The Toll/interleukin-1 receptor (TIR) signaling domain is distributed widely in mammalian Toll-like receptors and adaptors, plant nucleotide-binding leucine-rich repeat receptors, and specific bacterial virulence proteins. Proteins that possess TIR domain exhibit NADase activity which is distinct from the canonical signaling function of these domains. However, the effects of bacterial TIR domain proteins on host metabolic switches and the underlying mechanism of NADase activity in these proteins remain unclear. Here, we utilized Brucella TIR domain-containing type IV secretion system effector protein, BtpB, to explore the mechanism of NADase activity in host cells. We showed that using ectopic expression BtpB not only generates depletion of NAD+ but also loss of NADH and ATP in RAW264.7 macrophage cells. Moreover, immunoprecipitation-mass spectrometry, co-immunoprecipitation, and confocal microscope assays revealed that BtpB interacted with host protein disulfide isomerase A4 (PDIA4). The Brucella mutant strain deleted the gene for BtpB, significantly decreased PDIA4 expression. Furthermore, our data revealed that PDIA4 played an important role in regulating intracellular NAD+/NADH levels in macrophages, and PDIA4 overexpression restored the decline of intracellular NAD+ and NADH levels induced by Brucella BtpB. The results provide new insights into the metabolic regulatory activity of TIR domain proteins in the critical human and animal pathogen Brucella.

Autonomous robot-based orchard operations will become an alternative solution in the field of precision agriculture. One of the keys to robotic work is to achieve autonomous navigation that is as accurate as possible to ensure the most accurate working effect. In this work, we propose an orchard path fitting and navigation method based on the fusion of improved A-Star algorithm and Support Vector Machine Regression (SVR) to meet the requirements of autonomous navigation crawler platform for autonomous navigation in orchard environment and ensure accuracy. In this study, the actual speed and turning radius of the left and right tracks of the crawler platform were collected under 5 different slopes and 400 sets of different theoretical speed combinations of left and right tracks through the design nesting test, and the motion model of the crawler platform was constructed based on SVR. Orchard point cloud data were obtained by 3D solid-state LiDAR, and the improved A-star algorithm was used to fit the navigation path and calculate the turning curvature radius. Taking this curvature radius as the optimal navigation target value, the motion model predicts the optimal theoretical speed of left and right tracks, which is used as a reference for autonomous navigation. The comparison experiment of autonomous navigation was carried out in two modes: traditional and improved A-Star algorithm. The results show that the average values of the maximum lateral and longitudinal deviation of the improved automatic navigation method between orchards row are 6.90 cm and 9.88 cm, respectively. Compared with the method combined with the traditional A-Star algorithm and SVR, the values were 8.94 cm and 10.88 cm and were optimized by 29.57% and 10.12%, respectively. The autonomous navigation method proposed in this paper can meet the needs of orchards rows autonomous navigation, and can be widely applied to different orchard site environments (slope, ground obstacles, bad surface conditions), which can provide reference for the production practices of unmanned orchards.

Brucella is an intracellular parasitic bacterium lacking typical virulence factors, and its pathogenicity primarily relies on replication within host cells. In this study, we observed a significant increase in spleen weight in mice immunized with a Brucella strain deleted of the gene for alanine racemase (Alr), the enzyme responsible for alanine racemization (Δalr). However, the bacterial load in the spleen markedly decreased in the mutant strain. Concurrently, the ratio of white pulp to red pulp in the spleen was increased, serum IgG levels were elevated, but no significant damage to other organs was observed. In addition, the inflammatory response was potentiated and the NF-κB-NLRP3 signaling pathway was activated in macrophages (RAW264.7 Cells and Bone Marrow-Derived Cells) infect ed with the Δalr mutant. Further investigation revealed that the Δalr mutant released substantial amounts of protein in a simulated intracellular environment which resulted in heightened inflammation and activation of the TLR4-NF-κB-NLRP3 pathway in macrophages. The consequent cytoplasmic exocytosis reduced intracellular Brucella survival. In summary, cytoplasmic exocytosis products resulting from infection with a Brucella strain deleted of the alr gene effectively activated the TLR4-NFκB-NLRP3 pathway, triggered a robust inflammatory response, and reduced bacterial survival within host cells. Moreover, the Δalr strain exhibits lower toxicity and stronger immunogenicity in mice.

Brucella, a zoonotic facultative intracellular pathogenic bacterium, poses a significant threat both to human health and to the development of the livestock industry. Alanine racemase (Alr), the enzyme responsible for alanine racemization, plays a pivotal role in regulating virulence in this bacterium. Moreover, Brucella mutants with alr gene deletions (Δalr) exhibit potential as vaccine candidates. However, the mechanisms that underlie the detrimental effects of alr knockouts on Brucella pathogenicity remain elusive. Here, initially, we conducted a bioinformatics analysis of Alr, which demonstrated a high degree of conservation of the protein within Brucella spp. Subsequent metabolomics studies unveiled alterations in amino acid pathways following deletion of the alr gene. Furthermore, alr deletion in Brucella suis S2 induced decreased resistance to stress, antibiotics, and other factors. Transmission electron microscopy of simulated macrophage intracellular infection revealed damage to the cell wall in the Δalr strain, whereas propidium iodide staining and alkaline phosphatase and lactate dehydrogenase assays demonstrated alterations in cell membrane permeability. Changes in cell wall properties were revealed by measurements of cell surface hydrophobicity and zeta potential. Finally, the diminished adhesion capacity of the Δalr strain was shown by immunofluorescence and bacterial enumeration assays. In summary, our findings indicate that the alr gene that regulates amino acid metabolism in Brucella influences the properties of the cell wall, which modulates bacterial adherence capability. This study is the first demonstration that Alr impacts virulence by modulating bacterial metabolism, thereby providing novel insights into the pathogenic mechanisms of Brucella spp.

Brucella suis, the causative agent of brucellosis, poses a significant public health and animal husbandry threat. However, the role of the alanine racemase (alr) gene, which encodes alanine racemase in Brucella, remains unclear. Here, we analyzed an alr deletion mutant and a complemented strain of Brucella suis S2. The knockout strain displayed an unaltered, smooth phenotype in acriflavine agglutination tests but lacked the core polysaccharide portion of lipopolysaccharide (LPS). Genes involved in the LPS synthesis were significantly upregulated in the deletion mutant. The alr deletion strain exhibited reduced intracellular viability in the macrophages, increased macrophage-mediated killing, and upregulation of the apoptosis markers. Bcl2, an anti-apoptotic protein, was downregulated, while the pro-apoptotic proteins, Bax, Caspase-9, and Caspase-3, were upregulated in the macrophages infected with the deletion strain. The infected macrophages showed increased mitochondrial membrane permeability, Cytochrome C release, and reactive oxygen species, activating the mitochondrial apoptosis pathway. These findings revealed that alanine racemase was dispensable in B. suis S2 but influenced the strain's rough features and triggered the mitochondrial apoptosis pathway during macrophage invasion. The deletion of the alr gene reduced the intracellular survival and virulence. This study enhances our understanding of the molecular mechanism underlying Brucella's survival and virulence and, specifically, how alr gene affects host immune evasion by regulating bacterial LPS biosynthesis.

The phenotypic information of lettuce leaves can well reflect its health. In order to diagnose the nutrient deficiency types of hydroponic lettuce accurately, non-destructively and quickly in the mid-growth stage, a method for diagnosis of whole lettuce based on random forest algorithm (RF) was proposed. The images of lettuce under four different conditions, K-deficiency, Ca-deficiency, N-deficiency and Normal, were collected and segmented by Extra-green algorithm. Then, features of color, texture and shape were extracted. A RF classification model for the hydroponic lettuce nutrient deficiency diagnosis was constructed and compared with support vector machine (SVM) and back propagation neural network (BP). RF had the best classification effect among the three methods. The overall classification accuracy was 86.32%, Kappa coefficient was 0.82, and it can provide a basis for the prevention and remedies of lettuce deficiency and the scientific management of nutrient solutions.

Hydroponic lettuce was one of typical vegetables in plant factory, and its production scale is increasing rapidly. In order to extend the preservation time of for sale and thus increase the added value, effect of root lengths on preservation performance for hydroponic lettuce was studied. After that, a root cutting machine for hydroponic lettuce was developed. Four indexes (changes in relative chlorophyll content, changes in color of lettuce leaves, weight loss rate, and sensory quality) were employed to study the effects of different root lengths on hydroponic lettuce fresh-keeping performance, which concluded that keeping the lettuce root length 9 cm was good for lettuce preservation. The root cutting machine was then developed according to the preservation tests. The key components of the root cutting machine including a setting board conveying device, a root cutting device, and a lettuce pulling and carrying device, which were designed though preliminary tests and mechanical analysis. The root cutting machine was manufactured and tested by 90 lettuces which aimed to measure its success rate and operation efficiency. Experimental results showed that the success rate of root cutting was 82% and the operation efficiency for one lettuce was 7.7 s.