Arterial stiffness is a major risk factor and effective predictor of cardiovascular diseases and a common pathway of pathological vascular impairments. Homocysteine (Hcy) and uric acid (UA) own the shared metabolic pathways to affect vascular function. Serum uric acid (UA) has a great impact on arterial stiffness and cardiovascular risk, while the mutual effect with Hcy remains unknown yet. This study aimed to evaluate the mutual effect of serum Hcy and UA on arterial stiffness and 10-year cardiovascular risk in the general population. From the perspective of predictive, preventive, and personalized medicine (PPPM/3PM), we assumed that combined assessment of Hcy and UA provides a better tool for targeted prevention and personalized intervention of cardiovascular diseases via suppressing arterial stiffness.This study consisted of 17,697 participants from Beijing Health Management Cohort, who underwent health examination between January 2012 and December 2019. Brachial-ankle pulse wave velocity (baPWV) was used as an index of arterial stiffness.β: 30.76, 95% CI: 18.36–43.16 in males; β: 53.53, 95% CI: 38.46–68.60 in females). In addition, these individuals owned the highest 10-year cardiovascular risk . Of note, males with high homocysteine and low uric acid were significantly associated with increased cardiovascular risk , but not the high uric acid and low homocysteine group .Individuals with both high Hcy and UA had the highest baPWV, compared with those with low Hcy and low UA (This study found the significantly mutual effect of Hcy and UA on arterial stiffness and cardiovascular risk using a large population and suggested the clinical importance of combined evaluation and control of Hcy and UA for promoting cardiovascular health. The adverse effect of homocysteine on arteriosclerosis should be addressed beyond uric acid, especially for males. Monitoring of the level of both Hcy and UA provides a window opportunity for PPPM/3PM in the progression of arterial stiffness and prevention of CVD. Hcy provides a novel predictor beyond UA of cardiovascular health to identify individuals at high risk of arterial stiffness for the primary prevention and early treatment of CVD. In the progressive stage of arterial stiffness, active control of Hcy and UA levels from the aspects of dietary behavior and medication treatment is conducive to alleviating the level of arterial stiffness and reducing the risk of CVD. Further studies are needed to evaluate the clinical effect of Hcy and UA targeted intervention on arterial stiffness and cardiovascular health.The online version contains supplementary material available at 10.1007/s13167-022-00298-x. Arterial stiffness, which reflects arterial elasticity and function, increases with age and is affected by health conditions such as diabetes and hypertension –3. ArterAs an integrative strategy in health care system, the PPPM/3PM aims to promote health care-related research and management based on multidisciplinary expertise via early prediction of disease predisposition, timely prevention of disease onset or progression, and personalized treatment of diseases outcomes . DevelopFrom the perspective of PPPM/3 M, effective identification of risk factors for arterial stiffness is crucial for the lowering the health burden and adverse consequences. This study aims to investigate the mutual effect of serum Hcy and UA on arterial stiffness and 10-year cardiovascular risk using a cross-sectional design, and evaluate whether effects of homocysteine and uric acid on arterial stiffness and cardiovascular risk are distinct between males and females.This cross-sectional study enrolled participants from Beijing Health Management Cohort (BHMC) from January 2012 to December 2019. Details of the cohort design have been described in previous study . All parThe demographic characteristics and lifestyle behaviors factors were acquired by standard questionnaire interview. Educational level was categorized as illiteracy or primary school, middle school, high school or above. Taking moderate or intense physical activity “ ≥ 20 min each time and ≥ 4 times per week” was regarded as regular exercise. Smoking status was divided into never or former, and current smoker. Individual with an average alcohol consumption of more than 30 g per day was considered as current drinker.2 for the Chinese population [Physical examination information and laboratory tests data were acquired through the electronic information system. Body mass index (BMI) was calculated as weight (kg)/[height (m)*height (m)]. Systolic and diastolic blood pressures were calculated as the mean of two measurements using the mercury sphygmomanometer on the right arm after resting for at least 10 min. Laboratory tests were carried out using fasting venous blood samples including assays for triglyceride, total cholesterol, high density lipoprotein (HDL) cholesterol, low density lipoprotein (LDL) cholesterol, fasting plasma glucose, glycated hemoglobin (HbA1c), serum Hcy, and serum UA. According to a previous study based on the Chinese population, high level Hcy was defined as > 14 µmol/L in males and > 10 µmol/L in females, and high level UA was defined as > 420 µmol/L in males and > 320 µmol/L in females  or number (proportions), as appropriate. Differences between males and females were compared using chi-square test for categorical variables, and Student’s β) and corresponding 95% confidence interval (CI) for one-SD increase of Hcy and UA, respectively. To investigate the mutual effect between Hcy and UA, the population were divided into four groups: low Hcy/low UA, high Hcy/low UA, low Hcy/high UA, and high Hcy/high UA by sex specific cutoffs. In addition, the associations of Hcy (UA) concentration with baPWV were presented in people with low and high UA (Hcy) level. Then, we analyzed the mutual effect of Hcy and UA in subgroups stratified by age, obesity, hypertension, diabetes, and smoking status. We performed multiple sensitivity analyses. First, participants with cardiovascular diseases were excluded from the analysis; second, we excluded the individuals using any following medications: anti-hypertensive, anti-diabetic, lipid-lowering, anti-gout, and aspirin, to explore the stability of our findings. In addition, we used the logistics regression models to evaluate the association of Hcy and UA groups with 10-year cardiovascular risk.Unadjusted and adjusted linear regression models were used to estimate the association of Hcy and UA concentrations with baPWV level. Age, education level, BMI, physical activity, current smoking, current drinking, hypertension or not, diabetes or not, dyslipidemia or not, systolic pressure, fasting glucose, triglyceride, and HDL cholesterol were considered in the adjusted analyses. We calculated the regression coefficient . Similar result was found for women  as shown in Figure The final analysis enrolled a total of 17,697 participants, including 12,604 men and 5093 women. The mean age (± SD) was 58.95 (13.99) years in men and 54.81 (14.03) years in women. The medians [Pβ: 10.80, 95% CI: 1.85–19.74) or high UA . Similarly, female individuals with both high Hcy and high UA had a 53.53 (95% CI: 38.46–68.60) cm/s increase of baPWV in men compared with those with low Hcy and low UA, which was higher than those just with high Hcy  or high UA  as shown in Table In the adjusted model, one-SD increase in Hcy was associated with a 7.38 (95% CI: 3.98–10.79) cm/s increase of baPWV in men and an 11.00 (95% CI: 6.33–15.68) cm/s increase of baPWV in women. Men with high Hcy (> 14 µmol/L) had a 14.19 (95% CI: 6.60–21.78) cm/s higher baPWV than those with low Hcy; women with high Hcy (> 10 µmol/L) had a 21.61 (95% CI: 11.71–31.51) cm/s higher baPWV. Similarly, 1-SD increase in UA was associated with a 7.65 (95% CI: 4.11–11.18) cm/s increase of baPWV in men and a 16.60 (95% CI: 11.56–21.63) cm/s increase of baPWV in women. Men with high UA (> 420 µmol/L) had a 14.74 (95% CI: 6.85–22.63) cm/s higher baPWV than those with low UA; women with high UA (> 320 µmol/L) had a 38.16 (95% CI: 27.51–48.81) cm/s higher baPWV. Of note, individuals with both high Hcy and high UA had a 30.76 (95% CI: 18.36–43.16) cm/s increase of baPWV in men compared with those with low Hcy and low UA, which was higher than those just with high Hcy  in males and 7.61 (95% CI: 4.63–12.68) in females Table , and theThis study aimed to investigate the mutual effect of serum Hcy and UA on arterial stiffness in the general population. The results showed that high Hcy or high UA was significantly correlated with increased baPWV level. Individuals with both high Hcy and high UA had the highest level of arterial stiffness and 10-year cardiovascular risk than those with high Hcy or high UA alone. In addition, Hcy and UA have shown potential as biomarkers in the assessment of arterial stiffness and CVD risk. Of note, males of high Hcy and low UA had increased cardiovascular risk, but not high UA and low Hcy, which partly implies males suffer more from Hcy than females, and females are more sensitive to UA than males.From the perspective of PPPM/3 M, identification of cost-effective and reliable biomarkers is important for the prevention of diseases. The monitoring of these biomarkers could provide predictive information on the onset or prognosis of adverse events. People with high risk should adapt pharmaceutical or non-pharmaceutical interventions to alleviate the progression, which effectively contribute to the paradigm shift from reactive medicine to the advanced approach by utilizing PPPM/3 M concepts , 29. TheArterial stiffness is one of the pathological mechanisms of cardiovascular disease and an independent risk factor of all-cause mortality . A prospThis study showed that high Hcy and high UA are positively correlated with arterial stiffness in general population after adjusting for confounding factors. Consistent with our findings, Chen et al. found that serum Hcy level is positively correlated with cfPWV level . Wang etVascular endothelium maintains vascular homeostasis by regulating vascular tension, inflammation, and cell growth . EndotheStratified analysis showed that Hcy is positively associated with arterial stiffness in people with low UA levels, and UA is positively correlated with arterial stiffness regardless of Hcy level in females, but positively correlated with arterial stiffness only at low Hcy level in males (Hcy < 14 µmol/L). Similarly, Sugiura et al. found that UA is significantly correlated with cardio-ankle vascular index (CAVI) in both genders, and UA has a greater impact in females than males . Fang etGolubnitschaja et al. demonstrated that overweight or underweight can increase metabolic load and reduce adaptive capacity through up-regulation of inflammatory signals and increased ROS levels . AccordiIn our subsequent analysis of the association of UA and Hcy with 10-year CVD risk, people with both high UA and Hcy showed the highest risk, supporting our hypothesis that UA and Hcy can mediate CVD by causing arterial stiffness. The study by Zheng et al. suggests that ischemic stroke can be predicted based on routine hematological and biochemical features, and early identification of disease risk may facilitate the formulation of primary care strategies and improve the prognosis of the disease . In thisHcy and UA levels are regulated by both heredity and environmental factors , 51. ImpThis study found the mutual effect of Hcy and UA on arterial stiffness and cardiovascular risk based on a large sample population, and realized early identification of CVD high-risk groups based on Hcy and UA indicators, which contributes to the early comprehensive prevention and control of CVD. The results should be interpreted in the context of some limitations. First, this study is based on a cross-sectional design, and the causal associations of serum Hcy and UA with arterial stiffness cannot be claimed. And the combined effect of Hcy and UA on the dynamic changes of arterial stiffness needs further investigation using longitudinal cohort data. Moreover, the effect of combined control of Hcy and UA on arteriosclerosis and cardiovascular health warrants explanation in clinical trials. Second, the baPWV index measures stiffness of the elastic aorta and the muscular arteries, while the aortic stiffness and muscular stiffness tend to change in the opposite direction with aging. However, baPWV level still enhances the efficacy of predicting cardiovascular events over the Framingham risk score and reflects the cardiovascular risk especially in the Asian population. The association of Hcy and UA with arterial stiffness and screening methods for high-risk CVD patients need further validation in other populations and using other arterial stiffness index, such as cfPWV and the direct measurement of arterial stiffness like elastography. In addition, based on previous studies, we know that the primary prevention and treatment of arterial stiffness and CVD need to be integrated with diet, physical activity, and medication treatment, but how to target the abnormal metabolism of Hcy and UA to comprehensively prevent and treat arterial stiffness needs to be further explored.This study indicates the significant mutual effect of elevated Hcy and UA on arterial stiffness and 10-year cardiovascular risk in the general population. The adverse effect of Hcy on cardiovascular health should be underlined beyond UA, especially for males. Our study suggested the clinical importance of combined evaluation and control of Hcy and UA for preventing arterial stiffness and cardiovascular diseases, and quantified and visualized the risk of arterial stiffness and 10-year CVD by nomogram, which provides a basis for early identification of the progression of arterial stiffness and prediction of CVD risk in general population (Predictive).Hcy and UA could be novel indicators of arterial stiffness, providing new insights for the early predictive medical approach of CVD risk in the setting of PPPM/3PM health care system. The monitoring of Hcy and UA levels could provide a window opportunity for the primary targeted prevention for the progression of arterial stiffness and adverse CVD events. The health care providers should be aware of the CVD risk for people with Hcy/UA metabolic disorders (Preventive). Besides, pharmaceutic and non-pharmaceutic interventions are needed for alleviating the arterial stiffness progression from the perspective of Hcy and UA using PPPM/3PM strategy, such as balanced diet and physical activity. According to the risk assessment results based on clinical indicators including Hcy and UA, primary prevention or early treatment of arterial stiffness in high-risk groups is of great significance to control the progression of arterial stiffness and reduce the risk of CVD .Overall, there is a mutual effect of Hcy and UA on arterial stiffness and cardiovascular risk, and further studies should investigate the practical benefits of monitoring Hcy and UA levels for cardiovascular health promoting, including the predictive diagnostics, effective prevention, and personalized intervention. This supports the PPPM/3PM strategy and paradigm shift from reactive to advanced in cardiovascular health field. People with both high Hcy and UA should be aware of the cardiovascular health issues at an early stage and get personalized advice on social activity and support, family care, dietary behavior, physical activity, and even the pharmaceutical intervention. Hcy/UA metabolic disorders and cardiovascular adverse events could form a vicious circle, which should be underlined in the advanced PPPM/3PM paradigm.Supplementary file1 (DOCX 226 KB)Below is the link to the electronic supplementary material.


Inhibiting Notch signaling or knocking down the Her2 function reduced neural progenitor proliferation, whereas inactivating BMP signaling in Notch-Her2 deficient background restored the number of proliferating neural progenitors. These results reveal the time window for the proliferation of neural progenitors during zebrafish development and a fine balance between BMP and Notch signaling in regulating the proliferation of neural progenitor cells.Bone morphogenetic protein (BMP) signaling regulates neural induction, neuronal specification, and neuronal differentiation. However, the role of BMP signaling in neural progenitors remains unclear. This is because interruption of BMP signaling before or during neural induction causes severe effects on subsequent neural developmental processes. To examine the role of BMP signaling in the development of neural progenitors in zebrafish, we bypassed the effect of BMP signaling on neural induction and suppressed BMP signaling at different time points during gastrulation using a temporally controlled transgenic line carrying a dominant-negative form of Bmp receptor type 1aa and a chemical inhibitor of BMP signaling, DMH1. Inhibiting BMP signaling from 8 hpf could bypass BMP regulation on neural induction, induce the number of proliferating neural progenitors, and reduce the number of neuronal precursors. Inhibiting BMP signaling upregulates the expression of the Notch downstream gene hairy/E(spl)-related 2 ( Xenopus and zebrafish demonstrated that during early gastrulation, secreted neural inducers, such as Noggin, Follistatin, and Chordin, inhibit bone morphogenetic protein (BMP) signaling at the dorsal side of the animal cap, where they repress the epidermal fate and induce the neural fate of the ectoderm. In contrast, the ventral ectoderm expresses a high level of BMP and differentiates into the epidermis. This process, termed neural induction, specifies the neuroectoderm within the ectoderm and is conserved in vertebrates. However, other signals, such as fibroblast growth factor (FGF) and Wnt, have also been demonstrated as key modulators in neural induction . These iewed in ,4). TherId) and Msx [BMPs belong to the transforming growth factor-beta (TGF-β) superfamily, which mediates a diverse array of cellular processes, including proliferation, differentiation, and apoptosis . BMPs bi and Msx ,7. BMPs  and Msx ). BMP si and Msx ,10, revi and Msx ). Once t and Msx ). Furthe and Msx ,14. BMP2 and Msx , whereas and Msx . In cont and Msx  and redu and Msx ,19. HoweIn this study, we bypassed the effect of BMP signaling on the regulation of embryonic development before early gastrulation, including dorsoventral patterning, mesoderm formation, and neural induction, and examined the specific role of BMP signaling in the development of neural progenitors. We inhibited BMP signaling at distinct time points during zebrafish gastrulation using a transgenic fish line that carries the dominant-negative form of the Bmp type 1 receptor (DN-BmpR1aa) and confirmed the results using a chemical inhibitor of BMP signaling, dorsomorphin homologue 1 (DMH1). We defined the time point for the effect of BMP signaling during neural progenitor development and discovered that Notch-Her2 signaling underlies this regulation. bmpr1aa, during the gastrulation and early segmentation stages. The results of whole-mount in situ hybridization demonstrated that bmpr1aa was ubiquitously expressed in the entire embryo at 5.3 hpf and 6 hpf, and from 8 hpf onward, was expressed in the presumptive neuroectoderm and developing mesoderm  to 10 hpf). During this period, the neural progenitors are specified from the ectoderm  and thenmesoderm A. At thenal cord B. Durings system . This re(hsp70l: dn-bmpr1aa-eGFP)Tg  [eve1, which is a downstream target of BMP signaling and is decreased in bmp mutants [eve1 expression at 6 hpf and diminished eve1 expression at 7 hpf   which ca mutants ,25. Heatat 7 hpf A,C. Theseve1 expression  are direct downstream targets of the BMP-Smad signaling pathway. Upon binding of BMPs, BMP receptors phosphorylate Smad1/5/9 and the phosphorylated Smad1/5/9 and Smad4 complex directly bind to the 5ʹ regulatory elements of pression ,29,30. Tignaling .Tg (hsp70l: dn-bmpr1aa-eGFP) and DMH1 were used to inhibit BMP-BmpR1aa-Smad signaling. We performed heat-shock induction or treatment with DMH1 at 5.3, 8, or 9 hpf for one hour, and harvested the embryos at 10 hpf for examination. In situ hybridization analysis for the expression pattern of neuroectoderm markers sox2 and sox3 demonstrated that inactivating BMP-Bmpr1 signaling at 5.3 hpf caused the expansion of sox2 and sox3 expression at 10 hpf , and discovered that inhibiting BMP signaling at this time point was sufficient to inhibit the formation of surface ectoderm, as evidenced by reduced tp63 expression  by BMP inactivation , and discovered that pH3-positive neural progenitors were increased in BMP-signaling-inactivated embryos  to end-gastrulation (10 hpf) promotes the proliferation of neural progenitor cells and inhibits the differentiation of neuronal precursors. To our knowledge, this is the first in vivo study to define the time window of BMP-Bmpr1a-Smad signaling in regulating embryonic neural progenitor cell proliferation.her2 expression, suggesting an interaction between these two signaling pathways in regulating neural progenitor proliferation, inactivating BMP signaling upregulates neural progenitor proliferation in Her2 knockdown embryos, suggesting that these two signaling pathways may also regulate neural progenitor proliferation in a parallel manner. Taken together, a proper balance between BMP and Notch signaling is essential for the development of neural progenitors.We also discovered that BMP-Bmpr1a-Smad and Notch-Her2 signaling regulate the proliferation of neural progenitors in an opposite manner—inhibiting BMP-Bmpr1a-Smad signaling promotes neural progenitor proliferation, whereas inactivating Notch-Her2 signaling reduces neural progenitor proliferation. Although inactivating BMP signaling upregulates All experiments were performed in strict accordance with the standard guidelines for zebrafish work and were approved by the Institutional Animal Care and Use Committee of Chang Gung University .Tg (hsp70l:dnBmpr1aa-GFP) [AB (wild-type) zebrafish and transgenic line 1aa-GFP)  were pur1aa-GFP) .Batches of wild-type and transgenic embryos were strictly staged, as previously described  to ensurher2 morpholino (CTGGAAAGAGAAGGTAAAAGTTTGG) was purchased from Gene Tools LLC. . Morpholinos with a random sequence that does not bind to the zebrafish genome (CCTCTTACCTCAGTTACAATTTATA) and with 5 mismatched bases to the her2 morpholino  were used as controls. Microinjection of morpholinos into zebrafish blastomeres was performed during the one- to two-cell stages. A detailed procedure of her2 knockdown has been described by Cheng et al. [g et al. .sox2 expression by in situ hybridization, sox2-positive areas were selected and measured using ImageJ software. To detect apoptotic cells in the neuroectoderm, immunohistochemistry using rabbit monoclonal anti-active caspase-3 antibody  was applied to samples already stained with sox2 riboprobe using in situ hybridization. The caspase-3 antibody was detected using goat anti-rabbit IgG HRP , and 3, 3′-diaminobenzidine was used as a substrate for secondary antibody-conjugated HRP . Phospho-histone H3- and caspase-3-positive cells were manually counted.Digoxigenin-UTP-labeled riboprobes were synthesized according to the manufacturer’s instructions  and in situ hybridization was performed as described previously. Color reactions were conducted using NBT/BCIP substrate . To minimize variation between the control and experimental groups, we used embryos produced by a single pair of parents and used the same number of embryos for the control and experimental groups. The groups were compared under the same experimental conditions at the same time, and color reactions were initiated and stopped at precisely the same time. For immunohistochemistry, zebrafish embryos were blocked in 5% goat serum and incubated with a rabbit phospho-histone H3 antibody . Phosphorylated histone H3 was detected using red-fluorochrome-conjugated secondary antibodies . Embryos were mounted in the Vectashield mounting medium . Phospho-histone H3-positive cells were manually counted. To quantify 2O. The first strand of cDNA was reverse-transcribed using the SuperScript III First-Strand Synthesis System .RNA was isolated from 20 embryos in each experiment. Samples collected from each experiment were suspended in 500 μL TRIzol reagent  and homogenized using a 25-gauge needle, followed by the addition of 250 μL chloroform and centrifugation to separate the aqueous phase. The aqueous solution was mixed with a half-volume of acid phenol and chloroform and subjected to centrifugation for 15 min at 4 °C. The phenol-chloroform extraction was repeated twice. RNA was extracted using 250 μL isopropanol and 70% ethanol, and the mixture was centrifuged for 30 min at 4 °C. The ethanol was then removed, and the RNA pellet was resuspended in 12 μL DEPC Hsox2 , her2 , and GAPDH  were used. Gene expression levels were normalized to GAPDH and assessed using comparative Ct (40 cycles), according to the manufacturer’s instructions .For quantitative reverse transcriptase PCR (qRT-PCR), embryos were homogenized in TRIzol reagent , and total RNA was extracted using a standard method. cDNA was synthesized from total RNA through oligo(dT) priming using RevertAid First Strand cDNA synthesis kit . qPCR was performed on an ABI StepOneTM Real-Time PCR System  using SYBR Green . Primers for t-test with Microsoft Excel 2016. The significance level was set at p < 0.05. All reactions were performed in triplicate for each sample.Statistical analysis was performed using Student’s


The concept of Digital Twin is of fundamental importance to meet the main requirements of Industry 4.0. Among the standards currently available to realize Digital Twins there is the Digital Twins Definition Language. Digital Twin requires exchange of data with the real system it models and with other applications that use the digital replica of the system. In the context of Industry 4.0, a reference standard for an interoperable exchange of information between applications, is Open Platform Communications Unified Architecture. The authors believe that interoperability between Digital Twins and Open Platform Communications Unified Architectures communication standard should be enabled. For this reason, the main goal of this paper is to allow a Digital Twin based on the Digital Twins Definition Language to exchange data with any applications compliant to the Open Platform Communications Unified Architecture. A proposal about the mapping from Digital Twins Definition Language to the Open Platform Communications Unified Architecture will be presented. In order to verify the feasibility of the proposal, an implementation has been made by the authors, and its description will be introduced in the paper. Furthermore, the main results of the validation process accomplished on the basis of this implementation will be given. The fourth industrial revolution (Industry 4.0) has been aimed from its very beginning to the creation of more and more flexible, interoperable and innovative systems and services to achieve effective business models that enhance the quality of production ,2,3.Industry 4.0 is featured by a continuously-evolving digital transformation. It aims to automate all the traditional industrial practices, and it hopes to do so by bringing as much of the equipment from the physical space into the virtual domain. This is where the Digital Twin (DT) comes into play. Digital Twin emerged as an experimental technology set to enable replication of elements, functions, operations and dynamics of physical systems into digital world, with better control at testing, analysis, prediction and hazard prevention for sensitive processes .Digital Twins are used in different industrial settings, including health surveillance, agriculture, smart cities, smart grids, manufacturing, meteorology, education and automobiles. Digital Twins support the development of production processes making them reliable and flexible, enabling to visualise, monitor and optimize processes ,5,6,7.Different organisations are currently working aiming to standardize Digital Twin definition, interoperability and how to interact with these Digital Twins. Two notable projects in this area are the Asset Administration Shell (AAS) ,9,10 andOne of the main features of a Digital Twin is the communication with the physical world, from which a Digital Twin must receive the current state . At the same time a Digital Twin has the need to publish output data produced as a result of the processes accomplished on the physical system for which it realizes a replica. A Digital Twin may have also the need to exchange data with different applications according to the aims to be reached . For this reason, data exchange must be considered an important part of a Digital Twin; without data exchange, most of functions of a Digital Twin could not be realized . InteropIndeed, in the context of Industry 4.0, much effort has been spent and is currently being accomplished to fully integrate industrial applications in a multitude of production-related fields, safety and communication of machines, where they must interact and align their information models. An overview of the main trends about data integration and interoperability may be achieved by reading ,14. InteOpen Platform Communications Unified Architecture (OPC UA) , is consThe idea behind the paper is to enhance the interoperability of a Digital Twin through integration into the OPC UA domain. Among the available Digital Twins, the Digital Twin Definition Language model will be considered in this proposal. The paper will present the use of the OPC UA information model to structure and display a Digital Twin based on DTDL. The definition of a custom data structures in the OPC UA information model, able to represent each element of the original DTDL-based Digital Twin, will be introduced in this work. The proposed mapping has been implemented in order to be validated; the relevant implementation and validation will be introduced in the paper.The paper is organised as it follows. As said in the introduction, the main feature of the proposal is the use of OPC UA information model to realize a mapping from the representation of the data exposed by a DTDL-based Digital Twin and the OPC UA domain. Given this goal, this section aims to analyze the works present in the current literature in order to deal with the two important following issues.The use of OPC UA in this proposal is justified if the reader realizes that OPC UA is widely used in the current literature to realize mappings of information model between different domains; for this reason, the first part of this section is devoted to this demonstration giving to the reader the overview of the main publications related to the use of OPC UA to the mapping of different domains.The proposal of using OPC UA to map the DTDL-based Digital Twin, is justified if current literature does not present any other work about the same subject; for this reason, the second part of this section is devoted to this demonstration, giving an overview about the entire set of publications about the integration of OPC UA and the DTDL-based Digital Twin.Current literature provides a lot of publications pointing out the advantage in terms of interoperability of the use of OPC UA information model to structure and expose data coming from different domains of interest. An overview of examples of integration of OPC UA and other domains currently present in the literature, will be given in the following. In  a proposThe overview just given demonstrates that OPC UA is widely used to map different domains enhancing interoperability. One question remains unanswered; it is about the existence of mapping approaches between OPC UA and DTDL-based Digital Twins. This question is very important in order to point out the originality of the paper. For this reason, the second step accomplished in this section is that to give to the reader an overview about the entire set of publications about the integration of OPC UA and the Digital Twins and in particular about DTDL-based Digital Twins.Among the Digital Twins currently available, several works focus on the mapping of Asset Administration Shell DT and OPC UA. Mapping of AAS DT to OPC UA is proposed by . AnotherTo the best of authors’ knowledge, considering the integration of DTDL-based Digital Twin with OPC UA, only the software solution, called OPCUA2DTDL , is currIn conclusion, this section pointed out that OPC UA is one of the main standards used to enhance interoperability of different domains by mapping solutions, and to the best of authors’ knowledge, the proposed mapping solution between DTDL and OPC UA is original.The OPC UA communication standard is based on both client/server and publish/subscribe communication models and provides a semantically enriched information model in order to represent data.The OPC UA Information Model provides a standard way for servers to expose information to clients; publish/subscribe communication model is based on the same information model used for the client/server communication model. The set of information is maintained through OPC UA Nodes grouped together to compose the so-called OPC UA AddressSpace ,38,39. TEach OPC UA Node belongs to a class named NodeClass. Each NodeClass is derived from the Base NodeClass which defines the common attributes of OPC UA Nodes, among which: NodeId (which unambiguously identifies a Node in the OPC UA AddressSpace), Description , BrowseName (used to identify the OPC UA Node when browsing the OPC UA AddressSpace), WriteMask (exposing the possibilities of a client to write the attributes of the Node), NodeClass (which identifies the NodeClass of a Node) and DisplayName (which contains the name of the OPC UA Node that should be displayed in an user interface). In the following, a very concise overview of the main OPC UA NodeClasses will be given.Variable NodeClass is used to model data. It features an attribute named Value, containing the data, and an attribute named DataType specifying the type of the content of the attribute Value. Two types of Variables are defined: Property and DataVariable. Property contains information describing particular features of other OPC UA Nodes . A DataVariable maintains data values of a particular system .Method NodeClass allows to model callable functions that initiate actions within an OPC UA Server. One Method features two OPC UA Properties named InputArguments and OutputArguments, used to specify the input and output arguments of the Method.Object NodeClass is used to represent real-world entities such as system components, hardware and software components or even a whole system. An OPC UA Object is a container for other OPC UA Objects, Variables and Methods. As the Object Node does not provide for a value, an OPC UA DataVariable Node must be used to represent the data of an Object.OPC UA Information Model includes NodeClasses defining types. ObjectType NodeClass is used to hold type definition for OPC UA Objects. OPC UA defines the BaseObjectType which all the ObjectTypes must be extended from. OPC UA already defines several standard ObjectTypes derived from BaseObjectType. An example is FolderType whose instance, named Folder, is an Object organizing the AddressSpace into a hierarchy of OPC UA Nodes; it represents the root node of a subtree.VariableType NodeClass is used to provide for type definition of Variables. OPC UA standard defines the BaseVariableType, which all the VariableTypes must be extended from. Moreover, several standard VariableTypes derived from BaseVariableType are already defined by the standard. Among them there are the BaseDataVariableType and the PropertyType. The former is used to create an instance of a DataVariable Node, whilst the latter defines a Property Node.DataType NodeClass is used to provide type definition of the Value attribute of a Variable Node, as said before. DataType may be Built-in, Enumeration or Structured; arrays of elements are also allowed.Relationships may be defined between OPC UA Nodes; they are called References. The ReferenceType NodeClass is used to define different semantics for References. References may be classified in two different main categories: Hierarchical and NonHierarchical.Among the Hierarchical References, the following ones will be used in the paper: HasComponent, HasProperty and Organizes. The HasComponent is a Reference featuring an OPC UA Object or DataVariable as the source, and an OPC UA Object, a DataVariable or a Method as the target Node. If the source is an Object, the semantic associated to this reference is that the source is made up by the target OPC UA Object, DataVariable and Method Node. If the source OPC UA Node is a DataVariable, the target Nodes must be other OPC UA DataVariables; the meaning, in this case, is that the source variable is made up by a set of other variables. HasProperty Reference may connect a source OPC UA Node to an OPC UA Property Node; the meaning is that the source Node features a property described by the target Node. Organizes Reference allows to organize OPC UA Nodes inside a Folder.Among the NonHierarchical References, there are the HasTypeDefinition, HasSubtype and HasModellingRule. HasTypeDefinition is used to bind an OPC UA Object or Variable to its ObjectType or VariableType, respectively. HasSubtype Reference expresses a subtype relationship between types. The HasModellingRule Reference is used to describe how instances of a particular type should be created. The source of this Reference is named InstanceDeclaration, whilst its target is a ModellingRule Object. An InstanceDeclaration is an Object, Variable or Method that is the target of a Hierarchical Reference starting from an ObjectType or VariableType Node . A ModellingRule Object specifies what happens to the InstanceDeclaration with respect to instances of the relevant OPC UA type. Several ModellingRule Objects are defined in OPC UA. A Mandatory ModellingRule for a specific InstanceDeclaration specifies that instances of the OPC UA type referencing the InstanceDeclaration must have a counterpart of that InstanceDeclaration. This means that each instance must hold an OPC UA Node of the same NodeClass of the InstanceDeclaration; furthermore, the Reference targeting this OPC UA Node must be of the same ReferenceType of the one pointing the InstanceDeclaration. An Optional ModellingRule for a specific InstanceDeclaration, instead, specifies that instances of the OPC UA type may have a counterpart of that InstanceDeclaration, but it is not required. Other two ModellingRule Objects exist, named MandatoryPlaceholder and OptionalPlaceholder. The difference with the previous ones is that the counterparts of InstanceDeclaration may be more than one.OPC UA defines standard graphical representation for both Nodes and References. Some of them are summarized by In order to better understand the role of the InstanceDeclaration and ModellingRule Object, Very recently, a new feature has been added to the OPC UA Information Model, called AddIn ,39; it iIn The Digital Twins Definition Language (DTDL)  was bornThe DTDL implements six classes of metamodels: Interface, Telemetry, Property, Control, Relationship and Component. These classes are able to fully define the structure and behaviour of a Digital Twin.In DTDL, resources are called interfaces and can contain a set of telemetry, properties, commands, relationship and components. Telemetry describes data emitted by a resource, whether it is a regular stream of sensor readings or a calculated data stream, such as the raw data of sensors, processed data or data generated by DT models; Telemetry does not store any data. Properties define values within a Digital Twin; these values can be read-only or have read and write states. Properties have a backing storage; this allows us to read the value of the property at any time. However, the property can also be writable according to a particular setting; this allows us to store a value in the property.Commands correspond to functions that can be invoked with optional input and output parameters. Relationship describes a link to another digital twin and makes it possible to create graphs of digital twins. Component models the entities that exist in the DT, including sensors, gateways, and digital systems.This paper aims to present a proposal to map the DTDL metamodel classes  into corresponding elements in the OPC UA Information Model.As it was pointed out in In this proposal, the above-mentioned common practices have been taken into account to map the DTDL metamodel classes into OPC UA Information Model; custom OPC UA types have been defined through an extension of the current OPC UA Information Model, able to represents the DTDL metamodel classes and the relevant properties. The following subsections will present the mapping of the six DTDL metamodels classes, introducing the custom OPC UA types defined to realize the proposal.The metamodel class Interface allows to describe the contents of a digital twin, as told in It has been assumed to map the Interface metamodel class with a custom OPC UA ObjectType, called DTDLInterfaceType. The DTDLInterfaceType will inherit all the attributes of the BaseObjectType, including those of the OPC UA Base NodeClass. Some of these last attributes are used to represent a subset of the properties of the Interface class. The DTDL @id property is used to univocally identify each metamodel class. On the basis of the definition of NodeId given in In DTDL, the @type property is used to specify the type of the metamodel class; in this case, this property must be set to the “Interface” value. As said in The remaining properties of Interface class shown by The DTDL contents property is very important as it allows to define the set of objects representing the contents of an Interface; these objects belong to the other DTDL metamodel classes, i.e., Telemetry, Property, Command, Relationship and Component. As suggested in , an elemThe DTDL comment property allows to define a comment for model authors. It has been mapped through the OPC UA Node named Comment, which is an optional property of the ObjectType DTDLInterfaceType, as clearly shown by The DTDL schemas property represents the set of the descriptions of the data types in a digital twin interface. A full set of primitive data types are provided in DTDL, along with support for a variety of complex schemas in the forms of Arrays, Enums, Maps and Objects. In OPC UA the DataType NodeClass may be used to define the representation of the DTDL data types. As said in The last mandatory folder present in The metamodel class Telemetry describes the data emitted by any digital twin, whether the data is a regular stream of sensor readings or a computed stream of data, such as occupancy, or an occasional error or information message. Telemetry does not store any data. This class has several properties shown by Due to the lack of data stored within a DTDL Telemetry element, it has been assumed to map it with a custom OPC UA ObjectType, called DTDLTelemetryType. Some of the attributes of the OPC UA Base NodeClass are used to represent a subset of the properties of the DTDL Telemetry class, as described by The same considerations accomplished for the properties @id, description and displayName seen for the Interface class are still valid and can be applied to the Telemetry class. The DTDL name property is defined as the “programming” name of the telemetry; mapping with the OPC UA BrowseName attribute seems suitable.Considering the other DTDL properties shown by The DTDL @type property may be mapped as accomplished for the Interface class. In particular, for each OPC UA Object of OPC UA DTDLTelemetryType type used to represent the DTDL Telemetry, the @type property may be represented by the HasTypeDefinition Reference pointing to the OPC UA DTDLTelemetryType type.The other DTDL properties present in The ObjectType DTDLTelemetryType has two optional OPC UA properties, Comment and Unit, mapping the DTDL properties comment  and unit (which defines a semantic type of the Telemetry), respectively.The DTDL schema property represents the data type of the Telemetry; OPC UA DataType seems suitable to represent this property, as said in the previous subsection. The proposed approach is to map the DTDL schema property with a custom OPC UA PropertyType Node, which contains information about the OPC UA DataType chosen to represent the DTDL data type. The attribute Value of the OPC UA PropertyType Node is used to contain this information. On the basis of this assumption, a custom OPC UA PropertyType Node has been considered for the DTDLTelemetryType ObjectType; The metamodel class Property allows to store values within a digital twin. These values can be read-only or have read and write states. For example, a device serial number may be a read-only value that can be read at any time; the desired temperature on a thermostat may be a read-write value that can be updated. This class has several properties, shown by As the DTDL Property class is used to store values, it seems that it may be represented very well by the OPC UA DataVariable Node. For this reason, it has been assumed to map the DTDL Property class with a custom OPC UA DataVariableType, called DTDLPropertyType, derived from the BaseDataVariableType.Some of the basic attribute of the DTDLPropertyType may be used to represent properties of the Property class. About the other properties, mapping of @type has been accomplished using the same approach described in the previous subsections. The other properties have been mapped, as shown by It is important to point out that the proposed modelling of the DTDL Property class with the custom DTDLPropertyType DataVariableType allows to make available the OPC UA attribute Value, which may be used to maintain the current value of the digital twin property. According to the WriteMask attribute, the value may be read-only, or it may be updated. This is very important to be pointed out, as this feature allows to fully map the capability of DTDL Property class to store values within a Digital Twin.The metamodel class DTDL Command allows to describe a function or operation that can be performed on digital twins. It was assumed to represent the Command class through a custom OPC UA ObjectType called the DTDLCommandType. The reason of this mapping is due to the need to represent both properties and method; on account of what said about OPC UA, the only NodeClass suitable to represent complex structure is the ObjectType. The DTDLCommandType will inherit all the attributes of the OPC UA Base NodeClass. Some of these attributes are used to represent the properties of the Command class, as shown by About the other properties, mapping of @type has been accomplished using the same approach described for the DTDL classes seen before. In particular, it has been assumed that this property was represented by the HasTypeDefinition Reference pointing to the OPC UA DTDLCommandType ObjectType for each OPC UA Object used to represent a DTDL Command.Three properties of the Command class are still to be mapped into OPC UA, i.e., comment, request and response. Furthermore, the function or operation represented by the Command class must be modelled in OPC UA. In order to realize these mappings, the structure shown by The metamodel class DTDL Relationship describes a link to another (separate) digital twin and enables graphs of digital twins to be created. The class features the properties shown by As accomplished for other DTDL metamodel classes, this class has been mapped using an OPC UA ObjectsType. A custom ObjectType has been defined and called DTDLRelationshipType. About the other properties shown by It has been assumed that the DTDLRelationshipType ObjectType features three optional properties named Comment, MinMultiplicity and MaxMultiplicity. They model the DTDL properties comment, minMultiplicity and maxMultiplicity, respectively. The comment property is a comment for model authors, whilst minMultiplicity and maxMultiplicity represent the minimum and maximum multiplicity for the target of the relationship, respectively.The DTDL property named properties represents the set of Property classes that define relationship-specific state; in other words, they define the main features of the relationship, if required. In order to represent this set, a folder has been considered in OPC UA; in The last DTDL property to be mapped is the target, representing the link to a DTDL Interface. In OPC UA, the links between Nodes are realized using References, as explained in The metamodel class Component enables interfaces to be composed of other interfaces. Components are different from Relationships, because they describe contents that are directly part of the interface, whilst a relationship describes a link between two interfaces. A custom OPC UA ObjectType NodeClass has been used to represent the Component class; it was named DTDLComponentType ObjectType. The property @type has been modelled using the HasTypeDefinition Reference, as explained in the previous subsections. The properties comments and schema have been mapped using OPC UA Nodes, as shown by The custom OPC UA Information Model presented in the previous section has been implemented by the authors and the software implementation is freely available at the GitHub repository . In partThe implementation of the custom OPC UA information model presented in this paper, allowed the authors to validate the proposal. Several case studies were considered, and real mappings were realized and tested. In order to give a clear idea about the procedure used to validate the proposal, in the following subsections, details about the different steps followed in the validation procedure will be given. Details will refer to a single case study; a very simple example has been considered in order to be easily read and understood.It has been assumed to take into consideration a Digital Twin of a building, modelling each component and, in particular, each room. In particular, the DT of a room will be considered in this case study. A basic model of a Room has been defined, and its extension, called MeetingRoom, has been considered. The MeetingRoom model includes all the properties of the Room model, adding other ones.The DTDL Interface models shown by The DTDL “MeetingRoom” model shown by As it can be seen from According to OPC UA Server was implemented in NodeJS using NodeOPCUA , which iThe realization of the OPC UA Server must include the implementation of the custom AddressSpace proposed in this paper and the code of the suitable procedures to populate this AddressSpace with real values.Applying the mapping solution presented in In particular, the OPC UA Object “MeetingRoomA” is an instance of the DTDLInterfaceType ObjectType, representing the DTDL Interface model. It features a component (pointed by the HasComponent reference) made by the mandatory folder Contents; this folder organizes the folder Properties, organizing two OPC UA DataVariable Nodes instances of the DTDLPropertyType type. They model the two DTDL properties “occupied” and “tempValue”. The OPC UA Object “MeetingRoomA” features another component made up by the mandatory folder Extends. As explained in The OPC UA DTDLInterfaceType Object “Room” represents the Interface with name “Room”, and it features only one OPC UA DataVariable Node organized by the Properties folder. This Node represents the DTDL property named “setlight”, shown in It is important to point out that the other mandatory folders of the DTDLInterfaceType ObjectType see  are not The OPC UA AddressSpace shown by As said before, the Digital Twin instance “MeetingRoomA” features three properties  whose value changes over the time. Changes in the DT instance are generally due to updates of the real system for which the Digital Twin realizes a digital replica. Using the “Azure Digital Twin Explorer”, these values may be changed by the user in order to simulate the updates from the real system, as accomplished by the authors during the validation procedure . The authors have presented an overview of the current state-of-the-art, pointing out the originality of their work. Furthermore, a software implementation has been presented; it is freely available on the GitHub repository. Implementation was allowed to validate the proposed approach.The authors believe that the proposed mapping accomplishes the goals set by the fourth industrial revolution, creating systems and services increasingly flexible, interoperable and innovative, improving the quality and efficiency of industrial production, by using Digital Twins. They believe that the proposed solution opens up a lot of opportunities in the interoperability between applications based on Microsoft Azure Digital Twin and the OPC UA domain.


In our current study, we used a combination of biochemical and genetic techniques to dissect the role of Rdh54 on the exchange of genetic information during DNA repair. Our data indicate that RDH54 regulates DNA strand exchange by stabilizing Rad51 at an early HR intermediate called the displacement loop (D-loop). Rdh54 acts in opposition to Rad51 removal by the DNA motor protein Rad54. Furthermore, we find that expression of a catalytically inactivate allele of Rdh54, rdh54K318R, favors non-crossover outcomes. From these results, we propose a model for how Rdh54 may kinetically regulate strand exchange during homologous recombination.Homologous recombination (HR) is a double-strand break DNA repair pathway that preserves chromosome structure. To repair damaged DNA, HR uses an intact donor DNA sequence located elsewhere in the genome. After the double-strand break is repaired, DNA sequence information can be transferred between donor and recipient DNA molecules through different mechanisms, including DNA crossovers that form between homologous chromosomes. Regulation of DNA sequence transfer is an important step in effectively completing HR and maintaining genome integrity. For example, mitotic exchange of information between homologous chromosomes can result in loss-of-heterozygosity (LOH), and in higher eukaryotes, the development of cancer. The DNA motor protein Rdh54 is a highly conserved DNA translocase that functions during HR. Several existing phenotypes in  Homologous recombination is an important pathway in repairing DNA double strand breaks. For the purposes of this study, HR can be divided into two stages. The first is a DNA repair stage in which the broken DNA molecule is fixed. In the second stage, information can move from one DNA molecule to another. Enzymes that use the power of ATP hydrolysis to move along dsDNA aid in regulating both stages of HR. In this work we focused on the understudied DNA motor protein Rdh54. We combined genetic and biochemical approaches to show that Rdh54 regulates HR by stabilizing the recombinase protein Rad51 at early HR intermediates. Homologous recombination (HR) is a universally conserved DNA double-strand break repair (DSBR) pathway that functions to maintain chromosome integrity. Failure in, or mis-regulation of, HR can lead to the loss of genome integrity and the development of human cancers ,2. In euD-loops are reversible three strand DNA structures that form between donor and recipient DNA molecules. D-loop maturation is regulated by efficiency of DNA base pairing , and theAfter DNA extension, D-loops mature through Rad52 mediated annealing of the second end of DNA into an active extended D-loop –27, promin vitro, including DNA supercoiling [rdh54Δ diploids have a 10-fold reduction in interhomolog gene conversion but had little effect on inter-sister chromatid recombination [rdh54Δ/rad54Δ diploid strains have enhanced phenotypes over that of individual mutants implying partial redundancy during DNA repair. Whether this is due to direct or indirect effects on HR is unclear. Rdh54 is required to prevent the accumulation of Rad51 on undamaged dsDNA [Rdh54   is a parrcoiling –37, nuclrcoiling ,38, enharcoiling ,36,39,40rcoiling ,42. Genercoiling , 44. Whebination . Howevered dsDNA ,47 and Red dsDNA ,47. The in vitro [RDH54 and RAD54 activity manifests in cells during HR. RDH54 is also involved the process of inter-chromosomal template switching (ICTS) [There are also several differences between Rdh54 and Rad54. These include Rad54 preferentially initiating Rad51 mediated homology search in vitro . The stain vitro . Major dg (ICTS) ,45. ICTSg (ICTS) –52. Bothg (ICTS) ,19.RDH54 deletion resulting in an increase in BIR outcomes, and catalytically inactive Rdh54 altering the ratio of CO/NCO outcomes. Finally, we find that Rdh54 phenotypes have stronger association with short tract gene conversion outcomes. From these data we develop a ‘pace car’ model, to explain the regulatory role of Rdh54 in D-loop expansion and its influence on HR outcomes.Here we have conducted a study on the biochemical and genetic mechanism by which Rdh54 effects downstream HR outcomes through regulation of D-loop intermediates. We conclude that Rdh54 stabilizes Rad51 at D-loop intermediates by reducing the rate of Rad54 mediated D-loop maturation. The genetic consequence of Rdh54 mediated stabilization is an alteration in the balance between BIR and CO/NCO outcomes. With RDH54 during HR between homologous chromosomes [RDH54 was required for direct targeting of the homologous chromosome or may function through a more indirect mechanism. To better understand this question and the role of RDH54 in HR outcomes between homologous chromosomes, we initiated our studies using a defined genetic reporter that reports on DSB-induced recombination between homologs [RDH54 when both sister chromatids are cut [RDH54. Therefore, even if only a fraction of cells utilized RDH54 during HR, we would still be able to observe changes to that specific population. Finally, this reporter is well characterized [Previous reports have used diploid heteroallelic strains to detect rare spontaneous recombination events to define a role for omosomes –45. Theshomologs ,54. We scterized –55.ADE2 (ade2-I) gene located on chromosome XV (HPHMX). The homologous chromosome contains a non-cleavable inactive allele of ADE2 (ade2-n) that results from a 2 bp deletion in the Nde1 site [NATMX) located 154 kbp downstream of ade2-n . The antibiotic markers are used to determine LOH outcomes due to crossovers or BIR. The differences in the ADE2 alleles allow for one chromosome to act as a substrate for the I-SceI nuclease, and the homolog to serve as an uncut template for repair. Importantly, to ensure recombination outcomes are not due to loss of chromosomes, URA3 and MET22 markers located on the far end of chromosome XV act as markers to report on loss of chromosomes  and the other is resistant to nourseothricin (clonNAT), or vice versa. Crossovers without LOH will also form with an equal probability. Therefore, all counted LOH crossovers were multiplied by two. LOH can also result from BIR. Sectored colonies that have gone through BIR will have a white sector that is resistant to clonNAT, but not HPH. This results from the preferential replication of one sister chromatid due to failed second end capture ,57,58. SRDH54/rdh54Δ (83+/-4.4%), rdh54Δ/rdh54Δ (69+/-4.1%), RDH54-KanMX/RDH54-KanMX (83.3+/-10.6%), and rdh54K318R/rdh54K318R  (75.5+/-8%) strains . Surprisingly, this strain resulted in a distribution of 81.4±3.9%, 14.6+/-2.8%, and 3.8+/-1.2%, NCO, CO, and BIR outcomes, respectively  of CO outcomes in favor of an increase in NCO outcomes (p = 0.0036)  . This obRDH54 has an ATP independent function to reduce BIR and an ATP dependent function to regulate CO/NCO outcomes.To ensure that these outcomes were due to HR and not the loss of chromosomes, sectored colonies were scored for viability on -Ura/-Met plates. Colonies that retain both homologous chromosomes will grow, and if chromosome loss has taken place colonies will fail to grow. No significant increase in chromosome loss was observed for all strains tested . From thWe chose to analyze solid red and white colonies separately from sectored colonies. When analyzing red colonies, the number of non-recombinant events were determined by re-induction assays and then colonies that did not cut were subtracted from the total red cell population. Therefore, recombination outcomes are only evaluated for recombinant colonies. NCO, BIR, and CO outcomes were determined from antibiotic sensitivities after replica plating of un-selected colonies. There were several differences between sectored, solid red, and solid white colonies. In general NCO outcomes were the most substantial part of the solid red and solid white colony outcomes with 89+/-4.8% and 89+/-2.1 of outcomes in the WT strain for red and white respectively  Tables.RDH54/rdh54Δ  and rdh54Δ/rdh54Δ  strains (rdh54K318R/rdh54K318R strain (1.7+/-1.4% p = 0.5). CO outcomes were different from those observed in sectored colonies. RDH54/rdh54Δ exhibited a slight but insignificant drop in crossover outcomes compared to WT , and the rdh54Δ/rdh54Δ strain had a significant drop in crossovers . Finally, the rdh54K318R/rdh54K318R strain also had reduced crossover formation   . From thRDH54/rdh54Δ and rdh54Δ/rdh54Δ strains resulted in a significant increase in BIR or BIR like outcomes as compared to WT (1.1+/-1.1% versus 7.4+/-2.9 (p = 0.03) and 17.7+/-6.6% (p = 0.006)). These results were also complemented by the rdh54K318R/rdh54K318R  strain (rdh54Δ/rdh54Δ (n = 0/33 versus 10/133 (WT)) strain, and a significant drop in CO outcomes in the RDH54/rdh54Δ strain . Additionally, the rdh54K318R/rdh54K318R strain did not lose CO outcomes entirely but was severely compromised   strain . Surpris = 0.01) . These din vitro biochemical assay to monitor the formation and maturation of nascent D-loops [Rad54 acts as a motor during HR promoting Rad51 homology search followed by conversion of nascent D-loops into mature D-loops by acting as a dsDNA pump to remove Rad51 ,21,28. P D-loops ,21. To e D-loops ,60,61. HThe nascent D-loop is detected by a gel shift in which the recipient DNA migrates higher in the gel due to an interaction with the donor plasmid. This reaction peaks within the first 10 minutes . D-loop −3 min-1, 8 x 10−3 min-1, 4.9 x10-3 min-1, and 0.18 x 10−3 min-1 at 0, 3:1, 1:1, 1:3 molar ratios of Rad54:Rdh54, respectively  as a DNA polymerase to form extended D-loops  of the reaction for both  minutes . It shouenotypes . WhetherRad54NRdh54 mutant would fail to prevent 3’ end extension, and D-loop maturation. In contrast, the Rdh54NRad54 mutant would prevent the extension of the 3’ end. Consistent with this prediction, we observed that Rdh54NRad54 severely inhibited extended D-loop formation with 0%, 0%, 0%, and 1.4+/-1.2% extended D-loops observed at 10, 20, 30, and 45 min, respectively   or rdh54NRad54  strains  .We measured outcomes for individual red and white colonies. The major findings were like those observed for sectored colonies with several exceptions. For red colonies,  = 0.07) . In rad5 30+/-9%  of the tectively . In cont strains . In soli 0.0002) . These rrdh54Δ and rad54Nrdh54 strains in solid white colonies the frequency relative to WT was 17 and 30 times greater respectively in BIR or BIR like outcomes. This stands in contrast to red and sectored colonies in which BIR events occurred at only a 4 times greater frequency than WT in the rdh54Δ/rdh54Δ strain (red and sectored). For the rad54NRdh54 strain a 2.7 (red) and 3.4 (sectored) increased frequency was observed. By comparison the Rdh54NRad54 strain produced a 1.0-, 1.6-, and 1.26 increase in frequency for solid white, solid red, and sectored colonies respectively . After overnight expression, the cells were harvested and frozen at -80°C. After freezing for at least 30 minutes, cell pellets were re-suspended in 25 mL lysis buffer  per 3 liters of cell culture. Cells were sonicated 10 times with a 65% amplitude cycle for 15 seconds on and 45 seconds off. The lysates were clarified by ultracentrifugation for 45 minutes at 15,000 rpm. Clarified lysates were incubated with 2 mL settled volume of Ni-NTA Superflow Agarose for 1 hour at 4°C. The agarose was then washed by 10 column volumes (CV) of lysis buffer and 10 CV of wash buffer . After washing, the agarose was packed in a 10 mL poly-prep chromatography column and then drained. The protein was then eluted with 4 CV elution buffer (wash buffer + 190 mM Imidazole). Peak fractions were collected and then applied to a HiScreen SP FF column . The protein was bound to the column in buffer A . Then the column was washed by 3 CV buffer A. The protein was eluted by a gradient increasing buffer B (buffer A + 1000 mM KCl). The protein would be eluted at around 35% to 65% buffer B. Peak fractions were pooled and concentrated to 2 mL by a protein concentrator spin column at 3500 rpm. Then the protein was applied to a HiPrep Sephacryl S-300 HR column pre-equilibrated with SEC buffer . The peak fractions were collected and concentrated to around 500 μL by a protein concentrator spin column (10 kDa MWCO) at 3500 rpm. The protein was then quantified by the absorbance at 280 nm. Samples were aliquoted and stored at -80°C.Purification of Rad51, RPA, GST-Rad54, and GST-Rdh54 were carried out as previously described ,74–76. 6KanMX selectable marker with 50 nt of homology upstream and downstream of the ORF of the RDH54 gene and transformed into WT-15D with selection on YPD+G418 (500 μg/ml). Genetic knockouts were confirmed by amplification of yeast genomic DNA with primers that annealed 250 bp upstream or downstream of the RDH54 ORF. Subsequent deletions were generated by amplification of the region of the genome and transforming WT-11C. A similar strategy was used for RAD54 deletions. For genetic knock-ins, a KanMX marker was integrated 125 bp downstream of the RDH54 stop codon in a plasmid containing the RDH54 gene. WT-KanMX and rdh54K318R-KanMX were PCR amplified and used to replace the endogenous RDH54 locus in both WT-11C and WT-15D. Integration was confirmed by PCR. Full strain genotypes are in Gene deletions were generated by PCR amplification of 600 of 0.2 and allowed to reach an OD600 of 0.4–0.5 followed by the expression of I-SceI by the addition of 2% galactose. Cells were allowed to grow for an additional 1.5 hours after which they were plated on YP+2% dextrose and allowed to grow for 48 hours. After 48 hours they were placed in the 4°C overnight to allow further development of red color. The number of white, red, and sectored colonies was then counted followed by replica plating onto YPD+ hygromycin B (200 μg/ml) and YPD+ nourseothricin  for analysis of recombination outcomes. Technical duplicate plates were also plated onto YNBR (+ade) +2% galactose to measure reinduction of I-Sce1, indicating the fraction of cleaved sister chromatids. Finally, strains were also replica plated on YNB (-URA/-MET) + 2% dextrose to insure proper chromosome segregation. The data were analyzed by counting colonies that were red, white, or sectored, and then counting colony survival on different antibiotic sensitivities. The data for each category was then divided by the total population of sectored colonies. The standard deviation between biological replicates analyzed for at least three independent experiments. The red and white colonies were analyzed in similar ways with the exception that the number of red colonies that were not cleaved by I-Sce1(as assayed by reinduction) were subtracted from the total red cell population, and only recombinants were used in the analysis.The WT strain used in this assay as well as the procedure for diploid formation are described here ,55. The 2, 1 mM DTT, 0.2 mg/ml BSA) using an Atto647N labeled DNA duplex (15 nM) consisting of 21 nt overhang that was homologous to a region on the pUC19 plasmid. Rad51 (300 nM) was incubated with recipient DNA at 30°C for 15 minutes. The resulting Rad51 filaments were then mixed with indicated concentrations of Rad54, Rdh54 or both, RPA (500 nM) and pUC19 plasmid (0.3 nM). Reactions were quenched after indicated periods of time and treated with 1 unit of Proteinase K at 37°C for 20 minutes. The reactions were then resolved by electrophoresis on a 0.9% agarose gel and imaged for fluorescence using a Typhoon FLA 9500. The sequences of the oligos used in this study can be found in D-loop formation experiments were performed in HR buffer  and Klenow (exo-)(NEB) were added after 5 minutes of D-loop formation. Experiments were performed with either 21 nt of ssDNA homology or 65 nt of ssDNA homology to the pUC19 plasmid. The recipient DNA molecules were labeled by annealing a shorter non-homologous piece of DNA that was 5’ end labeled with Atto647N. The reactions were quenched as described above and incubated with 1 unit of Proteinase K for 37°C for 20 minutes. The reactions were then resolved by electrophoresis on a 0.9% agarose gel and imaged for fluorescence using a Typhoon FLA 9500. The sequences of the oligos used in this study can be found in 32P–ATP. All reactions were performed at 30°C and contained pUC19 plasmid dsDNA (50 ng/μl). Aliquots were removed at specified time points and quenched with 25 mM EDTA and 1% SDS. The quenched reactions were spotted on TLC plates  and resolved in 0.5 M LiCl plus 1 M formic acid. Dried TLC plates were exposed to phosphorimaging screen and scanned with a GE Healthcare Life Sciences Typhoon FLA 9500 biomolecular imaging system.ATP hydrolysis assays were performed in HR buffer  in the presence of 1 mM ATP and trace amounts of γRAD54-YFP (strain ML1067) or RDH54-YFP (strain ML906) cells were estimated by measuring total nuclear YFP fluorescence intensities using Volocity software.Total protein measurements were made as described in Silva et al. . BrieflyS1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S1 FigWT, RDH54/rdh54Δ, rdh54Δ/rdh54Δ, and rdh54K318R/rdh54K318R strains. The columns represent the mean, and the error bars represent the standard deviation of at least 3 independent experiments. (B). Quantification of sectored white colonies in which both sister chromatid has undergone LTGC for BIR or BIR like, CO, and NCO for WT, RDH54/rdh54Δ, rdh54Δ/rdh54Δ, and rdh54K318R/rdh54K318R strains. The columns represent the mean, and the error bars represent the standard deviation of at least 3 independent experiments.(A). Quantification of sectored red colonies in which both sister chromatid has undergone STGC for BIR or BIR like, CO, and NCO for (TIF)Click here for additional data file.S2 Fig(A). ATPase assay illustrating the fraction of Rad54 ATP hydrolysis in the presence and absence of Rad51 with and without Rdh54K318R. The bars represent the mean and standard deviation of three independent experiments. (B). Quantification of the number of fluorescently labeled Rad54 (N = 100) and Rdh54 (N = 100) molecules in yeast nucleus. The line and error bars represent the mean and 95% confidence interval of the data. The red number above the data is the mean molecules per cell.(TIF)Click here for additional data file.S3 Fig(A). Schematic illustrating chimeric constructs for Rdh54NRad54, and Rad54NRdh54. These mutants alter the position of translocase binding on Rad51 filaments. (B). Representative agarose gel illustrating the formation and disruption of D-loops in the presence of Rad54 (left), Rdh54NRad54 (middle left), Rdh54 (middle right), Rad54NRdh54 (right). The recipient DNA used in these studies is 21 nt of homologous ssDNA and 36 bp of non-homologous dsDNA. (C). Quantification of D-loop formation and disruption in the presence of Rad54 (left), Rdh54NRad54 (middle left), Rdh54 (middle right), Rad54NRdh54 (right). The columns and error bars represent the mean and standard deviation of independent experiments.(TIF)Click here for additional data file.S4 Fig(A) Graph representing the red/white/sectored colony outcomes for all strains used in this paper. Strains are labeled in the figure. The bars and error bars represent the mean and standard deviation of at least three independent experiments.(TIF)Click here for additional data file.S5 FigWT, rdh54Δ/rdh54Δ, rad54Nrdh54/rad54Nrdh54, andrdh54Nrad54/rdh54Nrad54 strains. The WT and rdh54Δ strains are reproduced from WT, rdh54Δ/rdh54Δ, rad54Nrdh54/rad54Nrdh54, and rdh54Nrad54/rdh54Nrad54 strains. The WT and rdh54Δ strains are reproduced from (A). Quantification of sectored red colonies in which both sister chromatids have undergone STGC for BIR or BIR like, CO, and NCO for (TIF)Click here for additional data file.S6 Figrdh54Δ/rdh54Δ, rad54NRdh54/rad54NRdh54, and rdh54NRad54/rdh54NRad54 for BIR outcomes in Solid White, Solid Red, and Sectored outcomes. (B). Bar graph representing the differences between rdh54Δ/rdh54Δ, rdh54K318R/rdh54K318R, rad54NRdh54/rad54NRdh54, and rdh54NRad54/rdh54NRad54 for CO outcomes in Solid White, Solid Red, and Sectored outcomes. The data was generated by dividing the appropriate mutant strain by the WT.(A). Bar graph representing the differences between (TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Fertility preservation in the cancer setting, known as oncofertility, is a fieldthat requires cross-disciplinary interaction between physicians, basicscientists, clinical researchers, ethicists, lawyers, educators, and religiousleaders. Funded by the National Institutes of Health, the OncofertilityConsortium (OC) was formed to be a scientifically grounded, transparent, andaltruistic resource, both intellectual and monetary, for building this new fieldof practice capable of addressing the unique needs of young patients withcancer. The OC has expanded its attention to include other nonmalignantconditions that can threaten fertility, and the work of the OC now extendsaround the globe, involving partners who together have created a community ofshared effort, resources, and practices. The OC creates materials that aretranslated, disseminated, and amended by all participants in the field, andlocal programs of excellence have developed worldwide to accelerate the pace andimprove the quality of oncofertility research and practice. Here we review theglobal oncofertility programs and the capacity building activities thatstrengthen these research and clinical programs, ultimately improving patientcare. Established in2007 as part of a National Institutes of Health center grant, the OncofertilityConsortium (OC) is an interinstitutional, interdisciplinary consortium to expandresearch in fertility loss in patients with cancer, accelerate clinical translationof fertility preservation techniques, and address the complex health care andquality-of-life issues that concern young patients with cancer whose fertility maybe threatened by their disease or its treatment.7 The termoncofertility was originally coined to describe a new discipline that bridgesoncology and reproductive medicine to discover and apply new fertility preservationoptions for young patients with cancer. However, as the OC worked to createfertility preservation technologies and clinical oncology management plans forpatients with cancer, it became clear that fertility concerns resulting fromnonmalignant diseases and iatrogenic causes were much broader than just thoseassociated with cancer. GI diseases, rheumatologic disorders, nonmalignanthematologic conditions , neurologicdisorders, renal disorders, gynecologic conditions, and metabolic diseases can alladversely affect fertility. By expanding its scope, the OC now ensures that allpatients facing a disease or treatment that limits reproductive function can benefitfrom the findings of basic and clinical reproductive research. The wordoncofertility was created when few options were available and now providesterminology for a medical field at the intersection of many iatrogenic causes ofinfertility.Survival rates among young patients with cancer have steadily increased over the pastthree decades, in part because of the development of more effective cancertreatments.8Since its inception, the OC has aimed to involve more partners to create anationwide community of shared resources and practices, with the ultimate goal ofimproving patient care. Today, there is wide acceptance that partnerships that bringresearch and clinical teams together catalyze progress, and the global partnershipsdiscussed here are moving quickly to provide broad reproductive care to anyoneexperiencing an iatrogenic impact on reproduction, fertility, orsexuality.2To facilitate sharing of knowledge and resources, the OC formed the NationalPhysicians Cooperative, which today represents > 60 centers across the UnitedStates that provide oncofertility services to men and women, as well as 19 centersfocused on pediatric patients.There are currently 19 countries engaged in the global oncofertility community  and other online resources are madeavailable to global partners, including the iSaveFertility mobile app (savemyfertility.org),patient navigator tools (preservefertility.northwestern.edu), decision aids (myoncofertility.org), andRepropedia (http://www.repropedia.org), anonline reproductive lexicon. Branding materials are available at oncofertility.northwestern.edu/branding-materials. Global partnersare encouraged to translate these materials and disseminate them to internationalaudiences, while in turn providing our team with new content and links to include onthe main OC Web site (oncofertility.northwestern.edu). A complete list of active globalpartners and the work they have contributed can be found here: http://oncofertility.northwestern.edu/global-oncofertility-partners.Making these materials available to all partners fosters interaction and a sharedlanguage and purpose among diverse groups, which enables our global partners toapply these resources, methodologies, and other experiences in the field. Guidelinesare not within the scope of the consortium, but the OC does provide a site foraggregation and dissemination of guidelines from formal medical societies, andguidelines for our field from oncology, fertility, pediatric, and nursing specialtygroups from around the globe can be found in one central location . Establishinga strong global network not only drives the collaborative nature of the OC, but alsohelps global partners build their own consortia and fertility preservationnetworks.9Global partners of the OC receive tools and guidance to set up their own localconsortia Fig . The admAs part of the global partners model Fig ,the OC 10 withstriking success in advancing knowledge.11 Similarly, extending the oncofertility network beyond theconfines of the United States and into the international realm not only advancesknowledge and promotes discovery, but also provides uniform access for all expertsand their unique perspectives. These collaborations benefit the scientific communityby accelerating the pace of discovery and decreasing the time to clinicalapplication. The capacity for making landmark breakthroughs in the field isenhanced. Furthermore, research has shown that the trend toward internationalcollaboration and network building attracts attention to issues and leads to agreater number of publications and greater support for basic research and clinicalstudies.12 Globalpartnerships, including research collaborations and joint authorships, strengthenthe dissemination of scientific results; as the size of the audience increases, sodoes the reach and influence of the research, thus increasing the potentialtranslational impact the research may have on future patient care.In recent decades, globalization has extended the reach of researchuniversities,Because of the intrinsic value in creating diverse networks and collaborations, theOC continues its efforts to connect local centers of excellence and create a strongglobal network of diverse collaborators, many of whom may not have worked togetherotherwise. The OC supports interaction between global and local partners to createmomentum for clinical activities , research , and meeting patient needs . By facilitating theseinteractions, the OC ensures the coordinated effort of the global oncofertilitycommunity in conducting cutting-edge research that can continue to be rapidlytranslated to the clinic and establish an evidence-based standard of care.Today, many global partners are contributing to the worldwide oncofertility networkTable . Here, wIn Australia and New Zealand, a number of cancer and fertility groups havedeveloped specific oncofertility services and are undertaking research studiesto bridge the intellectual, disciplinary, and logistic gaps between reproductivemedicine and oncology. The Fertility Society of Australia established a specialinterest group in 2008 with the aim of facilitating collaborative research andimproving communication and education between cancer and fertilityclinicians.http://www.futurefertility.com.au). They are currently in theprocess of developing e-learning tools that will also be available forclinicians early in the new year.The Australasian OC was established in 2014 and is committed to interdisciplinaryinnovation. The consortium endeavors to support the collaborative efforts ofcancer and fertility clinicians in Australasia  toimprove oncofertility practice and services. The consortium developed theAustralasian Oncofertility Charter, which outlines the gold-standard model forcare to be implemented at each service. Working closely with consumer groups,> 30 resources on the topics of fertility preservation, sexual health, andsexual dysfunction have been made available on our Web site  FertilityTeam is a binational group of researchers who set up the AustralasianOncofertility Registry using a Web-based platform. In 2015, this registry willstart collecting oncofertility population data from patients with cancer inAustralia and New Zealand ages 0 to 45 years. These data will reveal referralpatterns for fertility preservation, uptake, and use of fertility preservation,reproductive risk based on annual follow-up of patients, and pregnancy outcomes. This will be the firstpopulation-based national oncofertility registry and will produce invaluableinsights into oncofertility care that are highly relevant to the efforts ofinternational colleagues.The Brazilian OC (BOC) was officially created in 2012. Known nationally in Brazilas Rede Brasileira de Oncofertilidade and coordinated by Jhenifer K. Rodrigues,PhD, the group is a network of professional members who share experiences,materials, research protocols, and ideas and engage in collaborative researchprojects. The network includes the Pró-Criar Medicina Reprodutiva andHuman Reproduction Laboratory/Federal University of Minas Gerais from the stateof Minas Gerais (southeast), Huntington Medicina Reprodutiva and Medical Schoolof Ribeirão Preto/University of São Paulo from São Paulostate (southeast), Fertilitat Centro de Medicina Reprodutiva from Rio Grande doSul (south), Laboratory of Oocytes and Preantral Follicles Manipulation/StateUniversity of Ceará from Ceará (north), Gênesis Centro deAssistência em Reprodução Humana , and Cenafert Centro de Medicina Reprodutiva from thestate of Bahia  and theJapan Society for Fertility Preservation (JSFP). The BOC has also collaboratedon publications and research projects with the OC in the United States. Thegroup has already published a native-language oncofertility book withcontributions from experts across Brazil and some from abroad. Now the group isworking on developing more native-language resources to further connect withpatients in Brazil. Efforts in Brazil are being made to bridge the gap betweenoncologists and reproductive specialists to help inform patients with cancerabout their fertility preservation options and reproductive future.Working from an initiative in the Departments for Gynecological Endocrinology andReproductive Medicine at the Universities of Heidelberg and Bonn in Germany, theFertiPROTEKT network was founded in May 2006. Two years later, the network wasextended to Austria and Switzerland to include not only universities but alsoprivate centers offering fertility preservation.http://www.fertiprotekt.com), which has > 100,000 visitorsper year, can be accessed in German or English. Annual meetings involving allparticipating centers guarantee consistent therapy standards among the centers.The therapy standards of the network are constantly updated andpublished.15Since January 2014, approximately 100 centers across Germany, Austria, andSwitzerland have joined the FertiPROTEKT network. Similar to the OC, theFertiPROTEKT network seeks to improve the standard of care for all patients byimplementing standardized protocols and methods of quality control. The mainachievements of the network are the optimization of stimulation therapiesadministered before cytotoxic therapies, the combination of ovarian stimulationwith ovarian tissue cryopreservation, and the establishment of overnighttransportation of ovarian tissue. Thus far, network members have performed andanalyzed approximately 60 ovarian tissue transplantations. Furthermore, anoptimized network infrastructure has been established, with two centralizedcryobanks for ovarian tissue and 100 in vitro fertilization centers, whichperform > 1,000 documented counseling sessions, 250 ovarian stimulations,and 500 cryopreservations of ovarian tissue per year. The homepage of theFertiPROTEKT network (http://www.j-sfp.org) with theaim of improving both the survival and quality of life of young patients withcancer in Japan. As part of this effort, the society is currently building anetwork for coordinating health care providers, using the OC as a guide, toprovide accurate information about fertility preservation to young patients withcancer in a timely manner. The JSFP has hosted a number of conferences aimed atattracting the attention of all health care providers who are concerned withfertility preservation and oncofertility. To date, these conferences have beenattended by approximately 280 clinicians, nurses, pharmacists, scientists, andothers.In November 2012, the JSFP was founded as a nonprofit corporation . The JSFP has alsosecured funding from a Health and Labor Sciences research grant for a studytitled “Development of a Psychological Support System for FertilityPreservation Aimed at Achieving Improvements for Young Cancer Survivors”. The JSFP is also working to initiate a clinical study titled“Oncofertility! Psychological Education and Couple Enrichment (O!PEACE)Therapy: An Intervention Study Protocol for a Randomized Controlled Trial inJapan.” The aim of this study is to examine whether psychotherapy canreduce concerns about fertility, alleviate psychological distress, and improvecommunication between patients with cancer and their partners. To expand itsefforts, the JSFP is actively engaged in various activities with OC Japan, incooperation with the OC, which are aimed at expanding oncofertility programs inAsia in association with the KSFP and the Fertility Preservation Society ofIndia (FPI).The Portuguese Centre for Fertility Preservation  was created in 2010 to meet the reproductive needs ofpatients undergoing treatments that are possibly fertility threatening. Althoughmale fertility preservation had been available in several public institutionssince the 1990s, female fertility preservation options were not available inPortuguese public practice. This center is the only one in the country thatprovides all fertility preservation techniques to both men and women.The team at the center includes six physicians, one embryologist, onepsychologist, and one pharmacist. Through a multidisciplinary approach, the maingoal is to provide reproductive monitoring and counseling to male and femalepatients undergoing gonadotoxic treatments. This team supports thepatients' decision-making process about fertility preservation and supportstheir reproductive decisions after treatment through regular follow-upconsultations Fig .http://www.myoncofertility.org, and Repropedia tool) weretranslated into Portuguese to allow the center to better inform the generalpopulation and health professionals about fertility preservation, thereby aidingin the decision-making process. The Portuguese Centre for Fertility PreservationWeb site is about to be launched and will include information and tools tailoredspecifically to Portuguese patients and health professionals. The center isworking with the Portuguese Society of Reproductive Medicine to develop anetwork with other public Portuguese institutions that can provide fertilitypreservation techniques to improve patient referral processes. Finally, researchprojects are also being developed to examine the fertility preservationdecision-making process, the impact of this decision on future individualadaptation, the effect of cancer treatments on patients' reproductivefunction, and new fertility preservation techniques. Specifically in 2015, themain goals of the Portuguese Centre for Fertility Preservation are: to developthe first telemedicine network in oncofertility so that patients and oncologistsanywhere can reach out the center team to enroll in multidisciplinaryconsultations to make more informed, shared, and quick decisions about fertilitypreservation, to produce and disseminate oncofertility decision aidsspecifically to the pediatric population and to pediatricians, and to create andimplement protocols for the cryopreservation of ovarian and testicular tissuesfor prepubertal patients.Although most of the patients with cancer are referred to the center by theironcologists, a significant number of them ask for a consultation on their own.Thus, another goal of this center is to try to better inform patients, healthprofessionals, and the general population about the impact of cancer onfertility, the techniques available for fertility preservation, and how toassemble a team that can provide patients counseling and assistance in makingdecisions. To achieve this goal, the center has developed information factsheets for patients and, in collaboration with the Portuguese Society forReproductive Medicine, has organized postgraduate courses. Some materials fromthe OC  tofacilitate collaboration between medical physicians and researchers whospecialize in reproductive medicine and oncology. The ultimate purpose of thesociety is to help patients who are undergoing treatments that will affect theirfertility and reproductive function. The goals of the KSFP are academiceducation, networking, advocacy, discussion, and development of standardprotocols for fertility preservation.The KSFP was established in 2013  may limit activity in one region, buta common understanding regarding what is possible in another area of the work mayprovide insight and opportunities to overcome these local barriers. Ultimately,there is no precise formula to success. Because of a variety of factors, what worksfor one center may not work for another. Our success comes from simply engaging aninspired individual or group of individuals to act and our collective interest inthe people with iatrogenic fertility concerns that we serve. The OC has made itpossible for individual practices to tap into a worldwide network of knowledge,experience, and discovery to improve patient care. OC partners communicate and workacross disciplinary, institutional, and geographic boundaries. Training the nextgeneration of oncofertility collaborators to undertake this multidisciplinary,multinational approach, as well as to engage all stakeholders, is essential toensure continued rapid transfer of sound science to quality medical practice. Witheach new partner, particularly those outside the traditional institutionalboundaries, a variety of new scientific, cultural, ethical, and personalperspectives are brought to bear on oncofertility research and patient care.Multifaceted approaches to connect cultures, communities, and countries show themost promise for future success.