Monoklonale und Recombinante Antikörper gegen potyvirale Proteine und ihre Verwendung

Ziel war es, Variable-Fragment-Einzelketten-Antikörper (scFv) gegen konservierte Aminosäuremotive der RNA-abhängigen RNA-Polymerase von Potyviren zu gewinnen. Die Expression der scFv-Gene in Pflanzen ist ein Weg zur Gewinnung von Kulturpflanzen, die resistent gegen Potyvirus-Infektionen sind. Die scFv könnten Anwendung bei der Diagnose von Potyviren finden. Folgende Ergebnisse wurden erzielt: 1. Vier monoklonale Antikörper (MAb), die gegen synthetische Peptide, die zwei der drei hochkonservierten Regionen des Kerneinschlussproteins b (NIb) von Potyviren widerspiegeln, gewonnen wurden, reagierten mit verschiedenen Potyviren. 2. Die Sequenz des NIb des potato Y potyvirus (PVY) sowie für eine synthetisches Protein, das aus einer Fusion aller drei hochkonservierten NIb-Regionen besteht, wurde in E. coli überexpremiert. Sie wurden für die Testung der Reaktivität der MAb genutzt. Das überexpremierte NIb wurde als Antigen für die MAb-Gewinnung eingesetzt. 3. MAb gegen das rekombinante NIb sowie das Hüllprotein (CP) des PVY wurden hergestellt. Es wurde gezeigt, dass sie PVY-Infektionen nachweisen können. Besonders der NIb-spezifische MAb 2E11 ist gut für diesen Nachweis geeignet. 4. Die Dynamik der Expression des CP und NIb des PVY in infizierten Nicotiana glutinosa und N. occidentalis wurde untersucht. Beide Proteine konnten in systemisch infizierten Blättern ab 5 dpi nachgewiesen werden. 5. ScFv-Antikörper gegen CP und NIb wurden aus Hybridomazellen, die MAb sekretierten, hergestellt. Die Expression des scFv 1D6F1, spezifisch für das PVY CP, erreichte ein Maximum von 17 vom gesamten löslichen Protein in E. coli. 6. Sowohl Phagen- als auch lösliche scFv-Antikörper zeigten ähnliche Bindungsaffinitäten mit den Ziel-Antigenen. Die Nachweisempfindlichkeit mit scFv war geringer als mit den Ausgangs-MAb. 7. ScFv-alkalische Phosphatase (AP) Fusionsproteine wiesen eine geringe Sensitivität auf, wahrscheinlich bedingt durch die niedrige enzymatische Aktivität der bakteriellen AP.The objective of this work was to engineer single chain variable fragment antibodies (scFvs) to highly conserved aa motifs of the RNA-dependent RNA polymerase of potyviruses (RdRp). The expression of scFv genes in plants holds great promise to create novel resistant crops against potyviral infections. The following results were obtained: 1. Four monoclonal antibodies (MAbs) obtained from synthetic peptides covering two of the three highly conserved regions of the nuclear inclusion b protein (NIb) of potyviruses reacted with the NIb of several potyviruses in both Western blot and ELISA. 2. The sequence encoding for the NIb of potato Y virus (PVY) as well as for a synthetic protein, consisting of a fusion of all three highly conserved NIb regions, was overexpressed in and purified from Escherichia coli near to homogeneity by immobilized metal-ion affinity chromatography. The fusion proteins were used to test the reactivity of MAbs. The overexpressed NIb was used as an antigen for MAb production. 3. MAbs to the recombinant NIb as well as to the coat protein (CP) of PVY were obtained. It was demonstrated that they can be used to detect PVY infection. 4. Dynamics of the expression of CP and NIb in PVY-infected Nicotiana glutinosa and N. occidentalis plants was systematically investigated. Both proteins could be detected in systemically infected leaves from the fifth day post infection on by ELISA and Western blotting. 5. ScFv antibodies were generated against CP and NIb from hybridoma cells secreting MAbs. Expression of scFv 1D6F1, specific for the PVY CP, reached a maximum of 17 of total soluble proteins in E. coli. 6. Both phage-displayed and soluble scFv showed a similar binding affinity to the target antigens. The detection sensitivity using scFv was lower than that of the parental MAb. 7. ScFv-alkaline phosphatase (AP) fusion proteins only showed low sensitivity, probably, due to the low enzymatic activity of the bacterial AP

Learning to Sample: an Active Learning Framework

Meta-learning algorithms for active learning are emerging as a promising paradigm for learning the ``best'' active learning strategy. However, current learning-based active learning approaches still require sufficient training data so as to generalize meta-learning models for active learning. This is contrary to the nature of active learning which typically starts with a small number of labeled samples. The unavailability of large amounts of labeled samples for training meta-learning models would inevitably lead to poor performance (e.g., instabilities and overfitting). In our paper, we tackle these issues by proposing a novel learning-based active learning framework, called Learning To Sample (LTS). This framework has two key components: a sampling model and a boosting model, which can mutually learn from each other in iterations to improve the performance of each other. Within this framework, the sampling model incorporates uncertainty sampling and diversity sampling into a unified process for optimization, enabling us to actively select the most representative and informative samples based on an optimized integration of uncertainty and diversity. To evaluate the effectiveness of the LTS framework, we have conducted extensive experiments on three different classification tasks: image classification, salary level prediction, and entity resolution. The experimental results show that our LTS framework significantly outperforms all the baselines when the label budget is limited, especially for datasets with highly imbalanced classes. In addition to this, our LTS framework can effectively tackle the cold start problem occurring in many existing active learning approaches.Comment: Accepted by ICDM'1

Microscopic validation of macroscopic in vivo images enabled by same-slide optical and nuclear fusion

It is currently difficult to determine the molecular and cellular basis for radioscintigraphic signals obtained during macroscopic in vivo imaging. The field is in need of technology that helps bridge the macroscopic and microscopic regimes. To solve this problem, we developed a fiducial marker (FM) simultaneously compatible with 2-color near-infrared (NIR) fluorescence (700 and 800 nm), autoradiography (ARG), as well as conventional hematoxylin and eosin (H&E) histology.Methods: The FM was constructed from an optimized concentration of commercially available human serum albumin (HSA), 700 nm and 800 nm NIR fluorophores, 99mTc-pertechnatete, DMSO, and glutaraldehyde (GA). Lymphangioleiomyomatosis (LAM) cells co-expressing the sodium iodide symporter (NIS) and green fluorescent protein (GFP) were labeled with 700 nm fluorophore and 99mTc-pertechnatete, then administered intratracheally into CD-1 mice. After in vivo SPECT imaging, and ex vivo SPECT and NIR fluorescence imaging of the lungs, 30 μm frozen sections were prepared and processed for 800 nm NIR fluorophore co-staining, ARG, and H&E staining on the same slide using the FMs to co-register all data sets.Results: Optimized FMs, composed of 100 μM unlabeled HSA, 1 μM NIR fluorescent HSA, 15% DMSO, and 3% GA in PBS (pH 7.4) were prepared within 15 min, displayed homogeneity and stability, and were visible by all imaging modalities, including H&E staining. Using these FMs, tissue displaying high signal by SPECT could be dissected and analyzed on the same slide and at the microscopic level for 700 nm NIR fluorescence, 800 nm NIR fluorescence, ARG, and H&E histopathological staining.Conclusion: When multimodal FMs are combined with a new technique for simultaneous same-slide NIR fluorescence imaging, ARG, and H&E staining, macroscopic in vivo images can now be studied unambiguously at the microscopic level

Role of GABAAR in the Transition From Acute to Chronic Pain and the Analgesic Effect of Electroacupuncture on Hyperalgesic Priming Model Rats

Chronic pain is a costly health problem that impairs health-related quality of life when not effectively treated. Regulating the transition from acute to chronic pain is a new therapeutic strategy for chronic pain that presents a major clinical challenge. The underlying mechanisms of pain transition are not entirely understood, and strategies for preventing this transition are lacking. Here, a hyperalgesic priming model was used to study the potential mechanism by which γ-aminobutyric acid receptor type A (GABAAR) in the dorsal root ganglion (DRG) contributes to pain transition. Furthermore, electroacupuncture (EA), a modern method of acupuncture, was administered to regulate pain transition, and the mechanism underlying EA's regulatory effect was investigated. Hyperalgesic priming was induced by intraplanar injection of carrageenan (Car)/prostaglandin E2 (PGE2). The decrease in mechanical withdrawal threshold (MWT) induced by PGE2 returned to baseline 4 h after injection in NS + PGE2 group, and still persisted 24 h after injection in Car + PGE2 group. Lower expression of GABAAR in the lumbar DRG was observed in the model rats. Furthermore, activating or blocking GABAAR could reversed the long-lasting hyperalgesia induced by Car/PGE2 injection or produced a persistent hyperalgesia. In addition, GABAAR may be involved in Protein Kinase C epsilon (PKCε) activation in the DRG, a mark molecular of pain transition. EA considerably increased the mechanical pain thresholds of hyperalgesic priming model mammals in both the acute and chronic phases. Furthermore, EA upregulated the expression of GABAAR and inhibited the activation of PKCε in the DRG. In addition, peripheral administration of picrotoxin blocked the analgesic effect of EA on the model rats and abolished the regulatory effect of EA on PKCε activation. These findings suggested that GABAAR plays a key role in both the transition from acute to chronic pain and the analgesic effect of EA on hyperalgesic priming

Design Considerations for Tumor-Targeted Nanoparticles

Inorganic/organic hybrid nanoparticles are potentially useful in biomedicine, but to avoid non-specific background fluorescence and long-term toxicity, they need to be cleared from the body within a reasonable timescale1. Previously, we have shown that rigid spherical nanoparticles such as quantum dots can be cleared by the kidneys if they have a hydrodynamic diameter of approximately 5.5 nm and a zwitterionic surface charge2. Here, we show that quantum dots functionalized with high-affinity small-molecule ligands that target tumours can also be cleared by the kidneys if their hydrodynamic diameter is less than this value, which sets an upper limit of 5–10 ligands per quantum dot for renal clearance. Animal models of prostate cancer and melanoma show receptor-specific imaging and renal clearance within 4 h post-injection. This study suggests a set of design rules for the clinical translation of targeted nanoparticles that can be eliminated through the kidneys.National Science Foundation (U.S.) (NSF-0070319)National Institutes of Health (U.S.) (NIH GM68762)National Institutes of Health (U.S.) (NIH grant no. R33-EB-000673)National Institutes of Health (U.S.) ( NIH grant no. R01-CA-115296)National Institutes of Health (U.S.) (MIT-Harvard NanoMedical Consortium (1U54-CA119349, a Center of Cancer Nanotechnology Excellence))Bank of AmericaMedical Foundation, inc. (Charles A. King Trust Postdoctoral Research Fellowship Program)cance

Edge states in a two-dimensional honeycomb lattice of massive magnetic skyrmions

We study the collective dynamics of a two-dimensional honeycomb lattice of magnetic skyrmions. By performing large-scale micromagnetic simulations, we find multiple chiral and non-chiral edge modes of skyrmion oscillations in the lattice. The non-chiral edge states are due to the Tamm-Shockley mechanism, while the chiral ones are topologically protected against structure defects and hold different handednesses depending on the mode frequency. To interpret the emerging multiband nature of the chiral edge states, we generalize the massless Thiele's equation by including a second-order inertial term of skyrmion mass as well as a third-order non-Newtonian gyroscopic term, which allows us to model the band structure of skrymion oscillations. Theoretical results compare well with numerical simulations. Our findings uncover the importance of high order effects in strongly coupled skyrmions and are helpful for designing novel topological devices.Comment: 6 pages,4 figures,accepted by Physical Review B as a Rapid Communicatio

Real-Time Monitoring of Tumorigenesis, Dissemination, & Drug Response in a Preclinical Model of Lymphangioleiomyomatosis/Tuberous Sclerosis Complex

Background: TSC2-deficient cells can proliferate in the lungs, kidneys, and other organs causing devastating progressive multisystem disorders such as lymphangioleiomyomatosis (LAM) and tuberous sclerosis complex (TSC). Preclinical models utilizing LAM patient-derived cells have been difficult to establish. We developed a novel animal model system to study the molecular mechanisms of TSC/LAM pathogenesis and tumorigenesis and provide a platform for drug testing. Methods and Findings: TSC2-deficient human cells, derived from the angiomyolipoma of a LAM patient, were engineered to co-express both sodium-iodide symporter (NIS) and green fluorescent protein (GFP). Cells were inoculated intraparenchymally, intravenously, or intratracheally into athymic NCr nu/nu mice and cells were tracked and quantified using single photon emission computed tomography (SPECT) and computed tomography (CT). Surprisingly, TSC2-deficient cells administered intratracheally resulted in rapid dissemination to lymph node basins throughout the body, and histopathological changes in the lung consistent with LAM. Estrogen was found to be permissive for tumor growth and dissemination. Rapamycin inhibited tumor growth, but tumors regrew after the drug treatment was withdrawn. Conclusions: We generated homogeneous NIS/GFP co-expressing TSC2-deficient, patient-derived cells that can proliferate and migrate in vivo after intratracheal instillation. Although the animal model we describe has some limitations, we demonstrate that systemic tumors formed from TSC2-deficient cells can be monitored and quantified noninvasively over time using SPECT/CT, thus providing a much needed model system for in vivo drug testing and mechanistic studies of TSC2-deficient cells and their related clinical syndromes

Neural activity associated with attention orienting triggered by implied action cues

Spatial attention can be directed by the actions of others. We used ERPs method to investigate the neural underpins associated with attention orienting which is induced by implied body action. Participants performed a standard non-predictive cuing task, in which a directional implied action (throwing and running) or non-action (standing) cue was randomly presented and then followed by a target to the left or right of the central cue, despite cue direction. The cue-triggered ERPs results demonstrated that implied action cues, rather than the non-action cue, could shift the observers' spatial attention as demonstrated by the robust anterior directing attention negativity (ADAN) effects in throwing and running cues. Further, earlier N1 (100-170 ms) and P2 (170-260 ms) waveform differences occurred between implied action and non-action cues over posterior electrodes. The P2 component might reflect implied motion signal perception of implied action cues, and this implied motion perception might play an important role in facilitating the attentional shifts induced by implied action cues. Target-triggered ERPs data (mainly P3a component) indicated that implied action cues (throwing and running) speeded and enhanced the responses to valid targets compared to invalid targets. Furthermore, P3a might imply that implied action orienting may share similar mechanisms of action with voluntary attention, especially at the novel stimuli processing decision-level. These results further support previous behavioral findings that implied body actions direct spatial attention and extend our understanding about the nature of the attentional shifts that are elicited by implied action cues. (C) 2016 Elsevier B.V. All rights reserved