We hypothesised that co-delivery of vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) followed by bone morphogenetic protein-2 (BMP-2) could be more effective in stimulating bone repair than the delivery of BMP-2 alone. Poly(lactic-co-glycolic acid) (PLGA ) based microparticles were used as a delivery system to achieve a controlled release of growth factors within a medical-grade Polycaprolactone (PCL) scaffold. The scaffolds were assessed in a well-established preclinical ovine tibial segmental defect measuring 3 cm. After six months, mechanical properties and bone tissue regeneration were assessed. Mineralised bone bridging of the defect was enhanced in growth factor treated groups. The inclusion of VEGF and PDGF (with BMP-2) had no significant effect on the amount of bone regeneration at the six-month time point in comparison to BMP-2 alone. However, regions treated with VEGF and PDGF showed increased vascularity. This study demonstrates an effective method for the controlled delivery of therapeutic growth factors  Most bone fractures do not require intervention and heal spontaneously, stimulated by a choreographed combination of biological signals . Large bAutologous bone grafts contain a mixture of cells, mineralised matrix, and signalling molecules. Research within the field of regenerative medicine has focused on seeking out efficacious alternatives in an effort to mitigate the limitations of autologous sourcing. Growth factors (GFs) can be used to impart bioactive properties onto synthetic scaffolds and bone morphogenetic proteins have been successfully used clinically for the treatment of long-bone fractures and to enhance spinal fusion ,5. HowevSpine explored the evolving safety profile of using rhBMP-2 in the treatment of spinal injuries and reconstructive surgery . The human fracture haematoma has potent angiogenic activity, thought to be due primarily due to VEGF expression in response to the fracture process ,31. ReceIn this investigation in a well-characterised and validated large preclinical animal model, we studied an implant consisting of two elements: a biodegradable structurally supportive, yet highly porous scaffold made from polycaprolactone (PCL) which was combined with PLGA-based microparticles delivering therapeutic GFs immobilised in place with platelet rich plasma. The treatment of load bearing bones, such as the tibia, requires a mechanically supportive scaffold in combination with an internal fixation device to provide a structure with appropriate mechanical characteristic to support the growing bone matrix.This research was based on the hypothesis that the combined delivery of VEGF, PDGF and BMP-2 would generate more efficient bone regeneration than BMP-2 alone based on the rationale that this more closely mimics biological events. The angiogenic factors should drive early vasculature and the BMP-2 should drive bone formation. To test this we used a previously reported PLGA microparticle delivery system ,22 to dein vitro. A theoretical 100% release would have been 10 µg/mg microparticles. It can be seen that the fast releasing formulation is close to this at approximately 70% whereas the slower formulation is lower at around 30% . The torsion angle at failure indicated higher average values for the multiple GF group but no significant difference between groups was evident .Within the scaffold wall A, both g3 to 6013 mm3 (All defects exhibited some bone growth in the periphery region (outside the scaffold) ranging from 409 mm6013 mm3 B but theVisual inspection of the explants (prior to embedding) indicated that the PLGA based microparticles had completely degraded whereas the PCL scaffold remained in place providing structural stability. Once the tissue was processed through xylene, the remaining PCL was dissolved. Mechanical testing was carried out before histological processing and deformations in the tissue/scaffold are attributed to this destructive mechanical testing.Representative haematoxylin and eosin (H&E) images from decalcified samples within each experimental group demonstrated good integration between the scaffold and the host bone both proximally and distally . All theEntire slices  from each experimental group are shown . CalcifiThe Goldner’s Trichrome staining technique was used to provide a macroscopic overview and compliment the Von Kossa results. The Goldner’s staining results support the micro CT and Von Kossa data with respect to bone and soft tissue distribution within the defects.Several antibodies were selected to evaluate the newly formed bone tissue and identify any differences between groups. Von Willebrand Factor (vWF) is a blood glycoprotein involved in hemostasis and by identifying regions of vWF using immunohistochemistry (IHC), we were able to identify blood vessels within the defect/scaffold. The mid-region  indicated a difference between groups. In the GF-free and BMP-2 only groups, very few blood vessels could be identified, whereas the VEGF, PDGF and BMP-2 group had a higher density of blood vessels, both large and small .Osteocalcin, CD68, and Collagen 1 are markers associated with bone growth and these were present within the mineralised regions of all samples . There wMore bridging was anticipated for GF treated groups but although the high spread of data makes definitive conclusions difficult, the mechanical testing data chearly indicated that the GF treated samples exhibited greater mechanical integrity. An optimum dosing of controlled release GFs remains to be determined but we have demonstrated the successful sustained delivery of GFs to defect sites. This administration regime still has enormous potential to increase both safety and efficacy of GF delivery as developments continue.Callus formation and neovascularisation into the repair site is known to be important in facilitating the recruitment and distribution of stem cells and osteoblasts in the repair site . VEGF waWhile VEGF is important for angiogenesis, there are concerns that prolonged exposure may lead to aberrant effects . It has The ovine tibial critical size defect model is well characterized for bone tissue engineering applciations. It has previously been shown that a scaffold by itself in combination with a fixation plate will not result in clinically predicatble bone volumes for bridging the defct , further3 [3/day BMP-2 and 1 ng/mm3/day VEGF and 1 ng/mm3/day PDGF.Controlled release of the administered GFs was a novel aspect of this approach, this was also one of the most challenging aspects. Determining a sustained release therapeutic GF dose was an estimation based on physiological concentrations. The physiological concentration of BMP-2 in bone can be calculated as approximately 0.02 ng/mm3  and this3  so the tin vitro and issues of incomplete release have been previously reported [The polymer formulations selected for this study were successful in staggering the release of growth factors. Both VEGF and PDGF were released at an early stage (up to day 5) and a later-release of BMP-2 was achieved (Days 25–35). Due to the dense way the microparticles were packed within the scaffold lumen , we postreported  and widereported . We postIt was apparent from histological mineral staining that inclusion of controlled release GFs increased the depth of bone ingrowth into the scaffolds and increased the incidence of bridging . Enhanced bridging was also evident from mechanical testing with bridging localised within the scaffold region. This provided a physiological appearance to the regenerated bone. Localised osteogenesis was an indicator that the system provided a localised region of activity. Mechanical testing showed that the defects treated with VEGF, PDGF and BMP-2 had a greater torsional range than the BMP-2 alone. The cause of this torsional range remains unclear. A greater sample size would have been necessary to begin to draw statistically valid conclusions as the treated groups exhibited less regeneration that was anticipated.The inclusion of microparticles delivering a controlled dose of BMP-2 was able to increase the bridging of defects in contrast to a GF free scaffold. The addition of VEGF and PDGF to the BMP-2 made no significant improvement over the administration of BMP-2 alone in this ovine critical defect model at six-months.Unless otherwise stated, reagents and consumables were purchased from Sigma-Aldrich .et al. [et al. [Poly(lactic-co-glycolic acid) (PLGA) was supplemented with triblock copolymers consisting of PLGA and polyethylene glycol (PEG) to tailor protein release. PLGA-PEG-PLGA was prepared and analysed using previously described methods . Triblocet al.  and adap [et al. .water/oil/water double emulsion solvent evaporation technique [oil phase. A protein mixture consisting of 1 mg growth factor and 9 mg albumin (from human serum) (HSA), or 10 mg HSA for GF-free microparticles, was dissolved in deionised water (100 µL); this was added to the oil phase. This mixture was homogenised using an LM5 axial impeller mixer  for 2 min at 4000 rpm. This primary water/oil emulsion was quickly added to a 200 mL bath of polyvinyl alcohol (0.3%) and homogenised again for 2 min at 2000 rpm. This water/oil/water emulsion was stirred at 300 rpm for 4 h and the microparticles were then washed and collected before freeze drying. The finished microparticles were free-flowing and of a homogeneous consistency and a mean diameter of 100 µm.Microparticles were prepared according to a previously described method involving a echnique ,22. PLGAThree different polymer formulations were selected, one for each growth factor. Two variables were utilised to control the rate of growth factor release: the ratio of lactide to glycolide in the PLGA and the weight percentage of PLGA-PEG-PLGA added to the PLGA. The three formulations are detailed in 165) and recombinant human platelet derived growth factor (rhPDGF-BB) were purchased from PeproTech . Recombinant human bone morphogenetic protein-2 (rhBMP-2) was purchased from Professor Walter Sebald . The microparticles that contained HSA and no growth factor are referred to throughout as GF-free.Recombinant human vascular endothelial growth factor  of the microparticles (triplicate samples from each batch) were suspended in 1.5 mL phosphate buffered saline ; samples were gently rocked in a humidified incubator at 37 °C. At defined time intervals the entire amount of PBS was removed from the 1.5 mL microcentrifuge tubes and replaced with 1.5 mL fresh PBS; all liquid above the microparticles was collected without removing the microparticles. The collected supernatants were stored frozen until required and then assayed using a Micro BCA assay kit  with a standard curve consisting of human serum albumin (0–40 µg/mL).Bioresorbable cylindrical scaffolds of medical grade poly-caprolactone (mPCL),  were produced by melt extrusion using a BioExtruder  .The structural parameters of the scaffolds were tailored by computer-aided design and included specifications of 70% porosity with 100% pore interconnectivity within a pore size of 350–500 μm size and a 0/90° lay-down pattern A. This aPrior to surgery, all scaffolds were surface treated for six hours with 1M sodium hydroxide (NaOH) and washed five times with phosphate-buffered saline (PBS) to render the scaffold more hydrophilic. Scaffold sterilization was achieved by incubation in 70% ethanol (followed by complete evaporation) and UV irradiation for 90 min, rotating the scaffold 3 times.The microparticles were combined with the scaffold to provide a bioactive GF-containing element to the structurally supportive mPCL scaffold as shown in et al. [To produce platelet rich plasma (PRP) 80 mL of blood was collected from the jugular vein of each sheep and transferred into 3.5 mL monovettes supplemented with sodium citrate (3.8%) at a ratio of 9 volumes blood and 1 volume sodium citrate according to procedures reported by Anitua et al. . The citet al. . The pelEach sheep was implanted with the same mixture of microparticle formulations, the only difference being whether the microparticles contained growth factor or not. The microparticles .Fifteen mature merino sheep  were operated upon as approved by the University Animal Ethics Committee of the Queensland University of Technology, Brisbane, Australia . The experimental groups consisted of a scaffold group containing GF-free microparticles  which served as a negative control, plus two growth factor containing groups as detailed in et al. [The surgical procedure was carried out using the methods described by Reichert et al. . BrieflyClinical X-rays  were taken immediately after surgery and after six months healing time in two standard planes  to determine the healing progress of the defect in the different experimental groups over time.After six months the animals (which gained on average 20 to 30% weight) were euthanized by intravenous injection of 60 mg/kg sodium pentobarbital  and both the left (control tibiae) and the right tibiae (containing the scaffold) and 1 cm of surrounding tissue were explanted for subsequent mechanical testing, micro computed tomography and histological analysis.Mechanical testing was carried out at the same time post euthanasia for all samples and all samples were subjected to testing but only samples with enough mechanical strength to resist torque were suitable for testing. The fracture fixation plate was carefully removed from the experimental leg. Both ends of the tibia were embedded in stainless steel cups using 80 ml of dental acrylic  and the bone was then mounted in an Instron 8874 biaxial testing machine  according to published methods . By leavSamples were scanned in a micro computed tomography (microCT) scanner  with a voxel size of 36 μm. The X-ray tube was operated at 70kV and 114 μA. For further analysis, the scans were segmented using a threshold of 437.8 mg HA/ccm, a Gaussian filter width of 0.8 and filter support of 1.0 to identify mineralised tissue. The segmented scans were then analysed for bone volume and bone mineral density within the defect using the software supplied by the manufacturer. For bone volume, the regions of interest were determined as external callus formation , bone formation within the scaffold  and bone formation in the inner part of the scaffold (scaffold lumen). Defect bridging was defined as when new and continuous mineralised bone joined the proximal and distal regions of native bone.After micro CT scanning, tibial bone specimens were trimmed to five cm length  and fixed in 4% paraformaldehyde for one week before being transferred to 70% ethanol. For histological analysis, the mid-defect regions were sectioned in the transverse and sagittal plane. The sagittally sectioned samples were used for paraffin embedding. For processing decalcified samples into paraffin, bone samples were decalcified in 15% EDTA for 6–8 weeks at 4 °C. The samples were then dehydrated in increasing ethanol concentrations in a tissue processor , and embedded in paraffin. Five-micrometer sections were obtained using a rotary microtome .The mineralised bone samples to be cut on the sagittal plane were embedded in methylmethacrylate resin . Six-micrometer sections of the full length of the defect (5 cm long) were cut using a sledge microtome  and 40–50 µm sections were cut using an EXAKT cutting and grinding system . Sections were cut at two thicknesses because the thicker ones preserve gross morphology more accurately and the thin sections reveal more cellular detail.Staining using the Von Kossa method was used to identify mineralised tissue which appears black and a MacNeil’s Tetrachrome stain showed cellular detail in the soft tissue. Resin sections (6 µm) were deplastified using acetone and rehydrated with serial concentrations of ethanol. Sections were immersed in silver nitrate solution (5%) for 5 min then developed with a sodium carbonate formaldehyde solution for 2 min. Slides were rinsed and immersed in Farmer’s Diminisher solution for 30 s. After further washing, slides were immersed in MacNeal’s Tetrachrome solution for 5 min with freshly prepared solution. The slides were then dehydrated and mounted using Eukitt mountant.Resin sections (40–50 µm) were immersed in xylene followed by descending concentrations of ethanol. Samples were stained in Weigert’s Haematoxylin then washed. Slides were then stained in Ponceau-Fuchsin working solution (10 min) followed by acetic acid (1%). Slides were next immersed in Orange G solution (20 min) followed by acetic acid (1%). Finally, slides were immersed in Light Green solution (10 min) followed by acetic acid (1%). Slides were serially dehydrated and then coverslips were applied using Eukitt mountant.Paraffin sections were deparaffinized with xylene and rehydrated with decreasing concentrations of ethanol, before being stained with haematoxylin and eosin, dehydrated and mounted with Eukitt mountant.2O2 in Tris-HCl for 20 min. This was followed by three washes with Tris buffer (pH = 7.4) for 2 min each. Sections were incubated with Proteinase K  for 20 min and subsequently incubated with 2% bovine serum albumin in DAKO antibody diluent in a humidified chamber at room temperature for 20 min to block nonspecific binding sites. Paraffin sections were deparaffinised with xylene and rehydrated with decreasing concentrations of ethanol. Subsequently, sections were rinsed in distilled water and placed in 0.2 M tris(hydroxymethyl)aminomethane (Tris) buffer prepared at pH = 7.4. Endogenous peroxidase activity was blocked by incubating the sections in 3% HImmunohistochemical staining was performed using a primary antibody specific to the markers of interest. The sections were incubated with the specific antibody in humidified chambers at 4 °C overnight (detailed below). Sections were then washed three times for 2 min with Tris buffer (pH = 7.4) and incubated with peroxidase labelled dextran polymer conjugated to goat anti-mouse and anti-rabbit immunoglobulins  at room temperature in humidified chambers for 60 min. Colour was developed using a liquid 3,3-diaminobenzidine (DAB) based system (DAKO). Slides were stained with Haematoxylin to visualise cell nuclei then serially dehydrated and Eukitt coverslip mountant was used for coverslip mounting.The selected antibodies were for Osteocalcin , Collagen 1 , CD68  and Von Willebrand Factor .p < 0.05.Statistical analyses for the biomechanical results and for the micro computed tomography scans (micro CT) were carried out using a Mann-Whitney U test and p-values were adjusted using the Holm method . All stain vivo invironments. Biomimicry is the key to biologically relevant treatment approaches.The group administered with VEGF, PDGF and BMP-2 indicated greater blood vessel density in the mid-section of the scaffold. This apparent increased vascularisation had little effect on mineralised bone and bridging at the six-month timepoint. Further study would be required to quantitatively assess vasculature and ascertain if this increased vasculature results in a better long-term outcome. It appears that under the conditions evaluated, that the angiogenic GFs added showed no benefit to defect mineralisation and added significant costs to treatment groups. Further studies of controlled release BMPs alone are however a strong candidate for future study. By controlling the release of GFs we can more accurately mimic Although this study failed to determine the advantage of VEGF and PDGF administration during osteogenic regeneration, it did show that controlled release formations of BMP-2 can be effective when applied in a clinically relavent large animal model.


Elevated estradiol levels are correlated with male infertility. Causes of hyperestrogenism include diseases of the adrenal cortex, testis or medications affecting the hypothalamus-pituitary-gonadal axis. The aim of our study was to elucidate the effects of estradiol treatment on testicular cellular morphology and function, with reference to the treatment regimen received. Testes samples (n = 9) were obtained post-orchiectomy from male-to-female transsexuals within the age range of 26–52 years. Each patient had a minimum of 1–6 years estradiol treatment. For comparison, additional samples were obtained from microscopically unaltered testicular tissue surrounding tumors (n = 7). The tissues obtained were investigated by stereomicroscopy, histochemistry, scanning electron microscopy (SEM) and immunohistochemistry. Our studies revealed that estradiol treatment significantly decreased the diameter of the seminiferous tubules (p < 0.05) and induced fatty degeneration in the surrounding connective tissue. An increase in collagen fiber synthesis in the extracellular matrix (ECM) surrounding the seminiferous tubules was also induced. Spermatogenesis was impaired resulting in mainly spermatogonia being present. Sertoli cells revealed diminished expression of estrogen receptor alpha (ERα). Both Sertoli and Leydig cells showed morphological alterations and glycoprotein accumulations. These results demonstrate that increased estradiol levels drastically impact the human testis. Estrogens play an important role in ensuring proper function of the male reproductive tract. In males, the main biologically active estrogen is estradiol. The primary source of this hormone is from the aromatization of testosterone23567810111213141617181921222324262729in vitroin vivo mouse model, estradiol exposure resulted in premature capacitation of the cauda epididymal sperm, which could be correlated with decreased sperm viability in the reproductive tract of the female35Estradiol’s physiological importance in the male reproductive tract has been well documented in the literature both in humans and in animal model systems. However, it is mainly in animal models that the effects of elevated estradiol levels in males has been evaluated. Previous studies have demonstrated that estradiol significantly enhanced mouse sperm fertilizing capacity Studies conducted in the late 1970 s and 1980 s have previously assessed the effects of estradiol treatment on morphology of the human testis in male-to-female transsexuals. One study showed that estradiol treatment resulted in decreased testicular size and reduced germ cell numbers39The purpose of this study was therefore to assess the effects of estradiol treatment on the functional morphology of the testis and on spermatogenesis in male-to-female transsexuals, with special focus on the impact of different treatment regimens on testicular alterations. This knowledge is a pivotal prerequisite to create novel therapeutic strategies for treatment of patients with hormonal disorders to restore testicular function and fertility.t-test). The seminiferous tubules of patients treated with estradiol were, on average, 21% smaller in diameter than the patients without estradiol treatment (Estradiol treatment significantly decreased the diameter of the seminiferous tubules (reatment . Followi®) did not show vacuolation. Sudan Red staining confirmed that these vacuoles were fat droplets , which in turn leads to reduced serum testosterone concentrations5455Besides its distinct effects on the cells involved in spermatogenesis, treatment with estradiol also induces strong alterations in the connective tissue surrounding the seminiferous tubules. Considerable fatty degeneration occurred in the interstitial tissue surrounding the tubules after estradiol treatment. It has been shown that increased adipose tissue in obese individuals leads to marked elevation of extragonadal aromatase activity, which results in enhanced estradiol levelsIn addition to the occurrence of fatty degeneration, collagen synthesis within the ECM of the seminiferous tubules was increased after estradiol treatment. This is of particular interest because an increase in thickness of the ECM has been correlated with male infertility62In summary, our study has shown that treatment with estradiol results in severely impaired spermatogenesis which is correlated with increased synthesis of glycoproteins in Sertoli cells and Leydig cells and increased collagen synthesis and fatty degeneration in the testicular connective tissue. The results of this study have added new insights by quantifying the significant decrease in diameter of seminiferous tubules after hormonal treatment, by linking the different effects seen to different treatment regimens and by assessing the effects of estradiol treatment on ERα expression in the human testis. There is clear evidence from the data that treatment of patients with additional medications, such as androgen antagonists or GnRH agonists, alongside estradiol exacerbate the effects of estradiol treatment, particularly on spermatogenesis. This data might be of interest to doctors prescribing hormonal drugs to male-to-female transsexuals in order to optimise the treatment of each individual. Our results are also likely to provide novel concepts for treatment of males revealing excess estradiol levels. Male infertility is a condition attributed to hyperestrogenism and it has been postulated that it is more common than currently assumed47656667The study was ethically approved by the Ethic Commission of the University of Munich (Nr. 161–12) and was performed according to the declaration of Helsinki.Informed and written consent was obtained from all patients before surgery.®, Gynokadin Dosiergel®); the remaining patient was taking oral estradiol (Estrofem®). 8 patients received additional medication in form of a) GnRH analogues , b) antiandrogens , c) progestins (Duphaston®) or d) progesterone (Progestogel®). Treatment regimen, dose and duration of treatment are outlined in All patients were on estradiol treatment for 1–6 years prior to operation. The estradiol medication used by 8 patients was estradiol gel  and the sections from patients without estradiol treatment (4) using CellSens Standard (Olympus) image processing software. Only circular tubules were measured on each patient’s section and the entire slide was assessed (on average 144 tubules were measured per slide). Two slides were analysed per patient.Sections were fixed in Bouin’s fluid, dehydrated in graded dilutions of ethanols (70–100%) and embedded in paraffin. For staining, 3 μm sections were cut and mounted on Superfrost Plus slides . Cryosections were prepared as follows: testicular tissue samples (1 cm) were snap-frozen in liquid nitrogen immediately after removal. After cryotomy, sections (8 μm) were put on coated slides  and dried at room temperature. The slides were frozen at −20 °C until use.Hematoxylin and Eosin (H&E) staining was carried out to investigate testicular morphology. Following dewaxing in xylene , rehydration in descending alcohols  and distilled water , sections were put in hematoxylin , running water (15 min), eosin  and then running water (5 min). Sections were then dehydrated in ascending alcohol solutions , cleared in xylene , and mounted in DPX .Periodic acid-Schiff (PAS) staining was carried out with and without amylase digestion for detecting glycogen and glycoproteins, respectively. Following dewaxing in xylene , rehydration in descending alcohols  and distilled water , sections were put in 0.25% periodic acid , running water (10 min), distilled water , Schiff’s reagent , sulfite water , running water (10 min), hematoxylin , and running water (10 min). Sections were then dehydrated in ascending alcohol solutions (70–100%), cleared in xylene , and mounted in DPX . The same protocol was carried out with slides pretreated with amylase  before incubation in periodic acid. Amylase digests glycogen so that glycogen and glycoproteins can be differentiated.Picrosirius red staining was carried out for localization of collagen around the tubules. Sections were dewaxed in xylene , rehydrated in descending alcohols  and put in picrosirius red/fast green solution in the dark . They were placed in distilled water , dehydrated in a series of ascending alcohol solutions , cleared in xylene , and mounted in DPX .Sudan Red staining was carried out as follows:For localization of fat vacuoles in tissue outside of the tubules, cryosections were thawed, washed twice in phosphate buffered saline (PBS) , pre-treated with 60% isopropylene , and finally put in oil red . Following treatment the specimens were washed in distilled water and counterstained with hematoxylin .Paraffin sections were dewaxed in xylene  and rehydrated in descending alcohols (100–70%). Antigen retrieval was carried out using citrate acid buffer in a high pressure cooker . Slides were then permeabilized in 10% Triton X-100 , rinsed with PBS-Tween  and blocked in 10% goat serum . The slides were then incubated with a MAGEA4 primary antibody . Anti-MAGEA4 is a mouse monoclonal antibody purified from hybridoma 57B, and enables differentiation between spermatogonia and Sertoli cells® 488 conjugate; Thermo Scientific, Dublin, Ireland; dilution 1:500, 1 hr]. After incubation, the slides were washed in PBS , mounted with Fluoromount  and coverslipped. Microscopy was performed using the Olympus BX51 microscope and an Olympus DP71 camera . In order to be able to compare immunohistochemical staining and morphological pictures, phase contrast microscopy (Olympus BX51 microscope) was applied on the sections after fluorescent imaging. The pictures were merged using the software Adobe Photoshop.Paraffin sections were dewaxed in xylene  and rehydrated in descending alcohols (100–70%). Sections were rinsed in PBS . Antigen retrieval was carried out using citrate acid buffer in a microwave oven . Slides were left at RT for 8 min and washed in PBS . Slides were placed in bovine serum albumin  and washed in PBS . Slides were blocked in 10% normal goat serum  and incubated in the primary antibody . The slides were washed in PBS  and incubated in Alexa Fluor 488 labelled secondary antibody [goat anti-Rabbit IgG (H + L), Alexa Fluor2PO4, and 0.07 M Na2HPO4-2H2O) and fixed in 1% glutaraldehyde in Soerensen’s buffer . Tissues were washed again in Soerensen’s buffer and dehydrated in a series of ascending acetones . A union point dryer CPD 030  was used to dry the tissue samples. For visualisation of the lumina of the seminiferous tubules, the samples were dipped in liquid nitrogen and mechanically broken into several pieces using a forceps. The pieces were coated with 12-nm gold-palladium using the Union SCD 040 sputtering device . Analyses were performed with a Zeiss scanning electron microscope DSM 950 at magnifications of x200 to x15,000.After removal, the tissues were washed in Soerensen’s buffer twice .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


BRCA1/2 genes. However, through germline and tumor sequencing an understanding of the larger phenomenon of homologous recombination deficiency (HRD) has emerged. HRD impairs normal DNA damage repair which results in loss or duplication of chromosomal regions, termed genomic loss of heterozygosity (LOH). The list of inherited mutations associated with ovarian cancer continues to grow with the literature currently suggesting that up to one in four cases will have germline mutations, the majority of which result in HRD. Furthermore, an additional 5–7% of ovarian cancer cases will have somatic HRD. In the near future, patients with germline or somatic HRD will likely be candidates for a growing list of targeted therapies in addition to poly (ADP-ribose) polymerase (PARP) inhibitors, and, as a result, establishing an infrastructure for widespread HRD testing is imperative. The objective of this review article is to focus on the current germline and somatic contributors to ovarian cancer and the state of both germline and somatic HRD testing. For now, germline and somatic tumor testing provide important and non-overlapping clinical information. We will explore a proposed testing strategy using somatic tumor testing as an initial triage whereby those patients found with somatic testing to have HRD gene mutations are referred to genetics to determine if the mutation is germline. This strategy allows for rapid access to genomic information that can guide targeted treatment decisions and reduce the burden on genetic counselors, an often limited resource, who will only see patients with a positive somatic triage test.Until recently our knowledge of a genetic contribution to ovarian cancer focused almost exclusively on mutations in the  BRCA1 and BRCA2 mutations. However, there also are patients with germline mutations in other HR pathway genes and patients who do not carry an inherited germline mutation but have tumors with sporadic HRD mutations. Data from the Cancer Genome Atlas (TCGA) demonstrates that approximately fifty percent of high grade serous ovarian cancers have aberrations in HR repair [Ovarian cancer is the fifth leading cause of death in women in the United States with an estimated 22,280 new cases and 14,240 deaths in 2016 . The advR repair . PatientBRCA1 and BRCA2, proteins of the MNR complex (MRE11/RAD50/NBS1), CtIP, MRE11, RAD51, ATM, H2AX, PALB2, RPA, RAD52 and proteins of the Fanconi anemia pathway [BRCA1 or BRCA2 deficiency, they rely on other repair pathways like non-homologous end-joining (NHEJ), which is less precise and more error-prone [DNA damage is a constantly occurring phenomenon that necessitates a complex network of molecular repair pathways in order to maintain genomic integrity and prevent cell death. HR is an important pathway that allows repair of double-stranded DNA breaks. HR operates during the S and G2 phases of the cell cycle and relies on many proteins including  pathway , 9. Whenor-prone . NHEJ reor-prone , 11.BRCA1 and BRCA2, with a small contribution from mutations in the DNA mismatch repair (MMR) genes [RAD51C, RAD51D, BRIP1, PALB2, BARD1 and the MMR genes [Until recently, hereditary EOC was thought to be caused almost exclusively by mutations in R) genes . TCGA, hMR genes , 13. ThiMR genes .BRCA1 and BRCA2 are the most well-known ovarian cancer susceptibility genes, with germline genetic testing available since the 1990s [BRCA mutation-associated ovarian cancers have multiple distinct clinical features including earlier age at diagnosis, visceral distribution of disease, improved survival, enhanced sensitivity to platinum-based chemotherapies and sensitivity to PARP inhibitors [BRCA1/2 germline mutations in patients with epithelial ovarian cancer is estimated to be about 11–15% [When considering the genetic contribution to carcinogenesis, an important distinction is whether a mutation is germline or somatic. Germline implies that the mutation was inherited and is therefore present in all of the individuals’ cells. Somatic mutations are those mutations that are acquired and therefore occur exclusively in the tumor cells. he 1990s . BRCA muhibitors –18. The t 11–15% .BRCA1 and BRCA2 testing in the United States. Following this decision, many other clinical laboratories began offering BRCA1 and BRCA2 testing, both as single gene tests and in the form of comprehensive genetic panels [BRCA1 or BRCA2 [In June 2013 the Supreme Court ruled unanimously against Myriad Genetics, invalidating the exclusive license rights to c panels . Althougor BRCA2 , 24 , EMSY (8%), PTEN (7%), RAD51C (3%), ATM/ATR (2%) and Fanconi anemia genes (5%) [BRCA1 and BRCA2 sequencing of 235 unselected ovarian cancer tumors and found mutations in 19% of cases (31 mutations in BRCA1 and 13 in BRCA2). Germline DNA specimens were available for 28 of the patients harboring a BRCA1 or BRCA2 tumor mutation. Eleven (39.3%) of the mutations present in the EOC tumor were found to be somatic and 17 germline (60.7%) [BRCA1 (2.1%) and BRCA2 (1.4%). , PTEN %, RAD51Cnes (5%)  performe (60.7%) . When ev (60.7%)  discover (60.7%)  evaluateBRCA1 and BRCA2, to full panel testing of multiple cancer-associated genes.Genetic testing for germline mutations is recommended for all women with non-mucinous epithelial ovarian cancer by multiple professional societies including the American College of Medical Genetics and Genomics (ACMG) , the AmeCurrently, the guidelines that recommend germline testing do not make specific recommendations regarding which of the many available platforms to utilize . Cancer BRCA1/2-associated ovarian cancer has multiple distinct features affecting age at diagnosis, disease distribution, chemosensitivity and survival. Patients with known mutations have access to targeted therapy, for example PARP inhibitors for BRCA1/2 mutation carriers. In December 2014, the U.S. Food and Drug Administration (FDA) granted olaparib accelerated approval for monotherapy in patients with germline BRCA1/2 mutations and recurrent ovarian cancer with three or more prior lines of chemotherapy, making olaparib the first of the PARP inhibitors to receive FDA approval. On December 19, 2016 the FDA granted accelerated approval to rucaparib in patients with recurrent ovarian cancer with two or more prior lines of chemotherapy and either germline BRCA1/2 mutations or somatic BRCA1/2 mutations in the tumor detected by a next-generation sequencing-based companion diagnostic test. The European Commission granted marketing authorization for the PARP inhibitor olaparib as monotherapy in the maintenance treatment of adult patients with platinum-sensitive, relapsed BRCA-mutated (germline and/or somatic) high-grade serous epithelial ovarian, fallopian tube, or primary peritoneal cancer who are in complete response or partial response following platinum-based chemotherapy [Discovering a germline mutation in a patient with EOC has multiple significant clinical implications. As previously stated, otherapy . The recotherapy .In addition to treatment eligibility, many mutations are predictive of other cancers in addition to ovarian cancer, offering patients the opportunity for heightened awareness and screening. Finally, identifying a germline mutation can allow family members to undergo genetic counseling and testing, termed “cascade” testing. Informed relatives have the opportunity to pursue intensive cancer surveillance and risk-reducing options. Data from individuals with Lynch syndrome demonstrate that awareness of a mutation results in cancer prevention or early diagnosis of cancer, which can improve morbidity and prevent mortality –51.BRCA1/2 status and identifying HRD tumors [In addition to germline testing, multiple companies now offer tumor genetic profiling for somatic mutations. There are methods using NGS similar to that done for germline DNA. There are also independent DNA-based measures of genomic instability reflecting underlying tumor HRD on the basis of loss of heterozygosity (LOH), telomeric allelic imbalance (TAI) and large-scale state transitions (LST). Each individual metric and the combination is significantly associated with D tumors –54. SomaD tumors . FinallyD tumors . While dD tumors .BRCA1/2 mutation is a predictive biomarker of PARP inhibitor activity however the number of patients with somatic mutations that have been analyzed is low. In a phase II study of olaparib, Gelmon et al. found that 24% of BRCA-negative patients with high grade serous or undifferentiated ovarian cancer achieved a radiological objective response [BRCA1/2 mutations and without germline BRCA1/2 mutations but with HRD. Swisher et al. [BRCA1/2 mutations and patients who were BRCA1/2 wild-type but were found to be LOH high. LOH status was determined by next-generation sequencing using a cutoff of 14% or more of genomic LOH in pretreatment biopsy specimens to be considered LOH high. Studies that are currently ongoing or recently completed, and future planned studies will likely clarify this question, including SOLO-2 (NCT01874353) and ARIEL-3 (NCT01968213) [Early evidence suggests that a somatic response . In the response  niraparir et al.  found thNCT0187433 and ARI1968213) . See TabBRCA1/2 mutations, 61% were the result of germline inherited mutations. This presents an important ethical consideration regarding patient counseling and informed consent. Thorough genetic counseling and informed consent are required prior to germline genetic testing however this is not the case with tumor profiling. This is important given that almost two-thirds of patients with a mutation found on tumor profiling will have a germline mutation which carries significant clinical implications for the patient and her family members.Somatic tumor testing has two important implications: 1) identifying patients for targeted therapies like PARP inihibitors, 2) identifying patients who should be referred for genetic counseling and offered genetic testing. Mutations that are purely somatic and not found in the germline DNA are not inherited, cannot be passed to offspring, and therefore do not warrant cascade genetic counseling and testing. If a patient is found to have a mutation on tumor profiling, the only way to definitively classify it as a germline versus somatic mutation is through germline testing. As described previously, Hennessy et al.  found thIt has become clear that both somatic and germline mutation testing provide important information for patients with EOC. The question is whether or not both are necessary and if so what is the ideal chronology of testing. Table BRCA mutation status has led to an increasing demand for genetic counselors [BRCA1/2 mutations were referred to genetics [BRCA1/2 [This issue of genetic counseling as a limited resource is an important one. The exponential growth in diagnostic and treatment options that utilize genetic and genomic sequencing information coupled with public coverage of celebrity unselors , 62. A sgenetics . Another[BRCA1/2 . There c[BRCA1/2 . Video-a[BRCA1/2 .BRCA1/2 mutations) indicates a great expansion of the population who can benefit from PARP inhibitors. Interestingly Mirza et al. even found efficacy in the non-germline BRCA mutation and HRD-negative group . In an exploratory analysis of ARIEL2 Part 1, Swisher et al. [The recent approval of rucaparib for BRCA positive (germline or somatic) patients with recurrent ovarian cancer who have had two or more lines of therapy would argue for tumor testing to screen for therapeutic options and as a triage for germline testing. However, the success of niraparib in significantly extending the duration of progression-free survival in all platinum-sensitive recurrent ovarian cancer patienrs,  or microsatellite instability (MSI) analysis was used for initial screening of Lynch syndrome. Mismatch repair deficiency (MMR-D) occurs in 15–20% of colorectal tumors with one-quarter resulting from Lynch syndrome [Currently, as stated above, somatic tumor testing can provide information only about genetic aberrations in the tumor, however as NGS continues to become increasing widespread its applications will likely broaden. Stadler et al.  recentlysyndrome . Stadleysyndrome , 70.If somatic tumor profiling can replace MMR assessment, the next question is whether or not somatic tumor profiling can replace germline testing altogether. Currently none of the tumor profiling platforms offer information on germline mutational status however this may change. The feasibility of this question was evaluated with a study comparing deep, uniform NGS sequencing of tumor tissue compared to Clinical Laboratory Improvement Amendments (CLIA)-certified, NGS-based germline exon sequencing of 236-cancer related genes. A computational method could predict the somatic status of mutations from tumor tissue without the need for a matched normal control with an accuracy of greater than 95%. As tumor profiling for this indication is permitted under investigational use only and is not CLIA-certified, the authors concluded that NGS tumor sequencing can indicate which patients require additional work-up for germline testing . HoweverBRCA1/2 silencing can occur via indirect mechanisms including promoter methylation and interactions with other proteins involved in DNA repair. The clinical implications of epigenetic changes remain unclear with conflicting data in the literature. Chiang et al. [BRCA1 promoter hypermethylation resulted in significantly shorter survival when compared to BRCA1 germline mutations and BRCA1 wild-type without promoter hypermethylation. In contrast, TCGA found no survival difference with BRCA1 hypermethylation [BRCA1 methylated cases had a survival benefit compared to germline wild-type and similar to that of the germline mutation cases. Prospective studies are necessary to better address the clinical significance of epigenetic alterations in the HR pathway.Finally, an important remaining area of uncertainty is the role of epigenetic alterations in EOC. HRD can result from genomic processes that are distinct from germline and somatic mutations in HR genes. For example g et al.  found thhylation  and Cunnhylation  observedIt is clear that the knowledge of both germline and somatic mutation status is becoming increasingly important in the management of patients with ovarian cancer. The experience with PARP inhibitors in ovarian cancer clinical trials demonstrates that the targeted therapeutic effect is beyond just BRCA germline mutations and more widely applicable to HRD. Whether epigenetic modifications to HRD genes confer the same therapeutic sensitivity remains unclear however future studies will likely allow for a better understanding of their underlying mechanisms and clinical significance. The list of ovarian cancer-associated genes continues to grow and the literature currently suggests that up to one in four women with ovarian cancer will have a germline HR deficiency and an additional 5–7% will have a somatic HR deficiency. Currently, these patients with recurrent disease are candidates for approved therapy with PARP inhibitors, however in the near future there will likely be a growing list of targeted therapies available for ovarian cancer patients. As a result, infrastructure for widespread somatic HRD testing in routine clinical practice, as is the case for HER2 in breast cancer and epidermal growth factor receptor in lung cancer, should be supported . Further


Multicellular spheroids serve as an excellent platform to study tissue behavior and tumor growth in a controlled, three-dimensional (3D) environment. While molecular and cellular studies have long used this platform to study cell behavior in 3D, only recently have studies using multicellular spheroids shown an important role for the mechanics of the microenvironment in a wide range of cellular processes, including during tumor progression. Despite the well-established relevance of mechanical cues to cell behavior and the numerous studies on mechanics using 2D cell culture systems, the spatial and temporal variations in endogenous cellular forces within growing multicellular aggregates remain unknown. Using cell-sized oil droplets with controlled physicochemical properties as force transducers in mesenchymal cell aggregates, we show that the magnitude of cell-generated stresses varies only weakly with spatial location within the spherical aggregate, but it increases considerably over time during aggregate compaction and growth. Moreover, our results indicate that the temporal increase in cellular stresses is due to increasing cell pulling forces transmitted via integrin-mediated cell adhesion, consistent with the need for larger intercellular pulling forces to compact cell aggregates. Multicellular spheroids are 3D aggregates of adherent cells that adopt an overall spherical morphology and display key defining features of 3D tissues through cell-cell and cell-matrix interactions7. Additionally, chemical gradients established within spheroids  due to their 3D geometry make them suitable to model tumor formation, which are often subject to inner hypoxic regions and proliferative gradients9. In addition to chemical gradients, variations in physical quantities, such as cellular forces, can also play a key role in the formation and homeostasis of 3D multicellular systems8.Cell culture techniques have provided an excellent platform to perform molecular and cell biology studies with carefully controlled biochemical conditions, especially when compared to more complex 15, as well as during development18 and disease progression in 3D tissues23. Most of our understanding of how cells generate forces and respond to mechanical cues was enabled by crucial advances in measuring cell forces in 2D multicellular systems15 (mainly cell monolayers in culture conditions) and 3D systems with cells embedded in artificial gel matrices25. In contrast, our understanding of cellular force generation within 3D multicellular systems remains poorly understood. While several studies have focused on the role of the mechanical environment external to multicellular 3D aggregates, the endogenous distributions of cell-generated stresses that build and sustain multicellular aggregate formation and growth in 3D remain unknown.Both the application of external forces and changes in the mechanical properties of the cellular microenvironment significantly alter cell behavior in 2D cell culture systems27, but never observed directly. Previous studies estimated internal stresses by physically cutting excised tumors and using the resulting tissue deformation, along with mathematical modeling, to infer these forces28. Using this method, compressive stresses within murine tumors were estimated to range from 0.37–8.01 kPa. Less invasive methods have also been used to measure the maximal value of stresses generated by cancerous cell spheroids. The resistance to spheroid growth through use of elastic microcapsules29 or gels of varying stiffness31, as well as osmotic pressure applied to the surface of spheroids27, have been used to the measure bulk, isotropic mechanical stresses that spheroids generate on the surrounding space. More recently, elastic microbeads were used to measure effects of mechanical compression on the propagation of isotropic stresses within tumor spheroids32. While these studies investigate mechanical stresses within aggregates due to confinement and external compression, the spatiotemporal variations of endogenous stresses during aggregate growth and compaction have not been yet investigated.Internal, endogenous spatial variations in cellular stresses have been suggested to exist within 3D cellular spheroids and tumorsin situ, within 3D multicellular systems33. The technique relies on fluorescently labelled, cell-sized, oil droplets that are injected between the cells in a multicellular 3D system and used as forces transducers. While these droplets were shown in proof-of-principle experiments to be able to measure cell-generated stresses within cellular aggregates of mammary epithelial cells and tooth mesenchymal cells33, the limited control of the physical and chemical properties of the droplets, as well as the reliance on non-commercial components, hindered the practical use of droplets as force sensors in many situations.A recently developed technique employs oil droplets to measure cell-generated stresses 33. We use a two-surfactant system that enables independent control over the droplet interfacial tension and cell-droplet specific interactions, and allows stress measurements even in chemical environments with varying ionic strengths or in the presence of small surface-active molecules, such as cell culture media. By injecting these new droplets in growing spheroids of tooth mesenchymal cells at different spatial locations and stages, we measure the spatiotemporal changes in endogenous cell-generated stresses. While the magnitude of cell-generated anisotropic stresses does not vary strongly with location inside the aggregate, it considerably increases over time during spheroid growth and compaction. Comparing the stresses measured using droplets coated with and without ligands targeting integrin receptors, we show that pulling forces transmitted via integrin-mediated adhesion increase over time during spheroid compaction.Here we introduce a new system of droplet stress sensors based only on commercially-available components and with fully-controlled physical and chemical properties. Using these new droplets, we quantify the spatiotemporal changes in endogenous, cell-generated stresses within growing multicellular spheroids of mouse tooth mesenchymal cells, a cell type previously used in experiments reporting cellular forces33. In addition, control over the droplet’s interfacial tension enables stress measurements in different systems, such as in multicellular aggregates of different cell types or in distinct regions of embryonic tissues, in which cellular stresses may vary by orders of magnitude.In order to use oil droplets as force transducers it is important to control the droplet interfacial tension and prevent uncontrolled interfacial tension variations arising from changes in ionic strength or the presence of a variety of small proteins in the surrounding medium, as the value of the measured cellular stresses relies on its knowledge33 controlled the interfacial tension and the cell-droplet interactions using the same surfactant molecule. Using a single surfactant, even in the presence of co-surfactants33, presents several problems. Cell-induced surfactant aggregation or extraction of surfactants from the droplet surface can lead to uncontrolled variations in the droplet’s interfacial tension, which may affect the accuracy of mechanical stress measurements. Moreover, the droplet’s interfacial tension needs to be adjusted so that droplet deformations generated by cellular stresses can be readily measured with a good signal. Low droplet interfacial tensions provide better signal-to-noise ratio in the measurement of the droplet shape, as the droplet deformations are higher for lower interfacial tensions. However, very low interfacial tensions can lead to sharp variations in the droplet shape that are difficult to quantify accurately. Since actomyosin-generated tensions largely control cortical mechanics  and have been measured to be typically in the range 0.1–1.5 mN/m36, droplet interfacial tensions in the range 1–10 mN/m enable quantitative measurements with moderate droplet deformations. While this range of interfacial tension can be achieved using custom-made dodecylamine (DDA) co-surfactants, as previously shown33, it is preferable to have a readily available commercial surfactant system for this purpose. Ideally, such a new surfactant system would decouple the control of interfacial tension from the control of cell-droplet interactions and also shield the droplet’s interface  from uncontrolled chemical environments.The originally developed droplet force transducers37  or the type of fluorocarbon oil used  fluorosurfactant allows control of the droplet interfacial tension in environments with strongly varying ionic strengths and helps reduce the adsorption of other small molecules to the droplet interface. Indeed, varying concentrations of ionic strength over several orders of magnitude do not have a substantial effect on the measured interfacial tension , above approximately 0.1 M, affected the interfacial tension by reducing it slightly (6% reduction for CNaCl = 0.1 M). This small decrease may be either due to the presence of unreacted short PEG molecules or to the presence of a vary small fraction of the ionic unreacted materials used to synthesize Krytox-PEG surfactants37. Previous works showed that the interfacial tension of interfaces stabilized only with such ionic materials  decreases in the presence of phosphate buffered saline (PBS)38. In order to explore how a more complex chemical environment would affect the interfacial tension, we measured it in the presence of cell culture media containing 10% fetal bovine serum (FBS). This chemical environment is characterized by high levels of salts and the presence of several small molecules and proteins, including large concentrations of bovine serum albumin (BSA), a small surface-active molecule. Our measurements indicate that the presence of saturating concentrations (2% w/w) of Krytox-PEG(600) largely shield the interfacial tension, with only a 25% decrease from its measured value in deionized (DI) water. Therefore, the presence of the fluorosurfactant Krytox-PEG(600) alone at saturating concentrations sed Fig. . Equilibsed Fig. . Increassed Fig. . The minion Fig. . For a 233. To investigate if the presence of DSPE-PEG(2000)-biotin affects the droplet interfacial tension when the fluorosurfactant is present, we measured the interfacial tension of fluorocarbon droplets containing a 2% (w/w) concentration of Krytox-PEG(600) in the fluorocarbon phase and a high concentration (0.2 mM) of DSPE-PEG(2000)-biotin in the water phase. For all fluorocarbon oils studied, interfacial tension only slightly decreased in the presence of DSPE-PEG(2000)-biotin -biotin surfactants, as previously describedtin Fig. , which ctin Fig. . These r33. In many multicellular systems, cell size ranges between 10 and 20 μm, so it is important to be able to generate droplets with controlled diameters in the range of 20–40 μm. To this end, we used droplet microfluidics , whereas droplets much larger than cell size can perturb normal developmental processes and cell-cell interactions. Previous studies have shown that optimal droplet diameters to measure cell-generated stresses are approximately twice the cell sizeics Fig.  to generics Fig. . This wases Fig. , as prevror Fig. .Figure 240. Confocal images of droplets generated using microfluidics (as described above) and coated with AlexaFluor-streptavidin conjugates show that droplets are uniformly labeled by fluorescent streptavidin molecules, indicating the presence of DSPE-PEG(2000)-biotin surfactants at the droplet interface , enabling the measurement of pulling forces transmitted via integrin molecules in addition to compressive stresses : first, the droplets within the aggregates were imaged in 3D using confocal microscopy  and γ the interfacial tension of the droplet. Using Eq. (Multicellular aggregates (spheroids) of tooth mesenchymal cells were formed in culture using standard protocols, in non-adherent, round-bottom wells (Methods). In order to measure cell-generated stresses within the aggregate we used the oil droplets described above. By microinjecting droplets at different locations within the aggregate and at different stages (Methods), it is possible to obtain a stereotypical map of spatiotemporal variations in cell-generated stresses as the aggregate grows and compacts. Unless otherwise stated, streptavidin-coated droplets were functionalized with (biotinylated) RGD ligands to target integrin adhesion receptors  Fig. . Local nmentclass2pt{minim33: here we focus on the measurement of stress anisotropy as defined by the difference in stresses along the two spatial directions defined by the normal directions to the droplet at the points 33 corresponded to the deviations in normal stresses from the isotropic state.In our experiments, we used Novec 7700 oil droplets (coated as described above), which are characterized by an interfacial tension of 5.2 ± 0.1 mN/m in cell culture media with 10% FBS Fig. . For a gx⃗2  of 0.38 ± 0.03 μm/hr . Single syringe pumps (New Era Pump Systems) were used to deliver fluids at controlled flow rates, through the Fluidic Connect PRO Chip Holder (Micronit Microfluidics), which is custom-made to interface with the flow-focused droplet generator chip. To reduce wetting of droplets to the channel walls, the channels were coated with a 1% (w/w) solution of BSA by flowing it through the channels for 30 min and then subsequently rinsing with deionized water, purified through a Milli-Q purification system (Millipore) for at least 30 min before starting the process of droplet generation. To generate the fluorocarbon oil-in-water droplets, the continuous phase consisted of a solution containing 0.2 mM of DSPE-PEG(2000)-biotin (Avanti Polar Lipids) and the disperse phase consisted of a 2% (w/w) solution of the Krytox-PEG(600) fluorosurfactant (RAN Biotechnologies) in Novec 7700 oil (3 M). Droplets were collected off chip, and subsequently incubated in a glass vial containing a solution of DSPE-PEG(2000)-biotin at a 0.2 mM concentration.Generation of monodisperse fluorocarbon oil-in-water emulsions was achieved using glass microfluidic devices. Borosilicate, flow-focused droplet generators with a 20 40. Briefly, droplets were placed into a vial containing deionized water and rinsed by replenishing the vial with deionized water a total of three times. Droplets were then aspirated and dispensed via an Eppendorf microloader pipette tip into a swirling (via vortex mixing), aqueous solution of fluorescent-streptavidin conjugates at 2 μM. For all experiments performed with tooth mesenchymal cell aggregates, droplets were coated with streptavidin conjugated with Alexa Fluor 633 (Thermofisher). Droplets were incubated with fluorescent-streptavidin conjugates for at least 1 hour at 4 °C, and then rinsed with deionized water again three times. To mediate adhesion to tooth mesenchymal cells, droplets were coated with a biotinylated arginine-glycine-aspartate (RGD) peptide . To this end, droplets were gently mixed by hand  in a solution of RGD peptides at 20 μM, and subsequently incubated at 4 °C for 1 hour. To hinder adhesion of cells to the droplet surface, droplets were coated with biotinylated mPEG of 1 kDa in molecular weight (Creative PEGworks) by gently mixing them in a solution containing mPEG at 5 μM and incubating them for 1 hour at 4 °C. Finally, droplets were rinsed and stored in a new vial of deionized water at 4 °C.Droplets were further made fluorescent and functionalized with a biotinylated ligand for adhesion to cells as described in40. Briefly, an optical pendant drop tensiometer (Biolin Scientific) was used to obtain equilibrium interfacial tension values. Fluorocarbon oil droplets were suspended from 25 gauge syringe needles (Kimble Chase Life Science and Research Products) and immersed in aqueous media contained in disposable cuvettes . Equilibrium interfacial tension measurements using the fluorosurfactant and phospholipid surfactant were obtained from the tensiometer software (using the Young-Laplace equation) over several minutes to 0.5 hours, depending on the surfactant concentration used. The lab temperature was maintained at 21 °C. Data for equilibrium interfacial tension 44, namelyL is the Langmuir constant. For saturating fluorosurfactant concentrations (above the dilute regime), in which the interfacial tension Measurements of interfacial tension were done using a pendant drop tensiometer, as described in detail inμL of culture media at a density of 103 cells/well and placed into incubators. Culture media was replenished every other day by removing 100 μL of old media from round-bottom wells and replacing with 100 μL of fresh media.Mesenchymal cells, isolated from mice tooth rudiments from day 10 post-fertilization embryos (transfected with GFP expressed in the cytoplasm) were a kind gift from Dr. Tada Mammoto . Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin Streptomycin (PenStrep), and incubated at 37 °C with 5% CO2. Cell culture media was replenished every other day. Cells were grown to 70–80% confluence before passaging. To generate aggregates, low-attachment, round-bottom well plates were used (Corning). Cells were seeded with 200 μm OD; BioMedical Instruments), which was then used to partially intake a droplet at the tip of the microneedle. It should be noted that droplets smaller than 20 μm in diameter would be sucked up into the needle, rather than being held at the tip. In our experiments we found that droplets between 21–23 μm in diameter would avoid this issue. The droplet, held at the tip of the microneedle, was brought inside of the aggregate through a small hole previously made with the same microneedle. Then, the negative pressure was released (and often a slight positive pressure was applied). Upon removing the microneedle, the droplet remained embedded within the aggregate. Aggregates containing droplets were transferred back to the round-bottom well plates and incubated at 37 °C with 5% CO2. To prevent damage from microinjection, we used microneedles with OD of 20 μm, much smaller than the diameter of the aggregates (~200 μm), we performed the microinjections early (when cells are loosely connected) and we let the aggregate recover in culture conditions for at least 8 hours before imaging. No trace of the microinjection event is observed in imaged aggregates with droplets  with approximately 2 mL of cell culture media. A prepared emulsion with fluorescent, functionalized droplets of Novec 7700 oil was dispensed into the microwell dish next to the aggregates. Injection of droplets at 16 hours after cell aggregation enabled stresses to be measured starting at 24 hours after initial cell seeding. Injection at this early time point does not allow for stress measurements from droplets embedded within aggregates at later time points , as the fluorescent coating starts to diminish. Injection of droplets at 40 hours enables the measurement of stresses at later time points, namely at 48 and 60 hours in our experiments. In all cases, single droplets were injected using a modification of standard microinjection techniques. Briefly, utilizing a Pico-Liter Injector , a negative pressure was applied to a glass blunt pipette microneedle  into a glass-bottom microwell dish (MatTek) containing 2 mL of cell culture media. An inverted Zeiss LSM 710 confocal microscope, equipped with a 40x water immersion lens, was used to obtain 3D fluorescent images of droplets embedded within tooth mesenchymal aggregates. An incubation chamber on the confocal microscope maintained the temperature at 37 °C and 5% CO2 during image acquisition. For long acquisitions of droplet movements over time, aggregates were placed in custom round-bottom wells made from 1% agarose. A PDMS mold was used to create the wells inside the glass-bottom microwell dish. The agarose was heated and transferred to the microwell dish carefully, to avoid the generation of bubbles. The PDMS mold was applied over the agarose and lightly pressed down to create wells close to the glass-bottom surface of the microwell dish. The entire dish was then wrapped in parafilm and stored at 4 °C for at least 30 min. 2 mL of pre-warmed cell culture media was placed into microwell dish before transferring the aggregates into the custom round-bottom wells for imaging.In order to obtain the spatial variations of cellular stresses, it is necessary to determine the position of the droplet within the aggregate. The location of the aggregate surface was first obtained from confocal image stacks using Imaris (Bitplane). The same software was used to measure the minimal distance of the droplet center from this surface.33. Briefly, the surface of the droplet is first located and described by a point cloud, and the map of mean curvatures on the droplet surface is calculated from the point cloud using standard differential geometry, as described in details in45. We used both steerable and median filters (ImageJ plugins) to reduce high-frequency noise in the images before analysis.Droplet 3D surface reconstruction and analysis of the droplet deformations were done using a similar procedure as that described inWe have developed a controlled and versatile system of droplet stress sensors using only commercially-available components. These new droplet sensors enable the measurement of endogenous, anisotropic stresses within multicellular 3D systems, even in chemical environments with varying ionic strengths or in the presence of small surface-active molecules. Using these droplet stress sensors, we measured both the spatial and temporal changes in cell-generated stresses within multicellular aggregates during growth and compaction. These measurements provide a first glance into the spatiotemporal variations in cell-generated stresses within multicellular spheroids, a widely-used model system to study cell behavior in 3D environments.33 relied only on ionic surfactants and their interfacial tension was dependent on the ionic strength of the medium surrounding the droplet. The constant and controlled interfacial tension provided by the new two-surfactant system described herein, as well as the ability to functionalize the droplet surface with virtually any biotinylated molecule, enables the measurement of cell-generated stresses in a wide range of systems, including living embryos47, tissue explants48, embryoid bodies50, organoids52 and also multicellular spheroids of tumor cells53.The new, two-surfactant coatings of the droplets and the use of microfluidics for droplet generation allows full control over the relevant parameters. Despite a slight decrease of interfacial tension in the presence of cell culture media (approximately 8% difference for Novec 7700 fluorocarbon oil), the new two-surfactant system largely shields the interface from variations in ionic strength and the presence of small, surface-active molecules. Previous droplet stress sensors54. While our measurements compare droplets coated with RGD peptides targeting integrin receptors and non-adhesive droplets (coated with PEG), care must be taken in interpreting the measured temporal increase in anisotropic stresses as being purely caused by integrin-mediated cell-generated forces, as forces from different origins could partially contribute to the droplet deformations and are difficult to rule out.The observed weak spatial dependence of the magnitude of anisotropic stresses within the aggregate reveals that the magnitude of cell-generated stresses is similar throughout the aggregate. Comparison of the anisotropic stresses measured with drops coated with and without ligands targeting integrin receptors shows that cells generate increasing pulling, integrin-dependent forces over time, as the aggregate grows and compacts. This may be due to the fact that cells are not able to transmit forces through integrin receptors in loose aggregates (at 24 hours) as easily as in more compact aggregates (above 48 hours). However, several other mechanisms could cause this increase, including a mechanical feedback by which cells could generate larger stresses in response to either increased cellular contacts, increased presence of extracellular matrix or even to changes in the mechanical properties of the cellular microenvironment. Regardless of their origin, these increasing, integrin-mediated pulling forces can substantially contribute to the compaction and cohesiveness of the aggregate. These observations are in agreement with previous experiments showing that integrins mediate strong cohesion between mesenchymal cells in 3D environments33 reported values of the maximal (rather than average) anisotropic stresses of about 1.5 kPa. Inspection of the distribution of anisotropic stresses measured in our experiments  and the global isotropic stresses generated by the entire aggregate are different quantities and should therefore be expected to differ (Fig. 32. With an applied surface stress of 5 kPa, the isotropic stresses within the tumor spheroids ranged from <1 kPa to 4 kPa, moving from the periphery to the core. In contrast to the measurements reported herein, which quantify the spatiotemporal variations in the endogenous anisotropic stresses at the cellular scale (Fig. 32 correspond to the supracellular, isotropic stresses measured within the aggregate in response to the applied external stress.The magnitude of the average anisotropic stresses is on the order of 0.5 kPa in our experiments. Previous measurements of anisotropic stresses in mesenchymal cell aggregates using oil dropletsnts Fig.  shows thfer Fig. . Finallyale Fig. , the meain vitro systems like multicellular spheroids will help understanding how mechanical cues affect cell behaviour in 3D multicellular environments. Novel tools, such as droplet sensors or elastic microbeads, can provide complementary information about different physical quantities in 3D multicellular environments, complementing our existing knowledge on how molecular cues affect cell behaviour in these systems. Notably, these tools can provide new insights into the role of mechanics in tumor progression.The study of mechanical stresses in model


Identifying protein targets for a bioactive compound is critical in drug discovery. Molecular similarity is a main approach to fish drug targets, and is based upon an axiom that similar compounds may have the same targets. The molecular structural similarity of a compound and the ligand of a known target can be gauged in topological (2D), steric (3D) or static (pharmacophoric) metric. The topologic metric is fast, but unable to represent steric and static profile of a bioactive compound. Steric and static metrics reflect the shape properties of a compound if its structure were experimentally obtained, and could be unreliable if they were based upon the putative conformation data. In this paper, we report a pharmaceutical target seeker (PTS), which searches protein targets for a bioactive compound based upon the static and steric shape comparison by comparing a compound structure against the experimental ligand structure. Especially, the crystal structures of active compounds were taken into similarity calculation and the predicted targets can be filtered according to multi activity thresholds. PTS has a pharmaceutical target database that contains approximately 250 000 ligands annotated with about 2300 protein targets. A visualization tool is provided for a user to examine the result.Database URL: http://www.rcdd.org.cn/PTS Recent A drug target prediction method can be categorized into structure-based or ligand-based method. INDOCK  and TarFIn this work, we have implemented a pharmaceutical target seeker (PTS), which uses the experimental 3D structures of ligands with known targets to calculate the similarity of the ligand and a compound. For those ligands for which experimental structure data are not available, their energy-minimized conformations are generated for the 3D similarity calculations. The 3D similarity search engine is Weighted Gaussian Algorithm (WEGA) , which cThe data of bioactive compounds and their targets were collected from public databases.Target data were derived from therapeutic target database (TTD version 2015)  and referemoving obsolete UniProt IDs from TTD target data;removing counter ion moieties from bioactive ligands;removing compounds from ChEMBL20 data if their activity (IC50/Ki/Kd) values are greater than 50 μM;removing small compounds (heavy atoms < 6) and large compounds (MW > 1000 Da).This resulted in 266 866 ligands associated with 2298 protein targets, 537 095 bioactivity data points, 4391 crystal structures and 16 590 related articles in the PTS built-in database  shape matching, which is only the molecular volumetric overlay, 2) feature matching, which is the pharmacophore mapping of molecule pair, 3) combination matching, which integrates the advantage of the above two aspects and is the most precise approach. The detailed method of WEAG is described in reference .PTS uses a browser and server framework. Client interface was implemented in HTML and Javascript. The back-end is implemented in Golang language and MySQL database system. The molecule editor and chemical structure viewers are supported with Marvin JS and ChemDoodle web component. All tools have been summarized in PTS provides an intuitive interface to predict small molecule protein targets. A user can input a query molecule by uploading a file (mol/SDF format) or drawing a chemical structure with its built-in chemical structure editor . PTS wilBy clicking on the check button, a user can inspect the predicted template ligands for a query molecule . In the Afatinib is an irreversible kinase inhibitor targeting epidermal growth factor receptor (EGFR) and inhibiting tyrosine kinase auto-phosphorylation  to stop Experimental data indicate that Afatinib is an EGFR inhibitor (IC50 = 1 nM) . EGFR U. The preEstrogen receptors (ERs) are well-known targets for Tamoxifen –37. The In Chlorprothixene is an old antipsychotic drug. It antagonizes dopaminergic D1 (UniProt ID: P21728) and D2 (UniProt ID: P14416) receptors in the brain to exert its antipsychotic effect . ChlorprhERG  is known as a potassium (K+) ion channel mediating the repolarizing IKr current in the cardiac action potential. A drug that potentially interacts with hERG can result in lethal side-effect . FluoxetSodium-dependent serotonin transporter (UniProt ID: P31645) is the primary target of Fluoxetine, which is ranked at fourth in the list. By inspect the potential target list, we find the top-10 target is hERG. Further literature studies reveal that Fluoxetine is experimentally proved as hERG inhibitor (IC50 = 3.1 μM) .PTS predicts targets for a compound through superimposing the compound structure onto the 3D ligand structures of putative targets. This approach considers pharmacophore shape similarity that 2D approaches cannot do. Other 3D approaches use molecular docking techniques, while PTS does not employ ligand-receptor docking techniques and can still produce results where the receptor structure data are not available. Both PTS and SwissTargetPrediction are web-based target fishing tools. The four testing cases were tested on both tools, which produced similar results. Additionally, small scale of structurally diverse drugs that targeting four classes of biological systems  were extracted to test the success rate and applicability of PTS. Averagely, at least one known targets of each drug is found among the top 20 predicted targets for 70% of the ligands . The advTo date, PTS has received >500 queries from 11 countries in the world since 10 August 2016. It can be used for target identification, drug repurposing, toxic risk estimation and molecular interaction simulation pre-processing tool.National Science Foundation of China ; Guangdong Frontier & Key Technology Innovation Program (2015B010109004); Guangdong Natural Science Foundation (2016A030310228); the Fundamental Research Funds for the Central Universities (2013HGCH0015); Tianhe-2 based biomedical health data application support platform (U1611261); the Fundamental Research Funds for the Central Universities (17LGJC23). Funding for open access charge: National Science Foundation of China (81473138), funded by the National Natural Science Foundation of China.Database Online.Conflict of interest. None declared.Supplementary DataClick here for additional data file.


While there is abundant evidence for mild hypotensive effects of nitrate on acute application there is limited data on chronic intake in humans, and results from animal studies suggest no long-term benefit. This is important as nitrate can also promote the formation of nitrosamines. It is therefore classified as ‘probably carcinogenic to humans', although a beneficial effect on CVD risk might compensate for an increased cancer risk.Nitrate (NO2−) by oral commensal bacteria to contribute to the formation of nitric oxide (NO). The extensive crosstalk between NO and hydrogen sulfide (H2S) related metabolites may further affect nitrate’s bioactivity. Using nitrate and nitrite concentrations of drinking water − the only dietary source continuously monitored for which detailed data exist − in conjunction with data of >14,000 participants of the EPIC-Norfolk study, we found no inverse associations with blood pressure or CVD risk. Instead, we found a strong interaction with sulfate (SO42−). At low sulfate concentrations, nitrate was inversely associated with BP (−4 mmHg in top quintile) whereas this was reversed at higher concentrations (+3 mmHg in top quintile).Dietary nitrate requires reduction to nitrite (NOOur findings have a potentially significant impact for pharmacology, physiology and public health, redirecting our attention from the oral microbiome and mouthwash use to interaction with sulfur-containing dietary constituents. These results also indicate that nitrate bioactivation is more complex than hitherto assumed. The modulation of nitrate bioactivity by sulfate may render dietary lifestyle interventions aimed at increasing nitrate intake ineffective and even reverse potential antihypertensive effects, warranting further investigation. Nitrate is also formed by reaction of endogenously produced nitric oxide (NO) with oxygenated hemoglobin in red blood cells; it enjoys a relatively long half-life in blood (5–7 h in humans) due to renal reabsorption. About a fifth to a quarter of the circulating amounts of nitrate are actively secreted into saliva; here, dietary and endogenously produced nitrate is reduced to nitrite by facultative anaerobic bacteria that are part of our commensal oral microflora. Upon swallowing, nitrite ends up in the stomach where the protonated form .We therefore used household-level municipal water records to investigate potential associations between drinking water nitrite/nitrate concentrations and BP in 15,549 participants of EPIC-Norfolk 3Drinking water nitrite concentrations were below 0.5 mg/L for all participants. The ranges of nitrate and sulfate concentrations were considerably larger A, but th432−) or hydrogen sulfide (H2S/HS−) by sulfate-reducing gut bacteria, followed by either scavenging of NO and/or interference with NO2− metabolism in the host tissues. Alternatively, sulfate may compete with bacterial nitrate reduction to nitrite in the oral cavity, as described for the microbial flora in the large intestine. The mechanism of this interaction is currently unknown and requires further study. One possible explanation may be related to the crosstalk between sulfur and nitrogen metabolic pathways. Higher dietary sulfate intake may shift the balance between transsulfuration and methionine recycling towards the remethylation of homocysteine, thereby leading to an alteration of global redox status while attenuating endogenous NO formation following inhibition of endothelial nitric oxide synthase (eNOS) via S-adenosylmethionine-mediated asymmetric dimethylarginine formation 5acceptable daily intake (3.7 mg/kg body weight) for nitrate, are increasingly marketed for the primary prevention of hypertension and cardiovascular diseases, despite the lack of robust long-term data and animal experimental results suggesting no long-term beneficial effects. The significance of the oral microbiome for the efficacy of nitrate is well-known, in particular the potential of antibacterial mouthwashes to abolish its BP-lowering effects These findings have a potentially significant impact on public health and may also explain some of the variability observed in hemodynamic effects with (nitrite and) nitrate in pharmacological studies. Nitrate-rich foods, often exceeding the The authors declare no conflict of interest.


Zea mays lines carrying mutant alleles for CMT or RdDM-associated genes. While the majority of the transcriptome was not affected, we identified sets of genes and transposon families sensitive to context-specific decreases in DNA methylation in mutant lines. Many of the genes that are up-regulated in CMT mutant lines have high levels of CHG methylation, while genes that are differentially expressed in RdDM mutants are enriched for having nearby mCHH islands, implicating context-specific DNA methylation in the regulation of expression for a small number of genes. Many genes regulated by CMTs exhibit natural variation for DNA methylation and transcript abundance in a panel of diverse inbred lines. Transposon families with differential expression in the mutant genotypes show few defining features, though several families up-regulated in RdDM mutants show enriched expression in endosperm tissue, highlighting the potential importance for this pathway during reproduction. Taken together, our findings suggest that while the number of genes and transposon families whose expression is reproducibly affected by mild perturbations in context-specific methylation is small, there are distinct patterns for loci impacted by RdDM and CMT mutants.DNA methylation is a chromatin modification that can provide epigenetic regulation of gene and transposon expression. Plants utilize several pathways to establish and maintain DNA methylation in specific sequence contexts. The chromomethylase (CMT) genes maintain CHG  methylation. The RNA-directed DNA methylation (RdDM) pathway is important for CHH methylation. Transcriptome analysis was performed in a collection of  The epigenome describes the potential for additional heritable information that can be passed on through mitosis or meiosis   / rpd1 (rna polymerase d1) locus  in genomic regions with low, but detectable, CWA methylation  following manufacturer’s suggestions. The final library was quantified using PicoGreen and twelve libraries were pooled per Illumina lane. Library quality was checked using Agilent Bioanalyzer. Sequencing was performed on HiSeq2500 using 2 × 50 bp mode.Trim_glore was used to trim low-quality base from the 3′ end of the reads, as well as to remove adapters. Reads that passed quality control were mapped to B73 version 4 genome (2(FoldChange) > 1 were called differentially expressed (DE) genes. Detailed analysis was restricted to genes with consistent DE calls in at least two mutant contrasts in the same pathway (RdDM or CMT). Genes were considered expressed if at least 3 replicates in the libraries described had an RPM (reads per million) value >1.Raw read counts were input into DEseq2 (2(FoldChange) cutoff of 1 and FDR adjusted p-value cutoff of 0.05. Detailed analysis was restricted to TE families with consistent DE calls in at least two mutant contrasts in the same pathway (RdDM or CMT).B73v4  subtractThe WGBS datasets used in this study are detailed in Table S2 and SRA accession numbers for each sample are provided. One μg DNA was used to prepare libraries for whole-genome bisulfite sequencing using the KAPA library preparation kit. DNA was sheared to a peak between 200-250 bp. End repair was performed to make blunt-ended fragments, followed by adding base A to the 3′ end, and adapter ligation. Size selection was performed to enrich library with a size between 250-450 bp. Bisulfite conversion was then carried out using Zymo DNA methylation lightning kit according to user’s manual. Finally, the libraries were enriched using PCR amplification. Library quality was checked using the Agilent Bioanalyzer. Library quantification was performed with qPCR before sequencing. Sequencing was performed on HiSeq2000 with paired end 100 cycles.Analysis was performed as previously described  allowingDMRs were called using previously described criteria , and accession numbers for all libraries are listed in Table S1. Expression values, differential expression calls, list assignments, and DMR calls for all genes and TE families are listed in Tables S2 and S3, respectively. Unique mapping read counts and descriptors for each transposable element are listed in Table S4. Supplemental material available at Figshare: mop1-1 and mop3-1 (mop1-1 (RNA-seq was used to perform transcriptome profiling for maize lines carrying mutations in genes encoding CMT (this study) or RdDM components . For mutations that were identified in one background and then backcrossed into B73 , there were often a cluster of DE genes surrounding the locus of the mutation itself. For the other mutation (mop3-1) that was not backcrossed into another genetic background, there is less evidence for expression changes at linked genes . Based on these results we omitted DE genes located within 40 Mb of the mutations in subsequent analyses.The transcript abundance of individual genes was estimated from the RNA-seq data for each sample. Differentially expressed (DE) genes in each mutant line (relative to the appropriate control) were identified using DESeq2 followed by a requirement for a minimum of 2 fold-change and an FDR value of less than 0.05 (Table S2). The observed differences in gene expression in the mutant lines could be direct effects of the mutation on transcript abundance, indirect effects caused by direct targets, or could be the result of introgressions of linked loci that contain mop3-1 samples from mutants and controls in the B73 background, suggesting that tissue and genotype represent the strongest sources of differences in our transcriptomes  were identified in each mutant  comprise a large portion of the maize genome, and typically have high levels of CG and CHG methylation, with CHH methylation peaks at the edges of some TE families. There are two classes of TEs, Class I (retrotransposons) and Class II (DNA transposons), which transpose either through a copy-and-paste mechanism requiring an RNA intermediate (retrotransposons) or through a cut-and-paste mechanism (DNA transposons) (zmet2-m1/zmet2-m2/zmet5-m1) or RdDM mutants (mop1-1/mop3-1) . In order to understand the reproducible effects of these pathways on expression, we focused our analyses on the set of 237 genes and 104 TE families that exhibit consistent up- or down-regulation in multiple CMT or RdDM mutants. Hierarchical clustering of the fold-change of these genes or TE families relative to WT in all samples reveals a relatively consistent magnitude of expression changes between similar genotypes  but fewer examples of consistent down-regulation (7 genes), consistent with the greater number of up-regulated than down-regulated genes in RdDM mutants in general. An analysis of the RNA-seq data for rmr6-1 . A portion of the “genes” that are affected by CMT or RdDM mutations may represent mis-annotated transposons or pseudogenes. We assessed the proportion of genes with altered transcript abundance that are classified as syntenic based on a comparison with sorghum and rice genomes  shown in zmet2-m1 mutants is associated with increased expression, suggesting that CMT-dependent silencing of these genes depends on CHG but not CG methylation.The differential expression observed in each mutant background could result from direct changes in DNA methylation or chromatin at these loci or could result from indirect effects due to secondary effects from genes that are direct targets. Genes that are direct targets for silencing by DNA methylation would be expected to have high levels of DNA methylation in wild-type plants. The context-specific DNA methylation profiles were assessed in wild-type B73 for genes that were up- or down- regulated compared with all expressed genes . The genhylation . As in tMop1 and Mop3 (A large number of maize genes (∼60%) have been associated with the presence of a region of elevated CHH in the promoter region, termed a mCHH island . This provides evidence to support a requirement for RdDM and CHH methylation in the proper transcriptional control of this gene in maize.In some cases, the mCHH island itself may result in transcriptional regulation. Work in There is a substantial amount of natural variation for methylation levels among maize inbreds. We sought to determine the extent to which genes that are sensitive to CMT or RdDM mutations also display variable methylation levels in these lines. These would reflect examples of potential cryptic information in one inbred line that is expressed in other genotypes, reflecting natural epigenetic variation. We used WGBS data from B73 and 17 other diverse maize inbreds to document natural variation for DNA methylation among maize inbreds. Differentially Methylated Regions (DMRs) were identified in all three contexts  between B73 and the other inbreds. More than 200,000 DMRs were called in the CG and CHG contexts, with over 50,000 DMRs in the CHH context. Each maize gene was classified based on whether there was a DMR within 200 bp up or downstream of the transcription start site for each of the three sequence contexts. A relatively small portion (∼3–7%) of maize genes has CG, CHG or CHH DMRs near the promoter (Table S2). We proceeded to assess whether naturally occurring DMRs were more prevalent near genes that exhibit altered expression in CMT or RdDM mutants . Genes tzmet2-m1 mutant lines - blue dots) the DMR has low methylation and transcripts are observed.This suggests that many of the genes with altered transcript abundance in CMT or RdDM mutations may have pre-existing natural variation for DNA methylation that would affect expression levels in maize populations. RNA-seq data from leaf tissue for ten of the inbred lines with WGBS data were utilized to determine whether there was a significant association  between context-specific methylation level at the DMR and transcript abundance levels. We found that nearly 50% of the genes that exhibit altered expression in CMT or RdDM mutants that are located near DMRs had natural variation for gene expression levels that was significantly associated with DNA methylation levels. Two examples of CMT up-regulated genes that exhibit significantly correlated transcript abundance and CHG methylation at the bin overlapping the TSS among diverse lines are shown in t-test). We also tested the mean GC content of TEs within families to test whether families depleted in cytosines are more susceptible to subtle perturbations in methylation, as is the case for the ONSEN family in Arabidopsis (t-test).There are 104 TE families with altered expression in mutants that perturb RdDM or CMT components in maize. These included 32 families up- and 7 families down-regulated in at least two contrasts of RdDM mutants, and 48 families up- and 24 families down-regulated in at least two contrasts of CMT mutants . There aMop1 and Mop3 genes in these tissues  or CMT mutants (27 families) were assessed to determine if they exhibit distinct patterns of expression. Interestingly, approximately one third of the TE families that are up-regulated in RdDM mutants show higher expression in the endosperm than other tissues . In cont tissues . This revs. locus-specific expression . The unique mapping reads for these families were used to evaluate element-specific transcript abundance. Half of the testable TE families had expression of a single member of the family indicating locus-specific changes  mutants exhibited transcription changes from a larger portion of the genome but much of this was associated with increased transcriptional ‘noise’ at lowly expressed regions . Many of the genes or TEs with altered expression in rmr6-1 also exhibit altered expression in mop3-1 and/or mop1-1 . While relatively few genes exhibit major changes in expression there are a significant number of genes that exhibit similar expression changes in multiple RdDM or CMT mutants. These findings are compatible with the concept that there are a small number of genes in the maize genome that have epigenetic regulation that is solely dependent upon RdDM or CMT mediated regulation. While some of these “genes” lack synteny with other related species others do show syntenic genomic positions and likely represent potentially functional genes . It is likely that a much larger number of genes are redundantly regulated by the RdDM and CMT pathway along with MET1 mediated CG methylation.The effects of mutations in RdDM or CMT genes in maize are quite limited. Our evidence suggests that there is little effect on the overall transcriptome of these plants. This might be expected given the limited effect on overall plant phenotype for each of these mutations. A recent study found that zmet2-m1 mutations varied in different maize inbreds (Many of the genes that are up-regulated in CMT mutants exhibit high levels of CHG methylation. The CMT mutants reduce this methylation and may result in increased transcript abundance. Previous studies noted that the genes sensitive to Arabidopsis (DNA methylation is often considered to play a primary role in maintaining genome integrity by silencing transposable elements. Indeed, there are clear examples of release of transposon silencing in mutants affecting DNA methylation in This study defines a set of genes and TE families that are regulated by DNA methylation. The silencing of these genes and TEs relies solely upon RdDM or CMT based epigenetic regulation. These loci provide important insights into the mechanisms that allow for epigenetic regulation and the natural variation for epigenetic regulation in maize.