fig. . Additio−/−/LDL receptor−/− mice, we first analysed whether decreased testis and vessel volume, previously observed in old KO mice under Western diet, are inherent characteristics of this atherosclerosis model. Indeed, without particular diet overall testicular  to account for their interdependency reflecting the complexity of the microvascular network. Figure −/−/LDL receptor−/− mice. Furthermore, for WT mice the variability of capillary parameters appeared to increase from 40-week-old to older  animals (reflected in the slightly wider spread of the polylines in the higher age groups).Interestingly, the overlap of KO and WT polylines decreased from 40 over 60 to 87-week-old mice (being responsible for the respective increase in differences between the average polyline-trends of WT and KO), suggesting an age-dependent reduction of micro-vessels in ApoEThe multivariate mean values of absolute capillary parameters for all combinations of age group and strain are also presented as three-dimensional scatter plots in Fig. Likewise, the multivariate mean values of the relative  capillary parameters are shown in Fig. These structural changes were accompanied by increased gene expression levels of pro-inflammatory cytokines in KO opposed to WT mice with TNFα and VEGF reaching a level of statistical significance. Interestingly, the macrophage-specific marker F4/80 was upregulated Fig. .In summary, these findings suggest that testicular microcirculatory changes occur also at the capillary level in this atherosclerosis model and could contribute to the observed reduced serum testosterone levels.Next, it was tested, whether potential changes of Leydig cell number and size could also represent a factor for reduced testosterone levels in the KO mice.−/−/LDL receptor−/− mice (p < 0.001)  showed a significantly reduced Leydig cell number in ApoE01) Fig. . Analyzi01) Fig. .Figure 63, the mean KO Leydig cell was only about half the size of the WT Leydig cell with 1331.84 µm3. This reduction of the cell size was also readily visible in the histological pictures  smaller in KO mice when comparing data from pooled age groups  Fig. . Reduced−/−/LDL receptor−/− mice. In agreement with the outcomes of hormonal, vascular and microvascular parameters congenic homozygous back-crossings of young females and old males exhibited a significant difference to the litter size distribution of WT mice (p = 0.0117), and in particular a significantly different mean litter size (p < 0.001) . The testicular capillary network surrounds the seminiferous tubules and runs in the interstitial tissue of the testis comprising scarce connective tissue and the Leydig cells−/−/LDL receptor−/− mice may affect Leydig cells and their function at different levels. By limiting the supply of nutrients, general aspects of cell function may be jeopardized. Testosterone production, the Leydig cell specific function, depends on the local supply and uptake of cholesterol as a substrate48. Similarly, LH as the main stimulator of Leydig cell function is delivered to its target cell by capillary perfusion47 and in a model of Sertoli cell ablation, vascular disturbances prevented LH from reaching its target49.Capillary networks are differently influenced by atherosclerosis depending on nutritional conditions of endothelia. The testicular capillary network which is rarefied in ApoE51 supporting that the Leydig cell functions at the brink to hypoxia. In humans chronically exposed to high altitude thus hypobaric hypoxia, serum testosterone levels were significantly reduced52.In line with an adequate afflux of nutrients, substrates and stimulants, the Leydig cell also requires a sufficient oxygen supply to match the metabolic demand for testosterone synthesis. Interestingly, HIF1alpha is constitutively expressed in Leydig cells even under normoxic conditions53. Endothelial cells initiate vessel formation and arteriosclerotic disease has been shown to interfere with endothelial regenerative function in various organs. In ApoE−/− mice with chronically elevated cholesterol levels, endothelial barrier and repair function has been compromised after transient cerebral artery occlusion and VEGF-induced capillary formation of brain endothelial cells was attenuated54. Similarly, in a model of hindlimb ischemia abundant cholesterol inhibited endothelial proliferation and migration55. In the kidney, cortical microvascular remodeling was concomitant with increased intrarenal protein expression of MCP-1 and TNFα56 indicating associated inflammation. Increases of inflammatory cytokines TNFα and interleukin-6 were reported in regard to endothelial damage and are considered features of both endothelial dysfunction and early stages of plaque formation. Various markers for inflammation, endothelial dysfunction and plaque formation are therefore of interest and in our study, we have clearly identified increased inflammatory cytokine gene expression in ApoE−/−/LDL receptor−/− testis as compared to wild-type testis.Hypoxic conditions may be balanced by formation of new micro-vessels, this has been shown e.g. in the aortic wall57, this is in agreement with the testicular microvascular changes observed in the present study. Arteriosclerotic disease may prevent adequate formation of new micro-vessels in the testis and result in the observed rarefication of the capillary network in our ApoE−/−/LDL receptor−/− model.In atherosclerosis, vascular function is compromised by oxidized LDL which induces endothelial dysfunction and injury, not only in atherosclerotic plaques of large arteries, but also in micro-vessels−/−/LDL receptor−/− model reduces the surface area available for transendothelial exchange and thus likely limits testosterone transfer into the blood stream which results in lowered serum testosterone levels as also seen in a model of Sertoli cell ablation with consecutive vascular disturbances49. With limited perfusion of the testis, the local supply of sex hormone binding globulin (SHBG), the plasma transport protein for testosterone, may also be reduced and contribute to lower serum testosterone levels as suggested before in a xxy mouse model58.Adequate capillary perfusion in the testis is necessary for efflux of steroids to the general circulation. Rarefication of the capillary network in our ApoE2O2 or advanced glycation end products (AGEs) have been reported to interfere with testosterone production60.Similarly, the rarefied capillary network may interfere with removal of waste products and cause oxidative stress in the testis. Reactive oxygen species (ROS) like H−/−/LDL receptor−/− testis. Changes of Leydig cell parameters in the ApoE−/−/LDL receptor−/− mouse could be due to indirect (vasculature-mediated) or direct effects on the Leydig cells or a combination of both.In addition to reduced serum testosterone levels we also found a reduced number and size of Leydig cells in the ApoE62 indicating that atherosclerotic changes in ApoE−/−/LDL receptor−/− mice cause a reduction of Leydig cell number.The Leydig cell number of our control (wild type) mice is in agreement with previous findings−/−/LDL receptor−/− mice might reflect reduced (i) volume of smooth endoplasmic reticulum, previously shown to correlate with lower testosterone levels63, and (ii) fewer mitochondria as found in conditions of oxidative stress64.Reduced Leydig cell size in ApoE67.However, it is unlikely that LDL receptor mutation especially in Leydig cells represents the main cause for reduced Leydig cell number and size. In contrast to diminished testosterone levels, serum concentrations of another steroid hormone, corticosterone, were not reduced in KO mice, but instead, significantly elevated. It is conceivable that corticosterone release was stimulated by inflammatory cytokines and nutritional overflow which in turn accelerates the development of atherosclerotic changes in ApoE-deficient mice68, could argue for vasculature-mediated aspects of Leydig cell changes.Studies describing reduced total Leydig cell volume in rats exposed to hypobaric hypoxia69. Rarefication of the capillary network as observed in the ApoE−/−/LDL receptor−/− model would diminish the availability of progenitors and thus impair regeneration potential.It is also conceivable that the reduced Leydig cell population is due to the contribution of the testicular capillary network to the regeneration of Leydig cells. Pericytes located within the capillary walls have been shown to serve as Leydig progenitor cells during postnatal development and to regenerate the Leydig cell population after ablation by EDS−/−/LDL receptor−/− mice exhibit mutations of both the receptor and its ligand they are considered an appropriate model to investigate atherosclerosis without atherogenic diet23. As opposed to our previous study utilizing Western diet protocol27, in the present study mice were administered regular balanced rodent chow to discriminate the mutant phenotype from dietary effects. In addition, the experimental design of this study included defined age groups . The members in younger groups outnumbered oldest because some mice died before the age of 87 weeks. High death rate was described in ApoE-deficient mice before71. In this study KO mice were compared to WT mice at exactly the same age. Thus, animals with severest atherosclerosis might have escaped on time analysis resulting in negative experimental bias and rather underestimate changes in the capillary parameters.Since ApoE−/−/LDL receptor−/− mice also revealed a contribution of age to the reduction of capillary parameters. They decreased from 40 over 60 to 87-week-old mice. In addition, very young KO mice still showed normal testosterone levels indicating again that steroid synthesis was functional in spite of LDL receptor mutation.Against this background it is noteworthy that our stereological investigations in ApoE−/−/LDL receptor−/− mice27 and ApoE−/− mice72 on Western diet are predominantly due indirectly to Leydig cell / testosterone deficits or directly to rarefication of testicular vasculature. Since peritubular capillaries with their close proximity to undifferentiated spermatogonia were described as vascular niche for germ cells73 a negative effect on spermatogenesis may arise from capillary reduction. The relevance of intact testicular vasculature34 and perfusion74 for spermatogenesis was suggested before. Reduced testicular blood flow was shown to induce hypoxic conditions which result in degeneration of germinal epithelium with disturbed spermatogenesis76. Similarly, hypobaric hypoxia in the rat led to increased apoptosis of spermatogonia and spermatocytes76.It is not clear whether disturbances of spermatogenesis described in old ApoE78.The number of pups per litter was significantly reduced in KO mice, suggesting a direct relationship between disturbed blood supply and male hypogonadism. Interestingly, ageing alone had been shown not to reduce litter size despite of its negative effects on sperm number79.Although female mutants are known to be less affected by atherosclerosis, our data do not allow complete exclusion of female fertility disturbances as wellOur data offer new insights into atherosclerotic disease by adding the testis as a hitherto underappreciated organ where one of its integral functions, providing the body with testosterone, is disturbed. Data showed that rarefaction of the capillary microvasculature in the testis together with reduced number and size of testosterone-producing Leydig cells might be causal factors. Thus, atherosclerosis contributes to the pathological changes of the testis and needs to be integrated into the management of male infertility.Supplementary MovieSupplementary Information


STMN1 and TAF15 reduces cellular transformation and invasiveness, providing evidence that SAM targets are genes important for cancer growth and invasiveness. Taken together these data provide a molecular rationale for SAM as an anticancer agent.S-adenosyl methionine (SAM) is a ubiquitous methyl donor that was reported to have chemo- protective activity against liver cancer, however the molecular footprint of SAM is unknown. We show here that SAM selectively inhibits growth, transformation and invasiveness of hepatocellular carcinoma cell lines but not normal primary liver cells. Analysis of the transcriptome of SAM treated and untreated liver cancer cell lines HepG2 and SKhep1 and primary liver cells reveals pathways involved in cancer and metastasis that are upregulated in cancer cells and are downregulated by SAM. Analysis of the methylome using bisulfite mapping of captured promoters and enhancers reveals that SAM hyper-methylates and downregulates genes in pathways of growth and metastasis that are upregulated in liver cancer cells. Depletion of two SAM downregulated genes  Broad changes in DNA methylation are a hallmark of cancer and are hypothesized to play a role in cancer initiation, progression and metastasis , 2. ChanHeparanase[Mmp2[uPA[Studies revealed that hypomethylation of promoters of genes that are important for cancer metastasis is a common feature of cancer , 4 i.e. eparanase Mmp2[6–8[Mmp2[uPA. Genome [Mmp2[uPA revealedLiver hypertrophy or carcinogenesis could be precipitated by agents that deplete SAM such as ethionine , cholineSAM is a global methyl donor and is predicted to effect all methylation reactions in the cell including DNA methylation , it is aSAM is an attractive anticancer agent since it is an approved nutritional supplement, however in absence of an understanding of its genomic targets and its selectivity to cancer cells, it is hard to make a case for its clinical use in treating or preventing HCC. We therefore characterized in this paper the effect of SAM on the methylome in primary liver cells and hepatocellular carcinoma cell lines. Our results identify particular gene pathways that are targeted by SAM. We show phenotypic as well as epigenomic and transcriptomic selectivity of SAM to cancer cells. Our data provide molecular support for the hypothesis that SAM is reversing cancer and metastasis related epigenomic programs and provide justification for further examining SAM as a candidate agent for preventing and reversing hepatocellular carcinoma.We first examined whether SAM selectively effects growth, transformation and invasiveness  of liver cancer cells using two human liver cancer cell lines, HepG2 (HCC) and SKhep1 (liver adenocarcinoma) and primary untransformed liver cells (NorHep). SAM has a significant dose dependent effect on cell growth and anchorage independent growth (a measure of cellular transformation) . We seleSAM inhibits growth of the two liver cancer cell lines HepG2 Figure  and SKheTo understand the molecular mechanisms behind these effects of SAM and to delineate the potential functional consequences of SAM therapy on normal and cancer liver cells we performed mRNA sequencing of SAM treated and control HepG2, SKhep1 and NorHep cells. We used Cufflink  to delineate differentially expressed genes (DEGs) between cancer cell lines and NorHep and to determine the effect of SAM on mRNA expression. The differential expression of SAM/control (log2fold change) was plotted against baseline gene expression levels of the untreated control cells (log2FPKM) using ggplot2 in R  Figure . ExaminaWe examined how phenotypic differences between the effect of SAM on normal and cancer cell lines Figure  might beThen we compared using IPA the significance of enrichment (Fisher’ exact test) of SAM treatment DEGs that are involved in the functional pathways of cell survival and death, cell growth and cellular movement and metastasis. Whereas the cellular death and survival pathway shows similar enrichment in SAM treatment DEGs in both NorHep and cancer cell lines, the proliferation pathway was highly enriched in HepG2 and SKhep1 relative to NorHep, while cell movement pathway was enriched in invasive SKhep1 cells Figure , which iThe IPA pathway analysis suggests that SAM targets different pathways in cancer and normal liver cells. We reasoned that SAM might act on genes that are differentially expressed between cancer and normal cells and reverse this difference, shifting the transcriptome of cancer cells “closer” to normal. 3102 genes are upregulated and 3697 genes are downregulated in HepG2 cells when compared with NorHep while 3272 genes are upregulated and 3732 genes are downregulated in SKhep1 in comparison with NorHep Figure . IPA anaWe then determined whether SAM treatment reverses the differences in enrichment in these pathways between cancer cell lines and normal cells. SAM treatment downregulates genes that are expressed at higher levels in cancer cells versus normal cells and upregulates genes that are expressed at a lower level in cancer cells Figure . IPA anaSAM is the methyl donor of the DNA methylation reaction catalyzed by DNA methyltransferases and was previously reported to inhibit and reverse demethylation of several genes in cancer cells . ChangesAfter extracting the methylation values, the distribution of CG methylation levels was plotted for the different samples before and after SAM treatment  and examined their profiles in genes that are down regulated Figure  versus gWe then examined whether SAM treatment reversed the differences in DNA methylation (DMC) between cancer and NorHep cells. As shown in Figure We determined how reversal of differences in methylation in promoter regions between cancer and control cell lines following SAM treatment associated with reversal of gene expression differences. 748 genes in HepG2 cells and 1233 in SKhep1 cells are both hypermethylated and downregulated relative to NorHep  Figure . SAM treWe listed the genes that were both hypomethylated and upregulated in either HepG2, SKhep1 or both cell lines relative to NorHep and whose expression and state of methylation was reversed by SAM Figure  show thaDYNC1H1 and PEG10, RAN).We used QRT-PCR to validate changes in expression in genes whose a. function was shortlisted by IPA analysis as related to proliferation, b. were upregulated in both SKhep1 and HepG2 cells relative to NorHep and c. were downregulated by SAM. We also validated that SAM downregulated metastasis related genes that were upregulated in untreated SKhep1 relative to NorHep Table . We measQRT PCR validated that SAM downregulated 11 metastasis related genes in SKhep1 cells Figure . We valiSTMN1 and TAF15 and measured the effect of depletion of these genes on the transformation and invasive phenotypes. These two genes were selected for the biological plausibility of their involvement in oncogenesis. Stathmin1 (STMN1) is a candidate oncogene whose activity is influenced by p53, p27, and the P13K/Akt pathway and is involved in metastasis [TATA-box binding protein associated factor 15 (TAF15) is a member of the FET family of RNA- and DNA-binding proteins which play a role in gene transcription, is a member of the TFIID transcription initiation complex and was shown to undergo translocation in acute leukemias and sarcomas [STMN1 or TAF15 mRNA in SKhep1 cells , human untransformed primary hepatocytes  were obtained from Celprogen (33003-02). S-adenosylmethionine chloride (SAM) was prepared in buffer containing 0.005M sulphuric acid and 10% ethanol. 200μm SAM or equivalent volume of buffer (control) were added to regular culture medium. Media were refreshed daily over a period of 5 days.Cell viability was determined by the Trypan blue (Sigma-Aldrich) exclusion assay. Cells were harvested after 5 days of treatment. Cell invasiveness was evaluated by the Boyden Chamber Cell Invasion Assay Kit ECM550 (Chemicon Int.). Briefly, 50,000 cells resuspended in serum-free media were added to the inserts which were dipped in the lower chamber containing complete media. Following 24h incubation at 37°C, invasive cells were stained and counted under the microscope. Additionally, 50,000 viable cells (as determined by Trypan blue) resuspended in complete media were added to a six-well plate (in absence of the insert) and were counted concurrently following 24h incubation period to determine effects on cell viability.in vitro was determined as described [Anchorage-independent growth on soft agar, a measure of transformation escribed . 3,000 vDNA and RNA was extracted using AllPrep DNA/RNA/miRNA Universal Kit (Qiagen) according to the manufacturer’s protocol. Quantitative real-time PCR (QPCR) reaction was carried out in Light Cycler 480 machine (Roche) using forward and reverse primers listed in Western blot analysis was performed as described  using 50DNA samples (1μg) were subjected to bisulfite conversion using the EZ DNA Methylation-Gold Kit  according to the manufacturer’s protocol. Bisulfite converted target sequences were amplified with HotStar Taq DNA polymerase (Qiagen) using biotinylated primers listed in STMN1 was targeted with ShSTMN1#V3LHS_383505 and TAF15 was targeted with ShTAF15#V2LHS_172493 (For STMN1 and TAF15 depletion we used lentivirus-mediated human pGIPZ shRNA plasmids and control pGIPZ-scrambled shRNA (Open Biosystems) . shRNAs RNA (4 μg) was prepared for sequencing using the TruSeq RNA Sample Prep Kit  following the manufacturer’s protocol and sequenced in duplicate using Illumina HiSEQ2K platform (50bp pair-end reads). Around 30 million reads were obtained per sample .Fastqc was used to quality control (QC) RNAseq data, paired end reads were aligned to the human reference sequence  with TopHat 2.0.9  with defTarget DNA fragments were captured using the human SeqCap Epi CpGiant Enrichment Kit  interrogating 5.5 million CpG sites covering promoters and regulatory sequences in human genome according to the manufacturer’s recommendations. Sequencing was performed on the Illumina HiSEQ2K platform using a standard 50 cycle paired-end read sequencing protocol according to the manufacturer’s recommendations.https://sftp.rch.cm//diagnostics/sequencing/literature/nimblegen/07292163001_NG_SeqCap_TchNote_EvalEpiData.pdf). Methylation difference was calculated using methylKit [The raw data was processed as recommended by Sequencing Solutions Technical Note from Roche for SeqCap Epi CpGiant bisulfite sequencing data analysis  was used to compute enriched gene networks, functional categories, canonical pathways and upstream regulators. Heatmaps were created using GeneE (http://www.broadinstitute.org/cancer/software/GENE-E/doc.html). GSEA was used to examine gene-set enrichment of differentially methylated genes in response to SAM and their expression profile [Ingenuity Pathway Analysis (IPA) program ( profile .


A set of short peptide sequences susceptible to fibrillar aggregation produces sequneces capable of arresting elongation of amyloidfibrils. The "stop" signals are short helices customized for each individual target. Such a helix should exhibit high amphiphilicity,with differing conditions present on each side . Theemergence and elongation of fibrillary forms resulting from linear propagation of local hydrophobicity peaks is shown using thefuzzy oil drop model. An important voice in the discussion regarding Alzheimer'sdisease comes from the psychologist community, whichattempts to identify objective causes for the disase . CliniciThe fuzzy oil drop model describes the existence of ahydrophobic core, with hydrophobic residues congregating atthe center of the molecule and hydrophilic residues exposed onthe surface. This type of structure favors interaction with water, while aThe fuzzy oil drop model also reveals another type ofdiscordance versus the theoretical monocentric hydrophobiccore: linear propagation of local hydrophobicity peaksinterspersed by local minima. This situation occurs when thepolypeptide (or polypeptides) is unable to fold as an individualmolecule with a monocentric FOD-compliant hydrophobic core,and instead folds in a way, which is dependent only on theintrinsic hydrophobicity of each individual residue. If theresulting fragment is placed in the proximity of other similarlyfolded fragments, linear propagation becomes highly likely. Asshown in , once liWe base our study on the set of amyloid-forming popetidesdiscussed in  and treaThe pattern for the design of a "stop" signal is provided by alyase - bacterial chondroitinase b pectate lyase (PDB ID: 1DBG). This prThe fuzzy oil drop model has been described in detail elsewhere, 34. AccUnlike globular proteins, amyloid-like structures do not exhibita monocentric distribution of hydrophibicity. Instead, the localdistribution in each unit structure is determined solely by theintrinsic properties of its constituent residues. Environmentcontaining another peptide with a different sequence yet asimilar distribution of hydrophobicity results in complexation.Analysis of amyloid fibril structures  reveals A "stopper" fragment arrests linear aggregation. A putativedrug that exploits this concept would have to fulfill severalconditions and be adapted to the specific sequence of its"target" fragment. The proposed drug design model is based onnaturally occurring amyloid-like sequences, which haveevolved the corresponding "stop" signals preventingunrestricted propagation. The underlying mechanism isdiscussed in detail elsewhere .The proposed helical stopper should, as a rule, be compatiblewith the local hydrophobicity distribution of the unit structure of the amyloid fibril; however it must also fulfill anothercondition - propensity for adopting helical, β and random coilconformations for a range of sequences . We usedWe show the distribution of hydrophobicity in the 32-residueamyloid β a4 protein (2MXU - 11-42) . As showAnalysis of distribution profiles suggests linear propagation oflocal hydrophobicity peaks and troughs. This type of structure,devoid of any "stop" signal, would tend to propagateindefinitely. The observed attenuation of hydrophobicity peaksin terminal fragments of the complex and it is caused by thelack of another adjacent peptide. It does not ensure "closure"which would enable the structure to remain water-soluble.Such linear propagation of two distinct local maxima  discordaSolenoids exhibit linear propagation of local hydrophobicitydistributions, stretching along their axis. Since such forms arepresent in naturally occurring proteins, evolution must havecome up with a way to counteract their unrestrictedpropagation. This role falls to a "stop" signal, such as the onepresent in bacterial chondroitinase b pectate lyase (PDB ID:1DBG) . We use According to the fuzzy oil drop model, linear propagation offibrils is facilitated by the following phenomena: (1) lack of monocentric hydrophobic core described by a 3D Gaussian; (2)repeatable sequence with alternating hydrophobicity peaks andtroughs; (3) distribution of hydrophobicity dominated by theintrinsic properties of each residue; (4) linear propagation ofhydrophobicity along a given axisIn order to counteract propagation, the following conditiousshould be satisfied: (1) the fibri's terminal fragments, alongwith an adjoining "stop" signal, should be characterized by RD< 0.5, indicating a local distribution of hydrophobicityconsistent with the Gaussian model; (2) The "stop" signalshould adopt the form of an amphipathic helix; (3) the outward(water-facing) side of the helix should be strongly hydrophilic;(4) the inward (fibril-facing) side should have a distribution ofhydrophobicity consistent; with that of the target peptide. Weused peptides identified as strongly amyloidogenic as reportedelsewhere in this study .The sequence present in 2Y3J (PDB ID) is characterized by adistribution of hydrophobicity, which closely corresponds to theone in 1DBG (PDB ID). Accordingly, the 1DBG "stop" signalappears to work equally well in 2Y3J. In light of the above, the"stopper" sequence adapted for 2J3Y should be as follows:VNETLYQVVKEV . The resThe HSSNNF sequence is characterized by local maxima onboth sides, with relatively low hydrophobicity in the middle.The proposed stopper sequence is VNSNAAQAAKNV . The AAQThe next target peptide is LSFSKD (3LOZ), with a distributionshown in For the MMHFGN target peptide (3NVE), the matching stoppersequence is VNETTAQAVKEV . This seHere, a similar stoper to the one designed for 1DBG mightsuccessfully arrest propagation . The genIt is interesting to consider potential stoppers adapted toAβamyloids, such as the ones present in 2MVX and 2MXU.The proposed sequence for a helix which could potentiallyobstruct the 10-20 aa section in 2MVX is as follows:SNETLYQVVKE(V)ASNETLYQVVKEA(V). It is a fairly longhelix that can span the entire length of the β-fragment. Webelieve that any shorter fragment of this helix may be sufficientas a stopper to avoid unwanted immune reaction. The sequenceis based on the 1DBG template, with a single change marked bysquare brackets. The introduction of an Ala residue is dictatedby the need to reduce hydrophobicity compared to the originalVal residue. This substitution has been verified to increase thesequence's affinity for adopting helical conformations. In this amyloid we can discern two potential anchoring pointsfor a stop signal . The corOur analysis of the terminal sections of linearly orderedfragments revealed some very short bracketing folds, whoselength does not exceed half the length of the blocked peptide. It seems that such short β-fragments may also play the role of a"stop" signal are as long as they disrupt the regular ordering oflocal hydrophobicity peaks, preventing the attachment ofanother unit peptide .It is difficult to properly discuss the stoppers proposed in , 41, 42Drug like inhibitors: Peptides with short helix should (1) easilyinteract with fragments which sustain propagation in fibrils; (2)include hydrophilic elements which enable contact with waterand prevent indefinite propagation of linear forms; (3) avoidself-association; (4) exhibit a tendency to form an amphiphatichelical conformation, with the hydrophobic side attached to thefibril  and the hydrophilic side facing the waterenvironment - such as in 1DBG.An ampiphatic helix with hydrophobic residues facing the fibriland hydrophilic residues facing the water environment creates a"bridge" between the hydrophobic portion of the fibril and theenvironment. Note that a helical peptide is typically stable in itsisolated form, whereas the stability of an isolated β-strandcannot be ensured. Known peptides discussed elsewhere , 41, 42


Citrullination, or the post-translational deimination of polypeptide-bound arginine, is involved in several pathological processes in the body, including autoimmunity and tumorigenesis. Recent studies have shown that nanomaterials can trigger protein citrullination, which might constitute a common pathogenic link to disease development. Here we demonstrated auto-antibody production in serum of nanomaterials-treated mice. Citrullination-associated phenomena and PAD levels were found to be elevated in nanomaterials -treated cell lines as well as in the spleen, kidneys and lymph nodes of mice, suggesting a systemic response to nanomaterials injection, and validated in human pleural and pericardial malignant mesothelioma (MM) samples. The observed systemic responses in mice exposed to nanomaterials support the evidence linking exposure to environmental factors with the development of autoimmunity responses and reinforces the need for comprehensive safety screening of nanomaterials. Furthermore, these nanomaterials induce pathological processes that mimic those observed in Pleural MM, and therefore require further investigations into their carcinogenicity. Several isotypes of PAD exist, each with different tissue distribution5. Citrullinated proteins are recognized as non-self-proteins, and can subsequently induce the development of autoimmune conditions7. The autoimmune response is linked to the pathogenesis of several diseases including rheumatoid arthritis (RA) multiple sclerosis (MS) and psoriasis10. It has been also reported that the levels of citrullinated proteins and PAD4 were elevated in patients with various cancers, such as oesophageal cancer, breast cancer and lung adenocarcinoma15. A more recent work by Jiang and co-workers reported that citrullinated proteins were detected in liver, breast, lung, ovarian, cervical, kidney, pancreatic and gastric tumour cell lines15.Citrullination is involved in several pathological processes in the body, including autoimmunity and tumorigenesis. Citrullinated proteins are generated by a post-translational deimination or demethylimination of polypeptide-bound arginine by a family of Ca17. Indeed, previous studies have associated human exposure to environmentally presented nano-size and ultra-fine materials with various pathologic processes, such as chronic inflammation, pneumonia, chronic obstructive pulmonary disease, autoimmune conditions21, pleural malignant mesothelioma (MM)24 and lung cancer25. Given these links, the increasing presence and application of manufactured nanomaterials has intensified investigations to assess their potential impact on human health. We hypothesised that engineered nickel nanomaterials which can mimic environmental fibre-and filamentous-type nanomaterials, trigger similar pathological responses.It has also been shown that nanomaterials of diverse origin can trigger protein citrullination which might constitute a common pathogenic link to disease development17, this study uses well-characterised nanowires (NiNWs) of two defined lengths, to test the length-dependent pathogenicity hypothesis. A method of intrapleural injection of nanomaterials, described by Murphy et al.26, was used to measure organ specific response as well as the systemic response towards the nickel nanomaterials. This method is justified on the basis that a fraction of all inhaled particles and fibers that deposit in the peripheral lung translocate into the pleural space31. Subsequently, serum and tissue samples of NiNWs-treated mice were analysed for the presence of citrullinated proteins, the expression of PAD enzymes and formation auto-antibodies against citrullinated proteins. In parallel, we examined the expression profiles of PAD2, PAD4 and citrullinated proteins in human MM tissue samples.Further to the previous reportsin vitro and in vivo. Furthermore, we report autoantibody production following nickel nanomaterials exposure in in vivo mouse model. The expression of PAD2 and PAD4 and the induction of protein citrullination were also demonstrated in pleural and pericardial MM tissue sections. Based on the comparison of the data collected in experimental and clinical samples from pleural MM patients with known prior asbestos exposure, we suggest that both asbestos fibres and nickel nanomaterials trigger elevation of PAD enzymes thereby altering citrullination pathways and resulting in the development of autoimmune responses.Our results show that exposure to nickel nanomaterials increases the protein citrullination and the induction of PAD2 and PAD4 The differential ability of short and long nickel nanowires (NiNWs) to induce protein citrullination was studied in a human lung epithelial cell line A549 and a phagocytic cell line THP-1. Both cell lines showed induction of protein citrullination as examined by HCS Fig.  in signi16. In agreement with this, the treatment of A549 and THP-1 cells with nanowires resulted in the significant induction of calcium ion accumulation in both cells  following exposure to S-NiNWs, L-NiNWs and NiNPs in mouse sera. Faint bands were detected at the level of 150 kDa in all experimental groups, including vehicle and untreated controls, which may represent naturally occurring citrullinated proteins  paradigm since a high quantity of long nickel magnetic material (L-NiNW) is found in the lungs and similarly in the mediastinal lymph nodes; whereas for the short NiNW (S-NiNW) the accumulation is higher in the lymph nodes than the lung indicating a pleural barrier crossing, resulted to be statistically significant as shown Fig. Quantification of inorganic nickel magnetic material present in mice organs was carried out in three animals after 2-week-nanomaterials exposure, as shown in Fig. The measurements of PAD2 and PAD4 expression and protein citrullination in human pleural and pericardial MM with or without a history of patient’s exposure to asbestos  and benign Pleural Material (PM) was carried out using immunofluorescence method. As shown in Fig. 8. However certain genetic predisposition has been identified such as HLA-DRB133, however studies of external triggers that lead to disease manifestation have been inconclusive34. Furthermore, despite possible links between RA and environmentally imposed factors such as cigarette smoke, air pollution, and occupational exposure to nanomaterials such as silicon dioxide particles, the exact cellular events involved are still unclear35. Our group has previously demonstrated that nanomaterials of different origin are capable of promoting conversion of arginine residues to citrulline17, which may induce an autoimmune process and eventually lead to the development of RA. In this work, we tested the hypothesis that nickel nanomaterials of different geometrical size and aspect ratio could trigger a systemic citrullination response. Here we show for the first time, that in vivo exposure to nickel nanomaterials induces PAD2 and PAD4 expression, protein citrullination and production of auto-antibodies against citrullinated proteins.The production of anti-citrullinated protein antibodies is almost 100% specific for patients with RA, indicating an important role of protein citrullination in the pathogenesis of RA36. Therefore, these results reinforce the concept that nanomaterial-mediated citrullination could be associated with inflammation or with the development of autoimmune conditions such as RA.The analysis of LN tissue sections from mice intrapleurally injected with nickel nanomaterials showed sustainable increase in the levels of citrullinated peptide residues. This increase was associated with higher PAD2 and PAD4 expression level. PAD2 and PAD4 have gained attention as potential candidates that drive the citrullination of self- antigens in RA39. This is also coherent with previously published work where short nickel nanowires where found in lymphoid organs of mice26. In our study, a transient induction of intracellular protein citrullination was found in spleen tissue sections after exposure to nickel nanowires indicating the potential clearance of nanomaterials over time.Distribution of nickel nanomaterials into the spleen is also possible through the bloodstream following intrapleural injection. Specifically, it has been previously shown that in the murine spleens, polystyrene nanoparticles accumulated in the marginal zones of lymphoid follicles due to the macrophage activity40. In addition, characteristic arrangement of cytoskeletal proteins such as vimentin in podocytes is required to sustain the complex structures41. Also, due to high blood supply level and ability to concentrate toxins, the kidneys could be potentially affected by any material that reaches the circulatory system.We found that prolonged exposure to nickel nanowires induce a transient expression of PAD2 and PAD4 in mouse kidneys, however no induction of protein citrullination was detected. The kidneys are predominantly responsible for the exclusion of metabolic waste such as urea and ammonia. The filtration barrier is mainly created by the glomerular visceral epithelial cells or podocytes. The podocyte forms the filtration barrier by synthesizing the glomerular basement membrane, elaborating filtration slits, and producing the slit diaphragms2+ levels known to be pre-requisite for PAD activation are not sustained in the kidneys.Our findings of the transient expression PAD2 and PAD4 in the absence of induction of protein citrullination in the kidneys could be explained by relatively low enzymatic activity level in this organ insufficient to induce citrullination are of most abundant potential targets, such as for example podocyte vimentin. Other possibility is that the high Ca19. Another possibility stems from studies in autophagy, where cellular components are degraded in an auto-catabolic mechanism as a means of self-clearance. Different nanoparticles have been observed to induce autophagy in various conditions, perhaps from oxidative stress or a need to eliminate foreign bodies from cells, similarly to viral infections46. Considering the possibility that autophagy in APCs mediates citrullination and antigen presentation to T cells, nanomaterial uptake by macrophages and/or dendritic cells could contribute to the pathogenesis of ACPA-positive RA through these mechanisms48. Our work further supports the differences between the phagocytic mechanisms induced by long versus short NiNW, as shown in Fig. Therefore, it would be reasonable to suggest that the primary citrullination phenomena induced by nanoparticles are developing in other organs and tissues rich in residential macrophages. This also could involve the uptake, transport and presentation of nanomaterials by antigen presented cells (APCs). Indeed, macrophages have previously been shown to phagocytose NiNPs and S-NiNWs, while L-NiNWs are only partially engulfed and cause frustrated phagocytosis, leading to inflammation promotion and influx of inflammatory cellsin vivo assays also demonstrated the presence of citrullination residues in mice sera following systemic administration of nickel nanomaterials. Of note anti-thrombin, an anti-coagulant with a molecular weight of 52 kDa, has been shown to become citrullinated, causing a loss of normal physiological function that may promote symptoms and pathology of RA through lowered thrombin inhibition and increased coagulation, inflammation and angiogenesis14. Thus, mouse anti-thrombin could be one of candidate citrullinated proteins found in sera and observed here in response to nickel nanomaterials. In certain diseases, 46 kDa keratin-18 is found in serum and could also be citrullinated either in tissue or in the circulation by PAD enzymes50. Our findings demonstrating the presence of citrullinated proteins in mouse sera  and by Nomarski technique. Average lengths of nanowires were found to be 4.3 ± 1.0 µm , and 24.0 ± 7.2 µm ; this in accordance with previously published work32. Commercially available nickel nanoparticles were also used in the tests as an alternative type of nickel-based nanomaterial . These had a mean dry diameter of 15.0 ± 5.0 nm, (as declared by manufacturer’s description and further refereed to NiNP) and could be readily dispersed in saline  with a resulting hydrodynamic diameter of 57.0 ± 10.0 nm as reported in previously published work58.Engineered nickel nanowires of two discrete lengths were fabricated as previously reported2. THP-1 cells were treated with phorbol 12-myristate 13-acetate (25 ng/ml) before experimentation.Human lung epithelial cell line A549 and a phagocytic cell line THP-1  were cultured in DMEM and RPMI 1640 medium, respectively. Both types of media were supplemented with 10% (v/v) foetal bovine serum, 1% (v/v) L-glutamine/ penicillin/streptomycin. Cells were grown in a humidified incubator at 37 °C in 5% COin vivo work was carried out in accordance to the FELASA guidelines and recommendations by fully trained staff holding a valid UK Home Office personal licence. Ethical use of animal and were approved by the UK Home Office under approved project licence in accordance to the international FELASA guidelines. 40 female C57BL/6 mice  were housed in a group size of five in standard caging with sawdust bedding within a pathogen-free Home Office approved facility as previously described19. Mice were maintained on a normal 12-hour light and dark cycle. The animals were allowed 7 days to acclimatise prior to study commencement, after which groups of three animals were injected intrapleurally with 0.5 ml of 100 g/ml  of NiNPs, S-NiNWs, L-NiNWs or 0.5 ml of 0.5% phosphate buffered saline (PBS) as a vehicle; in accordance to previously published work19. Animals were immediately placed back into their cages and monitored to ensure normal behaviour and for signs of distress or issues. The animals were sacrificed at 24 h and 2 weeks, then blood samples were collected and tissue samples from liver, mediastinal lymph nodes (LN), spleen and kidneys were harvested, fixed in formalin and paraffin blocks were prepared and processed for immunohistochemistry staining. From every second animal, isolated organs were used for inorganic magnetic nickel detection by SQUID magnetometry. All in vivo work was carried out by staff holding a valid UK Home Office personal licence under a Home Office approved project licence. Untreated mice (N/T) were also used as control.The To facilitate identification of PAD2, PAD4 expression and the induction of protein citrullination in MM, four patients with known history of asbestos exposure (MM/Exp+) were included in this study. All patient samples were acquired in accordance to the Declaration of Helsinki based on the ethical principles for medical research involving human subjects. Patients gave full and informed consent and immunohistochemistry was conducted on all FFPE archival material as per St James’s Hospital Ethical Approval 041018/8804. These patients were identified from the archival pathology files of the Pathology Unit of the Regional Hospital of Mestre Venice, Italy. All were diagnosed with pleural MM based on World Health Organization criteria. The pleural MM was confirmed in all patients by clinical, morphological and immunohistochemistry data. The tissue samples were formaldehyde fixed and paraffin embedded. In addition, we also included a tissue microarray (TMA) containing 4 cases diagnosed with MM without any history of patient’s exposure to asbestos (MM/Exp−) and 2 cases with benign pleural tissue .Rabbit polyclonal antibodies to citrulline, PAD2 and PAD4 antibodies and FITC-linked goat anti-rabbit antibodies were purchased from Abcam . Horseradish peroxidase conjugated anti-rabbit IgG and anti-mouse IgG antibodies were obtained from Dako A/S . Peroxidase complex detection method was purchased from Novocastra Laboratories Ltd., UK.4 cells/well), exposed to various concentrations of nanomaterials for multiple time points and fixed using 3% paraformaldehyde as described16,17. They were immuno-stained for citrullinated proteins and high content screening assays (HCSA) were performed on IN Cell Analyser 1000 equipped with IN Cell Investigator software . Images were acquired and analysed as previously described17.Cells were seeded in 96-well plates  were boiled with 1×NUPAGE® LDS sample buffer and resolved on a pre-cast 4–12% Bis-Tris NUPAGE® SDS-PAGE gel using NUPAGE® MOPS buffer. The resolved proteins were transferred to a polyvinylidene fluoride (PVDF) membrane by the wet transfer method using the XCell II™ Blot Module for 2 h. PVDF membrane was blocked in 5% non-fat dry milk in TBST  Tween 20, 20 mM Tris-HCl) for 1 h at room temperature and incubated with the primary antibodies (specific to polymer chains of the amino acid citrulline) and then with a horseradish peroxidase conjugated secondary antibody. The bands were detected with the using LUMINATA Western HRP Substrate  enhanced chemiluminescent detection system and subsequent exposure to Kodak light-sensitive film. Densitometric analyses of the western blots were performed by using GeneTools software (Syngene). The relative values of the samples were determined by giving an arbitrary value of 1.0 to the respective control samples of each experiment.As previously describedSera from each mouse were collected from the tail vein on the initial day of nanomaterials exposure and 7 days thereafter. On the day of sacrifice, sera were collected by cardiac puncture. Anti-cyclic citrullinated proteins (anti-CCP3) antibodies were determined using the Quanta Lite Anti-CCP3 IgG ELISA test  following manufacturers’ protocol. Mouse sera were diluted 1:80 in sample diluent, and the secondary antibody was replaced with rabbit anti-mouse  diluted 1:1,000 in PBS.Anti-filaggrin antibody reactivity was determined by indirect immunofluorescence. Undiluted sera or sera diluted 1:10 in PBS from mice post exposed to the nickel nanomaterials, vehicles, and untreated mice were applied to rat oesophageal sections  for 1 hour at room temperature with humidity. The slides were then washed 3 times for 5 minutes in PBS. FITC conjugated goat anti-mouse IgG (BD Biosciences-Pharmingen) was applied for 1 hour at room temperature in a humid environment. The slides were again washed 3 times for 5 minutes in PBS then Dako mounting medium and cover slip placed on the slide. The samples were visualized using confocal microscopy .59. Immunohistochemical analysis for the presence of citrullinated proteins, PAD2 and PAD4 expression was conducted on 5 µm tissue sections of LN, kidneys, liver and spleen using the peroxidase complex detection procedure62.The tissue sections preparation including preservation and fixation was performed as previously described63. Slides were sealed with Dako Fluorescent Mounting Medium (Dako Diagnostics Ireland Ltd). Confocal images of immunostained tissue section were taken with the Zeiss LSM Meta-510 confocal microscope . Two channel qualitative imaging was carried out using the 63× oil immersion objective.5 µm tissue sections of pleural and pericardial malignant mesothelioma with no information of asbestos exposure , tissue sections from patients with known exposure to asbestos  and tissue section of normal pleural tissue were mounted on glass slides and stained with anit-PAD2, anti-PAD4 and anti-citrullination antibodies using immunofluorescence staining methods. Cell nuclei were visualised with Hoechst 33342  as previously described2/kg.Quantification of the inorganic magnetic nickel nanowire material present in different organs explanted from exposed mice was carried out by assessing the total magnetic signal collected from each organ sample. Magnetization measurements were made at room temperature using a Quantum Design MPMS XL SQUID magnetometer . Same volume of organ samples was loaded into gel capsules mounted in a non-magnetic straw. All measurements were made using a standard second-order gradiometer, around the maximum slope position of the second derivative curve, with no auto-tracking of the actual sample position. Prior and post measurement empty and full capsules were weighted  in order to then calculate the total magnetic nickel nanowire material mass against the full sample mass. Total mass of magnetic nickel nanowire material was calculated from the magnetisation measurement carried out compared to the magnetisation saturation of the same weight mass of magnetic bulk nickel powder, which as a magnetisation saturation of 55.4 A·m2-way ANOVA with Bonferroni post-test analysis. A p-value of <0.05 was considered to be statistically significant . KNIME data exploration platform and the screening module HiTS were used to visualize the data. Protein citrullination level induced by the nanomaterials was normalized to positive controls. Scoring was done by using Z score of the normalized values and summarized as described in previous work64. Heatmap-type graphical illustration in a colorimetric gradient table format was adopted as the most suitable schematic representation.Response of each cell type to various nanoparticles was analysed by Supplemental Information


Kisspeptin/Neurokinin B/Dynorphin (KNDy) neurons of the arcuate nucleus (ARC) play a key role in the regulation of fertility. The ability to detect features of KNDy neurons that are essential for fertility may require three-dimensional (3D) imaging of the complete population. Recently developed protocols for optical tissue clearing permits 3D imaging of neuronal populations in un-sectioned brains. However, these techniques have largely been described in the mouse brain. We report 3D imaging of the KNDy cell population in the whole rat brain and sheep hypothalamus using immunolabelling and modification of a solvent-based clearing protocol, iDISCO. This study expands the use of optical tissue clearing for multiple mammalian models and provides versatile analysis of KNDy neurons across species. Additionally, we detected a small population of previously unreported kisspeptin neurons in the lateral region of the ovine mediobasal hypothalamus, demonstrating the ability of this technique to detect novel features of the kisspeptin system. KISS1)1 and its receptor (GPR54/Kiss1R)3, as well as genes encoding NKB (TAC3) and its receptor (TAC3R)4, result in failure to enter puberty and subsequent infertility in humans. Similarly, the deletion of the genes encoding kisspeptin or the receptors for kisspeptin and NKB in the mouse results in subfertility6 or infertility8. Within the arcuate nucleus (ARC) there is a unique population of neurons that express both kisspeptin and NKB9. In rodent and ovine brains, these neurons are further co-expressed with the endogenous opioid dynorphin A12 and are now commonly referred to as KNDy neurons. KNDy neurons have become an intense area of focus in neuroendocrinology as regulators of gonadotropin-releasing hormone (GnRH) secretion and steroid hormone feedback. The vast majority of KNDy neurons express steroid hormone receptors17 and are thought to form a reciprocally interconnected network that projects to GnRH neuron cell bodies20 or distal dendrites and terminals22. Each of the three peptides are hypothesized to play individual roles at both the level of GnRH neurons and reciprocally connected KNDy neurons to form the pulsatile shape of GnRH release23.Neurons expressing the peptides kisspeptin or neurokinin B (NKB) play a critical role in the regulation of fertility. Mutations that inactivate the genes encoding kisspeptin . In contrast, three-dimensional (3D) analysis of the complete intact neuronal circuits has the potential to reveal novel features that are not discernable in sectioned tissue. Therefore, 3D visualization of the complete KNDy cell population and their projections under different physiological states may be necessary to detect anatomical and functional heterogeneity among these cells. Although it is possible to reconstruct 3D circuits from sectioned tissue, this is a time-consuming and intensive process. The last five years have seen an increase in the development and use of optical tissue clearing techniques that permits rapid imaging of fluorescent cell populations in intact organs. In particular, many clearing techniques, such as CLARITY34 and CUBIC35, are particularly efficient at imaging endogenous fluorescent proteins in transgenic animals. As transgenic technology is largely limited in mammalian species to the mouse, clearing techniques amenable to immunocytochemistry are required for use in other mammalian species. In addition to the mouse, the rat and sheep are the most commonly used models in neuroendocrine research and significant advances in knowledge on KNDy neuroanatomy has been achieved in these species36. The sheep has provided a particularly valuable model in which to study neuroendocrine networks given the ability to collect detailed hormonal profiles from both peripheral and portal blood in unanaesthetized animals38. However, given the size of the rat brain and sheep hypothalamus, it is necessary to adapt immunolabelling and clearing techniques for larger tissue volumes. iDISCO40, adapted from 3DISCO 41, permits immunolabelling and optical tissue clearing within multiple organs. 3DISCO and iDISCO protocols have been used to visualize cell populations in multiple mouse organs42, the spinal cord of the rat and non-human primate43, and human embryos45. We aimed to further modify iDISCO in order to visualize the complete KNDy population within the rat brain and sheep hypothalamus. To achieve this, we optimized the iDISCO technique for labelling of KNDy peptides and clearing of larger tissue sizes using the rat brain before applying the optimized iDISCO to the sheep hypothalamic block.Previous analyses of the KNDy neural network have relied largely on examination of coronal sections which represent a very limited sampling of the population and may have resulted in some apparent conflicting data on its characteristics  in the ewe, which activated kisspeptin neurons specifically in the caudal region of the ARC58. In future experiments, the complete kisspeptin population can be assessed in 3D under differing physiological and pathological conditions. For example, markers of activation, such as Fos, can be used to detect activated subpopulations of kisspeptin neurons during differential modes of GnRH release. Although this is achievable with the current resolution of the lightsheet microscope, the investigation of fine neuroanatomical details (such as dendritic spines) and subcellular markers (such as G-protein coupled receptors to mark the release of endogenous neurotransmitters) will require future technological advances to improve the lateral resolution of the lightsheet microscope, which is currently limited to 1 µm.Three-dimensional rendering of the arcuate kisspeptin population in the ewe demonstrated the sparse number of kisspeptin neurons in the rostral ARC when compared to the middle and caudal regions, consistent with previous reports59. Here, in addition to these previously identified populations, we detected a scattered, small number of kisspeptin-positive neurons within the lateral region of the mediobasal hypothalamus. The functional role of this kisspeptin population is not yet known, although there are reports of scattered GnRH neurons located within this brain region61.The ability to image large areas of the intact sheep hypothalamus revealed a previously unreported population of kisspeptin neurons. Besides the ARC, kisspeptin neurons in the ovine MBH and surrounding regions had previously been reported within the DMH and VMH62. Tissue shrinkage in clearing protocols has recently been taken advantage of for more rapid lightsheet imaging of large areas, such as described in the “ultimate DISCO” (uDISCO) protocol, which shrinks tissue by 65%63. Although this is an advantage for larger tissue samples such as the sheep hypothalamus, it is possible that distortion of the tissue can occur. The recently published iDISCO + protocol is reported to reduce shrinkage to 10% in cleared mouse brain samples by replacing post-immunolabelling dehydration of brain tissue by THF with methanol64. However, in our hands, the intact rat brain shrank by 26–27% with iDISCO+, indicating that shrinkage still occurs in larger tissue samples with methanol-based dehydration (data not shown).Dehydration of tissue with THF during the clearing process shrunk tissue volume by an average of 27.4%. This is in line with what has previously been reported using THF-mediated clearing, under which mouse brain samples have shrunk up to 50%Although the iDISCO system offers an important added 3D view of the complete distribution of a neuronal population there are a number of considerations when applying to larger animal models, which can be expensive and time consuming to produce. First, autofluorescent labelling of blood vessels were more pronounced in ovine tissue when compared to the rodent brain, although this did not prevent labelling of kisspeptin neurons. Second, not all antibodies are compatible with solvent-based tissue clearing. Importantly, traditional sectioning techniques provide multiple series for experimental analysis, whereas using a complete brain limits the number of antibodies that can be visualized per animal, and thus, the number of experimental data points. Therefore, iDISCO is likely to be used in conjunction with traditional sectioning techniques, but does not entirely replace 2D histological analysis.In conclusion, we report adaptation of the iDISCO technique for high resolution imaging of the complete KNDy neuron population in the transparent rat and sheep brain. This technique permits versatile analysis of the KNDy population, including the extraction of detailed 3D information regarding cell numbers and fiber distribution. In addition, the ability to render the intact rat brain and sheep hypothalamus transparent provides the broad opportunity to interrogate a variety of cell populations throughout the central nervous system of species other than mice that are commonly used in neuroscience, including large animal models.Adult female Sprague Dawley rats (Charles River) were housed in separate rooms, in same sex-pairs in standard Plexiglass cages with ad libitum access to food and water. Animals were maintained in temperature and humidity controlled rooms on a 12-hour dark/light cycle with lights off at 0900 hr. Adult black-faced ewes of mixed breeding were housed in an open barn. Ewes were given open access to water and fed once daily with a maintenance regimen of silage. Animals were moved into an indoor facility 3–5 days before surgery in which the duration of artificial lighting was adjusted to match that outdoors. They were fed a diet of alfalfa cubes, and had free access to water and minerals.65, containing either sesame oil vehicle (n = 4) or 100 µg/mL of 17β estradiol-benzoate dissolved in sesame oil (n = 4)66. Eight days following OVX, rats were deeply anesthetized using ip injection of sodium pentobarbital  and perfused intracardially with 10 mL of 0.9% saline, followed by 500 mL of 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB). Rats were decapitated post-perfusion, brains were extracted from the skull and post-fixed in 4% PFA overnight at 4 C. The lateral cortices were removed using vertical cuts lateral to the amygdala in OVX rats , prepared as described inats Fig.  so to en67 and treated with dexamethasone and penicillin pre- and post-operatively. Daily analgesia  was given starting at time of anesthesia through to 5 days post-surgery. Two weeks following surgery, OVX ewes were euthanized with iv sodium pentobarbital  and decapitated. The heads were perfused through both internal carotid arteries with 6L of 4% PFA in 0.1M PB (pH 7.3) mixed with 0.1% sodium nitrite and 10 U/mL heparin. Whole brains were removed from the skull post perfusion and placed into 0.1M PB with 0.01% sodium azide. Hypothalamic tissue blocks measuring approximately 1.5 cm (length) x 1.5 cm (width) x 1 cm (height) were prepared (n = 4). Meninges were removed from the tissue blocks, and the majority of the infundibular stalk was carefully removed for optimal visualization of the ARC under the lightsheet microscope. All procedures were approved by the University of Mississippi Medical Center Animal Care and Use Committee (rats) and the West Virginia University Animal Care and Use Committee (sheep) and followed National Institutes of Health guidelines for animal research.Ewes were bilaterally ovariectomized by midventral laparotomy under isoflurane anesthesia (2–4%) using sterile techniques69.The primary antibodies used in these protocols were polyclonal mouse anti-tyrosine hydroxylase , polyclonal rabbit anti-kisspeptin , polyclonal guinea pig anti-proNKB  and polyclonal mouse anti-GnRH . Lower concentrations of tyrosine hydroxylase (1:5000), kisspeptin and NKB  primary antibodies detected few cell bodies or fibers in cleared brains from OVX rats (data not shown). The secondary antibodies used were Alexa Fluor goat anti-rabbit 647, Alexa Fluor goat anti-mouse 647, Alexa Fluor goat anti-mouse 555, Alexa Fluor goat anti-guinea pig 555 and Alexa Fluor goat anti-mouse 555 . All antibodies have been previously characterized in rats and sheep and validated for specificity with target peptides39, rat and sheep brain tissue was dehydrated in methanol, bleached with hydrogen peroxide, rehydrated, and blocked using normal goat serum. The brain tissue was incubated with shaking in blocking serum, primary antibodies and secondary antibodies for 7 days at 37 C at each step to permit diffusion through the large tissue samples. Brains from ovary-intact rats (n = 4) were immunolabelled using mouse anti-TH serum followed by Alexa Fluor goat anti-mouse 647 secondary antibody. Brains from OVX + VEH and OVX + E2 rats were dual-labeled using rabbit anti-kisspeptin and guinea pig anti-NKB, which were detected using Alexa Fluor goat anti-rabbit 647 and Alexa Fluor goat anti-guinea pig 555 secondary antibodies, respectively. Sheep hypothalamic blocks were immunolabelled using either rabbit anti-kisspeptin or both rabbit anti-kisspeptin and mouse anti-GnRH antibodies, followed by goat anti-rabbit 647 or goat anti-mouse 555 secondary antibodies, respectively.As outlined inPost-immunolabelling dehydration of tissue using tetrahydrofuran (THF) was adapted for the larger size of the rat brain and sheep hypothalamic block, as outlined in Table Mosaic stacks of 16-bit TIFF images were stitched together using Fiji software to generate a single z-stack containing the imaged areas. To quantify the number of kisspeptin and NKB labelled neurons, users were blinded to experimental treatment and counts confirmed by a second user. In TIFF z-stacks, the rostral, middle and caudal regions of the ARC were outlined using the ‘regions of interest’ tool in Fiji. The ‘multi-point’ tool was used to count the number of kisspeptin or NKB positive cell bodies within each arcuate region in 200 µm segments from the ventral surface up to 1 mm. To count the number of neurons containing both peptides, the users switched between channels. Three-dimensional volume files and movie files were generated using IMARIS software (Bitplane). Stacks were converted to Imaris files (.ims) and 3D projections of z-stack images were generated using the volume rendering function. In ovine tissue, stacks of images measuring mm 1.14 mm  by 3.90 mm  by 1.74 mm  containing the ARC were projected in 3D. Regions of interest in 3D reconstructions were isolated using the 3D crop tool. In the ovine tissue, large autofluorescent blood vessels lateral and medial to the kisspeptin population in the caudal ARC, and a small region of autofluorescence along the third ventricle wall dorsal to the ARC, was removed using the surface tool and the mask function  cells were compared between OVX + VEH and OVX + E2 treated rats in the rostral, middle and caudal ARC using two-way ANOVA with Tukey post-tests (Sigma Plot). All data are expressed as mean + SEM. A p value of <0.05 was considered statistically significant.The datasets generated and analyzed during the current study are available from the corresponding author upon request.Supplementary informationVideo 1—Animated 3D rendering of hypothalamic tyrosine hydroxylase neuronsVideo 2—Animated 3D rendering of arcuate kisspeptin neurons in the ovine hypothalamus