Proton Beam Therapy in the Reirradiation Setting of Brain and Base of Skull Tumour Recurrences.

The therapeutic management of local tumour recurrence after a first course of radical radiotherapy is always complex. Surgery and reirradiation carry increased morbidity due to radiation-induced tissue changes. Proton beam therapy (PBT) might be advantageous in the reirradiation setting, thanks to its distinct physical characteristics. Here we systematically reviewed the use of PBT in the management of recurrent central nervous system (CNS) and base of skull (BoS) tumours, as published in the literature. The research question was framed following the Population, Intervention, Comparison and Outcomes (PICO) criteria: the population of the study was cancer patients with local disease recurrence in the CNS or BoS; the intervention was radiation treatment with PBT; the outcomes of the study focused on the clinical outcomes of PBT in the reirradiation setting of local tumour recurrences of the CNS or BoS. The identification stage resulted in 222 records in Embase and 79 in Medline as of March 2023. Sixty-eight duplicates were excluded at this stage and 56 were excluded after screening as not relevant, not in English or not full-text articles. Twelve full-text articles were included in the review and are presented according to the site of disease, namely BoS, brain or both brain and BoS. This review showed that reirradiation of brain/BoS tumour recurrences with PBT can provide good local control with acceptable toxicity rates. However, reirradiation of tumour recurrences in the CNS or BoS setting needs to consider several factors that can increase the risk of toxicities. Therefore, patient selection is crucial. Randomised evidence is needed to select the best radiation modality in this group of patients.

Assessing Equity of Access to Proton Beam Therapy: A Literature Review.

Proton beam therapy (PBT) is one of the most advanced radiotherapy technologies, with growing evidence to support its use in specific clinical scenarios and exponential growth of demand and capacity worldwide over the past few decades. However, geographical inequalities persist in the distribution of PBT centres, which translate into variations in access and use of this technology. The aim of this work was to look at the factors that contribute to these inequalities, to help raise awareness among stakeholders, governments and policy makers. A literature search was conducted using the Population, Intervention, Comparison, Outcomes (PICO) criteria. The same search strategy was run in Embase and Medline and identified 242 records, which were screened for manual review. Of these, 24 were deemed relevant and were included in this analysis. Most of the 24 publications included in this review originated from the USA (22/24) and involved paediatric patients, teenagers and young adults (61% for children and/or teenagers and young adults versus 39% for adults). The most reported indicator of disparity was socioeconomic status (16/24), followed by geographical location (13/24). All the studies evaluated in this review showed disparities in the access to PBT. As paediatric patients make up a significant proportion of the PBT-eligible patients, equity of access to PBT also raises ethical considerations. Therefore, further research is needed into the equity of access to PBT to reduce the care gap.

Outcomes of Patients Treated in the UK Proton Overseas Programme: Central Nervous System Group.

AIMS: In 2008, the UK National Health Service started the Proton Overseas Programme (POP), to provide access for proton beam therapy (PBT) abroad for selected tumour diagnoses while two national centres were being planned. The clinical outcomes for the patient group treated for central nervous system (CNS), base of skull, spinal and paraspinal malignancies are reported here. MATERIALS AND METHODS: Since the start of the POP, an agreement between the National Health Service and UK referring centres ensured outcomes data collection, including overall survival, local tumour control and late toxicity data. Clinical and treatment-related data were extracted from this national patient database. Grade ≥3 late toxicities were reported following Common Terminology Criteria for Adverse Events (CTCAE) v 4.0 definition, occurring later than 90 days since the completion of treatment. RESULTS: Between 2008 and September 2020, 830 patients were treated within the POP for the above listed malignancies. Overall survival data were available for 815 patients and local control data for 726 patients. Toxicity analysis was carried out on 702 patients, with patients excluded due to short follow-up (<90 days) and/or inadequate toxicity data available. After a median follow-up of 3.34 years (0.06-11.58), the overall survival was 91.2%. The local control rate was 85.9% after a median follow-up of 2.81 years (range 0.04-11.58). The overall grade ≥3 late toxicity incidence was 11.97%, after a median follow-up of 1.72 years (0.04-8.45). The median radiotherapy prescription dose was 54 GyRBE (34.8-79.2). CONCLUSIONS: The results of this study indicate the safety of PBT for CNS tumours. Preliminary clinical outcomes following PBT for paediatric/teen and young adult and adult CNS tumours treated within the POP are encouraging, which reflects accurate patient selection and treatment quality. The rate of late effects compares favourably with published cohorts. Clinical outcomes from this patient cohort will be compared with those of UK-treated patients since the start of the national PBT service in 2018.

Normal Tissue Complication Probability Modelling for Toxicity Prediction and Patient Selection in Proton Beam Therapy to the Central Nervous System: A Literature Review.

Normal tissue complication probability (NTCP) models can guide clinical decision making in radiotherapy. In recent years, they have been used for patient selection for proton beam therapy (PBT) for some anatomical tumour sites. This review synthesizes the published evidence regarding the use of NTCP models to predict the toxicity of PBT, for different end points in patients with brain tumours. A search of Medline and Embase using the Patients, Intervention, Comparison, Outcome (PICO) criteria was undertaken. In total, 37 articles were deemed relevant and were reviewed in detail. Nineteen articles on NTCP modelling of toxicity end points were included. Of these, 11 were comparative NTCP studies of PBT versus conventional photon radiotherapy (XRT), which evaluated differences in plan dosimetry and then assumed that XRT-derived literature estimates of NTCP would be applicable to both. Seven papers derived NTCP models based on PBT outcome data, two of which provided model parameters. Among analysed end points, the reduced risk of secondary tumours with PBT as compared with XRT is estimated - through modelling studies - to be considerable and was highlighted by most authors. For other analysed end points, the clinical benefit of PBT mainly depends on tumour location in relation to organs at risk as well as prescription doses. NTCP models can be useful tools for treatment plan comparison. However, most published toxicity data were derived from XRT cohorts; this review has highlighted the need for further studies relating dose-volume parameters to observed toxicity in PBT-treated patients. Specifically, there is a need for PBT-specific NTCP models that can be implemented in the clinical practice. NTCP models built on robust clinical data for the most common radiotherapy toxicities in the brain would potentially redefine the current indications for PBT.

Pseudomonas aeruginosa Uses c-di-GMP Phosphodiesterases RmcA and MorA To Regulate Biofilm Maintenance.

While the early stages of biofilm formation have been well characterized, less is known about the requirements for Pseudomonas aeruginosa to maintain a mature biofilm. We utilized a P. aeruginosa-phage interaction to identify rmcA and morA, two genes which encode bis-(3',5')-cyclic dimeric GMP (c-di-GMP)-degrading phosphodiesterases (PDEs) and are important for the regulation of biofilm maintenance. Deletion of these genes initially results in an elevated biofilm phenotype characterized by increased production of c-di-GMP, Pel polysaccharide, and/or biofilm biomass. In contrast to the wild-type strain, these mutants were unable to maintain the biofilm when exposed to carbon-limited conditions. The susceptibility to nutrient limitation, as well as subsequent loss of biofilm viability of these mutants, was phenotypically reproduced with a stringent response mutant (ΔrelA ΔspoT), indicating that the ΔrmcA and ΔmorA mutants may be unable to appropriately respond to nutrient limitation. Genetic and biochemical data indicate that RmcA and MorA physically interact with the Pel biosynthesis machinery, supporting a model whereby unregulated Pel biosynthesis contributes to the death of the ΔrmcA and ΔmorA mutant strains in an established biofilm under nutrient limitation. These findings provide evidence that c-di-GMP-mediated regulation is required for mature biofilms of P. aeruginosa to effectively respond to changing availability of nutrients. Furthermore, the PDEs involved in biofilm maintenance are distinct from those required for establishing a biofilm, suggesting that a wide variety of c-di-GMP metabolizing enzymes in organisms such as P. aeruginosa allows for discrete control over the formation, maintenance or dispersion of biofilms.IMPORTANCE Recent advances in our understanding of c-di-GMP signaling have provided key insights into the regulation of biofilms. Despite an improved understanding of how biofilms initially form, the processes that facilitate the long-term maintenance of these multicellular communities remain opaque. We found that P. aeruginosa requires two phosphodiesterases, RmcA and MorA, to maintain a mature biofilm and that biofilms lacking these PDEs succumb to nutrient limitation and die. The biofilm maintenance deficiency observed in ΔrmcA and ΔmorA mutants was also found in the stringent response-defective ΔrelA ΔspoT strain, suggesting that a regulatory intersection between c-di-GMP signaling, extracellular polysaccharide biosynthesis, and the nutrient limitation response is important for biofilm persistence. We uncover components of an important regulatory system needed for P. aeruginosa biofilms to persist in nutrient-poor conditions and provide some of the first evidence that maintaining a mature biofilm is an active process.

Evaluation of Ultra-low-dose Paediatric Cone-beam Computed Tomography for Image-guided Radiotherapy.

AIMS: In image-guided radiotherapy, daily cone-beam computed tomography (CBCT) is rarely applied to children due to concerns over imaging dose. Simulating low-dose CBCT can aid clinical protocol design by allowing visualisation of new scan protocols in patients without delivering additional dose. This work simulated ultra-low-dose CBCT and evaluated its use for paediatric image-guided radiotherapy by assessment of image registration accuracy and visual image quality. MATERIALS AND METHODS: Ultra-low-dose CBCT was simulated by adding the appropriate amount of noise to projection images prior to reconstruction. This simulation was validated in phantoms before application to paediatric patient data. Scans from 20 patients acquired at our current clinical protocol (0.8 mGy) were simulated for a range of ultra-low doses (0.5, 0.4, 0.2 and 0.125 mGy) creating 100 scans in total. Automatic registration accuracy was assessed in all 100 scans. Inter-observer registration variation was next assessed for a subset of 40 scans (five scans at each simulated dose and 20 scans at the current clinical protocol). This subset was assessed for visual image quality by Likert scale grading of registration performance and visibility of target coverage, organs at risk, soft-tissue structures and bony anatomy. RESULTS: Simulated and acquired phantom scans were in excellent agreement. For patient scans, bony atomy registration discrepancies for ultra-low-dose scans fell within 2 mm (translation) and 1° (rotation) compared with the current clinical protocol, with excellent inter-observer agreement. Soft-tissue registration showed large discrepancies. Bone visualisation and registration performance reached over 75% acceptability (rated 'well' or 'very well') down to the lowest doses. Soft-tissue visualisation did not reach this threshold for any dose. CONCLUSION: Ultra-low-dose CBCT was accurately simulated and evaluated in patient data. Patient scans simulated down to 0.125 mGy were appropriate for bony anatomy set-up. The large dose reduction could allow for more frequent (e.g. daily) image guidance and, hence, more accurate set-up for paediatric radiotherapy.

Anticancer Properties of Graviola (Annona muricata): A Comprehensive Mechanistic Review.

Graviola (Annona muricata) is a small deciduous tropical evergreen fruit tree, belonging to the Annonaceae family, and is widely grown and distributed in tropical and subtropical regions around the world. The aerial parts of graviola have several functions: the fruits have been widely used as food confectionaries, while several preparations, especially decoctions of the bark, fruits, leaves, pericarp, seeds, and roots, have been extensively used in traditional medicine to treat multiple ailments including cancers by local communities in tropical Africa and South America. The reported therapeutic benefits of graviola against various human tumors and disease agents in in vitro culture and preclinical animal model systems are typically tested for their ability to specifically target the disease, while exerting little or no effect on normal cell viability. Over 212 phytochemical ingredients have been reported in graviola extracts prepared from different plant parts. The specific bioactive constituents responsible for the major anticancer, antioxidant, anti-inflammatory, antimicrobial, and other health benefits of graviola include different classes of annonaceous acetogenins (metabolites and products of the polyketide pathway), alkaloids, flavonoids, sterols, and others. This review summarizes the current understanding of the anticancer effects of A. muricata and its constituents on diverse cancer types and disease states, as well as efficacy and safety concerns. It also includes discussion of our current understanding of possible mechanisms of action, with the hope of further stimulating the development of improved and affordable therapies for a variety of ailments.

Optimal vitamin D spurs serotonin: 1,25-dihydroxyvitamin D represses serotonin reuptake transport (SERT) and degradation (MAO-A) gene expression in cultured rat serotonergic neuronal cell lines.

BACKGROUND: Diminished brain levels of two neurohormones, 5-hydroxytryptamine (5-HT; serotonin) and 1,25-dihydroxyvitamin D3 (1,25D; active vitamin D metabolite), are proposed to play a role in the atypical social behaviors associated with psychological conditions including autism spectrum disorders and depression. We reported previously that 1,25D induces expression of tryptophan hydroxylase-2 (TPH2), the initial and rate-limiting enzyme in the biosynthetic pathway to 5-HT, in cultured rat serotonergic neuronal cells. However, other enzymes and transporters in the pathway of tryptophan metabolism had yet to be examined with respect to the actions of vitamin D. Herein, we probed the response of neuronal cells to 1,25D by quantifying mRNA expression of serotonin synthesis isozymes, TPH1 and TPH2, as well as expression of the serotonin reuptake transporter (SERT), and the enzyme responsible for serotonin catabolism, monoamine oxidase-A (MAO-A). We also assessed the direct production of serotonin in cell culture in response to 1,25D. RESULTS: Employing quantitative real-time PCR, we demonstrate that TPH-1/-2 mRNAs are 28- to 33-fold induced by 10 nM 1,25D treatment of cultured rat serotonergic neuronal cells (RN46A-B14), and the enhancement of TPH2 mRNA by 1,25D is dependent on the degree of neuron-like character of the cells. In contrast, examination of SERT, the gene product of which is a target for the SSRI-class of antidepressants, and MAO-A, which encodes the predominant catabolic enzyme in the serotonin pathway, reveals that their mRNAs are 51-59% repressed by 10 nM 1,25D treatment of RN46A-B14 cells. Finally, serotonin concentrations are significantly enhanced (2.9-fold) by 10 nM 1,25D in this system. CONCLUSIONS: These results are consistent with the concept that vitamin D maintains extracellular fluid serotonin concentrations in the brain, thereby offering an explanation for how vitamin D could influence the trajectory and development of neuropsychiatric disorders. Given the profile of gene regulation in cultured RN46A-B14 serotonergic neurons, we conclude that 1,25D acts not only to induce serotonin synthesis, but also functions at an indirect, molecular-genomic stage to mimic SSRIs and MAO inhibitors, likely elevating serotonin in the CNS. These data suggest that optimal vitamin D status may contribute to improving behavioral pathophysiologies resulting from dysregulation of serotonergic neurotransmission.

Graviola (Annona muricata) Exerts Anti-Proliferative, Anti-Clonogenic and Pro-Apoptotic Effects in Human Non-Melanoma Skin Cancer UW-BCC1 and A431 Cells In Vitro: Involvement of Hedgehog Signaling.

Non-melanoma skin cancers (NMSCs) are the leading cause of skin cancer-related morbidity and mortality. Effective strategies are needed to control NMSC occurrence and progression. Non-toxic, plant-derived extracts have been shown to exert multiple anti-cancer effects. Graviola (Annona muricata), a tropical fruit-bearing plant, has been used in traditional medicine against multiple human diseases including cancer. The current study investigated the effects of graviola leaf and stem extract (GLSE) and its solvent-extracted fractions on two human NMSC cell lines, UW-BCC1 and A431. GLSE was found to: (i) dose-dependently suppress UW-BCC1 and A431 cell growth, motility, wound closure, and clonogenicity; (ii) induce G0/G1 cell cycle arrest by downregulating cyclin/cdk factors while upregulating cdk inhibitors, and (iii) induce apoptosis as evidenced by cleavage of caspases-3, -8 and PARP. Further, GLSE suppressed levels of activated hedgehog (Hh) pathway components Smo, Gli 1/2, and Shh while inducing SuFu. GLSE also decreased the expression of pro-apoptotic protein Bax while decreasing the expression of the anti-apoptotic protein Bcl-2. We determined that these activities were concentrated in an acetogenin/alkaloid-rich dichloromethane subfraction of GLSE. Our data identify graviola extracts and their constituents as promising sources for new chemopreventive and therapeutic agent(s) to be further developed for the control of NMSCs.

[Target volume concepts in radiotherapy and their implications for imaging]. / Zielvolumenkonzepte in der Strahlentherapie und ihre Bedeutung für die Bildgebung.

CLINICAL ISSUE: Successful radiotherapy requires precise localization of the tumor and requires high-quality imaging for developing a treatment plan. STANDARD TREATMENT: Irradiation of the tumor region, including a safety margin. TREATMENT INNOVATIONS: The target volume consists of the gross tumor volume (GTV) containing visible parts of the tumor, the clinical target volume (CTV) covering the GTV plus invisible tumor extensions, and the planning target volume (PTV) to account for uncertainties. The non-GTV parts of the CTV are based on historical patient data. The PTV margins are based on a calculation of possible uncertainties during planning, setup, or treatment. Normal tissue deserves the identical care in contouring, since its tolerance may limit the tumor dose, taking into account the contours of organs at risk. Serial risk organs benefit from defining a planning organ of risk volume (PRV) to better limit the dose delivered to them. DIAGNOSTIC WORK-UP: The better the imaging, the more reliable the definition of the GTV and treatment success will be. Multiple imaging sequences are desirable to support the delineation of the tumor. They may result in different CTVs that, depending on their tumor burden, may require different doses. PERFORMANCE: The definition of standardized target volumes according to the ICRU reports 50, 62, and 83 forms the basis for an individualized radiation treatment planning according to unified criteria on a high-quality level. ACHIEVEMENTS: Radio-oncology is by nature interdisciplinary, the diagnostic radiologist being an indispensable team partner. A regular dialogue between the disciplines is pivotal for target volume definition and treatment success. PRACTICAL RECOMMENDATIONS: Imaging for target volume definition requires highest quality imaging, the use of functional imaging methods and close cooperation with a diagnostic radiologist experienced in this field.

Bioactive Dietary VDR Ligands Regulate Genes Encoding Biomarkers of Skin Repair That Are Associated with Risk for Psoriasis.

Treatment with 1,25-dihydroxyvitamin D3 (1,25D) improves psoriasis symptoms, possibly by inducing the expression of late cornified envelope (LCE)3 genes involved in skin repair. In psoriasis patients, the majority of whom harbor genomic deletion of LCE3B and LCE3C (LCE3C_LCE3B-del), we propose that certain dietary analogues of 1,25D activate the expression of residual LCE3A/LCE3D/LCE3E genes to compensate for the loss of LCE3B/LCE3C in the deletant genotype. Herein, human keratinocytes (HEKn) homozygous for LCE3C_LCE3B-del were treated with docosahexaenoic acid (DHA) and curcumin, two low-affinity, nutrient ligands for the vitamin D receptor (VDR). DHA and curcumin induce the expression of LCE3A/LCE3D/LCE3E mRNAs at concentrations corresponding to their affinity for VDR. Moreover, immunohistochemical quantitation revealed that the treatment of keratinocytes with DHA or curcumin stimulates LCE3 protein expression, while simultaneously opposing the tumor necrosis factor-alpha (TNFα)-signaled phosphorylation of mitogen activated protein (MAP) kinases, p38 and Jun amino-terminal kinase (JNK), thereby overcoming inflammation biomarkers elicited by TNFα challenge. Finally, DHA and curcumin modulate two transcription factors relevant to psoriatic inflammation, the activator protein-1 factor Jun B and the nuclear receptor NR4A2/NURR1, that is implicated as a mediator of VDR ligand-triggered gene control. These findings provide insights into the mechanism(s) whereby dietary VDR ligands alter inflammatory and barrier functions relevant to skin repair, and may provide a molecular basis for improved treatments for mild/moderate psoriasis.

Human Hairless Protein Roles in Skin/Hair and Emerging Connections to Brain and Other Cancers.

The mammalian hairless protein (HR) is a 130 kDa nuclear transcription factor that is essential for proper skin and hair follicle function. Previous studies have focused on the role of HR in skin maintenance and hair cycling. However, the hairless gene (HR) is also expressed in brain and other tissues, where its role remains poorly understood. HR has been reported to contain functional domains that potentially serve in DNA binding, histone demethylation, nuclear translocation and protein-protein interactions. Indeed, HR has been shown to interact with and repress the action of the nuclear receptors for vitamin D and thyroid hormone as well as RAR-related orphan receptor alpha, possibly via recruitment of histone deacetylases. HR may also have important functions in non-skin tissues given that nearly 200 HR mutations have been identified in patients with various cancers, including prostate, breast, lung, melanoma, uterine, and glioma. This suggests that HR and/or mutants thereof have relevance to the growth and survival of cancer cells. For example, the reported intrinsic histone H3K9 demethylase activity of HR may activate dormant genes to contribute to carcinogenesis. Alternatively, the demonstrated ability of HR to interact with p53 and/or the p53 DNA response element to influence p53-regulated pathways may explain, at least in part, why many cancers express mutated HR proteins. In this review, we summarize the current knowledge of HR bioactions, how HR mutations may be contributing to alopecia as well as to cancer, and, finally, outline future directions in the study of this largely enigmatic nuclear protein. J. Cell. Biochem. 119: 69-80, 2018. © 2017 Wiley Periodicals, Inc.

SIRT1 enzymatically potentiates 1,25-dihydroxyvitamin D3 signaling via vitamin D receptor deacetylation.

The hormonal metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), binds to the vitamin D receptor (VDR) and promotes heterodimerization of VDR with a retinoid-X-receptor (RXR) to genomically regulate diverse cellular processes. Herein, it is revealed for the first time that VDR is post-translationally acetylated, and that VDR immunoprecipitated from human embryonic kidney (HEK293) cells displays a dramatic decrease in acetylated receptor in the presence of 1,25D-ligand, sirtuin-1 (SIRT1) deacetylase, or the resveratrol activator of SIRT1. To elucidate the functional significance of VDR deacetylation, vitamin-d-responsive-element (VDRE)-based transcriptional assays were performed to determine if deacetylase overexpression affects VDR/VDRE-driven transcription. In HEK293 kidney and TE85 bone cells, co-transfection of low amounts (1-5ng) of a SIRT1-expression vector elicits a reproducible and statistically significant enhancement (1.3- to 2.6-fold) in transcription mediated by VDREs from the CYP3A4 and cyp24a1 genes, where the magnitude of response to 1,25D-ligand is 6- to 30-fold. Inhibition of SIRT1 via EX-527, or utilization of a SIRT1 loss-of-function mutant (H363Y), resulted in abrogation of SIRT1-mediated VDR potentiation. Studies with a novel, non-acetylatable VDR mutant (K413R) showed that the mutant VDR possesses enhanced responsiveness to 1,25D, in conjunction with reduced, but still significant, sensitivity to exogenous SIRT1, indicating that acetylation of lysine 413 is relevant, but that other acetylated residues in VDR contribute to modulation of its activity. We conclude that the acetylation of VDR comprises a negative feedback loop that attenuates 1,25D-VDR signaling. This regulatory loop is reversed by SIRT1-catalyzed deacetylation of VDR to amplify VDR signaling and 1,25D actions.

The Mammalian Hairless Protein as a DNA Binding Phosphoprotein.

The mammalian hairless (Hr) protein plays critical roles in skin and brain tissues, but how it interacts with DNA and partner protein is only now being defined. Our initial tests of four consensus response elements, revealed that rat Hr can specifically bind to a consensus p53 response element (p53RE), 5'-AGACATGCCTAGACATGCCT-3', but not to response elements for NF-κB, TCF4 or Sp1. We then employed ChIP assays which verified that human HR binds to a p53RE of the GADD45A gene in both HEK293 (embryonic kidney) and U87 (glioblastoma) cells. Further, HR was shown to interact directly with the p53 protein in a co-immunoprecipitation assay. Cotransfections with p53RE reporter gene constructs revealed that rat Hr can boost p53-mediated transactivation of a reporter gene linked to the GADD45A p53RE, but blunts p53-mediated transactivation when the reporter gene is linked to a p21 promoter fragment containing a p53RE, with implications for the regulation of these two cell cycle control genes. Finally, our investigations of HR phosphorylation revealed that rat Hr is a substrate for PKC, but not PKA, and that human HR is phosphorylated in intact U87 cells at Ser-416, located in a highly conserved region which partially fulfills the criteria of a PKC site. We propose that mammalian Hr is a phosphoprotein which can exert cross-talk with the p53 pathway with important implications for the regulation of cell proliferation and differentiation in tissues such as skin and brain where Hr is highly expressed. J. Cell. Biochem. 118: 341-350, 2017. © 2016 Wiley Periodicals, Inc.

Evolutionary and Functional Diversification of the Vitamin D Receptor-Lithocholic Acid Partnership.

The evolution, molecular behavior, and physiological function of nuclear receptors are of particular interest given their diverse roles in regulating essential biological processes. The vitamin D receptor (VDR) is well known for its canonical roles in calcium homeostasis and skeletal maintenance. Additionally, VDR has received an increased amount of attention due to the discovery of numerous non-calcemic functions, including the detoxification of lithocholic acid. Lithocholic acid is a toxic metabolite of chenodeoxycholic acid, a primary bile acid. The partnership between the VDR and lithocholic acid has been hypothesized to be a recent adaptation that evolved to mediate the detoxification and elimination of lithocholic acid from the gut. This partnership is speculated to be limited to higher vertebrates (birds and mammals), as lower vertebrates do not synthesize the parent compound of lithocholic acid. However, the molecular functions associated with the observed insensitivity of basal VDRs to lithocholic acid have not been explored. Here we characterize canonical nuclear receptor functions of VDRs from select species representing key nodes in vertebrate evolution and span a range of bile salt phenotypes. Competitive ligand binding assays revealed that the receptor's affinity for lithocholic acid is highly conserved across species, suggesting that lithocholic acid affinity is an ancient and non-adaptive trait. However, transient transactivation assays revealed that lithocholic acid-mediated VDR activation might have evolved more recently, as the non-mammalian receptors did not respond to lithocholic acid unless exogenous coactivator proteins were co-expressed. Subsequent functional assays indicated that differential lithocholic acid-mediated receptor activation is potentially driven by differential protein-protein interactions between VDR and nuclear receptor coregulator proteins. We hypothesize that the vitamin D receptor-lithocholic acid partnership evolved as a by-product of natural selection on the ligand-receptor partnership between the vitamin D receptor and the native VDR ligand: 1α,25-dihydroxyvitamin D3, the biologically active metabolite of vitamin D3.

1,25-Dihydroxyvitamin D and Klotho: A Tale of Two Renal Hormones Coming of Age.

1,25-Dihydroxyvitamin D3 (1,25D) is the renal metabolite of vitamin D that signals through binding to the nuclear vitamin D receptor (VDR). The ligand-receptor complex transcriptionally regulates genes encoding factors stimulating calcium and phosphate absorption plus bone remodeling, maintaining a skeleton with reduced risk of age-related osteoporotic fractures. 1,25D/VDR signaling exerts feedback control of Ca/PO4 via regulation of FGF23, klotho, and CYP24A1 to prevent age-related, ectopic calcification, fibrosis, and associated pathologies. Vitamin D also elicits xenobiotic detoxification, oxidative stress reduction, neuroprotective functions, antimicrobial defense, immunoregulation, anti-inflammatory/anticancer actions, and cardiovascular benefits. Many of the healthspan advantages conferred by 1,25D are promulgated by its induction of klotho, a renal hormone that is an anti-aging enzyme/coreceptor that protects against skin atrophy, osteopenia, hyperphosphatemia, endothelial dysfunction, cognitive defects, neurodegenerative disorders, and impaired hearing. In addition to the high-affinity 1,25D hormone, low-affinity nutritional VDR ligands including curcumin, polyunsaturated fatty acids, and anthocyanidins initiate VDR signaling, whereas the longevity principles resveratrol and SIRT1 potentiate VDR signaling. 1,25D exerts actions against neural excitotoxicity and induces serotonin mood elevation to support cognitive function and prosocial behavior. Together, 1,25D and klotho maintain the molecular signaling systems that promote growth (p21), development (Wnt), antioxidation (Nrf2/FOXO), and homeostasis (FGF23) in tissues crucial for normal physiology, while simultaneously guarding against malignancy and degeneration. Therefore, liganded-VDR modulates the expression of a "fountain of youth" array of genes, with the klotho target emerging as a major player in the facilitation of health span by delaying the chronic diseases of aging.

FGF23 gene regulation by 1,25-dihydroxyvitamin D: opposing effects in adipocytes and osteocytes.

In a closed endocrine loop, 1,25-dihydroxyvitamin D3 (1,25D) induces the expression of fibroblast growth factor 23 (FGF23) in bone, with the phosphaturic peptide in turn acting at kidney to feedback repress CYP27B1 and induce CYP24A1 to limit the levels of 1,25D. In 3T3-L1 differentiated adipocytes, 1,25D represses FGF23 and leptin expression and induces C/EBPß, but does not affect leptin receptor transcription. Conversely, in UMR-106 osteoblast-like cells, FGF23 mRNA concentrations are upregulated by 1,25D, an effect that is blunted by lysophosphatidic acid, a cell-surface acting ligand. Progressive truncation of the mouse FGF23 proximal promoter linked in luciferase reporter constructs reveals a 1,25D-responsive region between -400 and -200  bp. A 0.6  kb fragment of the mouse FGF23 promoter, linked in a reporter construct, responds to 1,25D with a fourfold enhancement of transcription in transfected K562 cells. Mutation of either an ETS1 site at -346  bp, or an adjacent candidate vitamin D receptor (VDR)/Nurr1-element, in the 0.6  kb reporter construct reduces the transcriptional activity elicited by 1,25D to a level that is not significantly different from a minimal promoter. This composite ETS1-VDR/Nurr1 cis-element may function as a switch between induction (osteocytes) and repression (adipocytes) of FGF23, depending on the cellular setting of transcription factors. Moreover, experiments demonstrate that a 1 kb mouse FGF23 promoter-reporter construct, transfected into MC3T3-E1 osteoblast-like cells, responds to a high calcium challenge with a statistically significant 1.7- to 2.0-fold enhancement of transcription. Thus, the FGF23 proximal promoter harbors cis elements that drive responsiveness to 1,25D and calcium, agents that induce FGF23 to curtail the pathologic consequences of their excess.

Neurocognitive Effects Following Cranial Irradiation for Brain Metastases.

About 90% of patients with brain metastases have impaired neurocognitive function at diagnosis and up to two-thirds will show further declines within 2-6 months of whole brain radiotherapy. Distinguishing treatment effects from progressive disease can be challenging because the prognosis remains poor in many patients. Omitting whole brain radiotherapy after local therapy in good prognosis patients improves verbal memory at 4 months, but the effect of higher intracranial recurrence and salvage therapy rates on neurocognitive function beyond this time point is unknown. Hippocampal-sparing whole brain radiotherapy and postoperative stereotactic radiosurgery are investigational techniques intended to reduce toxicity. Here we describe the changes that can occur and review technological, pharmacological and practical approaches used to mitigate their effect in clinical practice.

1,25-Dihydroxyvitamin D regulates expression of the tryptophan hydroxylase 2 and leptin genes: implication for behavioral influences of vitamin D.

To investigate vitamin D-related control of brain-expressed genes, candidate vitamin D responsive elements (VDREs) at -7/-10 kb in human tryptophan hydroxylase (TPH)2 were probed. Both VDREs bound the vitamin D receptor (VDR)-retinoid X receptor (RXR) complex and drove reporter gene transcription in response to 1,25-dihydroxyvitamin D3 (1,25D). Brain TPH2 mRNA, encoding the rate-limiting enzyme in serotonin synthesis, was induced 2.2-fold by 10 nM 1,25D in human U87 glioblastoma cells and 47.8-fold in rat serotonergic RN46A-B14 cells. 1,25D regulation of leptin (Lep), encoding a serotoninlike satiety factor, was also examined. In mouse adipocytes, 1,25D repressed leptin mRNA levels by at least 84%, whereas 1,25D induced leptin mRNA 15.1-fold in human glioblastoma cells. Chromatin immunoprecipitation sequencing analysis of the mouse Lep gene in response to 1,25D revealed a cluster of regulatory sites (cis-regulatory module; CRM) at -28 kb that 1,25D-dependently docked VDR, RXR, C/EBPß, and RUNX2. This CRM harbored 3 VDREs and single C/EBPß and RUNX2 sites. Therefore, the expression of human TPH2 and mouse Lep are governed by 1,25D, potentially via respective VDREs located at -7/-10 kb and -28 kb. These results imply that vitamin D affects brain serotonin concentrations, which may be relevant to psychiatric disorders, such as autism, and may control leptin levels and affect eating behavior.