A New Sandwich ELISA for Quantification of Thymidine Kinase 1 Protein Levels in Sera from Dogs with Different Malignancies Can Aid in Disease Management

Thymidine kinase 1 (TK1) is a DNA precursor enzyme whose expression is closely correlated with cell proliferation and cell turnover. Sensitive serum TK1 activity assays have been used for monitoring and prognosis of hematological malignancies in both humans and dogs. Here we describe the development of a specific sandwich TK1-ELISA for the quantification of TK1 protein levels in sera from dogs with different malignancies. A combination of rabbit polyclonal anti-dog TK1 antibody and a mouse monoclonal anti-human TK1 antibody was used. Different concentrations of recombinant canine TK1 was used as standard. Clinical evaluation of the ELISA was done by using sera from 42 healthy dogs, 43 dogs with hematological tumors and 55 with solid tumors. An established [3H]-dThd phosphorylation assay was used to determine the TK1 activity levels in the same sera. The mean TK1 activities in dogs with hematological tumors were significantly higher than those found in healthy dogs. In agreement with earlier studies, no significant difference was observed in serum TK1 activities between healthy dogs and dogs with solid tumors. However, the mean TK1 protein levels determined by new TK1-ELISA were significantly higher not only in hematological tumors but also in solid tumors compared to healthy dogs (mean ± SD = 1.30 ± 1.16, 0.67 ± 0.55 and 0.27± 0.10 ng/mL, respectively). Moreover, TK1-ELISA had significantly higher ability to distinguish lymphoma cases from healthy based on receiver operating characteristic analyses (area under the curve, AUC, of 0.96) to that of the activity assay (AUC, 0.84). Furthermore, fluctuations in TK1 protein levels during the course of chemotherapy in dogs with lymphoma closely associated with clinical outcome. Overall, the TK1-ELISA showed significant linear correlation with the TK1 activity assay (rs= 0.6, p<0.0001). Thus, the new TK1-ELISA has sufficient sensitivity and specificity for routine clinical use in veterinary oncology

A monoclonal antibody-based sandwich ELISA for measuring canine Thymidine kinase 1 protein and its role as biomarker in canine lymphoma

IntroductionDogs play an important role in society, which increased during the covid epidemics. This has led to a much higher workload for the veterinarians. Therefore, there is a need for efficient diagnostic tools to identify risk of malignant diseases. Here the development of a new test that can solve some of these problems is presented. It is based on serum Thymidine Kinase 1 (TK1), which is a biomarker for cell proliferation and cell lysis.MethodsAnti-TK1 monoclonal antibodies were produced against two different epitopes, the active site of the TK1 protein and the C-terminal region of canine TK1. The antibodies were developed with hybridoma technology and validated using dot blot, Quartz Crystal Microbalance (QCM) technology, western blots, immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA). Clinical evaluation of Canine TK1 ELISA was done by using sera from 131 healthy dogs and 93 dogs with lymphoma. The two selected Anti-TK1 monoclonal antibodies have Kd values in the range of 10−9 M and further analysis with dot and western blots confirmed the high affinity binding of these antibodies. A sandwich Canine TK1 ELISA was developed using the anti-TK1 antibodies, and TK1 concentrations in serum samples were determined using dog recombinant TK1 as a standard.ResultsSerum TK1 protein levels were significantly higher in dogs with lymphoma compared to those in healthy dogs (p < 0.0001). Receiver operating curve analysis showed that the canine TK1-ELISA obtain a sensitivity of 0.80, at a specificity of 0.95. Moreover, the Canine TK1 ELISA has a positive predictive value (PPV) of 97%, and the negative predictive value (NPV) of 83%, reflecting the proportion of test results that are truly positive and negative. Furthermore, Canine TK1 ELISA had significantly higher capacity to differentiate dogs with T-cell lymphoma from those with B-cell lymphoma compared to earlier used TK1 activity assays.DiscussionThese results demonstrate that the Canine TK1 ELISA can serve as an efficient tool in the diagnosis and management of dogs with lymphomas

A monoclonal antibody-based sandwich ELISA for measuring canine Thymidine kinase 1 protein and its role as biomarker in canine lymphoma

Introduction: Dogs play an important role in society, which increased during the covid epidemics. This has led to a much higher workload for the veterinarians. Therefore, there is a need for efficient diagnostic tools to identify risk of malignant diseases. Here the development of a new test that can solve some of these problems is presented. It is based on serum Thymidine Kinase 1 (TK1), which is a biomarker for cell proliferation and cell lysis. Methods: Anti-TK1 monoclonal antibodies were produced against two different epitopes, the active site of the TK1 protein and the C-terminal region of canine TK1. The antibodies were developed with hybridoma technology and validated using dot blot, Quartz Crystal Microbalance (QCM) technology, western blots, immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA). Clinical evaluation of Canine TK1 ELISA was done by using sera from 131 healthy dogs and 93 dogs with lymphoma. The two selected Anti-TK1 monoclonal antibodies have Kd values in the range of 10(-9) M and further analysis with dot and western blots confirmed the high affinity binding of these antibodies. A sandwich Canine TK1 ELISA was developed using the anti-TK1 antibodies, and TK1 concentrations in serum samples were determined using dog recombinant TK1 as a standard. Results: Serum TK1 protein levels were significantly higher in dogs with lymphoma compared to those in healthy dogs (p < 0.0001). Receiver operating curve analysis showed that the canine TK1-ELISA obtain a sensitivity of 0.80, at a specificity of 0.95. Moreover, the Canine TK1 ELISA has a positive predictive value (PPV) of 97%, and the negative predictive value (NPV) of 83%, reflecting the proportion of test results that are truly positive and negative. Furthermore, Canine TK1 ELISA had significantly higher capacity to differentiate dogs with T-cell lymphoma from those with B-cell lymphoma compared to earlier used TK1 activity assays. Discussion: These results demonstrate that the Canine TK1 ELISA can serve as an efficient tool in the diagnosis and management of dogs with lymphomas

Visual Rhyme Judgment in Adults With Mild-to-Severe Hearing Loss

Adults with poorer peripheral hearing have slower phonological processing speed measured using visual rhyme tasks, and it has been suggested that this is due to fading of phonological representations stored in long-term memory. Representations of both vowels and consonants are likely to be important for determining whether or not two printed words rhyme. However, it is not known whether the relation between phonological processing speed and hearing loss is specific to the lower frequency ranges which characterize vowels or higher frequency ranges that characterize consonants. We tested the visual rhyme ability of 212 adults with hearing loss. As in previous studies, we found that rhyme judgments were slower and less accurate when there was a mismatch between phonological and orthographic information. A substantial portion of the variance in the speed of making correct rhyme judgment decisions was explained by lexical access speed. Reading span, a measure of working memory, explained further variance in match but not mismatch conditions, but no additional variance was explained by auditory variables. This pattern of findings suggests possible reliance on a lexico-semantic word-matching strategy for solving the rhyme judgment task. Future work should investigate the relation between adoption of a lexico-semantic strategy during phonological processing tasks and hearing aid outcome

Treatment with Anti-HER2 Chimeric Antigen Receptor Tumor-Infiltrating Lymphocytes (CAR-TILs) Is Safe and Associated with Antitumor Efficacy in Mice and Companion Dogs

Patients with metastatic melanoma have a historically poor prognosis, but recent advances in treatment options, including targeted therapy and immunotherapy, have drastically improved the outcomes for some of these patients. However, not all patients respond to available treatments, and around 50% of patients with metastatic cutaneous melanoma and almost all patients with metastases of uveal melanoma die of their disease. Thus, there is a need for novel treatment strategies for patients with melanoma that do not benefit from the available therapies. Chimeric antigen receptor-expressing T (CAR-T) cells are largely unexplored in melanoma. Traditionally, CAR-T cells have been produced by transducing blood-derived T cells with a virus expressing CAR. However, tumor-infiltrating lymphocytes (TILs) can also be engineered to express CAR, and such CAR-TILs could be dual-targeting. To this end, tumor samples and autologous TILs from metastasized human uveal and cutaneous melanoma were expanded in vitro and transduced with a lentiviral vector encoding an anti-HER2 CAR construct. When infused into patient-derived xenograft (PDX) mouse models carrying autologous tumors, CAR-TILs were able to eradicate melanoma, even in the absence of antigen presentation by HLA. To advance this concept to the clinic and assess its safety in an immune-competent and human-patient-like setting, we treated four companion dogs with autologous anti-HER2 CAR-TILs. We found that these cells were tolerable and showed signs of anti-tumor activity. Taken together, CAR-TIL therapy is a promising avenue for broadening the tumor-targeting capacity of TILs in patients with checkpoint immunotherapy-resistant melanoma

Genetic variation of biomass recalcitrance in a natural Salix viminalis (L.) population

Background: Salix spp. are high-productivity crops potentially used for lignocellulosic biofuels such as bioethanol. In general, pretreatment is needed to facilitate the enzymatic depolymerization process. Biomass resistance to degradation, i.e., biomass recalcitrance, is a trait which can be assessed by measuring the sugar released after combined pretreatment and enzymatic hydrolysis. We have examined genetic parameters of enzymatic sugar release and other traits related to biorefnery use in a population of 286 natural Salix viminalis clones. Furthermore, we have evaluated phenotypic and genetic correlations between these traits and performed a genomewide association mapping analysis using a set of 19,411 markers. Results: Sugar release (glucose and xylose) after pretreatment and enzymatic saccharifcation proved highly variable with large genetic and phenotypic variations, and chip heritability estimates (h2 ) of 0.23–0.29. Lignin syringyl/guaiacyl (S/G) ratio and wood density were the most heritable traits (h2=0.42 and 0.59, respectively). Sugar release traits were positively correlated, phenotypically and genetically, with biomass yield and lignin S/G ratio. Association mapping revealed seven marker–trait associations below a suggestive signifcance threshold, including one marker associated with glucose release. Conclusions: We identifed lignin S/G ratio and shoot diameter as heritable traits that could be relatively easily evaluated by breeders, making them suitable proxy traits for developing low-recalcitrance varieties. One marker below the suggestive threshold for marker associations was identifed for sugar release, meriting further investigation while also highlighting the difculties in employing genomewide association mapping for complex trait

Strengths and weaknesses in executive functioning in children with intellectual disability

Children with intellectual disability (ID) were given a comprehensive range of executive functioning measures, which systematically varied in terms of verbal and non-verbal demands. Their performance was compared to the performance of groups matched on mental age (MA) and chronological age (CA), respectively. Twenty-two children were included in each group. Children with ID performed on par with the MA group on switching, verbal executive-loaded working memory and most fluency tasks, but below the MA group on inhibition, planning, and non-verbal executive-loaded working memory. Children with ID performed below CA comparisons on all the executive tasks. We suggest that children with ID have a specific profile of executive functioning, with MA appropriate abilities to generate new exemplars (fluency) and to switch attention between tasks, but difficulties with respect to inhibiting pre-potent responses, planning, and non-verbal executive-loaded working memory The development of different types of executive functioning skills may, to different degrees, be related to mental age and experience

Interannual variation in competitive interactions from natural and anthropogenic disturbances in a temperate forest tree species: Implications for ecological interpretation

Competition is a major determinant of plant growth and is often used in studies of tree growth and species coexistence. However, these approaches are usually temporally static, i.e., assessed at a single point or period in time. While constantly changing forest conditions due to natural and human-induced disturbances potentially alter competition among individuals, static approaches cannot qualify the temporal variability of competitive interactions. Herewepresent a longitudinal analysis of competitive interactions among trees and discuss the implication of our results for ecological interpretation. Spatially-explicit tree growth data were obtained from 18 study plots (0.4 ha each) in sugar maple (Acer saccharum Marsh.) stands in Quebec, Canada. During the studied period (1980–2003), these stands had been disturbed by insect outbreaks (forest tent caterpillar, Malacosoma disstria Hubner) and by commercial partial harvest. We analyzed radial growth rates (outcome of competition) on an annual basis and as a function of tree biology (bole diameter, crown position), competition (above- and belowground competition from neighbours) and environmental conditions (light availability, harvest disturbance). Competitive interactions changed throughout the studied period. Canopy disturbance from partial harvest interacted with defoliators and influenced competition symmetry by favoring smaller trees. Competitive interactions seemed to have switched from below- to above-ground following canopy recovery after harvest. Release from competition due to partial harvest increase neighbourhood size (radius of effective competition) and enhanced the competitive pressure from larger individuals. The temporal variability in parameter estimates may be used for setting confidence intervals on competitive success (growth rates), thereby yielding a more robust basis for ecological interpretation. Our results also show that temporal variability in competitive interactions could contribute to the maintenance of high tree species diversity and structural complexity in some ecosystems by temporally altering species-specific responses to environmental change and disturbance

Using developmental trajectories to examine verbal and visuospatial short-term memory development in children and adolescents with Williams and Down syndromes

Williams (WS) and Down (DS) syndromes have been associated with specifically compromised short-term memory (STM) subsystems. Individuals with WS have shown impairments in visuospatial STM, while individuals with DS have often shown problems with the recall of verbal material. However, studies have not usually compared the development of STM skills in these domains, in these populations. The present study employed a cross sectional developmental trajectories approach, plotting verbal and visuospatial STM performance against more general cognitive and chronological development, to investigate how the domain-specific skills of individuals with WS and DS may change as development progresses, as well as whether the difference between STM skill domains increases, in either group, as development progresses. Typically developing children, of broadly similar cognitive ability to the clinical groups, were also included. Planned between- and within group comparisons were carried out. Individuals with WS and DS both showed the domain specific STM weaknesses in overall performance that were expected based on the respective cognitive profiles. However, skills in both groups developed, according to general cognitive development, at similar rates to those of the TD group. In addition, no significant developmental divergence between STM domains was observed in either clinical group according to mental age or chronological age, although the general pattern of findings indicated that the influence of the latter variable across STM domains, particularly in WS, might merit further investigation