Characterizing monoclonal antibody structure by carboxyl group footprinting

Structural characterization of proteins and their antigen complexes is essential to the development of new biologic-based medicines. Amino acid-specific covalent labeling (CL) is well suited to probe such structures, especially for cases that are difficult to examine by alternative means due to size, complexity, or instability. We present here a detailed account of carboxyl group labeling (with glycine ethyl ester (GEE) tagging) applied to a glycosylated monoclonal antibody therapeutic (mAb). The experiments were optimized to preserve the structural integrity of the mAb, and experimental conditions were varied and replicated to establish the reproducibility of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include aspartic acid (D), glutamic acid (E), and the C-terminus (i.e., the target probes), with the experimental data in order to understand the accuracy of the approach. Data from the mAb were compared to reactivity measures of several model peptides to explain observed variations in reactivity. Attenuation of reactivity in otherwise solvent accessible probes is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. A comparison of results with previously published data by Deperalta et al using hydroxyl radical footprinting showed that 55% (32/58) of target residues were GEE labeled in this study whereas the previous study reported 21% of the targets were labeled. Although the number of target residues in GEE labeling is fewer, the two approaches provide complementary information. The results highlight advantages of this approach, such as the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling, reproducibility of replicate experiments (<2% variation in modification extent), the similar reactivity of the three target probes, and significant correlation of reactivity and solvent accessible surface area.     

Amino acid flooding doses for measuring rates of protein synthesis

Summary The development, advantages and disadvantages of using the amino acid flooding dose technique to determinein vivo rates of protein synthesis are examined in this review. A discussion of the use of this procedure in animals greater than 5 kg is included. The flooding dose procedure reduces the disparity between isotope enrichment in different amino acid precursor pools, which should theoretically improve the precision and accuracy of protein synthesis measurements. However, the possibility must be considered that the large doses of amino acids injected or infused in conjunction with this technique may influence protein turnover due to attendant metabolic effects. Therefore, a judicious choice of an amino acid and an evaluation of the experimental parameters involved in this procedure are required to optimize the accuracy of results obtained.Scientific Paper No. 710. Agriculture Canada, Lacombe Research Station, Bag Service 5000, Lacombe, Alberta T0C 1S0.     

Dual-energy CT imaging of orbits during episcleral brachytherapy with Ru-106 plaques: A phantom study on its potential for plaque position verification

PurposeTo investigate the potential of dual energy CT (DECT) to suppress metal artifacts and accurately depict episcleral brachytherapy Ru-106 plaques after surgical placement.MethodsAn anthropomorphic phantom simulating the adult head after surgical placement of a Ru-106 plaque was employed. Nine DECT acquisition protocols for orbital imaging were applied. Monochromatic 140 keV images were generated using iterative reconstruction and an available metal artifact reduction algorithm. Generated image datasets were graded by four observers regarding the ability to accurate demarcate the Ru-106 plaque. Objective image quality and visual grading analysis (VGA) was performed to compare different acquisition protocols. The DECT imaging protocol which allowed accurate plaque demarcation at minimum exposure was identified. The eye-lens dose from orbital DECT, with and without the use of radioprotective bismuth eye-shields, was determined using Monte Carlo methods.ResultsAll DECT acquisition protocols were judged to allow clear demarcation of the plaque borders despite some moderate streaking/shading artifacts. The differences between mean observers’ VGA scores for the 9 DECT imaging protocols were not statistically significant (p > 0.05). The eye-lens dose from the proposed low-exposure DECT protocol was found to be 20.1 and 22.8 mGy for the treated and the healthy eye, respectively. Bismuth shielding was found to accomplish >40% reduction in eye-lens dose without inducing shielding-related artifacts that obscure plaque delineation.ConclusionsDECT imaging of orbits after Ru-106 plaque positioning for ocular brachytherapy was found to allow artifact-free delineation of plaque margins at relatively low patient exposure, providing the potential for post-surgery plaque position verification.     

Shielding effectiveness of X-ray protective garment

PurposeCertification of the X-ray shielding garment is based on attenuation testing on flat material samples. We investigated the difference of shielding effectiveness compared to realistic use when the garment is worn on the body of a staff person.MethodsAttenuation factors of X-ray protective aprons have been evaluated for several clinical scenarios with Monte Carlo (MC) calculations based on the ICRP female reference model and an experimental setup. The MC calculated attenuation factors refer to the effective dose E, whereas the measured attenuation factors refer to the personal dose equivalent Hp(10). The calculated/measured factors were compared to the attenuation factors of the identical materials measured under the conditions of the standard IEC 61331-1 that is currently in use for the type testing of X-ray protective aprons.ResultsAs a result, for example, at a common tube voltage of 80 kV, the real attenuation factors of a 0.35 mm Pb apron worn by a 3-dimensional body were 38% to 76% higher than when measured under IEC conditions on flat samples. The MC-calculated organ doses show the maximum contribution to E being within the operatoŕs abdomen/pelvis region.ConclusionsWith our findings, personal X-ray protective garments could be improved in effectiveness.     

Early events in hepatitis B infection: the role of inoculum dose

The relationship between the inoculum dose and the ability of the pathogen to invade the host is poorly understood. Experimental studies in non-human primates infected with different inoculum doses of hepatitis B virus have shown a non-monotonic relationship between dose magnitude and infection outcome, with high and low doses leading to 100% liver infection and intermediate doses leading to less than 0.1% liver infection, corresponding to CD4 T-cell priming. Since hepatitis B clearance is CD8 T-cell mediated, the question of whether the inoculum dose influences CD8 T-cell dynamics arises. To help answer this question, we developed a mathematical model of virus–host interaction following hepatitis B virus infection. Our model explains the experimental data well, and predicts that the inoculum dose affects both the timing of the CD8 T-cell expansion and the quality of its response, especially the non-cytotoxic function. We find that a low-dose challenge leads to slow CD8 T-cell expansion, weak non-cytotoxic functions, and virus persistence; high- and medium-dose challenges lead to fast CD8 T-cell expansion, strong cytotoxic and non-cytotoxic function, and virus clearance; while a super-low-dose challenge leads to delayed CD8 T-cell expansion, strong cytotoxic and non-cytotoxic function, and virus clearance. These results are useful for designing immune cell-based interventions.     

In vivo monitoring of total skin electron dose using optically stimulated luminescence dosimeters

AimThis study retrospectively analysed the results of using optically stimulated radiation dosimeters (OSLDs) for in vivo dose measurements during total skin electron therapy (TSET, also known as TSEI, TSEB, TSEBT, TSI or TBE) treatments of patients with mycosis fungoides.BackgroundTSET treatments are generally delivered to standing patients, using treatment plans that are devised using manual dose calculations that require verification via in vivo dosimetry. Despite the increasing use of OSLDs for radiation dosimetry, there is minimal published guidance on the use of OSLDs for TSET verification.Materials and methodsThis study retrospectively reviewed in vivo dose measurements made during treatments of nine consecutive TSET patients, treated between 2013 and 2018. Landauer nanoDot OSLDs were used to measure the skin dose at reference locations on each patient, as well as at locations of clinical interest such as the head, hands, feet, axilla and groin.Results1301 OSLD measurements were aggregated and analysed, producing results that were in broad agreement with previous TLD studies, while providing additional information about the variation of dose across concave surfaces and potentially guiding future refinement of treatment setup. In many cases these in vivo measurements were used to identify deviations from the planned dose in reference locations and to identify anatomical regions where additional shielding or boost treatments were required.ConclusionsOSLDs can be used to obtain measurements of TSET dose that can inform monitor unit adjustments and identify regions of under and over dosage, while potentially informing continuous quality improvement in TSET treatment delivery.     

Dose to pelvic lymph nodes in image based high dose rate brachytherapy of carcinoma cervix

AimThe aim of this study is to analyse the dosimetry to the pelvic lymph nodes and its correlation to point B using CT based high dose rate brachytherapy of carcinoma cervix.BackgroundConventionally, dose to pelvic lymph nodes from intracavitary brachytherapy was reported by point B and by the reference points of the lymphatic trapezoid.Materials and methods30 consecutive CT based high dose rate applications were reviewed between February and March 2016. The high risk clinical target volume and the organs at risk and the pelvic nodal groups were contoured. DVH parameters for the right and left obturator nodal group, right and left external iliac nodal group and right and left internal iliac nodal group were recorded. Right and left point B doses were also recorded.ResultsOn analysis of the combined dose, it was found that all the DVH parameters were significantly different from point B, except the D100 obturator and D2cc internal iliac lymph node. There was a significant correlation between all DVH parameters and point B, except D2cc, D1cc and D0.1cc of external iliac. The obturator group received the highest dose contribution from brachytherapy. The mean D90 dose received per fraction for the obturator, external iliac and internal iliac nodes was 2.7 Gy, 1.17 Gy and 1.41 Gy, respectively.ConclusionsThere is a significant dose contribution to the pelvic lymph nodal groups during intracavitary brachytherapy. There is a low degree of correlation between point B dose and dosimetric parameters of the individual nodal groups. Hence, it is important to analyse the dose delivered to individual nodal groups during intracavitary brachytherapy, at least in patients with enlarged lymph nodes to calculate the cumulative dose delivered.     

The reduction of aboveground Calamagrostis epigeios mass and tiller number by enhanced UV-B in a dune-grassland ecosystem

Since early May 1997 dune-grassland vegetation in the Netherlands has been exposed to enhanced levels of ultraviolet-B (UV-B) radiation. Expected increases in the amount of biologically effective UV-B (UV-B_BE) upon a reduction of the stratospheric ozone layer with 15% were calculated and artificially supplemented.In June and September 1998, above- and belowground vegetation samples were taken. Of the dominant species Calamagrostis epigeios and Carex arenaria aboveground mass accumulation, leaf weight (LW), leaf area (LA), specific leaf area (SLA) and tiller number were assessed separate from the remaining vegetation.The results of our study indicate alterations in the vegetation structure due to UV-B supplementation. In June, a significant reduction due to UV-B supplementation in number of tillers and aboveground dry weight per soil area unit was found for C. epigeios. As C. epigeios is the most dominant species of the dune-grassland, these effects indicate a change in vegetation structure due to UV-B enhancement. Indications of UV-B effects on other parameters, such as the number of tillers of C. arenaria and aboveground plant dry weight of the group of species other than C. epigeios and C. arenaria, may also represent changes in vegetation structure. The LA and LW data show the same patterns as the mass accumulation trends. No significant UV-B effects on the SLA of the species or of the total vegetation could be found.Trends in patterns of species dry weight accumulation and partitioning of dry weight between species groups are different in June and September. This may indicate seasonal dependence of UV-B responses. Also, the consistently reducing trend in total and aboveground plant dry weight may indicate deleterious effects of UV-B on total plant matter accumulation. Possible causes of observed trends and effects are discussed.     

Prevalence of heavy metals and computation of its associated risk in surface water consumed in Ado-Odo Ota,South-West Nigeria

Heavy metal contamination levels, their potential sources and associated health risk of River Balogun – a major source of water for daily domestic activities–were investigated using statistical techniques and some health risk indices. To achieve this, 51 samples from 17 stations (STs) along the river were collected in December 2016 and analyzed for the presence of Lead (Pb), Zinc (Zn), Nickel (Ni), Arsenic (As), and Copper (Cu). The results of the study revealed the presence of As, Ni, and Pb concentration levels exceeding or equal to World Health Organization standards. In addition, the application of both methods used revealed that low pH values measured from the river could emanate from leachate of agrochemicals and carbonic acid from decayed plants while high Nickel and Arsenic concentrations were from the overapplication of fertilizers used in farms surrounding the river and found their way into the river after precipitation. Health risk assessment showed that all the water sampled at different STs would pose a serious threat to the health of children and adults overtime especially due to elevated arsenic concentration measured in all STs.