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Aim: Umbilical cord blood (UCB) sourced allografts are promising interventions for tissue regeneration. As applications of these allografts and regulations governing them continue to evolve, we were prompted to identify parameters determining their quality, safety and regenerative potential. Materials & methods: Flow-cytometry, mass-spectrometry, protein multiplexing, nanoparticle tracking analysis and standard biological techniques were employed. Results: Quality attributes of a uniquely processed UCB-allograft (UCBr) were enumerated based on identity (cell viability, immunophenotyping, proteomic profiling, and quantification of relevant cytokines); safety (bioburden and microbiological screening), purity (endotoxin levels) and potency (effect of UCBr on chondrocytes and mesenchymal stem cells derived exosomes). These attributes were stable up to 24 months in cryopreserved UCBr. Conclusion: We identified a comprehensive panel of tests to establish the clinical efficacy and quality control attributes of a UCB-sourced allograft.

Aiming at the problems of complex background and low accuracy in Thangka image detection tasks using deep learning algorithms, an improved object detection algorithm based on YOLOv4 is proposed. Considering that the Mish function used by YOLOv4 is static, which performs the same operation on all input samples, hence it is not enough to deal with complex scenarios. We use the DynamicReLU function to encode the global up and down into a hyperfunction, and dynamically adjust the piecewise linear activation function instead of the Mish function accordingly, which solves the defect that the activation function cannot be dynamically adjusted, and realizes the detection of the headgear and seat of the Thangka image. The experimental results in Thangka image detection show that the evaluation protocol mAP of the algorithm reaches 37.9%, which is an increase of 3.8% compared to the YOLOv4 algorithm. The algorithm improves detection accuracy without increasing the depth and width of the network.

ADAMTS-13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, is a zinc-containing metalloprotease that cleaves von Willebrand factor (vWf). Previous publications by our laboratory have shown that ADAMTS-13 may also be involved in angiogenesis. For this study, we report the successful transient knockdown of endogenous ADAMTS-13 in human umbilical vein endothelial cells (HUVEC) via siRNA and the effects of reduced endogenous ADAMTS-13 on HUVEC angiogenesis functions. 15nM of ADAMTS-13 siRNA reduced HUVEC ADAMTS-13 protein levels by 90% after 24h incubation, whereas control siRNA did not affect endogenous ADAMTS-13 levels. Furthermore, this transfection did not affect the HUVEC endogenous protein level of ADAMTS-1, a related family member of ADAMTS-13 indicating the specificity of the siRNA. Transfection of HUVEC with 15nM of ADAMTS-13 siRNA resulted in a 21% decrease in proliferation after 24h incubation. The effects of ADAMTS-13 knockdown on migration of HUVEC across a scratch wound were also evaluated. 24h after transfection with control siRNA, there was increased cell migration across the scratch wound. This dramatic migration did not occur with ADAMTS-13 knockdown cells. Decreased protein levels of endogenous ADAMTS-13 also affected angiogenesis as measured by endothelial cell tube formation using a Matrigel matrix method. The tube lengths, sizes and junction numbers of the ADAMTS-13 knockdown cells were all significantly lower compared to control cells by about 40%. The protein level of vascular endothelial growth factor (VEGF), a well-known regulator of angiogenesis, was significantly decreased by 45% upon knockdown of ADAMTS-13. Moreover, activity of the AKT pathway, one of the VEGF angiogenesis downstream signaling pathways was down-regulated by ADAMTS-13 siRNA. These data indicate that in cultured endothelial cells, one role of endogenous ADAMTS-13 is regulation of angiogenesis, mediated through VEGF and AKT signaling pathway. Overall, our data suggest an additional model of endogenous ADAMTS-13 functionality, beyond that of cleaving von Willebrand factor.

Aim: Umbilical cord blood (UCB) finds frequent applications in regenerative medicine. We evaluated the role of cytokines present in a uniquely processed, UCB-derived cellular allograft product (UCBp). Materials & methods: Luminex multiplex assay and standard cell biology methods were employed. Results: Study with allografts from 33 donors identified 44 quantifiable cytokines in the UCBp derived conditioned media (CM). The UCBp-CM elevated proliferation and migration rates of mesenchymal stem cells (MSCs) and bone marrow stromal cells. Moreover, UCBp-CM induced secretion of VEGF-A and osteoprotegerin, which promoted angiogenesis of endothelial cells and positively influenced the osteogenic differentiation of MSCs, respectively. Conclusion: Cytokines in UCBp stimulate cellular processes important for bone regeneration, making UCBp an excellent candidate for potential applications in orthopedic procedures like bone non-union and spinal fusion.

This study examined the effect of habitat types and ontogenetic stages on the diet shift of Coreius guichenoti (Sauvage and Dabry de Thiersant, 1874), a critically endangered fish species. Based on the stable isotope analysis method, the following was explored: the variations in δ13C and δ15N values, isotopic niche width and four basal food sources (Mollusks, Macrocrustaceans, Aquatic insect larvae and particulate organic matters (POMs)) among three essential habitat types (the spawning ground, natural riverine feeding and nursery area, and Three Gorges Reservoir area) and between two ontogenetic stages (immature and fully mature stages). A diet shift associated with habitat type changes was observed, but there were no obvious differences in diet composition between the two ontogenetic stages. Dietary plasticity and a preference for specific foods were the important determinants of feeding behavior through the life history of this species. POM was important for the survival of this species in the resource-limited spawning ground, but this species fed more heavily on higher-order consumers in resource-abundant areas. This study highlights the importance of maintaining free connectivity among different habitats (particularly spawning grounds) to ensure the long-term sustainability of potamodromous fish species as well as the full investigation of all types of critical habitats for understanding the trophic ecology of a single fish species.

Certain experiments involve the high-throughput quantification of image data, thus requiring algorithms for automation. A challenge in the development of such algorithms is to properly interpret signals over a broad range of image characteristics, without the need for manual adjustment of parameters. Here we present a new approach for locating signals in image data, called Segment and Fit Thresholding (SFT). The method assesses statistical characteristics of small segments of the image and determines the best-fit trends between the statistics. Based on the relationships, SFT identifies segments belonging to background regions; analyzes the background to determine optimal thresholds; and analyzes all segments to identify signal pixels. We optimized the initial settings for locating background and signal in antibody microarray and immunofluorescence data and found that SFT performed well over multiple, diverse image characteristics without readjustment of settings. When used for the automated analysis of multi-color, tissue-microarray images, SFT correctly found the overlap of markers with known subcellular localization, and it performed better than a fixed threshold and Otsu’s method for selected images. SFT promises to advance the goal of full automation in image analysis.

The fucose post-translational modification is frequently increased in pancreatic cancer, thus forming the basis for promising biomarkers, but a subset of pancreatic cancer patients does not elevate the known fucose-containing biomarkers. We hypothesized that such patients elevate glycan motifs with fucose in linkages and contexts different from the known fucose-containing biomarkers. We used a database of glycan array data to identify the lectins CCL2 to detect glycan motifs with fucose in a 3' linkage; CGL2 for motifs with fucose in a 2' linkage; and RSL for fucose in all linkages. We used several practical methods to test the lectins and determine the optimal mode of detection, and we then tested whether the lectins detected glycans in pancreatic cancer patients who did not elevate the sialyl-Lewis A glycan, which is upregulated in ∼75% of pancreatic adenocarcinomas. Patients who did not upregulate sialyl-Lewis A, which contains fucose in a 4' linkage, tended to upregulate fucose in a 3' linkage, as detected by CCL2, but they did not upregulate total fucose or fucose in a 2' linkage. CCL2 binding was high in cancerous epithelia from pancreatic tumors, including areas negative for sialyl-Lewis A and a related motif containing 3' fucose, sialyl-Lewis X. Thus, glycans containing 3' fucose may complement sialyl-Lewis A to contribute to improved detection of pancreatic cancer. Furthermore, the use of panels of recombinant lectins may uncover details about glycosylation that could be important for characterizing and detecting cancer.

The role of juvenile hormone (JH) in regulating the timing and nature of insect molts is well-established. Increasing evidence suggests that JH is also involved in regulating final insect size. Here we elucidate the developmental mechanism through which JH regulates body size in developing Drosophila larvae by genetically ablating the JH-producing organ, the corpora allata (CA). We found that larvae that lack CA pupariated at smaller sizes than control larvae due to a reduced larval growth rate. Neither the timing of the metamorphic molt nor the duration of larval growth was affected by the loss of JH. Further, we show that the effects of JH on growth rate are dependent on the forkhead box O transcription factor (FOXO), which is negatively regulated by the insulin-signaling pathway. Larvae that lacked the CA had elevated levels of FOXO activity, whereas a loss-of-function mutation of FOXO rescued the effects of CA ablation on final body size. Finally, the effect of JH on growth appears to be mediated, at least in part, via ecdysone synthesis in the prothoracic gland. These results indicate a role of JH in regulating growth rate via the ecdysone- and insulin-signaling pathways. National Science Foundation Grants: (IOS-0919855, IOS-084584), Howard Hughes Medical Institute, Fundação Calouste Gulbenkian, FCT : [SFRH/Bolsas de Pós-Doutoramento (BPD)/74313/2010].

2013年9月，中国国家主席习近平提出建设“丝绸之路经济带”的新构想。在这一新构想下，各国将利用全方位的区域合作实现共同发展和繁荣。其中，中俄作为相互毗邻的大国，在世界大国关系中具有重要的政治和经济地位，而能源合作又是中俄关系中最为关键、影响巨大的重点领域之一。本文在围绕中俄能源合作中具体问题探讨的基础上，以“丝绸之路经济带”为视角，对新构想下中俄能源合作的前景进行展望，并就深化中俄能源合作提出政策建议参考。 In September 2013, Chinese President Xi Jinping initially proposed the building of the Strategic Framework of The Silk Road Economic Belt. Against this background, the involved countries will achieve joint development and co-prosperity by carrying out all-round regional cooperation. As close neighbors, both China and Russia have very important political and economic positions in the world; meanwhile, energy cooperation is one of most significant and influential fields in the China-Russia relationship. This paper tries to analyze some concrete issues in the process of the China-Russia energy cooperation, illustrate the outlook of the cooperation between the two countries in terms of the Silk Road Economic Belt, and finally raise policy references to deepen the China-Russia energy cooperation in the future., Journal of China and International Relations, Vol 4 No 2 (2016)

Category: Ankle, Ankle Arthritis, Basic Sciences/Biologics, Hindfoot, Surgery Introduction/Purpose: Foot and ankle surgery with bone grafting relieves pain and improves function in patients with hind/midfoot conditions. Umbilical cord blood (UCB) derived allografts have gained recent prominence for their potential in tissue regeneration. UCB is enriched with cytokines which regulate autocrine and paracrine signaling. BioBurst Fluid (BBF) is a UCB derived cellular allograft carefully processed by the patent pending Progenokine™ technique to preserve the unique molecular heterogeneity of UCB. Clinical study is being conducted to determine the safety and efficacy of BBF administered in patients undergoing foot and ankle surgery (NCT03077256). Here we report our initial data from this ongoing study. In vitro biochemical characterization of BBF further established the osteogenic potential of this allograft and provided mechanistic link to its clinical efficiency. Methods: 57 patients undergoing surgery with concomitant BBF administration were enrolled in an IRB-approved study, the selection criteria being not to exclude anyone. Data obtained from 21 of the 57 enrolled patients completing their 6-month post- op is reported. The prospective registry recorded patient demographics, comorbidities, symptoms, fusion status, pain, pain medication use, and neurological status up to 6 months post-op. Surgical, immediate post-op, and follow-up complications were also recorded. Fusion was assessed at 3 and 6 months via anteroposterior and lateral x-rays. Pain and function were assessed with Visual Analogue Scale/VAS and modified Odom’s criteria. Neurological status was recorded as improved, maintained, or worse. Narcotic and NSAID use were rated as increased/same or decreased/discontinued. For in vitro characterization of BBF, standard cell-biology methods were employed using MSC as model system. Cytokine concentration in 30 random BBF lots was determined by Luminex multiplex assay. All data are analyzed using appropriate statistical tools. Results: No complications from administration of BBF were seen intra-operatively and during follow-up. Successful fusion (>50% incorporation) was achieved in 20/21 patients (95%) with 8 patients having fusion by 3 months. One non-union occurred in an HIV positive patient. VAS pain score improved clinically from pre-op at 3 months (-3.3, pConclusion: No adverse-event reports established the clinical safety of BBF. Extrapolation of this initial data suggests early and high rates of successful fusion and better than expected clinical outcomes in patients using BBF in foot and ankle procedures. These positive outcomes may be attributed to BBF’s molecular profile of anti-inflammatory cytokines which help in pain mitigation. Growth factors and chemokines present in BBF induce host MSC to participate in proliferation, recruitment, and osteogenic differentiation which contribute to bone regeneration and fusion.

Purpose Lectins are valuable tools for detecting specific glycans in biological samples, but the interpretation of the measurements can be ambiguous due to the complexities of lectin specificities. Here, we present an approach to improve the accuracy of interpretation by converting lectin measurements into quantitative predictions of the presence of various glycan motifs. Experimental design The conversion relies on a database of analyzed glycan array data that provides information on the specificities of the lectins for each of the motifs. We tested the method using measurements of lectin binding to glycans on glycan arrays and then applied the method to predicting motifs on the protein mucin 1 (MUC1) expressed in eight different pancreatic cancer cell lines. Results The combined measurements from several lectins were more accurate than individual measurements for predicting the presence or absence of motifs on arrayed glycans. The analysis of MUC1 revealed that each cell line expressed a unique pattern of glycoforms, and that the glycoforms significantly differed between MUC1 collected from conditioned media and MUC1 collected from cell lysates. Conclusions and clinical relevance This new method could provide more accurate analyses of glycans in biological sample and make the use of lectins more practical and effective for a broad range of researchers.

Molecular indicators to specify the risk posed by a pancreatic cyst would benefit patients. Previously we showed that most cancer-precursor cysts, termed mucinous cysts, produce abnormal glycoforms of the proteins MUC5AC and endorepellin. Here we sought to validate the glycoforms as a biomarker of mucinous cysts and to specify the oligosaccharide linkages that characterize MUC5AC. We hypothesized that mucinous cysts secrete MUC5AC displaying terminal N-acetylglucosamine (GlcNAc) in either alpha or beta linkage. We used antibody-lectin sandwich assays to detect glycoforms of MUC5AC and endorepellin in cyst fluid samples from three independent cohorts of 49, 32, and 66 patients, and we used monoclonal antibodies to test for terminal, alpha-linked GlcNAc and the enzyme that produces it. A biomarker panel comprising the previously-identified glycoforms of MUC5AC and endorepellin gave 96%, 96%, and 87% accuracy for identifying mucinous cysts in the three cohorts with an average sensitivity of 92% and an average specificity of 94%. Glycan analysis showed that MUC5AC produced by a subset of mucinous cysts displays terminal alpha-GlcNAc, a motif expressed in stomach glands. The alpha-linked glycoform of MUC5AC was unique to intraductal papillary mucinous neoplasms (IPMN), whereas terminal beta-linked GlcNAc was increased in both IPMNs and mucinous cystic neoplasms (MCN). The enzyme that synthesizes alpha-GlcNAc, A4GNT, was expressed in the epithelia of mucinous cysts that expressed alpha-GlcNAc, especially in regions with high-grade dysplasia. Thus IPMNs secrete a gastric glycoform of MUC5AC that displays terminal alpha-GlcNAc, and the combined alpha-GlcNAc and beta-GlcNAc glycoforms form an accurate biomarker of mucinous cysts.

ADAMTS-13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, is a zinc-containing metalloprotease that cleaves von Willebrand factor. Previous publications by our laboratory have shown that either endogenous or exogenous ADAMTS-13 may regulate angiogenesis in endothelial cells; for example, siRNA knockdown of endogenous endothelial cell ADAMTS-13 reduced angiogenic activities including cell migration, proliferation, and tube formation (Tang et al. Microvascular Res 2017; 113:85-70). Many tumor cells have the ability to develop a vascular phenotype, a process known as vasculogenic mimicry (VM). There is a multitude of evidence demonstrating that tumor cell-mediated VM may play a vital role in tumor development, independent of endothelial cell angiogenesis. For this study, we report that decreasing ADAMTS-13 levels inhibit vasculogenic functions in a glioblastoma cell line (LN-229), while increasing ADAMTS-13 levels had no effect. Incubating up to 30ng/ml of exogenous ADAMTS-13 did not promote cell proliferation and up to 200ng/ml did not change tube formation in LN-299 brain tumor cells. One possible explanation for this observation is that LN-299 brain tumor cells have almost twice the endogenous ADAMTS-13 expression than endothelial cells (~200pg/ml vs ~100pg/ml), so this cell line is not sensitive to exogenous ADAMTS-13. Using siRNA, we were able to successfully knockdown endogenous ADAMTS-13 in human brain tumor LN-229 cells. 50nM of ADAMTS-13 siRNA in a six-well plate inhibited HUVEC ADAMTS-13 expression levels by 75% after 48hr incubation, whereas control siRNA did not affect endogenous ADAMTS-13 levels. Additionally, expression of ADAMTS-1, a related family member of ADAMTS-13 was not affected by the knockdown. The effects of reduced endogenous ADAMTS-13 on glioblastoma vasculogenic functions were studied. Transfection of HUVEC with 50nM of ADAMTS-13 siRNA in a 24-well plate resulted in about 20% decrease in proliferation after 24hr and 48hr incubation. We demonstrated that decreased expression of endogenous ADAMTS-13 also affected vasculogenic mimicry as measured by glioblastoma cell tube formation using a Matrigel matrix method. The tube lengths, sizes and junction numbers of the ADAMTS-13 knockdown cells were all significantly lower compared to control cells by about 30-40%, respectively. Moreover, the expression of VEGF, a well-known regulator of angiogenesis, was significantly decreased by 25% after knockdown of endogenous LN-229 ADAMTS-13. Overall, these data suggest that endogenous ADAMTS-13 may regulate glioblastoma cell growth and function. Citation Format: Huiyuan Tang, Manfai Lee, Eun Ho Kim, Daniel Bishop, George M. Rodgers. Endogenous ADAMTS-13 regulates vasculogenic functions in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 81.

The sialyl-Lewis A (sLeA) glycan forms the basis of the CA19–9 assay and is the current best biomarker for pancreatic cancer, but because it is not elevated in ∼25% of pancreatic cancers, it is not useful for early diagnosis. We hypothesized that sLeA-low tumors secrete glycans that are related to sLeA but not detectable by CA19–9 antibodies. We used a method called motif profiling to predict that a structural isomer of sLeA called sialyl-Lewis X (sLeX) is elevated in the plasma of some sLeA-low cancers. We corroborated this prediction in a set of 48 plasma samples and in a blinded set of 200 samples. An antibody sandwich assay formed by the capture and detection of sLeX was elevated in 13 of 69 cancers that were not elevated in sLeA, and a novel hybrid assay of sLeA capture and sLeX detected 24 of 69 sLeA-low cancers. A two-marker panel based on combined sLeA and sLeX detection differentiated 109 pancreatic cancers from 91 benign pancreatic diseases with 79% accuracy (74% sensitivity and 78% specificity), significantly better than sLeA alone, which yielded 68% accuracy (65% sensitivity and 71% specificity). Furthermore, sLeX staining was evident in tumors that do not elevate plasma sLeA, including those with poorly differentiated ductal adenocarcinoma. Thus, glycan-based biomarkers could characterize distinct subgroups of patients. In addition, the combined use of sLeA and sLeX, or related glycans, could lead to a biomarker panel that is useful in the clinical diagnosis of pancreatic cancer. Precis: This paper shows that a structural isomer of the current best biomarker for pancreatic cancer, CA19–9, is elevated in the plasma of patients who are low in CA19–9, potentially enabling more comprehensive detection and classification of pancreatic cancers.

The validation of candidate biomarkers often is hampered by the lack of a reliable means of assessing and comparing performance. We present here a reference set of serum and plasma samples to facilitate the validation of biomarkers for resectable pancreatic cancer. The reference set includes a large cohort of stage I-II pancreatic cancer patients, recruited from 5 different institutions, and relevant control groups. We characterized the performance of the current best serological biomarker for pancreatic cancer, CA 19–9, using plasma samples from the reference set to provide a benchmark for future biomarker studies and to further our knowledge of CA 19–9 in early-stage pancreatic cancer and the control groups. CA 19–9 distinguished pancreatic cancers from the healthy and chronic pancreatitis groups with an average sensitivity and specificity of 70–74%, similar to previous studies using all stages of pancreatic cancer. Chronic pancreatitis patients did not show CA 19–9 elevations, but patients with benign biliary obstruction had elevations nearly as high as the cancer patients. We gained additional information about the biomarker by comparing two distinct assays. The two CA 9–9 assays agreed well in overall performance but diverged in measurements of individual samples, potentially due to subtle differences in antibody specificity as revealed by glycan array analysis. Thus, the reference set promises be a valuable resource for biomarker validation and comparison, and the CA 19–9 data presented here will be useful for benchmarking and for exploring relationships to CA 19–9.

Recent research is uncovering unexpected ways that glycans contribute to biology, as well as new strategies for combatting disease using approaches involving glycans. To make full use of glycans for clinical applications, we need more detailed information on the location, nature, and dynamics of glycan expression in vivo. Such studies require the use of specimens acquired directly from patients. Effective studies of clinical specimens require low-volume assays, high precision measurements, and the ability to process many samples. Assays using affinity reagents—lectins and glycan-binding antibodies—can meet these requirements, but further developments are needed to make the methods routine and effective. Recent advances in the use of glycan-binding proteins could meet that need. The advances involve improved determination of specificity using glycan arrays; the availability of databases for mining and analyzing glycan array data; lectin engineering methods; and the ability to quantitatively interpret lectin measurements. Here we describe many of the challenges and opportunities involved in the application of these new approaches to the study of biological samples. The new tools hold promise for developing methods to improve the outcomes of patients afflicted with diseases characterized by aberrant glycan expression.

In this study, we isolated and characterized thirteen polymorphic microsatellite markers for Jinshaia sinensis, a fish species endemic to the upper reaches of the Yangtze River in China. Each locus was screened in a population of 48 individuals. Number of alleles per locus ranged between five and nineteen. Observed heterozygosity ranged between 0.121 and 0.854, and expected heterozygosity between 0.722 and 0.928. No significant linkage disequilibrium was found between pairs of loci. However, three loci showed significant departures from Hardy-Weinberg equilibrium and four loci had evidence of null alleles. These markers presented here will be valuable tools to understand the genetic structure of J. sinensis populations in the Yangtze River.

ADAMTS-13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, is a zinc-containing metalloprotease that cleaves von Willebrand factor (vWf). Previous publications by our laboratory have shown that ADAMTS-13 may also be involved in angiogenesis; for example; addition of exogenous ADAMTS-13 enhances endothelial cell (Lee et al. Microvascular Res 2012; 84:109) and glioblastoma cell (Lee et al. Blood 2013; 122:#2306) migration and proliferation. For this study, we report the successful transient knockdown of endogenous ADAMTS-13 in human umbilical vein endothelial cells (HUVEC) via siRNA. 30pmols of ADAMTS-13 siRNA in a six-well plate inhibited HUVEC ADAMTS-13 expression levels by 90% after 24hr incubation, whereas control siRNA did not affect endogenous ADAMTS-13 levels. The knockdown effect was decreased over time: 72hr after the initial knockdown, ADAMTS-13 expression was reduced by only 50%. The effects of reduced endogenous ADAMTS-13 on HUVEC angiogenesis functions were studied. Transfection of HUVEC with 10pmols of ADAMTS-13 siRNA in a 24-well plate resulted in a 21% and 22% decrease in proliferation after 24hr and 48hr incubation, respectively. The effects of ADAMTS-13 knockdown on migration of HUVEC across a scratch wound were also evaluated. 24hr after transfection with control siRNA, there was robust cell migration across the scratch wound. This dramatic migration did not occur with ADAMTS-13 knockdown cells. Decreased expression of endogenous ADAMTS-13 also affected angiogenesis as measured by endothelial cell tube formation using a Matrigel matrix method. The tube lengths, sizes and junction numbers of the ADAMTS-13 knockdown cells were all significantly lower compared to control cells by about 40%. Activity of the AKT pathway, one of the angiogenesis downstream signaling pathways was down-regulated by ADAMTS-13 siRNA while ERK, a component of the MAP kinase pathway was not affected upon knockdown of ADAMTS-13. These data indicate that in cultured endothelial cells, one role of endogenous ADAMTS-13 is regulation of angiogenesis, mediated through the AKT signaling pathway. Overall, our data suggest an additional model of endogenous ADAMTS-13 functionality, beyond that of cleaving von Willebrand factor. Citation Format: Huiyuan Tang, Manfai Lee, Eun Ho Kim, Daniel Bishop, George M. Rodgers. Endogenous ADAMTS-13 regulates angiogenesis in cultured human endothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1816. doi:10.1158/1538-7445.AM2017-1816

Specific glycans or protein glycoforms are potential biomarkers of cancer, but variability in expression between people limits their value. A factor influencing the variation between people is the ...

A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers.

Background & Aims: The cancer antigen 19-9 (CA19-9) is the current best biomarker for pancreatic cancer, but it is not increased in approximately 25% of pancreatic cancer patients at a cut-off value that provides a 25% false-positive rate. We hypothesized that antigens related to the CA19-9 antigen, which is a glycan called sialyl-Lewis A (sLeA), are increased in distinct subsets of pancreatic cancers. Methods: We profiled the levels of multiple glycans and mucin glycoforms in plasma from 200 subjects with either pancreatic cancer or benign pancreatic disease, and we validated selected findings in additional cohorts of 116 and 100 subjects, the latter run with the investigators blinded to diagnoses and including cancers that exclusively were early stage. Results: We found significant increases in 2 glycans: an isomer of sLeA called sialyl-Lewis X, present both in sulfated and nonsulfated forms, and the sialylated form of a marker for pluripotent stem cells, type 1 N-acetyl-lactosamine. The glycans performed as well as sLeA as individual markers and were increased in distinct groups of patients, resulting in a 3-marker panel that significantly improved upon any individual biomarker. The panel showed 85% sensitivity and 90% specificity in the combined discovery and validation cohorts, relative to 54% sensitivity and 86% specificity for sLeA; and it showed 80% sensitivity and 84% specificity in the independent test cohort, as opposed to 66% sensitivity and 72% specificity for sLeA. Conclusions: Glycans related to sLeA are increased in distinct subsets of pancreatic cancers and yield improved diagnostic accuracy compared with CA19-9. Keywords: Biomarkers, Sialyl-Lewis A, Antibody Arrays, Lectins

The CA 19-9 assay is the current best serum/plasma marker for pancreatic cancer and can be an accurate indicator of cancer for many patients. However, it is not effective for patients who harbor germline DNA mutations that result in absent or weakened expression of CA 19-9. These mutations are present in the enzymes that synthesize the CA 19-9 antigen, which is a glycan structure called sialyl-Lewis A. Here we tested the hypothesis that the stratification of patients by specific genotypes and the use of optimized biomarkers for each genotype can yield improved diagnostic performance. We acquired the genotypes of 162 pancreatic cancer and control subjects for five different genes involved in the biosynthesis of the CA 19-9 antigen. The CA 19-9 levels in the blood plasma of these patients were accurately predicted by specific SNPs in the genes fucosyltransferase 2 and fucosyltransferase 3 (FUT2 and FUT3). The patients were subdivided into four groups based on these genotypes, and marker discovery and optimization was performed separately for each group using measurements from dozens of glycan-based markers. The FUT2 homozygous mutant (FUT2-/-) subjects were perfectly diagnosed (100% accurate discrimination of cancer from controls) using CA 19-9 alone, whereas FUT2+/- and FUT2 WT subjects were accurately diagnosed using CA 19-9 in combination with two other markers selected for each group. The FUT3-/- patients were accurately diagnosed using a new biomarker, a particular glycosylation variant of the MUC1 protein. The overall performance of the genotype-selected biomarker panel was 100% sensitivity (93/93 cancer cases correctly detected) and 96% specificity (66/69 controls correct). This performance significantly outperforms CA 19-9 alone (87% sensitivity and 84% specificity) and biomarker panels that do not incorporate genotype selection. Validation experiments using 40 additional unblinded samples and 200 additional blinded samples are underway. Genotype-selected biomarkers show the potential for significantly improving upon conventional biomarker strategies. Citation Format: Huiyuan Tang, Katie Partyka, Kevin Maupin, Randall Brand, Brain Haab. Genotype-selected biomarkers for the accurate diagnosis of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B6.

Individuals within species and populations vary. Such variation arises through environmental and genetic factors and ensures that no two individuals are identical. However, it is clear that not all traits show the same degree of intraspecific variation. Some traits, in particular secondary sexual characteristics used by males to compete for and attract females, are extremely variable among individuals in a population. Other traits, for example brain size in mammals, are not. Recent research has begun to explore the possibility that the extent of phenotypic variation (here referred to as "variability") may be a character itself and subject to natural selection. While these studies support the concept of variability as an evolvable trait, controversy remains over what precisely the trait is. At the heart of this controversy is the fact that there are very few examples of developmental mechanisms that regulate trait variability in response to any source of variation, be it environmental or genetic. Here, we describe a recent study from our laboratory that identifies such a mechanism. We then place the study in the context of current research on the regulation of trait variability, and discuss the implications for our understanding of the developmental regulation and evolution of phenotypic variation.

Cyclooxygenase (COX) isoforms catalyze the committed step in prostaglandin biosynthesis. The primary structures of COX-1 and COX-2 are very similar except that COX-2 has a 19-amino acid (19-AA) segment of unknown function located just inside its C terminus. Here we provide evidence that the major role of the 19-AA cassette is to mediate entry of COX-2 into the ER-associated degradation system that transports ER proteins to the cytoplasm. COX-1 is constitutively expressed in many cells, whereas COX-2 is usually expressed inducibly and transiently. In murine NIH/3T3 fibroblasts, we find that COX-2 protein is degraded with a half-life (t(1/2)) of about 2 h, whereas COX-1 is reasonably stable (t(1/2)12 h); COX-2 degradation is retarded by 26 S proteasome inhibitors. Similarly, COX-1 expressed heterologously in HEK293 cells is quite stable (t(1/2)24 h), whereas COX-2 expressed heterologously is degraded with a t(1/2) of approximately 5 h, and its degradation is slowed by proteasome inhibitors. A deletion mutant of COX-2 was prepared lacking 18 residues of the 19-AA cassette. This mutant retains native COX-2 activity but, unlike native COX-2, is stable in HEK293 cells. Conversely, inserting the COX-2 19-AA cassette near the C terminus of COX-1 yields a mutant ins594-612 COX-1 that is unstable (t(1/2) approximately 3 h). Mutation of Asn-594, an N-glycosylation site at the beginning of the 19-AA cassette, stabilizes both COX-2 and ins594-612 COX-1; nonetheless, COX mutants that are glycosylated at Asn-594 but lack the remainder of the 19-amino acid cassette (i.e. del597-612 COX-2 and ins594-596 COX-1) are stable. Thus, although glycosylation of Asn-594 is necessary for COX-2 degradation, at least part of the remainder of the 19-AA insert is also required. Finally, kifunensine, a mannosidase inhibitor that can block entry of ER proteins into the ER-associated degradation system, retards COX-2 degradation.

Phenotypic plasticity, the ability for a single genotype to generate different phenotypes in response to environmental conditions, is biologically ubiquitous, and yet almost nothing is known of the developmental mechanisms that regulate the extent of a plastic response. In particular, it is unclear why some traits or individuals are highly sensitive to an environmental variable while other traits or individuals are less so. Here we elucidate the developmental mechanisms that regulate the expression of a particularly important form of phenotypic plasticity: the effect of developmental nutrition on organ size. In all animals, developmental nutrition is signaled to growing organs via the insulin-signaling pathway. Drosophila organs differ in their size response to developmental nutrition and this reflects differences in organ-specific insulin-sensitivity. We show that this variation in insulin-sensitivity is regulated at the level of the forkhead transcription factor FOXO, a negative growth regulator that is activated when nutrition and insulin signaling are low. Individual organs appear to attenuate growth suppression in response to low nutrition through an organ-specific reduction in FOXO expression, thereby reducing their nutritional plasticity. We show that FOXO expression is necessary to maintain organ-specific differences in nutritional-plasticity and insulin-sensitivity, while organ-autonomous changes in FOXO expression are sufficient to autonomously alter an organ's nutritional-plasticity and insulin-sensitivity. These data identify a gene (FOXO) that modulates a plastic response through variation in its expression. FOXO is recognized as a key player in the response of size, immunity, and longevity to changes in developmental nutrition, stress, and oxygen levels. FOXO may therefore act as a more general regulator of plasticity. These data indicate that the extent of phenotypic plasticity may be modified by changes in the expression of genes involved in signaling environmental information to developmental processes., Author Summary The ability of an organism to respond to its environment is a defining quality of life. However, why are some characteristics or individuals sensitive to environmental change while others are not? We identified the mechanism that controls the response of growing organs to a particularly important environmental factor—developmental nutrition. In all animals, a decrease in developmental nutrition reduces final body and organ size. However, the size of some organs is less responsive to changes in nutrition than others. In a male fruit fly, it is the size of the genitals that is resistant to dietary restriction. This is achieved by the male fruit fly reducing expression of a key gene in their genitalia. This gene, FOXO, forms part of the insulin signaling system, which signals food levels to tissues in all animals. By lowering the production of FOXO, the genitalia are able to “ignore” hormonal signals that tell the rest of the body to grow slowly due to limited food. The ability of tissues to become insensitive to nutritional information is a characteristic of many tumors and also underlies type 2 diabetes. Our data may therefore provide insight into the origin and treatment of both conditions.