Neutron therapy was developed from neutron radiobiology experiments, and had identified a higher cell kill per unit dose and an accompanying reduction in oxygen dependency. But experts such as Hal Gray were sceptical about clinical applications, for good reasons. Gray knew that the increase in relative biological effectiveness (RBE) with dose fall-off could produce marked clinical limitations. After many years of research, this treatment did not produce the expected gains in tumour control relative to normal tissue toxicity, as predicted by Gray. More detailed reasons for this are discussed in this paper. Neutrons do not have Bragg peaks and so did not selectively spare many tissues from radiation exposure; the constant neutron RBE tumour prescription values did not represent the probable higher RBE values in late-reacting tissues with low α/β values; the inevitable increase in RBE as dose falls along a beam would also contribute to greater toxicity than in a similar megavoltage photon beam. Some tissues such as the central nervous system white matter had the highest RBEs partly because of the higher percentage hydrogen content in lipid-containing molecules. All the above factors contributed to disappointing clinical results found in a series of randomised controlled studies at many treatment centres, although at the time they were performed, neutron therapy was in a catch-up phase with photon-based treatments. Their findings are summarised along with their technical aspects and fractionation choices. Better understanding of fast neutron experiments and therapy has been gained through relatively simple mathematical models—using the biological effective dose concept and incorporating the RBEmax and RBEmin parameters (the limits of RBE at low and high dose, respectively—as shown in the  To understand the motivation for neutron therapy and its research history, readers from different academic backgrounds require a good working knowledge of radiobiology, as well as insights into the historical development of x-ray (or photon)—based radiotherapy. This is important because fast neutron therapy was essentially competing with best practice using x-rays (or photons).This review focuses on the clinical radiobiology aspects of fast neutron therapy, which considers the interaction between radiobiology discoveries and their applications in treatment, with radiobiological modelling used not only as analytical tool, but also as a guide to clinical practice. It is not meant to be a comprehensive review of treatment results or the quite separate topic of experimental neutron radiobiology.fast neutrons refers to neutrons that have sufficient energy to cause recoil protons that ionise materials. They should be distinguished from very low energy thermal neutrons found in nuclear reactors.It is essential to consider the progressive technical development of photon-based treatments , as well as the present shift to greater use of charged particle therapy and place neutron therapy within these different radiation modalities. Neutrons are hadrons and are sometimes included within the general concept of hadron therapy, which can cause confusion because neutrons have no electrostatic charge. Readers should be aware of the important aspects of dose distribution for neutrons and photons (a progressive attenuation with tissue distance or depth) but that protons and ions have Bragg peaks due not only to their charge but also a velocity reduction along particle tracks. The term In order to compare the effectiveness of neutrons with x-rays (photons), the following information must be understood, especially the fact that more sophisticated technical developments in treatment delivery occurred sooner in the case of x-rays than with neutrons. Technical developments were mainly based on the achievement of higher photon energies, which increased the range or tissue depth, while also for megavoltage photons reducing the skin or entry dose before attaining full secondary electronic equilibrium at the maximum dose, followed by pseudoexponential attenuation with increasing tissue depth. Increasing the photon energy to the megavoltage range was also accompanied by more uniform tissue attenuation depending on the electron density rather than the actual atomic composition at lower energies. Improvements in imaging techniques further improved radiotherapy targeting, and it became possible to superimpose radiation depth dose curves in three-dimensional (3-D) space on the relevant scan or even combination of fused scan images.During the 1950s and 1960s, external radiation beams progressed to the megavoltage energy range by use of 60-cobalt units and later increasing use of electron linear accelerators, which depended on the cavity magnetron principles originally used in radar applications  concept. RBE is the ratio of the dose of the reference radiation divided by the dose of the neutron radiation for the same biological effect. It follows that the reference radiation dose must be divided by the RBE in order to provide the equivalent (and lower) neutron dose, as shown:Z number of each element. Photons ionise by means of electronic interactions, forming secondary electrons in matter. Lower energy electrons converted by 10 to 100 keV photons have higher LET and biological effects than those in the megavoltage range , the chemical environment of the cells , and also the “quality” of the radiation. The latter refers to the linear energy transfer (LET) characteristics of a radiation, which depends not only on the nature of the radiation , its energy (lower energies confer higher LET), and the total nuclear electrostatic charge given by the ge range .3-(CH2)N-COOH, and exists in the connective tissues that surround many organs of the body and through which the vital blood vessels pass. The abdomen contains a large intraperitoneal fat pad called the omentum, which is in intimate contact with the bowel. The KERMA (kinetic energy released per unit mass) in lipid-containing tissues can exceed that in water, which contains a relatively high proportion of hydrogen (11.1% by weight), resulting in increased local dose and bioeffectiveness. Previously published data from a detailed study . Ignoring the facts that have emerged may lead to a repetition of disappointing clinical results and eventual reduction of referrals for proton and ion beam therapy. It is vital to understand that most of neutron ionisation occurs from recoil protons, as well as some nuclear fragments, so their radiobiological features, including their RBE values, will be similar to those of a proton beam in the Bragg peak region where the LET and dose increase substantially. This fact has not been sufficiently well-recognised within the proton therapy community until very recently , 12, andin vitro experimental evidence of more efficient cell killing per unit dose and a reduced dependency on tissue oxygen tension. The history of neutron therapy illustrates the problems that can arise when partial scientific knowledge is used in an attempt to improve the treatment of a complex biological condition such as cancer; it is self-evident that the sterilisation of cancer cells by a radiation technique in an in vitro laboratory experiment is more likely to be successful than the elimination of a malignant tumour situated close to essential organs/tissues of the body. For future radiotherapy developments, particularly the use of proton and ion beam therapy, this history is important.The medical rationale for fast neutron therapy of cancer was based on initial scientific In 1940, Gray et al.  had showFurther interest arose because of the discovery that high LET radiations, e.g., fast neutrons, with increased clustering of ionisation events along micrometre distances of their tracks, are less dependent than are x-rays on the presence of oxygen to produce cell death . The previous work of Gray and others had shown that many cancers contained zones of very low oxygen tension, which were considered an important cause of radioresistance. To overcome this problem, high-pressure oxygen (HPO) was used in experimental radiotherapy from the 1960s for several decades. In the United Kingdom, the initial results in animal experimental systems were impressive . In clinThe research efforts were complicated by there being several generations of technical equipment initially based on static beams, followed later with rotating gantries  and finaMany developed countries started neutron therapy research programs, often based in single institutions and concentrating on treating rare tumours such as sarcomas. Some encountered severe tissue complications and were discontinued. The usefulness of neutron therapy could not be gauged from the emerging data sets, containing small numbers of patients in each tumour class, so it eventually became necessary to perform randomised studies on more commonly occurring cancers in order to determine the role of fast neutrons in the treatment of cancer.The UK Medical Research Council (with other charitable contributions) funded three important sequential projects to investigate the usefulness of fast neutron therapy , from thAt Hammersmith (University of London), clinical studies were conducted by Dr. Mary Catterall with initial promise using a geometrically limited fixed horizontal beam , 22. DesArnott and Duncan at Edinburgh Royal Infirmary (University of Edinburgh) used stricter “in-house” randomised trials to compare megavoltage x-rays (with their superior tissue penetration) with relatively poorly penetrating fast neutrons (as in Hammersmith), but for both radiation classes, the beams could be rotated on a gantry. The photon-treated controls were consequently treated with fewer beams per treatment plan, and the neutron treatments contained up to seven fields per plan, thus increasing the integral dose of neutrons. Treatments were supervised by site-specialised cancer experts with good academic supervision. Although in some instances improved local cancer control was achieved, enhanced normal tissue toxicity was also reported. It is interesting to note that the neutron dose per fraction of 0.9 Gy, given five times per week, was lower than that used in Hammersmith, thus inevitably increasing the RBE and its normal tissue consequences.Errington and Warenius at Clatterbridge (University of Liverpool) used an extended fast neutron energy (obtained using 64-MeV protons from a cyclotron) with depth doses equivalent to 5-MeV x-rays in randomised trials, some of which were jointly undertaken with Seattle and Fermilab (USA). The dose per fraction chosen was the same as Hammersmith and treatment delivered 3 days per week to relatively advanced T3 head and neck and pelvic cancers. Compared with the photon treatments, neutron therapy toxicity was not increased, but there was no improvement in tumour control, and rather surprisingly, the metastatic rate appeared to be increased (perhaps due to the intermittent dose fractionation used).Taken together, all these trials showed that neutrons conferred no clinical advantage, as comprehensively summarised by Duncan , which sIn many other countries, relatively low energy neutrons had been tried without recourse to formal trials and with little convincing success, although a small randomised trial reported by Laramore et al.  showed bSome later neutron therapy research used polyethylene and iron MLCs, which initially started in Seattle , and theIn retrospect, neutron therapy failed to match its original promise for the following physical and radiobiological reasons:Routine absorbed dose computations did not include the highly efficient neutron interaction with hydrogen, resulting in higher energy release in hydrogen rich tissues such as brain white matter and fat, which surrounds most important organs and is closely associated with their blood supply.Dismissal of the well-established finding of RBE variations in different tissues and its important increase with a falling dose, which mitigates the effect of a reduction in physical dose beyond a cancer.The appreciation that RBE also varies with cell proliferation rate, so that slow-growing tumours have higher values. It is the slow-growing stem cells and vascular cells that contribute to the severe normal tissue damage at extended time periods after irradiation.Several generic improvements emerged as a consequence what was found useful in the neutron therapy trials. Nearly all clinical trials in radiotherapy after that time followed the Clatterbridge UK and North American RTOG neutron trials by inclusion of data monitoring committees, as well as rigorous quality assurance (QA) systems for dosimetry, as well as clinical standards. When the detailed QA systems established for neutrons were applied as a result of discussion within the PTCOG (Particle Therapy Co-operative Group), the study coordinated at Loma Linda showed potentially significant discrepancies in some major proton centres and which required correction by up to 7% . FurtherThe early neutron radiobiology was well summarised by Bewley  and remaThe standard low LET BED equation isD symbols refer to total doses and d the dose per fraction given in n treatments (or fractions) with subscripts L and H used to denote high and low LET radiations, respectively, the latter being the reference radiation for RBE estimations.where For high LET radiations, Equation 10 can be modified , 33 by iIsoeffect calculations then use the equality:DL = nL.dL, and DH = nH.dH, so that any two schedules with the same number of fractions can be either compared or equated for an isoeffect as in Equation 5.By definition, For single doses, Equation 5 is modified to be:Further details on obtaining an RBE at any dose per fraction are given in the Radiobiology studies had already shown high neutron RBE values, which varied inversely with dose, reductions in oxygen enhancement ratio (OER), and cell cycle phase dependency, and with greater dose per fraction insensitivity, with similar findings for heavier ions . Also, Bin vitro data can only be obtained from direct estimation of β values; the dose range cannot be as high as in vivo studies because of limited surviving colonies in the former.The graphical fits in these articles are informative, as linear-quadratic model theory does predict  that thein vivo jejunal crypt assay, which was mostly used to achieve this RBE value in mid-spread out Bragg peak (SOBP) (k (SOBP) ]. By thek (SOBP) .If the overall RBE is controlled by RBEmax and RBEmin and that these parameters are, respectively, inversely and directly related to the reference radiation α/β ratio , a complex binary therapy that involves a low-dose exposure to thermal or epithermal neutrons, which are selectively captured by boron-labelled amino acids that avidly enter rapidly growing tumour cells. The reaction creates more intense localised ionisation by generating an α particle and a lithium ion, with tissue ranges of only around 10 μm (about one-cell diameter). The RBE issues within BNCT are further complicated by the dose being dependent on the concentration of the boron-containing compound, which is reflected in a compound biological effectiveness or CBE . Some piFollowing the fast neutron clinical trials, the UK funding authorities decided not to invest in further ambitious radiation projects; and as a result a general decline in UK radiotherapy and radiobiology research followed, although the Clatterbridge cyclotron was successfully adapted for ocular proton therapy. There emerged a clinical scepticism regarding the use of cyclotrons in radiotherapy and high LET radiations in general. In other countries, more progress was achieved using charged particle therapy (protons or light ions) produced from cyclotrons or the larger synchrotrons. Some of these countries had no prior experience of high-LET radiations in the form of fast neutrons.In Japan, the government selected proton and carbon ion therapy as a patient-friendly alternative to fast neutrons, but interestingly applied neutron experimental data to link the LET and RBE for treatment prescriptions , althougA major radiotherapy research question over the next few decades will be whether carbon ions are superior to protons in specific cancers, because of better beam ballistics and dose localisation and/or the apparent greater reduction in the oxygen effect. Suit et al.  have poiThe process of reoxygenation in tumours during radiotherapy for schedules lasting several weeks may be the reason why hypoxic radioresistance is not especially important with photons and may explain why the control x-ray treatments, as in the Clatterbridge studies, produced results that were at least as good as with neutrons . HoweverBecause fast neutron energies are part of a spectrum, in principle it is possible to predict most of the RBE by considering the recoil proton energies and then applying proton RBE models , 39, 40 Research at the Gray Laboratory led by McNally had shown nonlinear effects on cell survival when photon and neutron irradiations were used sequentially, with a short temporal separation, which would not change DNA repair significantly . Lower dThe problem of neutron-induced cancers has already been mentioned . FurtherThe history of neutron experimental and clinical studies should be taught to doctors and physicists during their radiotherapy training. This is because the general principles learnt are educationally informing. These topics should be studied in greater detail by those engaged in particle beam therapies. At present, there is a curious lack of emphasis on RBE issues in proton therapy teaching courses, but regulatory bodies should insist on a good background of radiobiological understanding in high LET radiations in order to guide future clinical decision makers. Neutron experimental data sets were not examples of “wasted research,” because they have been further analysed 20 to 40 years after their original publication and have some impact on informing radiation modellers of how to improve proton and ion beam therapy. Health policy makers and research funding bodies should also study this overall neutron experience, because it contains many important lessons.The following conclusions can be made:It is important that the same fundamental errors that were made with neutrons are not repeated with charged particles. All, rather than some, of the scientific information needs to be included when deciding what may be best to apply in the clinic.As with neutrons, so also with all charged particles, an elevated RBE can be favourable only if the tumour RBE exceeds that of the prescription RBE, but can be a disadvantage when the critical normal tissue RBE exceeds the prescription RBE if accompanied by insufficient normal tissue dose sparing.For any form of radiation therapy, radiobiological testing should be sufficiently comprehensive to identify strengths and weaknesses and include testing in many cell lines and tissues with different radiobiological characteristics and not just a few or one.In retrospect, better radiobiological modelling would have alerted clinicians of at least some of the adverse features and might have prevented the widespread use of fast neutrons, or at least restricted their use to specially defined situations.All new forms of radiotherapy need to be tested in high-quality clinical centres, using the best input from physics, biology, and medicine and use randomised controlled studies wherever possible. These must include rigorous external dosimetry QA and data monitoring committees.The experimental fast neutron database remains important and has implications for proton therapy because neutrons mainly ionise by forming recoil protons.The neutron experimental and clinical experience should be taught to doctors and physicists embarking on careers in radiotherapy and should be studied in greater detail by those engaged in particle beam therapies, as well as health policy decision makers.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Transcranial magnetic stimulation (TMS) is a noninvasive neurophysiologic technique that can stimulate the human brain. Positioning of the coil was often performed based merely on external landmarks on the head, meaning that the anatomical target in the cortex remains inaccurate. Navigated transcranial magnetic stimulation (nTMS) combines a frameless stereotactic navigational system and TMS coil and can provide a highly accurate delivery of TMS pulses with the guidance of imaging. Therefore, many novel utilities for TMS could be explored due to the ability of precise localization. Many studies have been published, which indicate nTMS enables presurgical functional mapping. This review aimed to provide a comprehensive literature review on nTMS, especially the principles and clinical applications of nTMS. All articles in PubMed with keywords of “motor mapping,” “presurgical mapping,” “navigated transcranial magnetic stimulation,” and “language mapping” published from 2000 to 2018 were included in the study. Frequently cited publications before 2000 were also included. The most valuable published original and review articles related to our objective were selected. Motor mapping of nTMS is validated to be a trustful tool to recognize functional areas belonging to both normal and lesioned primary motor cortex. It can offer reliable mapping of speech and motor regions at cortex prior to operation and has comparable accuracy as direct electrical cortical stimulation. nTMS is a powerful tool for mapping of motor and linguistic function prior to operation, has high application value in neurosurgery and the treatment of neurological and psychiatric diseases, and has gained increasing acceptance in neurosurgical centers across the world. Since the introduction for stimulating the human motor cortex by transcranial magnetic stimulation (TMS) in 1985 –6. HowevIn the past decades, a combination of optically tracked stereotactic navigation systems and TMS technology was developed. This system can form a picture of the stimulation sites through the three dimensions (3D) rebuilt MRI data of the subjects’ brain, which was therefore named as navigated transcranial magnetic stimulation (nTMS) , 9. NTMSWe review the basic principles and clinical applications of nTMS, protocols for motor and language mapping, comparison between nTMS and other preoperative mapping modalities, and future applications of nTMS, so as to improve the understanding and clinical application of nTMS.Many techniques can be applied to the navigation of TMS. They can be divided into two groups, namely, line-navigated transcranial magnetic stimulation (Ln-TMS) and electric-field navigated transcranial magnetic stimulation (En-TMS). Ln-TMS exhibits the location of the coil while not showing coil angulation in relation to the patient’s head. The stimulation spot is assumed to be on the line that passes through the geometric center of the coil and is perpendicular to the coil surface , and if Preparation for the session:Upload individual MRI data and use them to generate a 3D head model. Match the 3D head model with the patients’ head Fig. .Fig. 1ThHot spot identification:The rapid changes of transcranial magnetic field produce electric current at a specific region at the cortex, leading to a corresponding motor evoked potential (MEP) . Hot spoRMT (resting motor threshold) determination:RMT refers to the lowest intensity of TMS which is able to elicit a 50-μV MEP in a relaxed small hand muscle in 5 out of 10 stimulations. The target muscle should be at rest, and monitoring of muscle activity is critical during the determination of the RMT .Set the stimulate mode to single pulse TMS and set the output of stimulator to 110% RMT when doing motor mapping. Stimulate the central sulcus, precentral sulcus, postcentral sulcus, and precentral gyrus with a 2–3-mm interval . Mark thMost language mapping protocols use object naming and navigated repetitive TMS (nrTMS) combined with video recording of the behavioral results. The stimulate strength is 100–120% of the RMT for the hand muscles. The stimulate spot is not restrained to conventional regions stated by the Geschwind’s language model , 28 The It has been proved that nTMS is safe in both normal subjects and patients with intracranial lesions. The only absolute contraindication of nTMS is the presence of metal or electronic devices, such as cochlear implants, pulse generators, and medical pumps, near the coil stimulation site, which are at risk of being destroyed . A cohorDES has always been accepted as the gold standard for motor mapping and language mapping . It was Language mapping conducted by NrTMS has shown close association with DES, and compared with DES, nTMS has a sensitivity value of 90%, specificity of 98%, negative predictive value of 99%, and positive predictive value of 69% , 39–41. NTMS mapping is highly reliable and can significantly shorten intraoperative DES mapping, but it still can not replace DES or interoperative monitoring (IOM) completely. On the contrary, nTMS serves as a valuable supplement for IOM but not a competing modality . They shAt present, fMRI is the most frequently applied imaging methods for motor and linguistic function localization before operation. Compared with fMRI, nTMS imaging has better time resolution and does not require complex post-processing analysis. It also can be applied to patients with claustrophobia who can not complete the MRI examination . The advResearches showed that fMRI was less reliable for motor mapping in patients with brain tumor, especially in the regions near the lesion , 47. NTMScientists have reported the combined use of nTMS and diffusion tensor imaging fiber tracking (DTI-FT) in the cortical spinal tract (CST) . The DTIThe identified language-positive nrTMS spots can also be used as regions of interest (ROIs) for nTMS-based DTI-FT. Sollmann et al. did preoperative nrTMS language mapping in 40 patients and defined each error categories separately as a ROI, which was used for function-specific nTMS-based DTI-FT. Their results suggested that using different error categories as ROIs could result in better guidance during operation .It is a great challenge for neurosurgeons and patients to perform a brain tumor resection located at or near the motor eloquent area. In brain tumor surgery, preserving neurological function and taking maximal resection are major principles, and neurosurgeons frequently encounter a dilemma between conservation of motor function and completeness of resection. Therefore, in order to reduce motor deficits, preoperative functional localization of nTMS is necessary. Intracranial lesions, such as ischemia or tumors, are likely to result in the displacement and remodeling of motor and speech areas. NTMS can accurately determine the displaced functional areas, thus providing assistance for surgical planning . It is rKrieg compared the surgical outcome of patients with motor eloquent metastatic lesions who received preoperative nTMS-based motor mapping with those that did not. Patients receiving nTMS had a lower rate of residual tumor, smaller craniotomies, shorter operation time, and decreased surgery-related paresis . Krieg cFunctional mapping with nTMS is available not only in tumorous brain lesions, but also within hypervascularized cortical areas . Many stThe mapping based on nTMS can visualize the language network more efficiently and can also detect cortical plasticity induced by an intra-hemispheric tumor-induced cortical plasticity. It can be used to formulate a tailored surgical plan which could preserve language function after the surgery. This tool could play a supplementary role for neurosurgeons in dealing with patients with potential language-eloquent tumors, but not applicable for awake surgery .Besides surgical planning, the nTMS data and nTMS-based tractography are far beyond its current application. Schwendner implemented nTMS motor mapping in patients with intracranial metastases during routine radiotherapy. The results showed that it can significantly reduce the dose applied to the motor cortex while not affecting the treatment doses for the planning target volume .NTMS can also be used in the prediction of recovery of paralysis. One study demonstrates that nTMS results before and after surgery have the potential to predict the motor function recovery after glioma resection. At 1 week after surgery, positive MEPs induced by nTMS signify a better recovery from postsurgical neurological deficits . Peters Single-pulse TMS can provide insight into the excitability and integrity of the CST , and paiStudies have used nTMS to evaluate the function of upper motor neurons in patients with amyotrophic lateral sclerosis (ALS). The RMT was significantly higher in ALS patients, and the mean motor mapping areas were smaller in patients with ALS than in controls. They found nTMS is a promising method for assessing upper motor neuron function in ALS and may clarify the pathogenic process of neurodegeneration and help establish novel diagnostic and prognostic models .In recent years, preservation of language and motor function had been prioritized over other necessary functions. Several centers also used TMS to map further essential brain functions , 67. SinAn international investigation obtained level A evidence in determining the efficacy of repetitive TMS (rTMS) on neuropathic pain , 71. In It should be noted that nTMS can produce mapping of cortical areas that are close to the surface and therefore reachable by the magnetic field. This signifies that mapping frontobasal and temporomesial gyri are not available, and similar to that, the mapping of the brain region covered by big meningiomas or big arachnoid cysts is unavailable.NTMS is the latest technology in the functional localization of the cerebral cortex, which plays an important role in surgical planning, patient consultation, and risk assessment. The results show that the accuracy of nTMS in locating motor and language functions is similar to that of DES, higher than that of fMRI, and it is easy to operate. Therefore, it is recommended that nTMS can be used routinely to locate functional areas in patients with occupied functional areas. NTMS is a useful supplement for established IOM workflows while planning and conducting surgery in suspected motor eloquent regions. NTMS has high application value in the treatment of neurological and psychiatric diseases.


We briefly summarize how parallel considerations of transcriptome, interactome, and epigenome data significantly elaborated the roles of key players derived from pre-omics era studies. Furthermore, the growing cohort of cNC multiomics data revealed contribution of the non-coding genomic landscape. As technological improvements are constantly being developed, we reflect on key questions we are poised to address by taking advantage of the unique perspective a multiomics approach has to offer.Neural crest ontogeny plays a prominent role in craniofacial development. In this Perspective article, we discuss recent advances to the understanding of mechanisms underlying the cranial neural crest gene regulatory network (cNC-GRN) stemming from  Coupled with an increasing catalog of sequenced genomes, genome-wide approaches heralded an explosion of exploratory studies that not only recapitulated previous knowledge but also increased the rate of identification of novel developmental players. In the avian NC, RNA-seq identified multiple genes not known previously to be expressed in the migratory cNC such as Lmo4, RxrG, Ltk, and Col9a3  factors such as snai1a, vent, vox, and cx43.4, suggesting that they may represent non-specified cNC progenitors maintained in premigratory cNC. This finding echoes an observation made by machine learning-based image analysis that clustered cNC cells based on expression of a selected panel of genes (including pluripotency and NC markers) within similar-staged avian embryos  of 406 cNC cells isolated from the avian embryo identified a fraction of invasive front cells (Trailblazers) with a distinct molecular signature that persisted through migration, therefore bringing to light subpopulations within one cell type with seemingly similar cell behavior  hinting at “multiple” NC-GRNs. In particular, comprehensive analysis of NC enhancer modules in the cranial region suggested that NC gene regulatory circuits controlling neuronal derivatives are established much earlier in the embryo and use non-exclusive Ciona intestinalis, where the resolution achieved enabled identification of an ancestral Six, Msx, and Pax regulatory module shared between cranial placodes and NC in vertebrates  but also enabled reverse engineering of gene regulatory circuits for every gene expressed, thus facilitating reconstruction of the global NC-GRN with unrivaled resolution  emerged following a study describing developmentally-regulated enhancers in mouse cortical neurons . Landmark findings describing lncRNAs in the HOX locus, HOTAIR and HOTTIP, served as important case studies of modern lncRNA biology  were actively differentiating into their neuronal derivatives. On the other hand, by isolating nuclei transcriptomes at the same developmental stage, we demonstrated that functional annotation of transcribed enhancers (eRNAs) and promoters reflected the molecular signature of migratory NC cells and derivatives  and where within the organism these players are involved, but also what non-coding element(s) are responsible for gene activation and where within a cell these new players are exerting their functions. Integrating this information is a next complex task on the agenda and is non-trivial given that NC cells transition from being a stem cell-like population to many subpopulations committed to different, not necessarily binary fates. It is therefore crucial to perform and integrate multiplex genetic lineage tracing analyses into this picture, interpret multiomics data at single cell and with subcellular resolution, as well as develop new, non-biased functional genomics integration tools based on artificial intelligence and deep learning approaches.The contrast between the two pictures painted from nuclei and polysomes of NC cells at the same developmental stage raised important questions relating to our interpretation of how development proceeds at the cellular level. Traditionally, cellular identity has perhaps been defined over-simplistically via the expression of all protein markers in a GRN. We are now in the position to expand this definition by not only taking into account what Embryology has progressed in leaps and bounds leading to the modern incarnation of developmental biology as we know it today. From embryological techniques to advances in genome biology, our understanding of animal development has reached impressive heights. Here, at the forefront of modern developmental genetics and genomics, we propose using a combination of “traditional” and “modern” methods to deepen our understanding of genetic programmes underlying cNC development encoded within the genome.The cNC is a multipotent population of cells key to vertebrate evolution. It is a versatile system for interrogation, as the genetic machinery underlying its biology reiterates throughout development and disease. This well-oiled system is also sensitive to fine-tuned regulation; disrupt a cog and development fails to proceed normally leading to neurocristopathies that account for roughly 1/3 of all birth defects. In order to discover ways to prevent or treat them, we first need to fully understand what the baseline scenarios are, at the level of genes within the context of a highly dynamic genome.in vivo decommissioning of NC enhancers for functional investigation (Williams et al., Tackling the non-coding genome has also uncovered non-coding RNA molecules that form part of the genetic regulation underlying cellular function. Previous work by many research groups has highlighted lncRNAs as molecular scaffolds that shuttle proteins to their target regions to regulate gene expression. eRNAs not only serve as “indicators” of when gene transcription onsets, but also have been proposed to facilitate chromosome-looping between enhancers and the promoters they regulate. Coupled with advances in gene editing including CRISPR/Cas, we are now in the position to design experiments with flexibility, efficiency and precision, from genome-wide screens of non-coding elements (Liu et al., In conclusion, we hope to not only propose fresh perspectives and potential avenues of investigation into the cNC-GRN but also challenge the reader to revisit how we study developmental biology as a whole. We are now ushering a new generation of scientists willing to embrace the exponential growth of molecular and computational tools at their disposal—the future is bright.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.VC-M and TS-S contributed to the writing and editing of the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nutritional constraints including not only caloric restriction or protein deficiency, but also energy-dense diets affect metabolic health and frequently lead to obesity and insulin resistance, as well as glucose intolerance and type 2 diabetes. The effects of these environmental factors are often mediated via epigenetic modifiers that target the expression of metabolic genes. More recently, it was discovered that such parentally acquired metabolic changes can alter the metabolic health of the filial and grand-filial generations. In mammals, this epigenetic inheritance can either follow an intergenerational or transgenerational mode of inheritance. In the case of intergenerational inheritance, epimutations established in gametes persist through the first round of epigenetic reprogramming occurring during preimplantation development. For transgenerational inheritance, epimutations persist additionally throughout the reprogramming that occurs during germ cell development later in embryogenesis. Differentially expressed transcripts, genomic cytosine methylations, and several chemical modifications of histones are prime candidates for tangible marks which may serve as epimutations in inter- and transgenerational inheritance and which are currently being investigated experimentally. We review, here, the current literature in support of epigenetic inheritance of metabolic traits caused by nutritional constraints and potential mechanisms in man and in rodent model systems. But so In contrast to retrospective studies outlined above, prospective studies in man have the advantage that they are specifically designed to address certain research questions and they have lent additional support to the idea that parental exposure to diverse environmental factors can affect the offspring’s metabolic health.For example, the Avon Longitudinal Study of Parents and Children (ALSPEC) showed that BMI was higher in 9-year-old sons the earlier the father started smoking and despite no significant association with BMI, daughters showed a reduction in total lean mass  and the hypomethylation of the Olfr151 gene in sperm of F0 and F1 males. Epigenetic inheritance via the parental gametes was evident due to the use of in vitro fertilization (IVF) and naïve and healthy fosters to produce F1 and F2 offspring , was transmitted to filial (F1) and grand-filial generations (F2)  diets, both of which were shown to significantly increase longevity and impact behavioral traits in adult rodents  undernutrition by feeding isocaloric low protein diet (LPD) during gestation and lactation effectively altered in utero environment  of adult rats after grand-maternal caloric restriction were transmitted only via the female germline  led to reduced insulin levels at weaning as well as reduced β-cell-to-pancreatic-mass ratio at birth and at weaning over three generations (F1–F3) via the maternal lineage. However, body weight at birth was only significantly lower in the first generation and by weaning all pups caught up in all generations  is still sparse  impaired insulin secretion and glucose tolerance in female offspring (F1) . The latter observation suggested that maternal mitochondria transmitted via oocytes did not represent the primary mode of inheritance. In addition, due to the use of IVF for the production of offspring, confounding factors were excluded  allele. The allele is hypermethylated in mothers that receive a diet supplemented with methyl donors. The resulting phenotype comprises a pseudoagouti coat color and protection against obesity and diabetes which is inherited epigenetically across generations . It is a precursor for the production of many different plastics used extensively, for example, to line water pipes and in diverse household products. Consequently, BPA is detectable in most humans  as well as in oocytes and liver of their female offspring (F1)  Ge . What is (F1) Ge  or other (F1) Ge .Small non-coding RNAs (sncRNAs) such as microRNAs (miRNAs), small-interfering RNAs (siRNAs) and piwi-interacting RNAs (piRNAs), have been recognized as functional regulators of gene transcription and translation, for example, by targeting messenger RNAs (mRNAs) for degradation. Apparently, also other non-coding RNAs such as ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) as well as their fragments function as regulators of gene expression. Therefore, non-coding RNAs were suggested as a third group of epigenetic marks that might transmit an environmentally induced phenotype from parents to subsequent generations  reversion of epigenetically inherited phenotypes may possibly be used as argument against a genetic contribution.Thus, there is persuasive evidence that non-coding RNAs play an important role in epigenetic inheritance of acquired metabolic phenotypes via sperm in rodents. Similar data for oocytes are still missing and the relevance for humans is awaiting formal proof. Also it should probably be pointed out that at least formally the contribution of acquired Do we have the option to avoid the transmission of potentially harmful epigenetic changes to our offspring? So far, there are only few studies dealing with this topic. Pharmacological compounds targeting epigenetic mechanisms are already in clinical use for cancer therapy. In diabetes, metformin is a widely used antidiabetic drug with potent glucose-lowering activity and it was recently documented that it also modulates epigenetic processes (Bridgeman et al. From reviewing the current literature there is plenty of evidence from animal studies and strong support from human prospective and epidemiological studies in favor of intergenerational epigenetic inheritance of metabolic disorders following dietary constraint. Results for transgenerational epigenetic inheritance, however, vary strongly depending on the experimental setup and are sometimes ambiguous. Human studies are always influenced by cultural and direct environmental factors and therefore more difficult to interpret. Confounding factors such as behavior learned from parents and the social context, shared microbiomes, heterogeneous genetic and environmental backgrounds and others are difficult to discern from epigenetic inheritance transmitted via gametes in humans.Certainly, direct in utero exposure and early postnatal exposure through lactation to maternal diet also can have profound effects on metabolism later in life. In addition, paternal and maternal nutrition before fertilization also affects metabolic health of the offspring. In experimental designs it is therefore important to exclude confounding factors, for example, by using IVF and healthy foster mothers. This way, true epigenetic inheritance through the parental gametes and potential mechanisms can be investigated. Interestingly, although both parents influence their offspring, the mode of action and the specific metabolic consequences for the offspring apparently vary between maternal and paternal epigenetic inheritance. In man overnutrition, obesity and diabetes are strong factors for the offspring’s metabolic health and make parents to some degree responsible for the health of their offspring. In this sense we have learned that not all inheritance is fatefully determined by the genes we inherited from mom and dad. Instead the discovery of epigenetic inheritance across generations brings some freedom into inheritance via gametes that is controlled by parental lifestyle. Unfortunately, we are still far from predicting the best type of intervention—be it exercise, change of diet or pharmacological intervention—for each individual parent to provide the best conditions for metabolic health of our offspring. Yet, we are positive that current and future research of epigenetic inheritance will generate the basic knowledge to make good decisions.


The last few years have seen considerable advances in our understanding of the genetic factors influencing osteoporosis, driven by a range of break-throughs. The seven papers comprising this Research Topic together provide a timely update, describing new insights into the genetic architecture of osteoporosis, application of genetic findings to study causal inference, and state-of-the-art approaches to functional genomics, paving the road for multi-omic applications.Makitie et al. discuss how recent advances in genetic methodology have led to a rapid increase in identification of monogenic causes of osteoporosis and related conditions associated with low bone mass and/or increased bone fragility. Osteogenesis Imperfecta (OI), the most common monogenic cause of bone fragility, is due to a defect in bone extracellular matrix, with 85% of cases harboring a mutation in type I collagen. Many other genes are now recognized to cause an OI-like skeletal disorder. Some of these perturbate type I collagen function, such as cartilage-associated protein (CRTAP), while others may act by impairing bone mineralization, as proposed for Plastin 3 (PLS3). There has also been considerable interest in the discovery of mutations which impair osteoblast differentiation and function, not the least since these may also prove useful therapeutic targets for osteoporosis in the wider population. These include genes involved in WNT signaling, which has an important role in skeletal homeostasis, such as WNT1, and the WNT inhibitor frizzled-related protein 4 (SFRP4); mutations in both these genes being implicated in rare monogenic cases of osteoporosis.The genetic architecture of osteoporosis comprises mutations affecting single genes responsible for rare monogenic causes of osteoporosis, and common genetic variants representing genetic susceptibility factors for osteoporosis in the wider population. Gregson and Duncan provides a comprehensive review of disorders associated with high bone mass (HBM). Even having excluded secondary causes such as degenerative changes, unexplained HBM is not uncommon . Several very rare mutations underlying HBM have been described, which cause a generalized increase in bone mass as a result of increased bone formation, and a reduction in bone fragility and fracture risk. These mainly arise from mutations leading to increased activation of the WNT/β-catenin signaling pathway. Sclerosteosis, van Buchem’s disease, LRP4 HBM, LRP5 HBM, and LRP6 HBM are all thought to involve this mechanism. Other signaling pathways may also contribute to HBM, as exemplified by HBM arising from a mutation in SMAD9, part of the TGF-β superfamily. Several monogenic HBM conditions are also recognized where increased bone mass arises from defective bone resorption, and is associated with increased bone fragility, such as osteopetrosis. In the great majority of HBM cases, no underlying monogenic disorder is evident. Although cases of unexplained HBM were found to be enriched for common high BMD alleles identified in GWASs, this does not exclude a role of rarer mutations yet to be discovered. Given the successful translation from identification of the genetic basis of sclerosteosis to new therapy in the case of Romosozumab . The article reviews genetic factors found to be associated with fracture risk, after comparing individuals with and without a history of fracture, as well as endophenotypes related to fracture risk including bone mineral density (BMD) measured by DXA scans, and BMD estimated from heel ultrasound (eBMD). To date, the majority of susceptibility loci for fracture have also been implicated in BMD. The authors anticipate that further progress will be helped by improved phenotyping. For example, whereas the great majority of GWAS fracture studies combine all fracture types, fractures at specific sites, like the spine, may have a specific genetic architecture. Progress may also be achieved in extending GWAS to endophenotypes other than BMD. A number of advanced imaging methods have been developed which are capable of ascertaining detailed components of bone structure, which may also have a specific genetic architecture, though a challenge is to assemble large enough study samples to conduct well powered GWASs.In terms of polygenic disease risk in osteoporosis, the paper by Zheng et al. discusses the application of common susceptibility loci to examine causal inference using Mendelian Randomization (MR). It provides a number of examples where MR has been applied to examine risk factors for osteoporosis, using either BMD or fracture risk as the outcome. This paper also reviews the main limitations and assumptions involved in MR, such as horizontal pleiotropy, whereby the genetic instrument is related to the outcome via a separate pathway to the exposure. A number of methods are discussed to exclude pleiotropy, including the use of bi-directional analyses. If bi-directional causal effects are observed, these usually reflect pleiotropy, for example as a result of genetic correlation between the exposure and outcome due to shared genetic instruments resulting from common biological pathways. That said, instances of true bi-directional pathways may exist, such as a proposed positive effect of BMD on sclerostin, whereas sclerostin also exerts a negative effect on BMD, possibly as part of a negative feedback loop. As well as examining causal relationships for associations initially identified in conventional epidemiological studies, more recent applications of MR are also discussed, such as hypothesis-free approaches to examine causal effects of a given risk factor on a range of outcomes, and application of MR for target validation and drug discovery.The paper by Maynard and Ackert-Bicknell discuss the availability of data from mouse models, as well as other online resources such as tissue expression panels and expression quantitative trait loci (eQTLs). The authors point out that over 500 susceptibility loci have been identified for osteoporosis, however these causative variants nearly all act to alter gene expression rather than representing the actual causative gene. Identifying the genes responsible for mediating the effects of genetic susceptibility loci on BMD is a prerequisite for identifying potential drug targets for new osteoporosis treatments, remains a major goal of functional genomic studies. Mouse models have proven enormously helpful in this regard, by providing a means of characterizing the function of candidate genes through studying the skeletal phenotype of mice where these have been deleted. The repertoire of mice with specific gene knockouts has increased massively over recent years, due to the work of the International Mouse Phenotyping Consortium (IMPC), which ultimately aims to making embryonic stem cells carrying a knockout allele for all protein coding genes. Mice generated from this program undergo phenotypic screens including limited assessment of skeletal phenotype, with more detailed skeletal phenotyping undertaken by BoneBase, and the Origins of Bone and Cartilage Disease (OBCD) projects, based in the US and UK respectively.Three articles in this Research Topic discuss recent advances in functional studies aiming to provide a basis for translation of human genetic studies into new treatments for osteoporosis. Bergen et al. review the emerging use of zebrafish as an animal model in functional follow up studies of osteoporosis. Zebrafish are vertebrates which show strong similarities in their skeletal physiology to mammals, and are highly suited to genetic studies since constructs which modify the genome can be directly injected into embryos at the single cell stage. Bone formed in zebrafish has the same skeletal cell types and modes of regulation as higher vertebrates, making them suitable for studying processes involved in osteoporosis, which can be carried out dynamically and imaged in vivo, for which a range of fluorescent reporter constructs are available. Several imaging methods have been applied to zebrafish which together enable highly detailed assessment of skeletal morphology. A number of zebrafish with mutations in skeletally relevant genes have been studied which re-capitulate a range of skeletal disorders, including osteoporosis and OI. As well as helping to identify causative genes by evaluating the effect of their deletion on the skeleton using methods such as CRISPR/Cas9 editing, zebrafish can be applied to osteoporosis genetics research by providing high throughput assays for compounds which target these genes, based on harvesting and culture of osteoblasts from elasmoid scales.Tobias et al.). One of the main conclusions from the workshop is that whereas many promising genetic signals have been identified for osteoporosis, to date it has only been possible to interrogate a small fraction of them in functional studies. Whereas financial and manpower resources remain an important limiting factor, functional studies in osteoporosis genetics will benefit from an expanding repertoire of on-line resources, such as the IFRMS knowledge portal which aims to bring together all relevant functional data (https://msk.hugeamp.org/). In addition, a number of multi-omic resources are available which, with the application of causal inference methods described above, can be applied to identify causative genes responsible for genetic association signals. The paper concludes that a roadmap of functional assessments needs to be established, aiming to integrate multi-omic resources with datasets from human genetics and animal models, in order to translate the wealth of genetic discoveries into new therapies for osteoporosis.The final article covers proceedings from a workshop held by the Royal Osteoporosis Society in the UK to review opportunities and challenges in functional genomics research in osteoporosis (All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This editorial paper accompanies a special series in the journal Global Mental Health focused on the topic of interpersonal violence and mental health. This series included 24 papers reporting on data from 31 countries, published between 2017 and 2019. This accompanying paper provides a short summary of findings in the special series and reflects on next steps in research and practice. Collectively, the series’ 24 papers suggest intricate bi-directional relationships between interpersonal violence and mental health, situated in particular contexts and varying across the life course. In order to study this complexity, an overarching theoretical framework is critical. This paper takes the social justice theory developed by Powers and Faden  as a starting point. It is argued that application of this social justice framework will be helpful to: strengthen conceptual clarity; provide a sense of direction for research and practice in the area of interpersonal violence and mental health; assist in conducting more fine grained analyses of contextually determined processes of disadvantage; and help situate disciplinary specific research and practice questions in their broader context, thereby strengthening multi-disciplinary research and multi-sectoral policy and programming efforts. In 2017, this journal published a call for papers for a special series on the topic of interpersonal violence and mental health. The invitation was received with much interest: from 2017 to 2019, 24 papers were published reporting on data from 31 countries. In this editorial paper my aims are to provide a short background to the series; provide a succinct overview of its content; and reflect on next steps. In doing so, I will draw on a social justice theoretical framework developed by Powers and Faden , (2019).Violence is a ‘slippery concept’  found that parents from households with reported Ebola virus disease sickness became harsher parents over a 12-month period compared to non-exposed parents   (on average 16 years old at the time of recruitment) found that exposure to sexual IPV  was a predictor for suicidal attempts 5 years later , IPV victimization at baseline was associated with higher levels of psychological distress, food insecurity, and rates of alcohol misuse  found mental health impacts of IPV and parental mental health concerns on adolescents  identified a pathway where men who had been tortured experienced a range of mental health concerns , which increased the chances that their partners reported IPV  victimization. Concerning the perpetration of interpersonal violence, findings in this series point to an important pathway between male alcohol misuse and perpetration of IPV   aimed to improve relationships with an intimate partner and management of unsettled infant behaviors. Follow-up findings showed lower anxiety scores at 12-months compared to the control condition  was effective in doing this, with few effects for cognitive processing therapy , personal security , knowledge and understanding, equal respect, personal attachments, and self-determination . A life et al., et al., This specification of the job of social justice aligns with the social determinants tradition in public  health [e.g. , resulting in inequities on all dimensions of wellbeing: impunity for violence perpetrated against them ; exclusion from education opportunities (knowledge and understanding); a lack of respect for women's social and economic contributions ; social exclusion from extra-familial relationships ; and exclusion from political liberties accorded to men (self-determination). The mental health consequences of IPV victimization for women , in turn further compromise opportunities for women to escape the odds stacked against them. For example, IPV may result in cognitive styles characterized by self-blame and low self-esteem, which may make it harder for women to act out of a position of strength and independence , or identify an overlap in social and economic drivers of other elements of wellbeing not previously considered. Moreover, situating our disciplinary-confined concerns in a broader analysis may open up conversations with actors in different practice spaces. Specifically, the emphasis of the above social justice theoretical framework on identifying interlocking patterns of disadvantage may help to stimulate conversations between actors across multiple sectors that are characterized by collaboration and coordination rather than competition. Prioritizing the analysis of how moral concerns interact as components of a shared goal (i.e. wellbeing) is likely more helpful than positioning moral concerns in a zero sum game.Applying a social justice lens will provide greater conceptual clarity and thus more effectively bridge research and practice. A social justice framework can put a check on some of the concerns about boundary creep in research that ultimately leads to diffuse intervention and policy recommendations. Defining mental health (as e.g. the WHO does) as an all-encompassing state of wellbeing makes almost all issues of moral importance mental health issues  towards a more fine-grained analysis of contextually determined patterns of disadvantage.n = 1415) identified a relationship between IPV victimization and adverse mental health outcomes   found a robust relationship between alcohol misuse by a male partner and female reports of IPV victimization. The latter study also suggests a contextual effect, in that living in a country with a higher prevalence of alcohol use was a contributing factor to female reports of IPV victimization independently of her partner's alcohol use  found that more equitable gender norms held by peers and community members were associated with higher levels of self-esteem amongst the young women  settings may help illustrate such a re-orientation in focus. Bajunirwe and colleagues’ cross-sectional study in five SSA sites  play in preventing or reducing IPV? In addition to addressing men's mental health, which other family-based and community-based interventions may be required to break the intergenerational transmission of violence victimization and subsequent perpetration in low-resource high-adversity contexts?Regarding the findings on inequitable gender norms and self-esteem, for example: What is the relationship between respect and self-esteem as measured by Stark and colleagues amongst refugee girls in Ethiopia? Is self-esteem a reflection of a cognitive style closely linked to depression , or better characterized as the result of living in socio-cultural contexts where women are systematically treated as morally inferior? And if both these scenarios are relevant, would a community-based intervention focused on changing inequitable norms related to IPV be sufficient to also address depressive cognitive styles associated with other types of adversities experienced more systematically by women?et al., et al., et al., Answering such questions will require a stronger emphasis on multi-level longitudinal epidemiological research, as well as a greater investment in multi-disciplinary research efforts. For example, ethnographic research will be critical to understand which connections between interpersonal violence and mental health across multiple social levels  will be worth investigating in multi-level quantitative epidemiological studies. Qualitative research will also be helpful to understand identified statistical relationships in quantitative studies between more generic variables (such as age and gender) and mental health outcomes. For example, qualitative research would be helpful to identify the particular social processes that resulted in girls and boys experiencing different consequences from family dysfunction in conflict-affected DRC . They advocate for a paradigm shift characterized by the image of ‘Chinese boxes’ (i.e. nested systems), and for epidemiology to take advantage of major advances in both social epidemiology and neuropsychological research  will be helpful to replace more simplistic models of causal analysis  provide a sense of direction to focus on key inequalities that prevent people from obtaining wellbeing with more fine grained analyses of contextually determined processes of disadvantage; (2) situate our own specific research and practice questions in their broader context ; and (3) strengthen conceptual clarity – without losing the forest for the trees. It is likely only through collective efforts – across academic disciplines, governmental departments, and non-governmental agency mandates – that the interlocking patterns of disadvantage that systematically marginalized whole population groups can be effectively identified and remedied.


Ensuring an adequate food supply in systems that protect environmental qualityand conserve natural resources requires productive and resource-efficientcropping systems on existing farmland. Meeting this challenge will be difficultwithout a robust spatial framework that facilitates rapid evaluation andscaling-out of currently available and emerging technologies. Here we develop aglobal spatial framework to delineate ‘technology extrapolationdomains’ based on key climate and soil factors that govern crop yieldsand yield stability in rainfed crop production. The proposed frameworkadequately represents the spatial pattern of crop yields and stability whenevaluated over the data-rich US Corn Belt. It also facilitates evaluation ofcropping system performance across continents, which can improve efficiency ofagricultural research that seeks to intensify production on existing farmland.Populating this biophysical spatial framework with appropriate socio-economicattributes provides the potential to amplify the return on investments inagricultural research and development by improving the effectiveness of researchprioritization and impact assessment. However, extrapolation of findings from theseexperiments to facilitate adoption by farmers is limited by the lack of a robustspatial framework to identify cropland ‘cohorts’ with similar soilsand climate where a comparable response to a given set of technologies would beexpected. Likewise, the ability to utilize these results to support more effectiveresearch prioritization and impact assessment is limited by the lack of anappropriate spatial upscaling method to estimate outcomes of technology adoption oncrop production and natural resources at regional, national, and global scales andsocio-economic  attributes. A unique combination of biophysical andsocio-economic circumstances is hereafter referred to as a ‘technologyextrapolation domain’ (TED). As a first step towards identifying a suitableTED framework, we focus on the biophysical attributes that define a TED for rainfedcrop production while acknowledging the need to supplement this biophysicalframework with appropriate socio-economic attributes.8). Recent advances in databasemanagement and public accessibility of climate and soil databases with completeterrestrial coverage now make it possible to overcome this deficiency . Third, and perhaps mostimportant, is the need to validate performance of a TED framework for ability topredict crop and cropping system performance. While several previous studies haveevaluated the robustness of crop yield extrapolations and their uncertainty usingboth crop simulation and statistical models ,that capability is currently a bridge too far. Current knowledge and models are notsufficiently robust for development of such customized spatial frameworks,especially when multiple interacting technologies are involved. Until suchcustomization is possible, the TED framework proposed here provides an initial, butsubstantive step towards the goal of greater efficiency and impact from investmentsin agricultural research worldwide, the more so because it is possible to make itmore customized by adding other variables, including socio-economic variables, thatare relevant for out-scaling and adoption of technology.The objectives of this study were to: (i) develop extrapolation domains fortechnology transfer in cropping systems; (ii) perform a quantitative validation ofthe TED scheme for its ability to represent spatial variation in rainfed crop yieldand the associated temporal variability; and (iii) demonstrate potentialapplications of the TED scheme. Although the TED framework can be applied worldwide,the evaluation of the TEDs requires detailed and spatially explicit data of weather,soil, crop management and yields. Hence, this paper reports initial testing of theframework in data-rich regions, including the US, Argentina, and Australia.www.yieldgap.org)  annual total growing degree-days, which gives an indication of the length oftime during the year that crop growth is not limited by cold temperature; (ii)aridity index, which largely defines the degree of water limitation in rainfedcropping systems; (iii) annual temperature seasonality, which differentiates betweentemperate and tropical climates; (iv) PAWHC, which determines the capacity of a soilto store water to support crop growth during rain-free periods . At issue, then, is the degree of detail in PAWHC, asdetermined by the number of class intervals for this variable, to optimize spatialresolution for a robust TED framework. Hence, we develop TEDs at two levels ofspatial resolution as determined by degree of detail in PAWHC categories . The trade-off between degree of detail used to delineate TEDsand their applicability is illustrated by estimating the number of zones (TEDs)required to achieve a desired coverage of crop area.et alWe assess the performance of the spatial framework to define TEDs in terms of cropperformance by evaluating average yields and temporal yield variability of maizeacross the US Corn Belt under the hypothesis that a robust spatial framework willadequately represent yield differences across a wide range of climate and soil types. The Corn Belt provides an appropriateregion of focus to test this hypothesis because it represents 11% of continental USland area, accounts for 30 and 28% of global maize and soybean production (based on2010– 2014 period) FAOSTAT , and inc−1 in regions like the Corn Belt where pre-plantrainfall is sufficient to fully recharge soil water holding capacity in mostyears but rainfall during the growing season does not meet crop waterrequirements versus field experimentsin only 16 and 18 unique TEDs for the moderate resolution scheme ). In the NW to SE direction, both average yieldand yield stability increase towards the SE due to longer growing season andsmaller aridity index (i.e. greater water supply). In the SW to NE direction,yields and yield stability likewise increase due to smaller aridity index.Similarly, counties located in the same climate zone, but with different PAWHCexhibited increasing yields and decreasing CVs in TEDs with greater PAWHC  with different climate but with soil in the same PAWHC class a and (c)er PAWHC d). TheseHC et al. In addi<20%of the variation in all cases. To summarize, the TED framework wassatisfactorily evaluated on its ability to distinguish regions with differentyield level, yield stability, and management practices for two crops (maize andsoybean), across two spatial scales (county and field-level), and two dimensions over a large geographic region with diversity in climateand soil that account for about one-third of global maize and soybeanproduction. We are not aware of previous efforts to quantitatively evaluate theeffectiveness of a spatial framework for characterizing performance of cropproduction in this manner across both time and space.Likewise, temporal analysis of county-level maize yield indicated that the TEDframework accounts for differences in yield across the majority of yearsevaluated . This finding agrees withresults from the analysis of field level soybean yield and management practicesacross the Corn Belt , which shows that80%–99% of the variance (excluding the error term) in yield andmanagement can be explained by the TEDs alone, while the year term (includingTED x year) explained These results suggest that the moderate resolution TED scheme is robust forcapturing the influence of key biophysical factors on crop productivity and itsvariability and, by extension, to also capture differences in crop response tocrop and soil management practices that depend on the amount and reliability ofwater supply and length of growing season in rainfed cropping systems. Bycontrast, a random selection of counties did not identify differences in yieldand CV among regions(supplementaryfiguresS1 andS2).And while a higher resolutionTED scheme would increase precision in discerning the directional trends shownin Given the high cost of time and labor to implement replicated field studies incommercial production fields, the TED framework presented here can help (i)optimize the number of environments covered by a field trial to maximize thecrop area coverage in unique TEDs for a given number of sites or, alternatively,to reduce the number of sites without sacrificing crop area coverage in uniqueTEDs, (ii) select specific environments for testing a technology where it ismost likely to have the greatest impact based on biophysical attributes of theselected TEDs, (iii) delineate the extrapolation domain for specific fieldtrials, allowing up-scaling of expected impact from trial locations to TEDs inwhich the trials were conducted, and (iv) facilitate technology transfer acrossanalog TEDs located in different geographic regions. Potential to improveefficiency of a field experimentation program is illustrated in 9 (supplementary figure 4). The96 sites were located within 19 TEDs  which  19 TEDs a). Inco 19 TEDs c), ordo 19 TEDs d).This case study relies on a number of assumptions. It assumes that one site perTED is enough to capture crop response to a given technology within that TED. Italso assumes no risk of losing sites to unforeseen events such as flooding,hail, or heavy yield loss from factors such as diseases, insect or pests. It maybe worthwhile to have more than one site per TED to account for unforeseenevents and to have a strategic focus on TEDs with largest crop area or where thetechnology being tested is expected to have greatest potential impact on yields,profit, and environmental quality. In addition, this TED framework for siteselection can easily be expanded to include other variables that have influenceon performance of a given technology or its adoption by farmers, includingirrigation, other biophysical factors such as soil pH, organic matter content,or terrain slope as well as socio-economic factors such as distance to market,farm size, and so forth.etalAnother application of the described spatial framework is to compare croppingsystems in the same TED across different regions, countries and continents thatshare a unique TED. For example, it is possible to evaluate promisingtechnologies to improve resource capture and productivity of land and waterresources that have been widely adopted by farmers in one region but have notyet been tested or are not widely used, in an analog TED elsewhere. Thishypothesis was explored for a TED that is present in both Argentina andAustralia . In Austet alGiven this large difference in cropping systems within the same TED on differentcontinents, we investigated the feasibility of increasing annual productivityand resource capture in the Australian TED by increasing crop intensity throughinclusion of a summer legume (mungbean) in the traditional single crop perseason . Compared to soybean, mungbeanis more suited to this Australian TED because its growth period, from sowing toharvestable maturity, better fits the period of time when there is sufficientstored soil moisture and rainfall to support crop growth. Simulation analysisrevealed that an annual rainfed double-crop of wheatmungbean (i.e. 2 crops peryear) would be a superior alternative to the traditional crop-fallow system thatcurrently dominates in the analogue Australian TED. While the wheat-fallowsystem exhibited highest yield for a single wheat crop, it had much lower annualnet income relative to the wheat-mungbean rotation . And althttp://nutrientstar.org/about-teds/. The shapefile delineating theTED spatial framework is available for downloading at: www.yieldgap.org/cz-ted.Combining high yields with efficient use of resources and small environmentalfootprint represents a major challenge and will require adequate investments inresearch and development, as well as efficient and effective prioritization of theseinvestments. Likewise, once new technologies are developed, new approaches areneeded to drive more rapid technology transfer to achieve widespread adoption inother regions. A robust spatial framework for evaluating the ‘extrapolationdomain’ for new technologies represents an essential tool for achieving thesegoals. Such a framework for technology testing and transfer must account for themost important factors governing crop productivity and environmental performancewithout requiring an excessive number of variables and categories. To that end,results from evaluation of the TED framework presented here show promise forcapturing effects of dominant climate and soil factors responsible for variation inrainfed crop yields, and for facilitating greater efficiency in testing of newtechnologies and gaining adoption of those that improve yields, yield stability, andprofits while reducing negative environmental impacts through delineating the areawhere they are likely to work best. An online version of the TED framework thatfocuses on US maize production, including tools to select TEDs based upon differentattributes , isavailable at Click here for additional data file.


Advanced sarcomas have yet to undergo improved outcomes seen in other cancer subtypes. Verteporfin has the potential to show landmark change in sarcoma due to its anti-proliferative properties: inhibition of the Hippo pathway and as photodynamic therapy. The effect of verteporfin on the Hippo pathway is reviewed specifically in the setting of sarcoma due to increased activation of this pathway in multiple subtypes. Role and efficacy of photodynamic therapy in other malignancies is also reviewed, with additional discussion of preclinical studies demonstrating synergistic effects of photodynamic therapy within current sarcoma standard of care treatment. Future investigations of the feasibility of incorporating verteporfin into sarcoma treatment are discussed.Sarcoma is a widely varied and devastating oncological subtype, with overall five-year survival of 65% that drops to 16% with the presence of metastatic disease at diagnosis. Standard of care for localized sarcomas is predicated on local control with wide-local resection and radiation therapy, or, less commonly, chemotherapy, depending on tumor subtype. Verteporfin has the potential to be incorporated into this standard of care due to its unique molecular properties: inhibition of the upregulated Hippo pathway that frequently drives soft tissue sarcoma and photodynamic therapy-mediated necrosis due to oxidative damage. The initial anti-proliferative effect of verteporfin is mediated via binding and dissociation of YAP/TEAD proteins from the nucleus, ultimately leading to decreased cell proliferation as demonstrated in multiple in vitro studies. This effect has the potential to be compounded with use of photodynamic therapy to directly induce cellular necrosis with use of a clinical laser. Photodynamic therapy has been incorporated into multiple malignancies and has the potential to be incorporated into sarcoma treatment. Sometimes unexpectedly, repurposing forgotten or outdated knowledge reveals progress for exciting breakthroughs. One example in the field of oncology is photodynamic therapy (PDT), a technology utilized as remotely as ancient Egypt and ancient Greece . PDT useAs PDT demonstrated promise as well as increased feasibility with the advent of laser technology, there have been a variety of studies undertaken to determine the effectiveness and safety of using PDT in treating cancers. Phase I trials in the setting of localized prostate adenocarcinoma WST11) using PDT found minimal side effects with respect to urinary retention and sexual function, and ultimately moved into phase II trials ,11. An e using PDThe multifactorial nature of PDT therapy delivery necessitates highly specified protocols to maintain the efficacy seen in preclinical settings. Huggett et al. performed a phase I/II trial evaluating the efficacy of the photosensitizer verteporfin in pancreatic adenocarcinoma that underscored the importance of accurate delivery of light exposure to exclusively identified tumor tissue . The triHahn et al. evaluated the PDT agent photofrin delivered during surgical resection of primary tumors in metastatic ovarian, gastrointestinal, and peritoneal sarcoma . This phMultiple phase II clinical trials have evaluated the efficacy of PDT in the setting of cholangiocarcinoma, demonstrating promise in the mechanistic potential of PDT as well as its potential for synergistic effects with current oncological treatment regimens ,22. InclOne of the key challenges of PDT in clinical use is the controlled delivery of light in order to maximize therapeutic benefit while minimizing side effects. As seen in experimentation performed by Huggett et al., more light delivered led to a higher level of necrosis, but the extent of cellular damage did not correlate to the dose of light given and was unexpectedly high . These dIn a report by Bown et al., solid organ was dosed unintentionally in a patient when a fiber moved after positioning . DespiteAdverse events (AEs) have been reported in using verteporfin as a PDT in cancer treatments. In a single arm single site Phase 1 study in 16 patients with recurrent resectable head and neck cancer, grade 1 and 2 edema lasting seven to 10 days and pain lasting two to four weeks was reported. These AEs were possibly attributed to PDT. There were phototoxicity reactions including grade 2 photophobia, which was likely related to prolonged exposure to operating room spotlights and two skin burns reported due to prolonged exposure from pulse oximeter for an extended period . A numbeOne field in which similar advancement would be welcome is soft tissue sarcoma (STS), in which the current standard treatment of localized disease still results in relapse rates of 20–30% within three years . AccordiGiven that PDT requires the use of a molecular potentiator to begin its cytotoxic effects, it allows for additional opportunity to evolve and be used in different clinical settings involving STS management. For example, PDT with verteporfin can be utilized intraoperatively to improve negative margin rate in STS located around critical structures such as sciatic nerve or femoral artery during surgical resection. Although there is a potential for damage to surrounding normal tissue, this can be mitigated with careful planning the wavelength of light used with known depth of penetration.Neoadjuvant radiation is routinely used for management of large, high risk soft tissue extremity sarcomas . Higher Drosophila, the Hippo pathway consists of a series of kinase cascades with impacts in tissue regeneration and malignancy through its downstream transcriptional activation via Yorkie (Yki) working in concert with Scalloped (Sd) proteins [The Hippo pathway was stumbled upon during investigations related to organogenesis, and the intrinsic regulatory mechanisms affecting end-organ size . Originaproteins . In mammproteins .The Hippo pathway is activated under multiple different conditions, some of which include cells in a detached state or contact inhibition from neighboring cells. Once activated, the serine/threonine kinases MST1/2 phosphorylate LATS1/2, which ultimately phosphorylate TAZ and YAP, leading to their degradation . In DrosIn addition to their tumor promoting capabilities, the Hippo pathway end effectors TAZ and YAP have also been shown to have the capacity to drive metastasis in a broad spectrum of cancers such as breast, melanoma, lung, gastric, and colorectal, as well as sarcomas . This isFurthermore, TAZ and YAP have been shown to drive tumorigenesis in many sarcomas in pre-clinical studies. A PAX3-FOXO1A fusion protein increases TAZ expression in an alveolar rhabdomyosarcoma, and xenograft mouse models show TAZ suppression inhibits tumor growth via a decrease in proliferation and a slight increase in apoptosis . In miceThe primary cause of mortality in sarcoma patients is metastasis, resulting in a five-year survival rate of approximately 15%, making it an essential target for therapeutic intervention . TAZ/YAPPDT requires the presence of three components: light, photosensitizer, and oxygen. A wide range of light sources can be used for PDT, including light emitting diodes, lasers, and fluorescent lamps . The lig3) implantable device and wireless powering system for light delivery [Furthermore, recently there have been innovative approaches reported to address the low penetration of light during PDT. Bansal et al. reported a wireless photonic approach to PDT using a miniaturized .One of the more encouraging aspects of verteporfin therapy is its possible synergistic effects with current standard of care treatments in STS, with doxorubicin and radiation therapy. This coupled with the anti-proliferative effect shown in several in vivo studies even in the absence of PDT would by itself be encouraging to explore in advanced sarcomas given the degree of limited treatment options and the morbidity and mortality experienced with this cancer. However, given the mechanistic promise verteporfin offers as well as the wealth of preclinical studies demonstrating its worth as a translational agent, exploring minimally invasive light therapy delivery mechanisms in concert with pharmaceutical administration is an appropriate step forward .We propose to initially assess single agent verteporfin in a dose finding Phase 1b/2 study to determine its safety in relapsed refractory metastatic sarcomas (both STS and bone sarcomas). Verteporfin efficacy will be determined using RECIST criteria in metastatic target lesions and will be correlated with YAP/TAZ expression on the archival tumor specimen. If tumor response correlates with the YAP/TAZ expression, this may be considered as an eligibility criterion in a future larger randomized trial. Ideally, the effect of verteporfin on YAP/TAZ dependent gene expression should be tested on metastatic tumors; however, as many STS tumors metastasize to the lungs, tumor biopsies can be challenging. As an alternative approach, YAP/TAZ gene expression could be evaluated in peripheral blood mononuclear cells obtained prior to and serially during verteporfin treatment as a surrogate marker. Once the single agent activity and toxicity is determined, then combination strategies with chemotherapy or targeted therapy or immunotherapy could be explored. Verteporfin as a PDT agent can be explored in the preoperative setting together with standard of care external beam radiation therapy to the primary tumor located in either the extremity or trunk. However, one of the biggest challenges is the effective light delivery at varying depths of the tumor tissue using optical fibers. Recently, interstitial PDT has been developed to address this challenge by using multiple optical fibers during PDT, which are precisely inserted using dosimetry, at different locations of the large tumor volume . Pre-cliConsiderations of light therapy in sarcoma include the depth to which the light exposure must reach, the assurance the entire tumor is treated, and minimizing possible harm to the surrounding normal tissue induced by this intervention. Discovering novel light delivery techniques with fiberoptic light sources to go along with verteporfin use would be essential, as transcutaneous delivery methods in other cancer trials cannot be reproduced to the tissue depth needed for similar success in deep seated sarcomas. Despite these challenges, the next steps forward will likely prove beneficial for both provider and patient.


Despite the widespread study of how injured nerves contribute to chronic pain, there are still major gaps in our understanding of pain mechanisms. This is particularly true of pain resulting from nerve injury, or neuropathic pain, wherein tactile or thermal stimuli cause painful responses that are particularly difficult to treat with existing therapies. Curiously, this stimulus-driven pain relies upon intact, uninjured sensory neurons that transmit the signals that are ultimately sensed as painful. Studies that interrogate uninjured neurons in search of cell-specific mechanisms have shown that nerve injury alters intact, uninjured neurons resulting in an activity that drives stimulus-evoked pain. This review of neuropathic pain mechanisms summarizes cell-type-specific pathology of uninjured sensory neurons and the sensory ganglia that house their cell bodies. Uninjured neurons have demonstrated a wide range of molecular and neurophysiologic changes, many of which are distinct from those detected in injured neurons. These intriguing findings include expression of pain-associated molecules, neurophysiological changes that underlie increased excitability, and evidence that intercellular signaling within sensory ganglia alters uninjured neurons. In addition to well-supported findings, this review also discusses potential mechanisms that remain poorly understood in the context of nerve injury. This review highlights key questions that will advance our understanding of the plasticity of sensory neuron subpopulations and clarify the role of uninjured neurons in developing anti-pain therapies. Neuropathic pain, or pain resulting from nerve injury, can be induced by any range of causes spanning from traumatic to chemical to metabolic insults. For example, a nerve innervating the skin may be severely damaged, eventually resulting in loss of innervation of that target region. However, instead of a numb, insensate phenotype that one might expect to see with a loss of innervation, stimuli applied to the adjacent skin often result in exaggerated or prolonged pain in response to noxious stimuli, termed hyperalgesia, or even painful responses to innocuous stimuli, termed allodynia. Curiously, this stimulus-driven pain relies upon intact primary sensory neurons to conduct the signals that are ultimately interpreted as painful. In conditions where the peripheral terminals of injured neurons die off, how do intact, uninjured sensory neurons go from being spared bystanders of injury to become drivers of neuropathic pain?This review examines the plasticity of uninjured sensory neurons through the lens of the cell-specific mechanisms of pain. Key studies in sensory biology have unearthed the vast diversity of sensory neurons, also termed primary afferents. In oversimplified terms, primary afferent neurons include several subtypes of mechanoreceptors that encode tactile information, nociceptors that encode painful information, pruriceptors that encode itch, and subsets of neurons that encode a combination of modalities. Sensory ganglia such as the dorsal root ganglion (DRG) house the cell bodies of mixed populations of sensory neurons, each with a pseudounipolar morphology whose axon bifurcates to innervate tissue with the peripheral branch and the spinal cord or brainstem with the central branch. Each sensory neuron is believed to be specifically tuned to particular types of stimuli given a combination of its receptive field, receptors expressed on peripheral axon terminals, axon diameter, intrinsic membrane properties, level of myelination, communication with adjacent glia and other neurons within the DRG, distribution of central axon terminals, and communication between central terminals and spinal cord neurons.Though central sensitization arising from altered spinal cord circuits is an important contributor to pain mechanisms, it will not be addressed in detail in this review article. However, the central terminals of sensory neurons do deserve some consideration in the context of primary afferent plasticity. The majority of C-fibers terminate primarily at the corresponding level of the spinal cord. However, up to ~25% of C-fiber nociceptors extend terminals one to two segments rostral or caudal to the segment of the entry  model is typically characterized in mice by ligation of the L4 spinal nerve just distal to the L4 DRG, affecting many fibers that innervate the distal hind limb  model  is a modulator of neuronal plasticity that is thought to be pro-nociceptive  and its receptor, the p75 neurotrophin receptor (p75NTR). After L5 SNL injury, p75NTR is upregulated in small and medium diameter uninjured L4 DRG neurons while it is decreased in injured DRGs neurons  have decreased mechanical threshold, increased receptive field area, faster AP rise times, small AHP amplitude, and increased excitability, while uninjured C-fibers have decreased mechanical threshold with no intrinsic membrane changes detected Neurotransmission of sensory information is also dependent upon proper myelination of myelinated fibers. Induced demyelination of whole nerve or selective demyelination of A-fibers can lead to pain  chloride channels, like the GABA(A) receptor, up to 30 mV above resting membrane potential (Price et al., Historically, the majority of pain research has occurred in male rodents or in studies that are not statistically powered to detect sex differences. Thus, there are still significant gaps in what we know about sex differences in neuropathic pain mechanisms; this is especially true of uninjured neurons. Some insight into potential sex differences has come from transcriptomic studies of sex differences in the CCI model of inflammatory nerve injury. Shared changes seen in rats of both sexes included upregulated gene expression of ATF3 and VIP (Stephens et al., In summary, there are several mechanisms by which uninjured neurons contribute to chronic neuropathic pain, some more well understood than others, especially in the context of nerve injury. These include molecular changes in the type and amount of neurotransmitters produced, the ion channels that mediate neurophysiological properties, and the gap junctions that mediate intraganglionic communication, in addition to several other potential mechanisms. A notable trend in discerning studies is that uninjured and injured neurons often have divergent changes. Further, uninjured mechanoreceptors are frequently implicated in such alterations, quite often with distinctive changes opposite to those seen in nociceptors or that are absent in nociceptors altogether. These are all changes that can be obfuscated in studies that combine injured and uninjured neurons into a single group. A mechanistic understanding of plasticity in uninjured neurons may shed light on the changes that occur contralateral to injury as well. For example, exposure to mediators in the systemic circulation or alterations in descending signals from central circuits may contribute to changes in contralateral, uninjured neurons after injury, as has been suggested of NGF in the cerebrospinal fluid (Cheng et al., What are the mechanisms that underlie alterations in uninjured neurons?Do emerging molecular markers that are upregulated in uninjured neurons contribute to pain phenotypes? Does this role differ between females and males?Do neuronal-glial interactions differ for uninjured neurons vs. injured neurons?Do inflammatory mediators and other signaling molecules have a selective effect on uninjured neurons? If so, is it due to anatomic features of the microenvironment, intracellular pathways that have been primed by evoked activity, or other potential mechanisms?Do the peripheral axon terminals of uninjured neurons show changes that parallel the phenotype switching seen in the cell body? After nerve injury can mechanoreceptors release ectopically expressed peptides in response to mechanical stimulation?Can we improve the diagnosis of neuropathic pain or track the therapeutic effects of anti-pain therapies using pain-associated markers of uninjured afferents?Can receptor agonists or receptor-mediated ablation strategies be used to target uninjured neurons that show ectopic expression of the targeted receptor? Can selective targeting of uninjured neurons help to treat secondary hyperalgesia or allodynia?Addressing the gaps in our knowledge of primary afferent plasticity and dysfunction will be crucial in propelling the study of neuropathic pain and generating novel diagnostic and therapeutic strategies. This will yield insight into nuanced mechanisms of pain and endogenous analgesia, highlight novel endpoints that can improve the diagnosis of distinct types of pain, and develop interventions that can treat specific modalities of pain as well as complex pain syndromes. Novel strategies can take advantage of the fact that DRGs sit outside of the blood-brain-barrier with their axons sitting in peripheral tissues, which yield pharmacologic access to molecular targets and anatomic access to peripheral devices that are unparalleled by strategies that rely on the central nervous system. Enhanced understanding of how both injured and uninjured neurons are altered in pain states can also potentially prevent unintended off-target side effects or identify opportunities for protective adjunct therapies to strengthen the benefit of existing therapies.LC developed the concept of the manuscript. ET and LC conducted the analysis of the literature and then composed and edited the manuscript text, figures, and tables. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.