In Vitro and In Vivo Effects of the Bumped Kinase Inhibitor 1294 in the Related Cyst-Forming Apicomplexans Toxoplasma gondii and Neospora caninum.

We report on the in vitro effects of the bumped kinase inhibitor 1294 (BKI-1294) in cultures of virulent Neospora caninum isolates Nc-Liverpool (Nc-Liv) and Nc-Spain7 and in two strains of Toxoplasma gondii (RH and ME49), all grown in human foreskin fibroblasts. In these parasites, BKI-1294 acted with 50% inhibitory concentrations (IC50s) ranging from 20 nM (T. gondii RH) to 360 nM (N. caninum Nc-Liv), and exposure of intracellular stages to 1294 led to the nondisjunction of newly formed tachyzoites, resulting in the formation of multinucleated complexes similar to complexes previously observed in BKI-1294-treated N. caninum beta-galactosidase-expressing parasites. However, such complexes were not seen in a transgenic T. gondii strain that expressed CDPK1 harboring a mutation (G to M) in the gatekeeper residue. In T. gondii ME49 and N. caninum Nc-Liv, exposure of cultures to BKI-1294 resulted in the elevated expression of mRNA coding for the bradyzoite marker BAG1. Unlike in bradyzoites, SAG1 expression was not repressed. Immunofluorescence also showed that these multinucleated complexes expressed SAG1 and BAG1 and the monoclonal antibody CC2, which binds to a yet unidentified bradyzoite antigen, also exhibited increased labeling. In a pregnant mouse model, BKI-1294 efficiently inhibited vertical transmission in BALB/c mice experimentally infected with one of the two virulent isolates Nc-Liv or Nc-Spain7, demonstrating proof of concept that this compound protected offspring from vertical transmission and disease. The observed deregulated antigen expression effect may enhance the immune response during BKI-1294 therapy and will be the subject of future studies

Neospora caninum calcium-dependent protein kinase 1 is an effective drug target for neosporosis therapy.

Despite the enormous economic importance of Neospora caninum related veterinary diseases, the number of effective therapeutic agents is relatively small. Development of new therapeutic strategies to combat the economic impact of neosporosis remains an important scientific endeavor. This study demonstrates molecular, structural and phenotypic evidence that N. caninum calcium-dependent protein kinase 1 (NcCDPK1) is a promising molecular target for neosporosis drug development. Recombinant NcCDPK1 was expressed, purified and screened against a select group of bumped kinase inhibitors (BKIs) previously shown to have low IC50s against Toxoplasma gondii CDPK1 and T. gondii tachyzoites. NcCDPK1 was inhibited by low concentrations of BKIs. The three-dimensional structure of NcCDPK1 in complex with BKIs was studied crystallographically. The BKI-NcCDPK1 structures demonstrated the structural basis for potency and selectivity. Calcium-dependent conformational changes in solution as characterized by small-angle X-ray scattering are consistent with previous structures in low Calcium-state but different in the Calcium-bound active state than predicted by X-ray crystallography. BKIs effectively inhibited N. caninum tachyzoite proliferation in vitro. Electron microscopic analysis of N. caninum cells revealed ultra-structural changes in the presence of BKI compound 1294. BKI compound 1294 interfered with an early step in Neospora tachyzoite host cell invasion and egress. Prolonged incubation in the presence of 1294 interfered produced observable interference with viability and replication. Oral dosing of BKI compound 1294 at 50 mg/kg for 5 days in established murine neosporosis resulted in a 10-fold reduced cerebral parasite burden compared to untreated control. Further experiments are needed to determine the PK, optimal dosage, and duration for effective treatment in cattle and dogs, but these data demonstrate proof-of-concept for BKIs, and 1294 specifically, for therapy of bovine and canine neosporosis

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Numerical study on flow and heat transfer of aviation kerosene at supercritical pressure in cooling channels with surface corrugations

In this paper, turbulent flow of aviation kerosene at supercritical pressure through cooling channels respectively with streamwise sinusoidal corrugation, spanwise sinusoidal corrugation and streamwise and spanwise coupled sinusoidal corrugations are numerically studied. The Reynolds-averaged Navier-Stokes method with RNG k -epsilon turbulence model is used to perform numerical simulation. The independence of grids is first examined, and the numerical results are compared with experimental data for validation. The present results show that the streamwise corrugation significantly improves convective heat transfer performance of cooling channels, the increase ratio of Nusselt number and Thermal Performance Factor reaches 20.44% and 6.7%. With the streamwise and spanwise corrugations coupling, the increase ratio of Nusselt number and Thermal Performance Factor are both larger than those in cooling channels with only streamwise corrugation. Furthermore, the results of flow and heat transfer of aviation kerosene at supercritical conditions with corrugations effect show that corrugations can eliminate heat transfer deterioration occurring at supercritical conditions. The present study is aimed to provide useful references for cooling optimization with wall microstructures for engineering applications

Exploiting Polydopamine Nanospheres to DNA Computing: A Simple, Enzyme-Free and G‑Quadruplex-Free DNA Parity Generator/Checker for Error Detection during Data Transmission

Molecular logic devices with various functions play an indispensable role in molecular data transmission/processing. However, during any kinds of data transmission, a constant and unavoidable circumstance is the appearance of bit errors, which have serious effects on the regular logic computation. Fortunately, these errors can be detected via plugging a parity generator (pG) at the transmitting terminal and a parity checker (pC) at the receiving terminal. Herein, taking advantage of the efficient adsorption/quenching ability of polydopamine nanospheres toward fluorophore-labeled single-stranded DNA, we explored this biocompatible nanomaterial to DNA logic computation and constructed the first simple, enzyme-free, and G-quadruplex-free DNA pG/pC for error detection through data transmission. Besides, graphene oxide (GO) was innovatively introduced as the “corrective element” to perform the output-correction function of pC. All the erroneous outputs were corrected to normal conditions completely, ensuring the regular operation of later logic computing. The total operation of this non-G4 pG/pC system (error checking/output-correction) could be completed within 1 h (about ¹/₃ of previous G4 platform) in a simpler and more efficient way. Notably, the odd pG/pC with analogous functions was also achieved through negative logic conversion to the fabricated even one. Furthermore, the same system could also perform three-input concatenated logic computation (XOR-INHIBIT), enriching the complexity of PDs-based logic computation

Traffic Context Aware Data Augmentation for Rare Object Detection in Autonomous Driving

Detection of rare objects (e.g., traffic cones, traffic barrels and traffic warning triangles) is an important perception task to improve the safety of autonomous driving. Training of such models typically requires a large number of annotated data which is expensive and time consuming to obtain. To address the above problem, an emerging approach is to apply data augmentation to automatically generate cost-free training samples. In this work, we propose a systematic study on simple Copy-Paste data augmentation for rare object detection in autonomous driving. Specifically, local adaptive instance-level image transformation is introduced to generate realistic rare object masks from source domain to the target domain. Moreover, traffic scene context is utilized to guide the placement of masks of rare objects. To this end, our data augmentation generates training data with high quality and realistic characteristics by leveraging both local and global consistency. In addition, we build a new dataset, Rare Object Dataset (ROD), consisting 10k training images, 4k validation images and the corresponding labels with a diverse range of scenarios in autonomous driving. Experiments on ROD show that our method achieves promising results on rare object detection. We also present a thorough study to illustrate the effectiveness of our local-adaptive and global constraints based Copy-Paste data augmentation for rare object detection. The data, development kit and more information of ROD are available online at: \url{https://nullmax-vision.github.io}.Comment: The IEEE Conference on Robotics and Automation, ICRA 202

Molecular Targets of the 5-Amido-Carboxamide Bumped Kinase Inhibitor BKI-1748 in Cryptosporidium parvum and HCT-8 Host Cells.

Cryptosporidium parvum is an apicomplexan parasite causing persistent diarrhea in humans and animals. Issuing from target-based drug development, calcium-dependent protein kinase 1 inhibitors, collectively named bumped kinase inhibitors (BKIs), with excellent efficacies in vitro and in vivo have been generated. Some BKIs including BKI-1748 share a core structure with similarities to the first-generation antiprotozoal drug quinine, which is known to exert notorious side effects. Unlike quinine, BKI-1748 rapidly interfered with C. parvum proliferation in the human colon tumor (HCT) cell line HCT-8 cells and caused dramatic effects on the parasite ultrastructure. To identify putative BKI targets in C. parvum and in host cells, we performed differential affinity chromatography with cell-free extracts from non-infected and infected HCT-8 cells using BKI-1748 and quinine epoxy-activated sepharose columns followed by mass spectrometry. C. parvum proteins of interest were identified in eluates from columns coupled to BKI-1748, or in eluates from both BKI-1748 and quinine columns. However, no C. parvum proteins could be identified binding exclusively to BKI-1748. In contrast, 25 BKI-1748-specific binding proteins originating from HCT-8 cells were detected. Moreover, 29 C. parvum and 224 host cell proteins were identified in both BKI-1748 as well as in quinine eluates. In both C. parvum and host cells, the largest subset of binding proteins was involved in RNA binding and modification, with a focus on ribosomal proteins and proteins involved in RNA splicing. These findings extend previous results, showing that BKI-1748 interacts with putative targets involved in common, essential pathways such as translation and RNA processing