Internal Marker Research Articles

A Framework for ExacTrac Dynamic Commissioning for Stereotactic Radiosurgery and Stereotactic Ablative Radiotherapy.

This paper aims to provide guidance and a framework for commissioning tests and tolerances for the ExacTrac Dynamic image-guided and surface-guided radiotherapy (SGRT) system. ExacTrac Dynamic includes a stereoscopic X-ray system, a structured light projector, stereoscopic cameras, thermal camera for SGRT, and has the capability to track breath holds and internal markers. The system provides fast and accurate image guidance and intrafraction guidance for stereotactic radiosurgery and stereotactic ablative radiotherapy. ExacTrac Dynamic was commissioned on a recently installed Elekta Versa HD. Commissioning tests are described including safety, isocenter calibration, dosimetry, image quality, data transfer, SGRT stability, SGRT localization, gating, fusion, implanted markers, breath hold, and end-to-end testing. Custom phantom designs have been implemented for assessment of the deep inspiration breath-hold workflow, the implanted markers workflow, and for gating tests where remote-controlled movement of a phantom is required. Commissioning tests were all found to be in tolerance, with maximum translational and rotational deviations in SGRT of 0.3 mm and 0.4°, respectively, and X-ray image fusion reproducibility standard deviation of 0.08 mm. Tolerances were based on published documents and upon the performance characteristics of the system as specified by the vendor. The unique configuration of ExacTrac Dynamic requires the end user to design commissioning tests that validate the system for use in the clinical implementation adopted in the department. As there are multiple customizable workflows available, tests should be designed around these workflows, and can be ongoing as workflows are progressively introduced into departmental procedures.

The Role of Stereotactic-Body-Radiation-Therapy in Neuroendocrine Pancreatic Tumors

Introduction: Surgery is standard treatment for pancreatic-neuroendocrine-tumors (TNP) although it is occasionally associated with high-rates of morbid-mortality. Radiotherapy can be an effective alternative in selected patients. Our objective is to evaluate the efficacy of SBRT (Stereotactic-body-radiation-therapy) as a radical treatment for TNP. Materials and Methods: Between December 2017 and November 2020, we prospectively analyzed 13 patients with pathological diagnosis of TNP of <2cm treated with SBRT as a part of an IRB-approved study. The endpoints of the study are local-control, tolerance, progression-free-survival and overall-survival. Before SBRT, two internal fiducial markers were placed into the tumor by endoscopic ultrasound puncture and a simulation-CT was obtained. Clinical-target-volume (CTV) included pancreatic tumor. Planning-target-volume (PTV) was CTV with 5mm expansion. Patients received 40Gy in 5 fractions on every other day. Results: Patients’ characteristics: 6 female (46%) and 7 males (54%) with a mean age of 55.6 years-old. Tumor location: head 3p (23%), neck 3p (23%), body 6p (46%) and tail of pancreas 1p (8%). Octreoscan prior to radiotherapy was positive in 8p (61.5%). FDG-PET was negative for all but one patient (SUV <4). With a median follow-up of 13.6 months (range 1.8- 34.4), all patients are without evidence of relapse. Acute tolerance was excellent. Only 3p (23%) showed asthenia. One p developed peptic ulcer on follow-up. Conclusions: SBRT is feasible and well tolerated and could be an alternative in selected patients with diagnoses of TNP, although further studies are necessary.

Quantitation of IgG Subclasses in Serum Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Serum IgG subclasses (IgGSC) are measured for a number of indications, but the most common are the identification of selective immunodeficiency disease and the diagnosis of IgG4-related disease (IgG4RD). Traditional nephelometric (IN) assays can suffer from two issues impacting the accuracy of the results: (1) hook effect and (2) antibody cross-reactivity between the subclasses. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is not vulnerable to these modes of interference and therefore serves as an excellent and relatively inexpensive means of diagnosing and/or monitoring the relevant clinical conditions.We describe a semiautomated and simple method for the accurate and precise measurement of IgGSC from 20μL of serum using a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method following digestion of serum proteins in 96-well plate format. Due to the high abundance of the target proteins, no specialized sample preparation (such as solid phase extraction) is required. Twenty microliters are injected to the LC-MS/MS system. Quantitation is performed against a five-point duplicate linear calibration curve prepared in blank matrix. The assay calibration range is 0.38-7.74g/L for IgG1, 0.24-4.46g/L for IgG2, 0.038-0.752g/L for IgG3, 0.025-0.435g/L for IgG4, and 0.62-15.5g/L for total IgG. Total IgG is used as an internal quality control marker and is compared to the sum of the four subclass results. Total imprecision in clinical production has been observed to be 5.1-10.6% for in-house prepared control materials having IgGSC mean values in the range of 0.38-8.43g/L for IgG1, 0.22-3.76g/L for IgG2, 0.0387-0.721g/L for Ig3, and 0.0279-1.46g/L for IgG4. Limit of quantitation (LoQ) was determined to be 0.29g/L for IgG1, 0.22g/L for IgG2, 0.019g/L for IgG3, and 0.0067g/L for IgG4.

Four-Dimensional Computed Tomography-Based Correlation of Respiratory Motion of Lung Tumors With Implanted Fiducials and an External Surrogate

PurposeOur purpose was to assess the suitability of airway-implanted internal fiducial markers and an external surrogate of respiratory motion for motion management during radiation therapy of lung tumors. Methods and MaterialsWe analyzed 4-dimensional computed tomography scans acquired during radiation therapy simulation for 28 patients with lung tumors who had anchored fiducial markers bronchoscopically implanted inside small airways in or near the tumor in a prospective trial. We used a linear mixed model to build population-based correlative models of tumor and surrogate motion. The first 24 of the 28 patients were used to build correlative models, and 4 of the 28 consecutive patients were excluded and used as an internal validation cohort. Of the 24 patients from the model building cohort, all were used for the models based on the internal fiducial. The external surrogate was completely visualized in 11 patients from the model building cohort, so only those were used for the models based on the external surrogate. Furthermore, we determined the predicted residual error sum of squares for our correlative models, which may serve as benchmarks for future research. ResultsThe motion of the internal fiducials was significantly associated with the tumor motion in the anterior-posterior (P < .0001) and superior-inferior (SI) directions (P < .0001). We also observed a strong correlation of the external surrogate anterior-posterior motion to the tumor dominant SI motion (P < .0001). In the validation cohort, the internal fiducial SI motion was the only reliable predictor of lung tumor motion. ConclusionsThe internal fiducials appear to be more reliable predictors of lung tumor motion than the external surrogate. The suitability of such airway-implanted internal fiducial markers for advanced motion management techniques should be further investigated. Although the external surrogate seems to be less reliable, its wide availability and noninvasive application support its clinical utility, albeit the greater uncertainty will need to be compensated for.

Use of internal markers to estimate feed intake and selection of forage in sheep fed grass and legume hay

The accurate prediction of feed intake is the most important information required to assess the feed conversion efficiency of individual grazing animals. The objective of this study was to evaluate the accuracy of three approaches using the combined performance of four internal markers: indigestible dry matter (iDM); indigestible neutral detergent fiber (iNDF); titanium (Ti); and the isotopic signature of carbon (δ13C). These markers were used to predict the individual intakes of sheep that were fed grass and forage legumes. Twenty-one Santa Inês sheep were randomly divided according to three diets: 100% Megathyrsus maximus cv. Aruana grass (CTL diet); 25% inclusion (as fed basis) of Macrotyloma axillare legume (MAC diet); and 50% inclusion (as fed basis) of Stizolobium aterrimum legume (MUC diet). After the 10-day diet adaptation period, the animals were transferred to individual metabolic cages, where they were kept for seven days with daily measurements and collection of feed offerings, feed leftovers, and feces samples. The iDM and iNDF fractions of the samples were determined after 240 h of in situ incubation. The natural Ti concentration was determined by benchtop energy dispersive X-ray fluorescence spectrometry (EDXRF). The abundance of 13C was determined by mass spectrometry. The accuracy of the predictions of digestibility and intake was increased when the fecal recovery rate of the internal markers (P < 0.05) was corrected. The proportion of grass and legumes in the feces was accurately predicted by the 13C method. The combined use of iNDF and 13C markers could be an alternative for predicting individual digestibility and intake of grasses and legumes by animals that are fed mixed diets.

Proximity hybridization induced DNA assembly for label-free surface-enhanced Raman spectroscopic detection of carcinoembryonic antigen

In our research, label-free and surface-enhanced Raman dyes-free Raman spectroscopy which was used to detect carcinoembryonic antigen (CEA) according to poly adenine (Poly A)-regulated self-assembly methods was developed and studied. CEA induced partial hybridization of Ab-H2 and Ab-H1, and Ab-H1-CEA-Ab-H2 (a sandwich proximity CEA-DNA complex) was formed, which unfolded molecular beacon 1 (MB1) and modified the substrate. Subsequently, MB2-AuNPs were hybridized with MB1, and Ab-H1-CEA-Ab-H2 was released via toehold regulated displacements of DNA strands. Therefore, hybridization processes of MB2 and MB1 were induced and promoted by CEA-DNA complexes which worked as catalysts. The misplaced target then induced a next round of strand exchange, and the signals for determination of CEA were amplified by AuNPs absorbed on the substrate. It was indicated that the spectral characteristics of adenine at 736 cm−1 were consistent with the SERS spectrum of DNA. Adenine acted as an internal marker for label-free SERS detection of CEA. Moreover, satisfactory stability and reproducibility were found. Meanwhile, the antibody could specifically recognize the corresponding antigen. Since adenine was dominant in SERS spectra, which was also proximal to Au surface, the sensitivity of the novel method was high without modifications. The analytical performance of this method in determining serum CEA was satisfactory.

Intrafraction Accuracy and Efficiency of a Surface Imaging System for Deep Inspiration Breath Hold in Ablative Pancreatic Cancer Treatment

Beam gating with deep inspiration breath hold (DIBH) has been widely used for motion management in radiotherapy. It commonly relies on some external surrogate for internal target motion, while the exact internal motion is unknown. In this study, we tracked internal targets and characterized the residual motion during DIBH treatment for pancreatic cancer patients guided by a surface imaging system. We also report the treatment time statistics.Eight consecutive patients with pancreatic cancer treated with surface-guided DIBH volumetrically-modulated arc therapy in 2019-2020 were selected for this study, each with some radiopaque markers (fiducial markers or surgical clips) implanted near or inside the target. Five patients were treated in 25 fractions, two were treated in 15 fractions, and one was treated in 10 fractions. The surface imaging system monitored a three-dimensional surface with gating thresholds of 3 mm translation and 3° rotation. During dose delivery, kV images were automatically taken every 20˚ or 40˚ gantry rotation, from which internal markers were identified. The marker displacement from their initial positions and the residual motion amplitudes were calculated. For the analysis of treatment efficiency, the treatment time of every session was obtained from imaging and treatment timestamps recorded at the treatment console.Even though the external surface was limited to 3 mm / 3°, significant residual internal target motion was observed for some patients. The residual motion ranged 3-21 mm. The average target displacement relative to the initial position was 0-3 mm for the cohort. The magnitude of instantaneous displacement was > 5 mm in 13% of the images analyzed. The average treatment time was 16 min with standard deviation 3 min.Internal target motion may differ significantly from external surrogate motion in DIBH treatment, even with surface image guidance. Radiographic verification of target position at the beginning and during each fraction is essential for accurate delivery of radiotherapy.

339 Effects of a Blend of Mannan and Glucan on Growth Performance and Immunocompetence of Newly Weaned Beef Steers

Abstract We examined the effects of dietary supplementation of a blend of mannan and glucan on the growth performance, diet digestibility, and whole-blood immune gene expression of newly weaned beef steers during a 42-d receiving period. Forty-eight newly weaned Angus crossbred steers (2 d post-weaning; 199 ± 13 kg of initial BW) were randomly assigned to 1 of 2 treatments: basal diet with no additive (CON; n = 24) or a basal diet top-dressed with 5 g of a blend of mannan and glucan (MANGLU; n = 24). Average daily gain (ADG) was calculated from weekly BW. Blood samples were collected on days 0, 14, and 42 for measurement of plasma glucose and NEFA. Expression of 84 immune-related genes was analyzed on blood samples collected on day 42. Beginning on days 37 to 42, total mixed ration, refusals, and fecal samples were collected once daily to determine apparent total tract digestibility of DM, CP, NDF, and ADF using indigestible NDF as an internal marker. Immune gene expression data was analyzed using a GeneGlobe Data Analysis Center. All other data were analyzed as a randomized complete block design using the GLIMMIX procedure of SAS. Over the 42-d feeding trial, MANGLU tended to increase final BW (P = 0.07) and ADG (P = 0.06). Compared to CON, beef steers fed supplemental MANGLU had greater DM digestibility (P = 0.03) and tended to have greater NDF digestibility (P = 0.09). No treatment effects (P &amp;gt; 0.10) on plasma glucose and NEFA on d 14 and 42 were detected. Compared with CON, MANGLU increased (P ≤ 0.05) the expression of five immune-related genes involved in recognition of and mounting immune defense against microbial pathogens. In conclusion, this study demonstrated that supplemental MANGLU enhances beef cattle immunocompetence and productivity during feedlot receiving period.

331 Effects of Dried Distillers’ Grains Cube Supplementation Rate on Voluntary Intake and Digestibility of Bermudagrass Hay Fed to Growing Heifers

Abstract The objective of this study was to evaluate supplementation rates of dried distillers’ grains cubes (DDGS) on voluntary intake (DMI), rate and extent of digestibility, and blood parameters of growing Charolais-cross heifers (BW = 286 ± 28.9 kg) fed ad libitum bermudagrass (Cynodon dactylon) hay. For the 29-d study, heifers were randomly assigned to one of four supplementation treatments: 1) Control, no supplement (n = 6); 2) Low, supplemented 0.90 kg/d (n = 6); 3) Intermediate, supplemented 1.81 kg/d (n = 5); or 4) High, supplemented 3.62 kg/d (n = 6). Heifers were offered supplement each morning in individual stalls. From d 15 to 25, heifers were dosed twice daily with titanium dioxide in gelatin capsules (10 g/d), and fecal samples were collected via rectum at time of dosing from d 22 to 25 to estimate fecal output. Post-dosing, fecal sampling continued over 96-h from d 25 to 29 to determine passage rate (Kp) via titanium dioxide analysis. Blood was collected from each animal on d 26 via jugular venipuncture before supplementation, and 4 and 8-h post-supplementation for analysis of blood urea N (BUN), lactate, and glucose. Samples of feces, hay, and supplement were incubated in rumen-cannulated Holstein steers (n = 4; BW = 281 ± 29.5 kg) for 576-h to estimate digestibility with indigestible neutral detergent fiber as an internal marker. Data were analyzed by ANOVA using the mixed procedure of SAS (SAS Inst. Inc. Cary NC), with treatment least-squares means separated using orthogonal polynomial contrasts. Increasing DDGS linearly (P &amp;lt; 0.01) decreased forage DMI linearly, but linearly increased Kp, total diet DMI, and total diet digestibility. Linear increases (P ≤ 0.02) in glucose, lactate, and BUN were likewise observed with increasing DDGS. Increasing DDGS supplementation to growing calves consuming bermudagrass hay may increase productivity while reducing forage intake.

305 Methane Emissions and Apparent Total Tract Nutrient Digestibility of Feedlot Beef Steers Under Intensive Management with or Without an Added Nutritional Packet

Abstract The effects of a nutritional packet were evaluated on CH4 emissions and apparent total tract nutrient digestibility of feedlot beef steers. Thirty Angus-crossbred steers (BW = 542 ± 8.4 kg) were used in a randomized complete block design and allocated into pens equipped with SmartFeed (C-Lock; 15 steers/treatment). Steers were consuming a steam-flaked corn-based diet (88% concentrate DM basis) ad libitum for the last 65 d on feed, and received the following treatments: 1) control and 2) a nutritional packet [0.29% DM basis; live yeast (Saccharomyces cerevisiae; 8.7 Log CFU/g); Vitamin C (5.4 g/kg); Vitamin B1 (13.33 g/kg); NaCl (80 g/kg); KCl (80 g/kg)]. Methane emissions and apparent total tract nutrient digestibility were measured during 3 periods with 5-d of collections each. Gas emissions from steers were measured utilizing the SF6 tracer technique. Feed and fecal samples were collected once and twice (0700 h and 1600 h) daily, respectively, to determine digestibility of nutrients using iNDF as an internal marker. Steer was considered the experimental unit. Data were analyzed as repeated measures using the MIXED procedure of SAS with the fixed effects of treatment, period, and their interaction, and the random effect of block. No treatment × period interactions (P ≥ 0.125) were observed for DMI and any of the CH4 production variables (g/day, g/kg BW0.75, g/nutrient intake, and g/nutrient digested). Moreover, treatments did not affect digestibility of DM, OM, or ADF (P ≥ 0.300); however, digestibility of NDF was increased for treated cattle (P = 0.013), which resulted in a tendency (P = 0.098) to decrease CH4 production in g per kg NDF intake and decreased (P = 0.020) grams CH4 per kg NDF digested. The nutritional packet may be altering ruminal fermentation on intensively managed steers and improving fiber digestibility, which can have benefits on CH4 emission intensity.

309 Nutrient Digestibility of Beef Steers Consuming Finishing Diets with an Added Nutritional Packet

Abstract The effects of a nutritional packet fed to beef steers during the final 64 d of the feedlot finishing phase on apparent total tract nutrient digestibility were evaluated. Angus-crossbred steers (n =120; initial BW = 544 ± 52 kg) were assigned to 30 pens (4 steers/pen; 15 pens/treatment; pen = experimental unit) in a randomized complete block design. A steam-flaked corn-based finishing diet was fed ad libitum and treatments applied as follows: 1) control and 2) 30 g/steer-daily (DM-basis) of the nutritional packet [containing live yeast (Saccharomyces cerevisiae; 8.7 Log CFU/g), vitamin C (5.4 g/kg), vitamin B1 (13.33 g/kg), NaCl (80 g/kg) and KCl (80 g/kg)]. The nutritional packet used ground corn as a carrier and was included at 1% of diet DM. Orts were quantified daily (if any) and subtracted from the total dietary DM offered to calculate DM intake. Fecal samples were collected twice daily (0700 and 1700 h) for 5 consecutive days (d 47 to 51) from at least 3 steers within pen. Feed samples were collected daily at time of feeding (0630 h). Fecal and feed samples were composited to represent the collection period, dried (55oC), ground (1 mm), and analyzed to assess DM, OM, NDF, ADF, and hemicellulose. In situ (288 h) iNDF was used as an internal marker to measure apparent total tract nutrient digestibility. Data were analyzed using the GLIMMIX procedure of SAS. Intake of DM, OM, and fiber components during the digestibility period was not affected (P ≥ 0.44) by treatment. Steers fed the nutritional packet had increased apparent total tract digestibility of OM (P = 0.02), DM (P = 0.01), NDF (P = 0.02), ADF (P = 0.01), and hemicellulose (P = 0.08; tendency). Improved nutrient digestibility without affecting nutrient intake may warrant an enhanced energy deposition in the carcass.

Diet, marker and fecal sampling method interactions with internal and external marker pairs when estimating dry matter intake in beef cattle

The use of markers is currently the most used technique for estimating feed intake in production animals when direct measurement is not possible. Twelve growing Nellore bulls were used in a crossover design aiming to evaluate the accuracy of internal markers to estimate dry matter digestibility (DMD), the accuracy of external markers to estimate fecal output (FO), and the combination of internal and external markers to estimate dry matter intake (DMI) at different fecal sampling procedures. Animals received one of the four diets which varied in forage source (corn silage or Tifton-85 hay) and forage to concentrate ratio (F:C; 60:40 or 40:60) in a 2 × 2 factorial arrangement. The internal markers, acetyl bromide lignin (ABL), indigestible aNDFom (iNDF), indigestible ADFom (iADF) and cutin, are naturally present in the feedstuffs and the external markers, Cr2O3 and TiO2, were daily fed to the animals. Three fecal sampling procedures were tested: bulk 72-h (continuous feces collection for 72 h), grab 9 × 3 (sample collection every 9 h over 3-d period), and grab 4 × 4 (4 fecal samples collected during daylight hours over 4-d period). None of the internal or external markers evaluated had complete fecal recovery (FR), being necessary to establish the FR of the markers in order to obtain correct estimates. The FR of ABL, iNDF and iADF were close to 100% when animals received a hay-based diets but close to 50% when feeding a corn silage-based diets, regardless the F:C ratio of the diet. However, ABL produced accurate DMD estimates only with both grab sampling procedures across all diets, while iNDF and iADF produced accurate DMD estimates across all sampling procedures and diets. Cutin failed to produce accurate DMD consistently. Both external markers produced accurate FO under both grab sampling procedures, except for TiO2 when the grab 4 × 4 sampling procedure was performed for animals receiving the diet with high silage proportion. In general, the grab sampling procedures yielded accurate DMD, FO and DMI estimates, which were similar or better than bulk 72 h sampling procedure. The combination of internal and external markers to estimate DMI produced satisfactory and accurate results, particularly when Cr2O3 or TiO2 was paired with iNDF or iADF under both grab sampling procedures. The grab sampling procedures can replace TFC, opening new possibilities for collectively housed animals.

QTL Mapping of a Novel Genomic Region Associated with High Out-Crossing Rate Derived from Oryza longistaminata and Development of New CMS Lines in Rice, O. sativa L.

High seed cost due to poor seed yield severely limits the adoption of hybrid rice by farmers. Increasing the out-crossing rate is one of the key strategies to increase hybrid seed production. Out-crossing rate is highly influenced by the size of female floral traits, which capture pollen grains from male donor plants. In the current study, we identified 14 QTLs derived from the perennial wild rice Oryza longistaminata by composite interval mapping for five key floral traits: stigma length (five), style length (three), stigma breadth (two), stigma area (one), and pistil length (three). QTL analysis and correlation studies revealed that these stigma traits were positively correlated and pleiotropic to the stigma length trait. We selected the major-effect QTL qSTGL8.0 conferring long stigma phenotype for further fine mapping and marker-assisted selection. The qSTGL8.0 (~ 3.9 Mb) was fine mapped using newly developed internal markers and was narrowed down to ~ 2.9 Mb size (RM7356–RM256 markers). Further, the flanking markers were validated in a segregating population and in progenies from different genetic backgrounds. The markers PA08-03 and PA08-18 showed the highest co-segregation with the stigma traits. The qSTGL8.0 was introgressed into two cytoplasmic male sterile (CMS) lines, IR58025A and IR68897A, by foreground, background, and trait selection approaches. The qSTGL8.0 introgression lines in CMS backgrounds showed a significantly higher seed setting rate (2.5–3.0-fold) than the original CMS lines in test crosses with their corresponding maintainer lines. The newly identified QTLs especially qSTGL8.0, will be quite useful for increasing out-crossing rate and this will contribute to increase seed production and decrease seed cost.

Association between serum markers of the humoral immune system and inflammation in the Swedish AMORIS study

BackgroundAlthough the onset of inflammatory cascades may profoundly influence the nature of antibody responses, the interplay between inflammatory and humoral (antibody) immune markers remains unclear. Thus, we explored the reciprocity between the humoral immune system and inflammation and assessed how external socio-demographic factors may influence these interactions. From the AMORIS cohort, 5513 individuals were identified with baseline measurements of serum humoral immune [immunoglobulin G, A & M (IgG, IgA, IgM)] and inflammation (C-reactive protein (CRP), albumin, haptoglobin, white blood cells (WBC), iron and total iron-binding capacity) markers measured on the same day. Correlation analysis, principal component analysis and hierarchical clustering were used to evaluate biomarkers correlation, variation and associations. Multivariate analysis of variance was used to assess associations between biomarkers and educational level, socio-economic status, sex and age.ResultsFrequently used serum markers for inflammation, CRP, haptoglobin and white blood cells, correlated together. Hierarchical clustering and principal component analysis confirmed the interaction between these main biological responses, showing an acute response component (CRP, Haptoglobin, WBC, IgM) and adaptive response component (Albumin, Iron, TIBC, IgA, IgG). A socioeconomic gradient associated with worse health outcomes was observed, specifically low educational level, older age and male sex were associated with serum levels that indicated infection and inflammation.ConclusionsThese findings indicate that serum markers of the humoral immune system and inflammation closely interact in response to infection or inflammation. Clustering analysis presented two main immune response components: an acute and an adaptive response, comprising markers of both biological pathways. Future studies should shift from single internal marker assessment to multiple humoral and inflammation serum markers combined, when assessing risk of clinical outcomes such as cancer.

Real-time CT image generation based on voxel-by-voxel modeling of internal deformation by utilizing the displacement of fiducial markers.

To show the feasibility of real-time CT image generation technique utilizing internal fiducial markers that facilitate the evaluation of internal deformation. In the proposed method, a linear regression model that can derive internal deformation from the displacement of fiducial markers is built for each voxel in the training process before the treatment session. Marker displacement and internal deformation are derived from the four-dimensional computed tomography (4DCT) dataset. In the treatment session, the three-dimensional deformation vector field is derived according to the marker displacement, which is monitored by the real-time imaging system. The whole CT image can be synthesized by deforming the reference CT image with a deformation vector field in real-time. To show the feasibility of the technique, image synthesis accuracy and tumor localization accuracy were evaluated using the dataset generated by extended NURBS-Based Cardiac-Torso (XCAT) phantom and clinical 4DCT datasets from six patients, containing 10 CT datasets each. In the validation with XCAT phantom, motion range of the tumor in training data and validation data were about 10 and 15mm, respectively, so as to simulate motion variation between 4DCT acquisition and treatment session. In the validation with patient 4DCT dataset, eight CT datasets from the 4DCT dataset were used in the training process. Two excluded inhale CT datasets can be regarded as the datasets with large deformations more than training dataset. CT images were generated for each respiratory phase using the corresponding marker displacement. Root mean squared error (RMSE), normalized RMSE (NRMSE), and structural similarity index measure (SSIM) between the original CT images and the synthesized CT images were evaluated as the quantitative indices of the accuracy of image synthesis. The accuracy of tumor localization was also evaluated. In the validation with XCAT phantom, the mean NRMSE, SSIM, and three-dimensional tumor localization error were 7.5±1.1%, 0.95±0.02, and 0.4±0.3mm, respectively. In the validation with patient 4DCT dataset, the mean RMSE, NRMSE, SSIM, and three-dimensional tumor localization error in six patients were 73.7±19.6 HU, 9.2±2.6%, 0.88±0.04, and 0.8±0.6mm, respectively. These results suggest that the accuracy of the proposed technique is adequate when the respiratory motion is within the range of the training dataset. In the evaluation with a marker displacement larger than that of the training dataset, the mean RMSE, NRMSE, and tumor localization error were about 100 HU, 13%, and <2.0mm, respectively, except for one case having large motion variation. The performance of the proposed method was similar to those of previous studies. Processing time to generate the volumetric image was <100ms. We have shown the feasibility of the real-time CT image generation technique for volumetric imaging.

Association between carotid artery perivascular fat density and cerebral small vessel disease.

Studies aiming to identify the significance of the carotid artery perivascular fat density are limited. The present study investigated the distribution pattern of pericarotid fat and its association with imaging markers of cerebral small vessel disease (CSVD). In total, 572 subjects who underwent both neck computed tomography angiography and cranial magnetic resonance imaging were analyzed. The pericarotid fat density near the origin of the internal carotid artery (ICA) and imaging markers of CSVD, such as lacunes, white matter hyperintensities (WMHs) and dilated perivascular spaces (PVSs), were assessed. We found that an increased pericarotid fat density was associated with the presence of lacunes and a higher WMH grade in all subjects, but in the patients with acute ischemic stroke, there was a difference only among the WMH grades. There was no significant difference in the pericarotid fat density in different grades of PVSs. The patients with acute ischemic stroke had a significantly higher mean pericarotid fat density than those without stroke. In conclusion, our study provides evidence suggesting that an increased pericarotid fat density is associated with the presence and degree of WMHs and lacunes. Our findings suggested that features that appear to extend beyond the vessel lumen of the ICA may be linked to CSVD.

Validating the n-alkane technique for determining herbage dry matter intake in sheep offered perennial ryegrass harvested at varying growth stages and seasons

Optimising nutrition for animals requires accurate estimates of feed intake. In research studies determining feed intake in grazing animals can be problematic with many methods requiring individual housing. The n-alkane technique whereby long chain alkanes are used as internal markers for predicting dry matter intake (DMI), facilitates estimation of feed intake from animals while grazing. The aim of this study was to determine the suitability of the n-alkane technique for estimating grass DMI from sheep grazing perennial ryegrass at three different herbage masses and across three seasons. A total of 242 in vivo DMI measurements were obtained from 50 Texel wethers across spring, summer and autumn from 2012 to 2014 using the total collection method; whereby herbage offered and refused plus total faeces produced was measured for each animal daily. Animals were offered perennial ryegrass ad libitium which was harvested at three different pre-grazing herbage masses (HM; low (<1600 kg DM/ha), medium (1601−2999 kg DM/ha), and high (>3000 kg DM/ha), across three separate seasons (spring, summer and autumn). All animals were dosed once daily with a C32 n-alkane bolus for 12 days with a subsample of faeces collected for the final six days of each measurement phase. Lin’s concordance correlations were used to examine the relationship between each n-alkane pair when predicting DMI. The Lin’s concordance correlation coefficients between n-alkane estimated DMI using the ratio of C31 / C32 and C33 / C32 and measured DMI were 0.46 and 0.59, respectively. The r-squared value between measured DMI and n-alkane estimated DMI was 0.76 using the n-alkane pair C31 / C32 and 0.79 for the n-alkane pair C33 / C32. Herbage mass affected DMI with animals offered the low HM treatment having higher intakes (P < 0.05). Offering animals low and medium HM provided a more accurate estimate of DMI using the n-alkane technique. Thus, this research provides evidence that the n-alkane technique can be used as a reliable predictor of perennial ryegrass intake in sheep, when offered HM less than 2999 kg DM/ha.

Prediction of target position from multiple fiducial markers by partial least squares regression in real-time tumor-tracking radiation therapy.

The purpose of this work is to show the usefulness of a prediction method of tumor location based on partial least squares regression (PLSR) using multiple fiducial markers. The trajectory data of respiratory motion of four internal fiducial markers inserted in lungs were used for the analysis. The position of one of the four markers was assumed to be the tumor position and was predicted by other three fiducial markers. Regression coefficients for prediction of the position of the tumor-assumed marker from the fiducial markers’ positions is derived by PLSR. The tracking error and the gating error were evaluated assuming two possible variations. First, the variation of the position definition of the tumor and the markers on treatment planning computed tomograhy (CT) images. Second, the intra-fractional anatomical variation which leads the distance change between the tumor and markers during the course of treatment. For comparison, rigid predictions and ordinally multiple linear regression (MLR) predictions were also evaluated. The tracking and gating errors of PLSR prediction were smaller than those of other prediction methods. Ninety-fifth percentile of tracking/gating error in all trials were 3.7/4.1 mm, respectively in PLSR prediction for superior–inferior direction. The results suggested that PLSR prediction was robust to variations, and clinically applicable accuracy could be achievable for targeting tumors.

In vivo and in vitro Digestibility of an Extruded Complete Dog Food Containing Black Soldier Fly (Hermetia illucens) Larvae Meal as Protein Source.

Growing attention is being directed toward insects as a novel and sustainable source of protein for pet food. The aim of the study was to evaluate nutrient digestibility of a diet containing black soldier fly larvae as its main protein source. Moreover, the purpose of the study was to compare the traditional in vivo total collection method with the in vivo marker method and in vitro digestibility method. Two isonitrogenous and isoenergetic dry diets containing either venison meal (CTRL diet) or black soldier fly larvae meal (BSF diet) as their primary sources of proteins were fed to six adult dogs, according to a Latin square design. The digestibility of nutrients was determined using both in vivo (“total collection” and “internal marker” approaches) and in vitro methods. The two diets showed similar nutrient digestibility values for dry matter, organic matter, ether extract, ash, and phosphorus. However, a statistical trend (p = 0.066) was observed indicating greater protein digestibility in the BSF diet compared with the CTRL diet. Calcium digestibility was higher in the BSF diet compared with the CTRL diet (p = 0.018). On the contrary, fiber digestibility was lower in the insect-based diet compared with the venison diet (p < 0.001). There was no difference between total collection and internal marker methods in the assessment of in vivo digestibility for any of the nutrients considered. The in vitro digestibility values for dry matter, organic matter, and crude protein, as well as the estimated in vivo digestibility of organic matter and crude protein by the means of the predictive equation, were aligned with the in vivo results, although in vitro estimations were consistently higher compared with those obtained by in vivo analysis. Digestibility analysis of a dog food containing insect meal as the sole source of protein (36.5% inclusion) showed promising results in terms of it presenting similar values as a meat-based diet, indicating its suitability as a sustainable protein source for pet food. Moreover, the study showed that both the in vivo marker method and the in vitro method could be possible alternatives to the traditional total collection method in digestibility trials.

Benchtop Isolation and Characterisation of Small Extracellular Vesicles from Human Mesenchymal Stem Cells.

The objective of this study is to develop a simple protocol to isolate and characterise small extracellular vesicles (sEVs) from human umbilical cord-derived MSCs (hUC-MSCs). hUC-MSCs were characterised through analysis of morphology, immunophenotyping and multidifferentiation ability. SEVs were successfully isolated by ultrafiltration from the conditioned medium of hUC-MSCs. The sEVs' size distribution, intensity within a specific surface marker population were measured with zetasizer or nanoparticle tracking analysis. The expression of surface and internal markers of sEVs was also assessed by western blotting. Morphology of hUC-MSCs displayed as spindle-shaped, fibroblast-like adherent cells. Phenotypic analysis by flow cytometry revealed that hUC-MSCs expressed MSC surface marker, including CD90, CD73, CD105, CD44 and exhibited the capacity for osteogenic, adipogenic and chondrogenic differentiation. Populations of sEVs with CD9, CD63 and CD81 positive were detected with size distribution in the diameter of 63.2 to 162.5nm. Typical sEVs biomarkers such as CD9, CD63, CD81, HSP70 and TSG101 were also detected with western blotting. Our study showed that sEVs from hUC-MSCs conditioned medium were successfully isolated and characterised. Downstream application of hUC-MSCs-sEVs will be further explored.