, 2004). Among the living organisms that produce phospholipase-D, Loxosceles spiders check details (brown spiders) are remarkable in producing a mixture of isoforms of these molecules in their venom ( da Silva et al., 2004; Kalapothakis et al., 2007). Among the different toxins found in brown spider venom, isoforms of phospholipase-D

(referred to as dermonecrotic toxins because of the involvement of these molecules as a hallmark of dermonecrosis) are the most widely biologically and biochemically studied toxins. When purified under laboratory conditions, these molecules can reproduce the major biological effects triggered by crude venom, such as dermonecrosis, red blood lysis, dysregulated inflammatory responses, platelet aggregation, increased vessel Epigenetics inhibitor permeability and acute renal failure ( Chaim et al., 2006; da Silveira et al., 2006, 2007; Kusma et al., 2008; Chaves-Moreira et al., 2009, 2011; Chaim et al., 2011). Previous studies have characterized the dermonecrotic toxin found in brown spider venoms

as a sphingomyelinase D molecule based on its ability to hydrolyze the phospholipid sphingomyelin into choline and ceramide 1-phosphate ( Kurpiewski et al., 1981). However, based on the hydrolysis of different purified phospholipids mediated by brown spider venom toxins, the term sphingomyelinase D has been replaced with phospholipase-D as a more accurate and broader denomination because these toxins hydrolyze not only sphingophospholipids but also lysoglycerophospholipids, generating ceramide 1-phosphate or lysophosphatidic acid (LPA) ( Lee and Lynch, 2005; Chaim et al., 2011; Chaves-Moreira et al., 2011). It has been postulated that by hydrolyzing phospholipids that generate ceramide 1-phosphate

or lysophosphatidic acid, dermonecrotic toxins activate signaling pathways in different cells causing pathophysiological changes, such as inflammatory responses, red blood cell hemolysis, acute renal disease, platelet aggregation, and increased blood vessel permeability ( da Silveira et al., 2007; Kusma et al., 2008; Chaves-Moreira et al., 2009, 2011; Chaim et al., 2011). The idea that there is a family of phospholipase-D proteins in the venoms of Loxosceles species was further supported by the cloning and expression of phospholipase-D toxins from a variety of Loxosceles spiders. Kalapothakis et al. (2002) eltoprazine performed studies with a recombinant phospholipase-D from Loxosceles intermedia. Ramos-Cerrillo et al. (2004) cloned, expressed and analyzed recombinant phospholipase-D proteins from Loxosceles reclusa and Loxosceles boneti venoms. Binford et al. (2005) reported three cDNA sequences of phospholipase-D in Loxosceles arizonica. Chaim et al. (2006), da Silveira et al., 2006 and da Silveira et al., 2007, and Appel et al. (2008) used a cDNA library obtained from the venom gland of L. intermedia to clone and express these toxins and observed differential functionality for six related toxins classified as phospholipase-D proteins. Catalán et al.

The murine C3H10T1/2 and

ST2 pre-osteoblast cell lines were obtained from ATCC (Manassas, VA) and cultured as described below. Recombinant human heparanase (rHPSE) and heparanase antibodies were kindly provided by Dr. Israel Vlodavsky (Technion, Haifa, Israel). Dickkopf1 (DKK1) inhibitor was purchased from Millipore (Billerica, MA); active and total β-catenin antibodies were purchased from Cell Signaling (Danvers, MA); human osteocalcin and mouse peroxisome proliferator-activated receptor gamma (PPARγ) antibodies were obtained from Abcam (Cambridge, MA); and mouse Runt-related transcription factor 2 (Runx2) antibody was purchased from MBL (Woods Hole, MA). Human and mouse DKK1 ELISA ABT-199 datasheet kits were obtained from R&D Systems (Minneapolis, MN). ALP and Oil Red O staining kits and β-actin antibody were purchased from Sigma (St. Louis, MO); and the Von Kossa staining kit was from Polysciences (Warrington, PA). All animals were used in this study according to the NIH Guide for the Care and Use of Laboratory Animals and were approved under local institutional guidelines for the humane use of animals in research. SCID (CB.17 scid/scid) and

C57BL/6 mice were purchased from Harlan Laboratories, Inc. (Indianapolis, IN) and housed in individual cages (5 KU-57788 mw per cage) in temperature (22 °C) and humidity (50%) controlled rooms having a 12 h light/12 h dark cycle with food and water ad libitum. All animal experiments were performed under a UAB IACUC approved protocol. The SCID-hu is a well described animal model in which human fetal long bones (Advanced Bioscience Resources, Inc., Alameda, CA) are implanted subcutaneously on each side of the dorsum of SCID mice [33], [34] and [37]. 105 CAG HPSE-low or HPSE-high cells were injected directly into the cut end of one human bone graft (primary bone) in each mouse, whereas the contralaterally implanted human bones were not injected with tumor cells

MG-132 nmr (7 mice in each group). Eight weeks after the injection of tumor cells, the mice were euthanized. Tumor-injected human bones and non-injected contralateral human bones were collected and fixed in 10% neutral-buffered formalin and embedded in paraffin as described [36]. The paraffin-embedded bone sections were then stained with human osteocalcin antibody according to the manufacturer’s recommendations and the numbers of osteocalcin positive osteoblasts on the surface of trabecular bones were counted [25] and [33]. Twenty eight paraffin-embedded bone marrow core biopsy specimens of myeloma patients, obtained from the Department of Pathology at UAB, were stained for both heparanase and osteocalcin. The experimental procedures and protocols were approved by the UAB Institutional Review Board.

Using human breast cancer as a model, researchers found that half of the sporadic basal-like cancers were characterized by duplication of the active X chromosome and loss of the inactive X chromosome [19]. While these abnormalities did not contribute to global increases of gene expression

from the X chromosome, it was associated with overexpression of a subset of genes. In addition, another paper provided evidence that the inactive X chromosomes accumulates more mutations than any other autosome in cancer genomes compared Veliparib chemical structure to non-tumorigenic samples [20], suggesting an inability to successfully repair damage. If this inactive X chromosome later becomes active, it could further contribute to genetic mutation load during Selleckchem Copanlisib cancer progression. An elegant and convincing study in mouse showed direct evidence that Xist loss causes cancer. Researchers conditionally knocked out Xist in vivo in mouse hematopoietic stem cells after random X chromosome inactivation had already taken place. A female specific, fully penetrant, lethal blood cancer developed that

began killing mice at 1.5 months. After two years, only 10 percent of the mice were still alive and neither homozygous nor heterozygous female mice have escaped the lethal phenotype at the time the research was published [ 21••]. While this was only demonstrated in one lineage in the mouse, other data suggest that the loss of Nintedanib (BIBF 1120) XIST in human iPSCs is strongly correlated with increased expression of X-linked oncogenes [ 22••]. Interestingly, male iPSCs, compared to female iPSCs, are more homogeneous and do not overexpress these genes suggesting a potential increased risk of tumorigenesis in female stem cells. This is a major hurdle in the clinical translation of female stem cells and will require much

more work to understand the different potentials of stem cells with different XCI states ( Table 1). Early mouse studies have revealed simple binaries: pluripotent cell types have two active X chromosomes (XaXa) (extensively reviewed in [2 and 23]), and somatic cell types have one active and one inactive X chromosome (XaXi) [24]. Differentiation of a mouse pluripotent cell into a somatic cell results in the inactivation of one X chromosome [25]. This is true for both embryonic stem (ES) cells and iPSCs in the mouse with the exception of ES cells derived from the epiblast. Epiblast stem cells (EpiSC) are thought to represent a distinct state of pluripotency, as they cannot contribute to blastocyst chimeras, have variable differentiation bias, and are characterized by an inactive X chromosome [26 and 27]. However, they can be converted to ES, reactivating the inactive X chromosome in the process [28]. These relationships in mouse have not directly translated to human biology. There is no universal rule governing the X chromosome state in human pluripotent cell types; indeed, a range of states are common (Figure 1).

The molecular and cellular mechanisms of this action remain elusive, however, it is clearly dependent on the function of Cd81, a tetraspanin molecule present on fibroblast exosomes [19••]. Interestingly, fluorescently tagged Cd81 was utilized to track fibroblast exosomes, which, upon endocytosis by BCCs,

could be visualized to colocalize with Wnt11 in endocytic vesicular structures. Whether the MVB is the nature of these vesicular structures needs further investigation. Furthermore, analysis of a published gene expression dataset indicated that CD81 expression is enhanced in breast cancer-associated stroma, suggesting that stromal Cd81-exosomes might correlate with disease progression [19••]. The PCP signaling pathway in BCCs was stimulated following the exosome-mobilized secretion

of Wnt11 [19••]. find more The activated BCCs display increased protrusions with asymmetric distributions of PCP signaling components, which are functionally critical for Buparlisib cost the migration and metastasis of BCCs. Intriguingly, although they lack de novo production of Cd81-positive exosomes, BCCs could secrete Wnt11 in the absence of fibroblast-derived exosomes. However, PCP signaling and migration were not activated in BCCs in the absence of fibroblast exosomes [19••]. This suggests that Wnt11 mobilized by Cd81-exosomes might have a distinct activity from autocrine Wnt11 secreted in other forms by BCCs. The mechanism of this difference may lie in the function of Cd81, a member of the family of tetraspanins that have essential roles in exosomal biology, such as membrane fusion and cargo sorting [40 and 41]. It will be necessary Anidulafungin (LY303366) to explore the activity of Cd81, which might directly facilitate exosomal sorting of Wnt11 or regulate exosome trafficking. As an emerging signaling platform, exosomes play an important role in facilitating Wnt secretion and transport (Figure 1) [19••, 35••, 36• and 37•]. Exosome-bound Wnts and their signaling activities have been functionally

implicated in Drosophila development as well as in fibroblast-promoted cancer metastasis. However, our knowledge about the underlying mechanisms remains rudimentary. Currently the primary challenge is to understand how the exosome biogenesis/trafficking pathway is dynamically integrated with the Wnt secretion pathway. To overcome this, we will first need to systematically profile molecular markers on exosomes that facilitate Wnt secretion. Given the complexity of exosomal biogenesis and Wnt biology, it will not be surprising to identify stage-specific markers for Wnt-exosomes during formation, secretion, and extracellular trafficking. Importantly, it will be necessary to validate these markers/mechanisms in different developmental and cancer model systems. Second, it is crucial to develop more sophisticated exosomal isolation techniques with one ultimate goal being to directly purify them from the body fluid, which will assist in disease diagnosis and prognosis.

chem.qmul.ac.uk/iubmb/enzyme/), enzymes are classified into six main classes: oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. Hence, lipases are hydrolases. Aldol condensation, on the other hand, is carried out by lyases, aldehyde-lyases has been assigned the number 4.1.2 (Nomenclature Committee of IUBMB, 1992). However, lipases have now been shown to catalyze not only aldol condensation, but also the Mannich reaction, Michael addition, Morita–Baylis–Hillman reaction as well (Hult and Berglund, 2007, Kapoor and Gupta, 2012, Lai et al., 2010 and Li et al., 2008)! So, to start with we have a problem with

the classification. Khersonsky and Tawfik (2010) have made some suggestions in the regard. In many cases, these find protocol promiscuous reactions involve high catalytic efficiency which is in the same range as seen in

normal enzyme catalyzed reactions. Babtie et al. (2010) have discussed this and point out that rate accelerations (kcat/Km)/k2 of up to 1018-fold are known. In many other cases, protein engineering and directed evolution has been successfully used to induce catalytic promiscuity ( Khersonsky and Tawfik, 2010). Many of these reactions are industrially important. Large number of reports regarding catalytic promiscuity deal with reactions carried out with industrial preparations of lipases ( Busto et al., 2010 and Kapoor and Gupta, 2012). While catalytic promiscuity involves the active site of the enzyme, moonlighting Sirolimus datasheet by proteins can involve different parts of the enzyme molecule (Jeffery, 1999). The phenomenon of moonlighting constitutes a definite shift from the well-known one gene-one protein-one function paradigm. The different functions of a moonlighting protein can be displayed: PtdIns(3,4)P2 in two different locations in the cell (one can be even intracellular and another extracellular); by a change from the monomer to oligomer structure, in different cell

types or even with a change in ligand or substrate concentrations (Jeffery, 2009). While few examples of moonlighting involve different catalytic activities, in larger number of cases the different activities encompass non-catalytic functions like repressor, growth factor, receptor, inhibitor, chaperone and regulator activities (Jeffery, 1999, 2009). Apparently new enzymes continue to evolve. Atrazine chlorohydrolase (which degrades herbicide atrazine) has evolved (from melamine hydrolase) between 1950 and 1990 (Janssen et al., 2005). Directed evolution, of course, is being extensively used to obtain enzymes which tailored specificity (Arnold and Georgiou, 2003a and Arnold and Georgiou, 2003b). All the different phenomena and observations discussed in this section have a common message: old classification and old way of reporting data on enzyme catalyzed reactions may not be adequate. In some cases, a little tweaking of guidelines may work. Eventually, we would need to evolve new guidelines (see also Tipton et al., 2014).

This would require further investigation. However, methamidophos was chosen as the biomarker in this study to reflect the risk of exposure to methamidophos, rather than a detoxification metabolite. Diet is likely to be a source of exposure to the general population and it has been shown that metabolites can be present as residues, therefore measuring methamidophos itself better reflects the risk from food. One of our volunteers showed exceptionally low excretion of methamidophos

following dosing; this may be due to differences in metabolism but this has not check details been investigated further. Alternatively, methamidophos may be hydrolyzed to its metabolites in the acidity of the stomach and then absorbed into the body, although available data suggests that methamidophos is stable under acidic conditions (IPCS, 2014). Due to research priorities, samples for five of the six volunteers were stored frozen for five years prior to analysis. In order to check stability, samples from a further volunteer were collected prior to analysis. Volunteers A–E (except C) showed comparable excretion to volunteer F (analyzed immediately after collection) indicating that the earlier samples were stable. This supports data from Montesano et al. (2007) showing methamidophos stability

at −20 °C. It is therefore unlikely that the results from the anomalous volunteer C are due to degradation. find more With such rapid elimination it would be appropriate to collect samples soon after exposure or at the end of each shift for occupational studies. For environmental studies, the short half-life means that estimates

of exposure using biomonitoring are likely to be highly variable (Aylward et al., 2012). Significant inter-individual variability in excretion is also 2-hydroxyphytanoyl-CoA lyase likely, judging by volunteer C in our cohort. Three environmental studies have been reported in the literature (Table 5). The number of positive samples in all three of the studies was low (<1.5% in all three studies), probably reflecting the rapid excretion and intermittent exposures of methamidophos. When compared with our own results (particularly taking into account the extent of negative results in the environmental surveys) it shows that general population exposure in countries where methamidophos is still in use is likely to be well within the ADI. The authors declare that there are no conflicts of interest. The authors would like to thank the volunteers who participated in this study. This publication and the work it describes were funded by the Health and Safety Laboratory. The authors declare that there are no conflicts of interest. "
“Human biological monitoring (HBM) has been used as a tool for prevention in occupational and environmental medicine for several decades.

This work was supported in part by the Grant-in-Aid for Scientific Research C (KAKENHI: 23500848) from Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan and by the Grant of Kao Research Council for the Study of Healthcare Science. The PI3K inhibitor funders had no roles in study design, data collection, data analysis, decision to publish, or preparation of the manuscript. “
“The nucleus incertus (NI), better known for its

status as the principal source of neuronal relaxin-3, is a brainstem nucleus located ventral and medial to the posterodorsal tegmental nucleus (PDTg) in the pontine periventricular grey (Burazin et al., 2002, Goto et al., 2001 and Olucha-Bordonau et al., 2003). The NI neurons were initially shown to express the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) (Ford et al., 1995) and have later been revealed to co-express the recently discovered neuropeptide, relaxin-3 (Ma et al., 2007). This small and distinct group of neurons has sparked off renewed interest due to its high expression of the Osimertinib supplier corticotropin releasing hormone receptor type 1 (CRF1) (Bittencourt and Sawchenko, 2000 and Potter et al., 1994), which suggests a role of the NI in the stress response. Subsequent studies reported the presence of other receptors in the NI, e.g. 5-hydroxytryptamine (5-HT or serotonin) receptor subtype 1A (5-HT1A) (Miyamoto

et al., 2008), and relaxin/insulin-like family peptide receptor 3 (RXFP3) (Sutton et al., 2004). Other neuropeptides expressed by nucleus incertus include neuromedin B (Chronwall et al., 1985 and Wada

et al., 1990) and cholecystokinin (Kubota et al., 1983 and Olucha-Bordonau et al., 2003). Of particular interest, expression of relaxin-3 is highly specific and most abundant in the NI and only a small number of neurons in other nearby regions such as the pontine raphe nucleus, periaqueductal grey and dorsal substantia nigra express relaxin-3 (Tanaka et al., 2005). As such, the NI – serves as a key point of regulation for functioning of the relaxin-3 neural circuitry. The NI possibly exerts its actions through numerous projections to several parts of the brain, including the prefrontal cortex, medial septum (MS), hippocampus, ADAM7 amygdala, hypothalamus and the raphe nuclei (Goto et al., 2001) – areas that are implicated in various psychiatric conditions. Relaxin-3 binds to the RXFP3, which belongs to the family of relaxin peptide receptors that was recently elevated from orphan receptor status (van der Westhuizen et al., 2008). The precise functions of the NI and relaxin-3 remain largely undetermined but intriguing reports have been published in the past decade demonstrating the alleged role of the NI/relaxin-3/RXFP3 system in feeding behaviour (McGowan et al., 2006 and McGowan et al., 2005), stress (Burazin et al., 2002 and Smith et al., 2012), anxiety (Watanabe et al., 2011) and cognition (Cervera-Ferri et al., 2011, Farooq et al., 2013 and Ma et al., 2009).

Nevertheless, tiered testing strategies for assessing metabolism have been suggested and reviewed previously ( ECVAM, 2002 and Coecke et al., 2005a). Models used to identify ADME properties (as well as organ-specific toxicities of chemicals) are summarised in Table 1, together with information regarding recommendations by the regulatory authorities and validation status. There is also a number of Quantitative Structure Activity Relationship (QSAR) models that

are available to both industry and academia and these include but are not limited to the OECD toolbox ( Table 2). Supporting Ku-0059436 mouse activities from industry, European Commission and Academia to enable the development of non-animal models are summarised in Table 3. An EPAA workshop was held in Duesseldorf learn more on 24th/25th November, 2008, and was attended by scientific experts in the pharmaceutical, chemical, pesticide and cosmetic industries,

by regulators, as well as by academia. Participants included representatives of the Scientific Committee on Consumer Safety (SCCS), European Centre for the Validation of Alternative Methods (ECVAM), European Food Safety Authority (EFSA) and Directorate General (DG) for Research. The aim of the workshop was to discuss how to implement in vitro ADME test systems as part of Integrated Testing Strategies (ITS) for the testing of cosmetics, pharmaceuticals and industrial chemicals and pesticides (including agro-chemicals such as herbicides, fungicides, or insecticides). The present report presents the outcome of the break-out group discussions in describing how in vitro assays may be used within different industry sectors

and how regulators view in vitro data. It also outlines international projects aimed at developing alternative test models. In addition, BCKDHA the break-out sessions discussed the suitability of in vitro approaches to systemic toxicity and hazard identification for target organs and steps required to attain regulatory acceptance. Emphasis is placed on in vitro assays and their use in risk assessment issues including preliminary risk assessment such as for prioritisation and deprioritisation, rather than in targeted risk assessment per se, since this is markedly different between industry sectors and is out of the scope of this paper. The use of animal assays is different across industries, whereby in vivo studies are required in one sector but banned in another. An overview of these differences and the agencies which affect the use of animals is described below. The European Medicines Agency (also known as the EMA) is a European agency which evaluates pharmaceuticals. In the USA, the equivalent agency is the Food and Drug Administration (FDA).

As shown in Table 1, the peripheral epithelial odontogenic cells of all studied tumours were positive for podoplanin while the central ones were negative. The exceptions were plexiform ameloblastoma, adenomatoid odontogenic tumour and ameloblastic fibroma. The plexiform ameloblastoma resembles the tooth germ in the dental lamina stage,17 when the differentiation process of the odontogenic epithelium has not initiated.

This lack of cellular differentiation may reflect the homogeneity of podoplanin expression found in those benign epithelial tumours, confirming previous results obtained by other authors.6 and 14 All nine adenomatoid odontogenic tumours showed membranous and cytoplasmic podoplanin expression in the central epithelial odontogenic cells, including the duct-like structures (Fig. Buparlisib cell line 1C). These results are contradicting the previous12 and 13 reports, which described negative podoplanin immunostaining in superficial and luminal epithelia of duct-like structures of only two adenomatoid odontogenic tumours. The explanation for this apparent discrepancy may be associated with the proliferative activity of the epithelial cells with duct-like appearance within the benign odontogenic tumour. However, these results demand further confirmation by other series of representative adenomatoid odontogenic tumour with characteristic cellular duct-like pattern. In ameloblastic

fibromas, moderate podoplanin expression by reticulum stellate-like cells was observed, which might indicate cellular activity of these selected cells. The selleck Org 27569 ectomesenchymal cells of mixed odontogenic tumour presented absent or weak immunoreaction for anti-podoplanin antibody, except for odontoblasts of ameloblastic fibro-odontoma. Although podoplanin distribution in benign odontogenic tumours has been recently identified,5, 6, 8, 12,

13 and 14 its precise biologic relevance and significance in tumoral or even in normal odontogenic tissues remains source of debate. The podoplanin expression accelerates cell motility in vitro and induces cell invasion and metastasis of the malignant epithelial tumour. 18 Furthermore, overexpression of podoplanin promotes the formation of elongated cell extensions and increases adhesion and migration of MDCK cells 3 and suggests a role for podoplanin in cytoskeletal reorganization. In ameloblastic fibro-odontoma, secreting ameloblasts expressed podoplanin while in non-secreting and reduced ameloblasts this immunoreaction was absent. Our findings are in line with González-Alva et al.’s 5 and we agree with them when they suggest that podoplanin, with its ability to remodel the actin cytoskeleton and form filopodia, is involved in the movement of ameloblasts away from odontoblasts and vice versa. Once enamel deposition has been completed and the ameloblasts no longer move, they lose their podoplanin expression.

All patients treated at our institution and for

whom medical records were available were selected for inclusion in this retrospective study. Initial locoregional staging included a clinical evaluation performed by a gynecologic surgeon and radiation oncologist (according to the 1995 FIGO (International Federation of Gynecology and Obstetrics) classification (7)). Abdominopelvic MRI was obtained for 168 patients (74.3%) and CT imaging for 160 patients (70.8%). FDG-PET (fluorine-18-fluorodeoxyglucose positron emission tomography) scan was not systematically mTOR inhibitor performed, and no decision has been taken based only on its results; 148 patients (65.4%), mostly Stages I and II, with a good health status and without suspicious lomboaortic nodes at CT or MRI were selected to receive pelvic lymphadenectomy by coelioscopy first. Only 1 patient had a para-aortic lymphadenectomy. Nodal involvement was determined if histologically proven (65 patients) or suspected on CT (24 patients). All patients with positive lymph nodes, including IB1 stage, received first external beam radiation therapy and are included in the study. Stage IB1 patients treated with preoperative intracavitary PDR BT followed with colpohysterectomy were excluded from the analysis (19 patients). Institutional review board approval

was obtained for this study, and it was conducted in compliance with the Helsinki Declaration. All patients received 45 Gy pelvic external beam radiotherapy (EBRT) before PDR BT with a standard four-field technique (190 patients) Buspirone HCl or a two anterior/posterior opposing fields technique (36 patients) using high buy Gemcitabine megavoltage photons from a linear accelerator (photons × 18 and 25 MeV). EBRT included the para-aortic area when the CT showed enlarged common iliac or para-aortic nodes. When the nodal involvement was histologically proved or suspected on CT, a complementary boost irradiation was delivered after BT to reach a minimum of 60 Gy to the parametria and/or involved pelvic nodes and 55 Gy to the para-aortic

nodes, taking into account the dose contribution of BT. From 1999, based on the results of randomized trials [8], [9], [10], [11] and [12], chemotherapy was given during EBRT for all stages ≥IB2, with intravenous cisplatin 40 mg/m² once a week for 5 weeks in 150 of 226 patients (66.4%). Chemotherapy courses were not delivered during the hospitalization for the BT procedure. After EBRT, the PDR BT boost was delivered during a single hospitalization, using the PDR Selectron (Elekta, Stockholm, Sweden). At the beginning of the BT procedure, a careful clinical examination was carried out under general anesthesia to assess clinical response to EBRT. A Fletcher applicator was used, and no patient underwent interstitial BT. Pulses were delivered hourly during night and day. Before 1999, the BT treatment planning dosimetry was based on orthogonal radiographs, in accordance with International Commission on Radiation Units (ICRU) 38 (13).