Importance of controlling the degree of saturation in soil compaction

n the typical conventional fill compaction, the dry density ρd and the water content w are controlled in relation to (ρd)max and wopt determined by laboratory compaction tests using a representative sample at a certain compaction energy level CEL. Although CEL and actual soil type affect significantly the values of (ρd)max and wopt, they change inevitably in a given earthwork project while CEL in the field may not match the value used in the laboratory compaction tests. Compaction control based on the stiffness of compacted soil in the field has such a drawback that the stiffness drops upon wetting more largely as the degree of saturation, Sr, of compacted soil becomes lower than the optimum degree of saturation (Sr)opt defined as Sr when (ρd)max is obtained for a given CEL. In comparison, the value of (Sr)opt and the ρd/(ρd)max vs. Sr - (Sr)opt relation of compacted soil are rather insensitive to variations in CEL and soil type, while the strength and stiffness of unsoaked and soaked compacted soil is controlled by ρd and “Sr at the end of compaction”. It is proposed to control not only w and ρd but also Sr so that Sr becomes (Sr)opt and ρd becomes large enough to ensue soil properties required in design.Fundação para a Ciência e Tecnologia (FCT)info:eu-repo/semantics/publishedVersio

Assessment of EGFR/HER2 dimerization by FRET-FLIM utilizing Alexa-conjugated secondary antibodies in relation to targeted therapies in cancers

The expression level of the HER family is unreliable as a predictive marker for targeted therapies in cancer. Thus, there is a need to develop other biomarkers, which can be used to accurately select responsive patients for targeted therapies. The HER dimerization status may be more important than HER receptor expression per se in determining sensitivity or resistance to a given therapeutic agent. The aim of the study is to develop a FRET assay using dye conjugated secondary antibodies to assess HER receptor dimerization. Using primary antibodies from different species in conjunction with Alexa488 and Alexa546 conjugated secondary antibodies, we validated our EGFR/HER2 dimerization assay in three cell lines, EGFR positive A431 cells as well as HER2 positive breast cell lines BT474 and SKBR3 cells. Finally, we applied our assay to assess EGFR/HER2 dimerization in paraffin embedded cell pellets. Our results show promise for the assay to be applied to tumor samples in order to assess the prognostic significance and predictive value of HER receptor dimerization in various cancers

Monitoring beam charge during FLASH irradiations

In recent years, FLASH irradiation has attracted significant interest in radiation research. Studies have shown that irradiation at ultra-high dose rates (FLASH) reduces the severity of toxicities in normal tissues compared to irradiation at conventional dose rates (CONV), as currently used in clinical practice. Most pre-clinical work is currently carried out using charged particle beams and the beam charge monitor described here is relevant to such beams. Any biological effect comparisons between FLASH and CONV irradiations rely on measurement of tissue dose. While well-established approaches can be used to monitor, in real time, the dose delivered during CONV irradiations, monitoring FLASH doses is not so straightforward. Recently the use of non-intercepting beam current transformers (BCTs) has been proposed for FLASH work. Such BCTs have been used for decades in numerous accelerator installations to monitor temporal and intensity beam profiles. In order to serve as monitoring dosimeters, the BCT output current must be integrated, using electronic circuitry or using software integration following signal digitisation. While sensitive enough for FLASH irradiation, where few intense pulses deliver the requisite dose, the inherent insensitivity of BCTs and the need for a wide detection bandwidth makes them less suitable for use during CONV “reference” irradiations. The purpose of this article is to remind the FLASH community of a different mode of BCT operation: direct monitoring of charge, rather than current, achieved by loading the BCT capacitively rather than resistively. The resulting resonant operation achieves very high sensitivities, enabling straightforward monitoring of output during both CONV and FLASH regimes. Historically, such inductive charge monitors have been used for single pulse work; however, a straightforward circuit modification allows selective resonance damping when repetitive pulsing is used, as during FLASH and CONV irradiations. Practical means of achieving this are presented, as are construction and signal processing details. Finally, results are presented showing the beneficial behaviour of the BCT versus an (Advanced Markus) ionisation chamber for measurements over a dose rate range, from <0.1 Gys−1 to >3 kGys−1

A Variable-Energy Soft X-Ray Microprobe to Investigate Mechanisms of the Radiation-Induced Bystander Effect.

The Gray Cancer Institute has pioneered the use of X ray focussing techniques to develop systems for micro irradiating individual cells and sub cellular targets in vitro. Cellular micro irradiation is now recognised as a highly versatile technique for understanding how ionising radiation interacts with living cells and tissues. The strength of the technique lies in its ability to deliver precise doses of radiation to selected individual cells (or sub cellular targets). The application of this technique in the field of radiation biology continues to be of great interest for investigating a number of phenomena currently of concern to the radiobiological community. One important phenomenon is the so called ‘bystander effect’ where it is observed that unirradiated cells can also respond to signals transmitted by irradiated neighbours. Clearly, the ability of a microbeam to irradiate just a single cell or selected cells within a population is well suited to studying this effect. Our prototype ‘tabletop’ X-ray microprobe was optimised for focusing 278 eV C-K X rays and has been used successfully for a number of years. However, we have sought to develop a new variable energy soft X-ray microprobe capable of delivering focused CK (0.28 keV), Al-K (1.48 keV) and notably, Ti-K (4.5 keV) X rays. Ti-K X rays are capable of penetrating several cell layers and are therefore much better suited to studies involving tissues and multi cellular layers. In our new design, X-rays are generated by the focussed electron bombardment of a material whose characteristic-K radiation is required. The source is mounted on a 1.5 x 1.0 metre optical table. Electrons are generated by a custom built gun, designed to operate up to 15 kV. The electrons are focused using a permanent neodymium iron boron magnet assembly. Focusing is achieved by adjusting the accelerating voltage and by fine tuning the target position via a vacuum position feedthrough. To analyze the electron beam properties, a custom built microscope is used to image the focussed beam on the target, through a vacuum window. The X-rays are focussed by a zone plate optical assembly mounted to the end of a hollow vertical tube that can be precisely positioned above the X ray source. The cell finding and positioning stage comprises an epi-fluorescence microscope and a feedback controlled 3 axis cell positioning stage, also mounted on the optical table. Independent vertical micro positioning of the microscope objective turret allows the focus of the microscope and the X ray focus to coincide in space (i.e. at the point where the cell should be positioned for exposure). The whole microscope stage assembly can be precisely raised or lowered, to cater for large differences in the focal length of the X ray zone plates. The facility is controlled by PC and the software provides full status and control of the source and makes use of a dual-screen for control and display during the automated cell finding and irradiation procedures

Cahiers d’études médiévales, 2 : La Science de la nature : théories et pratiques, Montréal, Bellarmin et Paris, Vrin, 1974, 199 p.

Background: Sentinel Lymph Node (SLN) sampling may significantly reduce surgical morbidity by avoiding needless radical lymphadenectomy. In gynaecological cancers, the current practice in the UK is testing the accuracy of SLN detection using radioactive isotopes within the context of clinical trials. However, radioactive tracers pose significant logistic problems. We, therefore, conducted a pilot, observational study to assess the feasibility of a novel optical imaging device for SLN detection in gynaecological cancers using near infrared (NIR) fluorescence. Methods: A novel, custom-made, optical imaging system was developed to enable detection of multiple fluorescence dyes and allow simultaneous bright-field imaging during open surgery and laparoscopic procedures. We then evaluated the performance of the system in a prospective study of 49 women with early stage vulval, cervical and endometrial cancer who were scheduled to undergo complete lymphadenectomy. Clinically approved fluorescent contrast agents indocyanine green (ICG) and methylene blue (MB) were used. The main outcomes of the study included SLN mapping detection rates, false negative rates using the NIR fluorescence technique and safety of the procedures. We also examined the association between injection sites and differential lymphatic drainage in women with endometrial cancer by fluorescence imaging of ICG and MB. Results: A total of 64 SLNs were detected during both open surgery and laparoscopy. Following dose optimisation and the learning phase, SLN detection rate approached 100 % for all cancer types with no false negatives detected. Fluorescence from ICG and MB detected para-aortic SLNs in women with endometrial cancer following uterine injection. Percutaneous SLN detection was also achieved in most women with vulval cancer. No adverse reactions associated with the use of either dyes were observed. Conclusions: This study demonstrated the successful clinical application of a novel NIR fluorescence imaging system for SLN detection across different gynaecological cancers. We showcased the first in human imaging, during the same procedure, of two fluorescence dyes in women with endometrial cancer. </p

Ultrasound Mediated Gemcitabine Delivery Reduces the Normal-Tissue Toxicity of Chemoradiation Therapy in a Muscle-Invasive Bladder Cancer Model

Purpose: Chemoradiation is the standard of care in muscle-invasive bladder. While agents such as gemcitabine can enhance tumour radiosensitisation, their side effects can limit patient eligibility and treatment efficacy. Here we investigate ultrasound and microbubbles for targeting gemcitabine delivery to reduce normal tissue toxicity in a murine orthotopic MIBC model. Materials and Methods: CD1-nude mice were injected orthotopically with RT112 bladder tumour cells. Conventional chemoradiation involved injecting gemcitabine (10 mg/kg) before 6 Gy targeted irradiation of the bladder area using a Small Animal Radiation Research Platform (SARRP). Ultrasound-mediated gemcitabine delivery (10 mg/kg gemcitabine) involved either co-administration of microbubbles with gemcitabine or conjugating gemcitabine onto microbubbles followed by exposure to ultrasound (1.1 MHz centre frequency, 1 MPa peak negative pressure, 1% duty cycle and 0.5 Hz pulse repetition frequency), prior to SARRP irradiation. The effect of ultrasound and microbubbles alone was also tested. Tumour volumes were measured by 3D ultrasound imaging. Acute normal tissue toxicity from 12 Gy to the lower bowel area was assessed using an intestinal crypt assay in mice culled. 3.75 days post-treatment. Results: Significant tumour growth delay was observed with conventional chemoradiation and both microbubble groups (p Conclusions: Ultrasound and microbubbles offer a promising new approach for improving chemoradiation therapy in muscle-invasive bladder cancer, maintaining tumour growth delay but with reduced acute intestinal toxicity compared to conventional chemoradiation therapy.</p

MicroRNA-210 Regulates Mitochondrial Free Radical Response to Hypoxia and Krebs Cycle in Cancer Cells by Targeting Iron Sulfur Cluster Protein ISCU

BACKGROUND: Hypoxia in cancers results in the upregulation of hypoxia inducible factor 1 (HIF-1) and a microRNA, hsa-miR-210 (miR-210) which is associated with a poor prognosis. METHODS AND FINDINGS: In human cancer cell lines and tumours, we found that miR-210 targets the mitochondrial iron sulfur scaffold protein ISCU, required for assembly of iron-sulfur clusters, cofactors for key enzymes involved in the Krebs cycle, electron transport, and iron metabolism. Down regulation of ISCU was the major cause of induction of reactive oxygen species (ROS) in hypoxia. ISCU suppression reduced mitochondrial complex 1 activity and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. Cancers with low ISCU had a worse prognosis. CONCLUSIONS: Induction of these major hallmarks of cancer show that a single microRNA, miR-210, mediates a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation

HER2-HER3 heterodimer quantification by FRET-FILM and patient subclass analysis of the COIN colorectal trial

BACKGROUND: The phase 3 MRC COIN trial showed no statistically significant benefit from adding the EGFR-target cetuximab to oxaliplatin-based chemotherapy in first-line treatment of advanced colorectal cancer. This study exploits additional information on HER2-HER3 dimerization to achieve patient stratification and reveal previously hidden subgroups of patients who had differing disease progression and treatment response. METHODS: HER2-HER3 dimerization was quantified by 'FLIM Histology' in primary tumor samples from 550 COIN trial patients receiving oxaliplatin and fluoropyrimidine chemotherapy +/-cetuximab. Bayesian latent class analysis (LCA) and covariate reduction was performed to analyze the effects of HER2-HER3 dimer, RAS mutation and cetuximab on progression-free survival (PFS) and overall survival (OS). All statistical tests were two-sided. RESULTS: LCA on a cohort of 398 patients revealed two patient subclasses with differing prognoses (median OS: 1624 days [95%CI=1466-1816] vs 461 [95%CI=431-504]): Class 1 (15.6%) showed a benefit from cetuximab in OS (HR = 0.43 [95%CI=0.25-0.76]; p = 0.004). Class 2 showed an association of increased HER2-HER3 with better OS (HR = 0.64 [95%CI=0.44-0.94]; p = 0.02). A class prediction signature was formed and tested on an independent validation cohort (N = 152) validating the prognostic utility of the dimer assay. Similar subclasses were also discovered in full trial dataset (N = 1,630) based on 10 baseline clinicopathological and genetic covariates. CONCLUSIONS: Our work suggests that the combined use of HER dimer imaging and conventional mutation analyses will be able to identify a small subclass of patients (>10%) who will have better prognosis following chemotherapy. A larger prospective cohort will be required to confirm its utility in predicting the outcome of anti-EGFR treatment

Controlling the rates of reductively-activated elimination from the (indol-3-yl)methyl position of indolequinones

A series of substituted 3-(4-nitrophenyloxy)methylindole-4,7-diones (Q) were synthesised. The effects of substitution patterns on the indole core on rates of elimination of 4-nitrophenol as a model for drug release following fragmentation of a phenolic ether linker were studied. After reduction to either the radical anion (Q ؒϪ ) or hydroquinone (QH 2 ) elimination of 4-nitrophenol occurred from the (indol-3-yl)methyl position. The half-lives , typical of tumour hypoxia, were t1 -2 ≈ 0.3-1.8 ms, the higher values associated with higher reduction potentials. Half-lives for the autoxidation of the QH 2 were markedly longer at the same oxygen concentration (t1 -2 ≈ 8-102 min) and longer still in the presence of 4 µmol dm Ϫ3 superoxide dismutase (t1 -2 ≈ 8-19 h). Although the indolequinones were able to eliminate 4-nitrophenol with high efficiency only Q ؒϪ radicals of the 3-carbinyl substituted derivatives did so with sufficiently short half-lives (t1 -2 ≈ 41-2 ms) to compete with electron transfer to oxygen and therefore have the potential to target the leaving group to hypoxic tissue. The hydroquinones are not sufficiently oxygen sensitive to prevent the elimination of 4-nitrophenol (t1 -2 ≈ 1.5-3.5 s) even at oxygen concentrations expected in normal tissue. By incorporating electron rich substituents at the indolyl carbinyl position it is possible to control the rate of reductive fragmentation. This may prove an important factor in the design of an indolequinone-based bioreductive drug delivery system

Intramolecular and Intermolecular Interactions of Protein Kinase B Define Its Activation In Vivo

Protein kinase B (PKB/Akt) is a pivotal regulator of diverse metabolic, phenotypic, and antiapoptotic cellular controls and has been shown to be a key player in cancer progression. Here, using fluorescent reporters, we shown in cells that, contrary to in vitro analyses, 3-phosphoinositide–dependent protein kinase 1 (PDK1) is complexed to its substrate, PKB. The use of Förster resonance energy transfer detected by both frequency domain and two-photon time domain fluorescence lifetime imaging microscopy has lead to novel in vivo findings. The preactivation complex of PKB and PDK1 is maintained in an inactive state through a PKB intramolecular interaction between its pleckstrin homology (PH) and kinase domains, in a “PH-in” conformer. This domain–domain interaction prevents the PKB activation loop from being phosphorylated by PDK1. The interactive regions for this intramolecular PKB interaction were predicted through molecular modeling and tested through mutagenesis, supporting the derived model. Physiologically, agonist-induced phosphorylation of PKB by PDK1 occurs coincident to plasma membrane recruitment, and we further shown here that this process is associated with a conformational change in PKB at the membrane, producing a “PH-out” conformer and enabling PDK1 access the activation loop. The active, phosphorylated, “PH-out” conformer can dissociate from the membrane and retain this conformation to phosphorylate substrates distal to the membrane. These in vivo studies provide a new model for the mechanism of activation of PKB. This study takes a crucial widely studied regulator (physiology and pathology) and addresses the fundamental question of the dynamic in vivo behaviour of PKB with a detailed molecular mechanism. This has important implications not only in extending our understanding of this oncogenic protein kinase but also in opening up distinct opportunities for therapeutic intervention