Graded or threshold response of the tet-controlled gene expression: all depends on the concentration of the transactivator

BACKGROUND: Currently, the step-wise integration of tet-dependent transactivator and tet-responsive expression unit is considered to be the most promising tool to achieve stable tet-controlled gene expression in cell populations. However, disadvantages of this strategy for integration into primary cells led us to develop an “All-In-One” vector system, enabling simultaneous integration of both components. The effect on tet-controlled gene expression was analyzed for retroviral “All-In-One” vectors expressing the M2-transactivator either under control of a constitutive or a new type of autoregulated promoter. RESULTS: Determination of luciferase activity in transduced cell populations indicated improvement of the dynamic range of gene expression for the autoregulated system. Further differences were observed regarding induction kinetics and dose–response. Most notably, introduction of the autoregulated system resulted in a threshold mode of induction, whereas the constitutive system exhibited pronounced effector-dose dependence. CONCLUSION: Tet-regulated gene expression in the applied autoregulated system resembles a threshold mode, whereby full induction of the tet-unit can be achieved at otherwise limiting doxycycline concentrations

Improved Tet-responsive promoters with minimized background expression

<p>Abstract</p> <p>Background</p> <p>The performance of the tetracycline controlled transcriptional activation system (Tet system) depends critically on the choice of minimal promoters. They are indispensable to warrant low expression levels with the system turned "off". On the other hand, they must support high level of gene expression in the "on"-state.</p> <p>Results</p> <p>In this study, we systematically modified the widely used Cytomegalovirus (CMV) minimal promoter to further minimize background expression, resulting in an improved dynamic expression range. Using both plasmid-based and retroviral gene delivery, our analysis revealed that especially background expression levels could be significantly reduced when compared to previously established "standard" promoter designs. Our results also demonstrate the possibility to fine-tune expression levels in non-clonal cell populations. They also imply differences regarding the requirements for tight regulation and high level induction between transient and stable gene transfer systems.</p> <p>Conclusions</p> <p>Until now, our understanding of mammalian transcriptional regulation including promoter architecture is limited. Nevertheless, the partly empirical modification of <it>cis</it>-elements as shown in this study can lead to the specific improvement of the performance of minimal promoters. The novel composite Ptet promoters introduced here will further expand the utility of the Tet system.</p

Early Detection of Stripe Rust in Winter Wheat Using Deep Residual Neural Networks

Stripe rust (Pst) is a major disease of wheat crops leading untreated to severe yield losses. The use of fungicides is often essential to control Pst when sudden outbreaks are imminent. Sensors capable of detecting Pst in wheat crops could optimize the use of fungicides and improve disease monitoring in high-throughput field phenotyping. Now, deep learning provides new tools for image recognition and may pave the way for new camera based sensors that can identify symptoms in early stages of a disease outbreak within the field. The aim of this study was to teach an image classifier to detect Pst symptoms in winter wheat canopies based on a deep residual neural network (ResNet). For this purpose, a large annotation database was created from images taken by a standard RGB camera that was mounted on a platform at a height of 2 m. Images were acquired while the platform was moved over a randomized field experiment with Pst-inoculated and Pst-free plots of winter wheat. The image classifier was trained with 224 × 224 px patches tiled from the original, unprocessed camera images. The image classifier was tested on different stages of the disease outbreak. At patch level the image classifier reached a total accuracy of 90%. To test the image classifier on image level, the image classifier was evaluated with a sliding window using a large striding length of 224 px allowing for fast test performance. At image level, the image classifier reached a total accuracy of 77%. Even in a stage with very low disease spreading (0.5%) at the very beginning of the Pst outbreak, a detection accuracy of 57% was obtained. Still in the initial phase of the Pst outbreak with 2 to 4% of Pst disease spreading, detection accuracy with 76% could be attained. With further optimizations, the image classifier could be implemented in embedded systems and deployed on drones, vehicles or scanning systems for fast mapping of Pst outbreaks

Optimized Deep Learning Model as a Basis for Fast UAV Mapping of Weed Species in Winter Wheat Crops

Weed maps should be available quickly, reliably, and with high detail to be useful for site-specific management in crop protection and to promote more sustainable agriculture by reducing pesticide use. Here, the optimization of a deep residual convolutional neural network (ResNet-18) for the classification of weed and crop plants in UAV imagery is proposed. The target was to reach sufficient performance on an embedded system by maintaining the same features of the ResNet-18 model as a basis for fast UAV mapping. This would enable online recognition and subsequent mapping of weeds during UAV flying operation. Optimization was achieved mainly by avoiding redundant computations that arise when a classification model is applied on overlapping tiles in a larger input image. The model was trained and tested with imagery obtained from a UAV flight campaign at low altitude over a winter wheat field, and classification was performed on species level with the weed species Matricaria chamomilla L., Papaver rhoeas L., Veronica hederifolia L., and Viola arvensis ssp. arvensis observed in that field. The ResNet-18 model with the optimized image-level prediction pipeline reached a performance of 2.2 frames per second with an NVIDIA Jetson AGX Xavier on the full resolution UAV image, which would amount to about 1.78 ha h−1 area output for continuous field mapping. The overall accuracy for determining crop, soil, and weed species was 94%. There were some limitations in the detection of species unknown to the model. When shifting from 16-bit to 32-bit model precision, no improvement in classification accuracy was observed, but a strong decline in speed performance, especially when a higher number of filters was used in the ResNet-18 model. Future work should be directed towards the integration of the mapping process on UAV platforms, guiding UAVs autonomously for mapping purpose, and ensuring the transferability of the models to other crop fields

Terahertz optically pumped silicon lasers

Stimulated terahertz (THz) emission from silicon single crystals doped by group-V donors has been obtained by optical excitation with pulsed infrared lasers. Pumping by a conventional TEA CO2 laser results in lasing on discrete lines between 1.3 and 7 THz (see figure). Laser thresholds can be as low as 10 kW/cm2. They depend on the donors species and the laser mechanism. Intracentre population inversion is realized between particular excited states which are large-spaced due to the chemical shift of the donor binding energy. The lifetime of an electron in an excited state (up to ~70 ps) is determined by the efficiency of phonon-assisted nonradiative relaxation. Optical excitation by the emission of a frequency-tunable free electron laser results in two different types of lasing. At relatively low pump intensities (~1 kW/cm2) the intracentre mechanism of lasing is dominating. At pump intensities above ~100 kW/cm2 stimulated scattering of pump photons on transverse acoustic intervalley phonons can occur in the vicinity of an impurity atom. This results in laser emission in the frequency range from 4.6 to 5.8 THz. In this case the laser frequency can be tuned proportionally to the pump frequency

The BRCT domain of mammalian Rev1 is involved in regulating DNA translesion synthesis

Rev1 is a deoxycytidyl transferase associated with DNA translesion synthesis (TLS). In addition to its catalytic domain, Rev1 possesses a so-called BRCA1 C-terminal (BRCT) domain. Here, we describe cells and mice containing a targeted deletion of this domain. Rev1(B/B) mice are healthy, fertile and display normal somatic hypermutation. Rev1(B/B) cells display an elevated spontaneous frequency of intragenic deletions at Hprt. In addition, these cells were sensitized to exogenous DNA damages. Ultraviolet-C (UV-C) light induced a delayed progression through late S and G2 phases of the cell cycle and many chromatid aberrations, specifically in a subset of mutant cells, but not enhanced sister chromatid exchanges (SCE). UV-C-induced mutagenesis was reduced and mutations at thymidine–thymidine dimers were absent in Rev1(B/B) cells, the opposite phenotype of UV-C-exposed cells from XP-V patients, lacking TLS polymerase η. This suggests that the enhanced UV-induced mutagenesis in XP-V patients may depend on error-prone Rev1-dependent TLS. Together, these data indicate a regulatory role of the Rev1 BRCT domain in TLS of a limited spectrum of endogenous and exogenous nucleotide damages during a defined phase of the cell cycle

Strand-biased defect in C/G transversions in hypermutating immunoglobulin genes in Rev1-deficient mice

Somatic hypermutation of Ig genes enables B cells of the germinal center to generate high-affinity immunoglobulin variants. Key intermediates in somatic hypermutation are deoxyuridine lesions, introduced by activation-induced cytidine deaminase. These lesions can be processed further to abasic sites by uracil DNA glycosylase. Mutagenic replication of deoxyuridine, or of its abasic derivative, by translesion synthesis polymerases is hypothesized to underlie somatic hypermutation

Aspects of high hydrostatic pressure food processing: Perspectives on technology and food safety

The last two decades saw a steady increase of high hydrostatic pressure (HHP) used for treatment of foods. Although the science of biomaterials exposed to high pressure started more than a century ago, there still seem to be a number of unanswered questions regarding safety of foods processed using HHP. This review gives an overview on historical development and fundamental aspects of HHP, as well as on potential risks associated with HHP food applications based on available literature. Beside the combination of pressure and temperature, as major factors impacting inactivation of vegetative bacterial cells, bacterial endospores, viruses, and parasites, factors, such as food matrix, water content, presence of dissolved substances, and pH value, also have significant influence on their inactivation by pressure. As a result, pressure treatment of foods should be considered for specific food groups and in accordance with their specific chemical and physical properties. The pressure necessary for inactivation of viruses is in many instances slightly lower than that for vegetative bacterial cells; however, data for food relevant human virus types are missing due to the lack of methods for determining their infectivity. Parasites can be inactivated by comparatively lower pressure than vegetative bacterial cells. The degrees to which chemical reactions progress under pressure treatments are different to those of conventional thermal processes, for example, HHP leads to lower amounts of acrylamide and furan. Additionally, the formation of new unknown or unexpected substances has not yet been observed. To date, no safety-relevant chemical changes have been described for foods treated by HHP. Based on existing sensitization to non-HHP-treated food, the allergenic potential of HHP-treated food is more likely to be equivalent to untreated food. Initial findings on changes in packaging materials under HHP have not yet been adequately supported by scientific data

Is radical surgery of an inverted papilloma of the maxillary sinus obsolete? a case report

Abstract Background Sinonasal inverted papilloma is a locally aggressive tumor arising from the Schneiderian membrane which lines the nasal cavity and paranasal sinuses. Aggressive surgical approaches, such as lateral rhinotomy, were used until recently for complete removal of the inverted papilloma. Currently, endoscopic resection is the gold standard in the treatment of inverted papilloma. However, there are situations that justify an open approach. For example there are studies that report a higher postoperative recurrence rate after endonasal endoscopic resection, particularly in the treatment of recurrent diseases. While endoscopic resection performed by an experienced surgeon is definitely a minimally invasive therapy, an open approach is not necessarily associated with functional and aesthetic disadvantages. This case report describes the treatment of inverted papilloma by an open approach. This has been described before but the new gold standard of endoscopic resection has to be taken into account before any treatment decision is made nowadays. Case presentation Contrast-enhanced magnetic resonance imaging of the head and neck area was indicated in a 72-year-old white German man who presented with suspected squamous cell carcinoma of his lower lip. Magnetic resonance imaging additionally revealed a 3×2 cm2 polycyclic arranged mucosal thickening with cystic and solid contrast affine shares at the antral laterocaudal area of his right maxillary sinus, extending from his right lateral nasal wall to his maxillary sinus floor. He received antral polypectomy with medial maxillectomy via a unilateral LeFort I osteotomy approach. His pterygoid plate was preserved. A histological examination demonstrated a tumor composed of hyperplastic squamous epithelium protruding into the stroma (surface epithelial cells grew downward into the underlying supportive tissue), thus producing a grossly convoluted cerebriform appearance. Two weeks later, the patient regained a well-formed maxilla without any restrictions. He has remained disease-free for 25 months following the surgery and surveillance was continued in our tumor clinic. Conclusions Endoscopic resection of an inverted papilloma continues to be the gold standard. However, some cases require a radical approach. This does not necessarily increase patient morbidity