Farmland boundary extraction based on the AttMobile-DeeplabV3+ network and least squares fitting of straight lines.

The rapid extraction of farmland boundaries is key to implementing autonomous operation of agricultural machinery. This study addresses the issue of incomplete farmland boundary segmentation in existing methods, proposing a method for obtaining farmland boundaries based on unmanned aerial vehicle (UAV) remote sensing images. The method is divided into two steps: boundary image acquisition and boundary line fitting. To acquire the boundary image, an improved semantic segmentation network, AttMobile-DeeplabV3+, is designed. Subsequently, a boundary tracing function is used to track the boundaries of the binary image. Lastly, the least squares method is used to obtain the fitted boundary line. The paper validates the method through experiments on both crop-covered and non-crop-covered farmland. Experimental results show that on crop-covered and non-crop-covered farmland, the network's intersection over union (IoU) is 93.25% and 93.14%, respectively; the pixel accuracy (PA) for crop-covered farmland is 96.62%. The average vertical error and average angular error of the extracted boundary line are 0.039 and 1.473°, respectively. This research provides substantial and accurate data support, offering technical assistance for the positioning and path planning of autonomous agricultural machinery.

Contribution of the NLRP3/IL-1ß axis to impaired vasodilation in sepsis through facilitation of eNOS proteolysis and the protective role of melatonin.

Endothelial dysfunction is a typical characteristic of sepsis. Endothelial nitric oxide synthase (eNOS) is important for maintaining endothelial function. Our previous study reported that the NLRP3 inflammasome promoted endothelial dysfunction by enhancing inflammation. However, the effects of NLRP3 on eNOS require further investigation. Therefore, the present study aimed to investigate the role of NLRP3 on eNOS expression levels in cecal ligation and puncture-induced impaired endothelium-dependent vascular relaxation and to determine the protective effects of melatonin. eNOS expression levels were discovered to be downregulated in the mesenteric arteries of sepsis model mice. Inhibiting NLRP3 with 10 mg/ kg MCC950 or inhibiting IL-1ß with 100 mg diacerein rescued the eNOS expression and improved endothelium-dependent vascular relaxation. In vitro, IL-1ß stimulation downregulated eNOS expression levels in human aortic endothelial cells (HAECs) in a concentration- and time-dependent manner, while pretreatment with 1 µM of the proteasome inhibitor MG132 reversed this effect. In addition, treatment with 10 mg/kg MG132 also prevented the proteolysis of eNOS and improved endothelium-dependent vascular relaxation in vivo. Notably, treatment with 30 mg/kg melatonin downregulated NLRP3 expression levels and decreased IL-1ß secretion, subsequently increasing the expression of eNOS and improving endothelium-dependent vascular relaxation. In conclusion, the findings of the present study indicated that the NLRP3/IL-1ß axis may impair vasodilation by promoting the proteolysis of eNOS and melatonin may protect against sepsis-induced endothelial relaxation dysfunction by inhibiting the NLRP3/IL-1ß axis, suggesting its pharmacological potential in sepsis.

Disrupted eNOS activity and expression account for vasodilator dysfunction in different stage of sepsis.

AIMS: Sepsis is a severe endothelial dysfunction syndrome. The role of endothelial nitric oxide synthase (eNOS) in endothelial dysfunction induced by sepsis is controversial. To explore the role of eNOS in vascular dysfunction. MAIN METHODS: The effect of sepsis on vasodilation and eNOS levels was examined in septic mouse arteries and in cell models. KEY FINDINGS: In early sepsis mouse arteries, endothelium-dependent relaxation decreased and phosphorylation of the inhibitory Thr495 site in endothelial nitric oxide synthase increased. Mechanically, the phosphorylation of endothelial nitric oxide synthase at Thr497 in bovine aortic endothelial cells occurred in a protein kinase C-α dependent manner. In late sepsis, both nitric oxide-dependent relaxation responses and endothelial nitric oxide synthase levels were decreased in septic mice arteries. Endothelial nitric oxide synthase levels expression levels decreased in tumor necrosis factor-α-treated human umbilical vein endothelial cells and this could be prevented by the ubiquitin proteasome inhibitor (MG-132). MG-132 could reverse the decrease in endothelial nitric oxide synthase expression and improve nitric oxide-dependent vasodilator dysfunction in septic mice arteries. SIGNIFICANCE: These data indicate that vasodilator dysfunction is induced by the increased phosphorylation of endothelial nitric oxide synthase in early sepsis and its degradation in late sepsis.

Regulation of O-GlcNAcylation on endothelial nitric oxide synthase by glucose deprivation and identification of its O-GlcNAcylation sites.

As an energy-sensitive post-translational modification, O-GlcNAcylation plays a major role in endothelial nitric oxide synthase (eNOS) activity regulation. However, effects of glucose deprivation on eNOS O-GlcNAcylation and the presence of novel O-GlcNAcylation sites of eNOS under glucose deprivation remain unknown. Hence, we aim to determine the effects of glucose deprivation on O-GlcNAcylation and novel O-GlcNAcylation sites of eNOS. Bovine aortic endothelial cells (BAECs) and Sprague-Dawley rats were induced by glucose deprivation and their eNOS O-GlcNAcylation was subjected to immunoblotting. eNOS and transfected eNOS were purified by pull-down assay and immunoprecipitation respectively. Novel O-GlcNAcylation sites of eNOS were predicted by HPLC-MS and MS/MS Ion and determined by immunoblotting. eNOS activity was detected by Elisa and isotope labeling method. In BAECs and rat thoracic aorta, low glucose-associated activation of eNOS was accompanied by elevated O-GlcNAcylation, which did not affect O-linked serine phosphorylation at 1179/1177 residues. Changes in this post-translational modification were associated with increased O-GlcNAc transferase (OGT) expression and were reversed by AMPK knockdown. Immunoblot analysis of cells expressing His-tagged wild-type human eNOS and human eNOS carrying a mutation at the Ser1177 phosphorylation site confirmed an increase in O-GlcNAcylation by glucose deprivation. A marked increase in O-GlcNAcylation indicated that eNOS contained novel O-GlcNAcylation sites that were activated by glucose deprivation. Immunoblot analysis of cells expressing His-tagged human eNOS carrying a mutation at Ser738 and Ser867 confirmed an increase in O-GlcNAcylation by glucose deprivation. Conversely, in His-tagged human eNOS carrying a mutation at Thr866, O-GlcNAcylation was unaffected by glucose deprivation. Differences in culture conditions were identified using two-way analysis of variance (ANOVA), one-way ANOVA, and unpaired Student's t-test. Glucose deprivation increases O-GlcNAcylation and activity of eNOS, potentially by the AMPK-OGT pathway, suggesting that Thr866 is a novel O-GlcNAcylation site involved in glucose-deprivation mediated eNOS activation.

N-Glycosylation at Asn695 might suppress inducible nitric oxide synthase activity by disturbing electron transfer.

Inducible nitric oxide synthase (iNOS) plays critical roles in the inflammatory response and host defense. Previous research on iNOS regulation mainly focused on its gene expression level, and much less is known about the regulation of iNOS function by N-glycosylation. In this study, we report for the first time that iNOS is N-glycosylated in vitro and in vivo. Mass spectrometry studies identified Asn695 as an N-glycosylation site of murine iNOS. Mutating Asn695 to Gln695 yields an iNOS that exhibits greater enzyme activity. The essence of nitric oxide synthase catalytic reaction is electron transfer process, which involves a series of conformational changes, and the linker between the flavin mononucleotide-binding domain and the flavin adenine dinucleotide-binding domain plays vital roles in the conformational changes. Asn695 is part of the linker, so we speculated that attachment of N-glycan to the Asn695 residue might inhibit activity by disturbing electron transfer. Indeed, our NADPH consumption results demonstrated that N-glycosylated iNOS consumes NADPH more slowly. Taken together, our results indicate that iNOS is N-glycosylated at its Asn695 residue and N-glycosylation of Asn695 might suppress iNOS activity by disturbing electron transfer.

A calibration transfer optimized single kernel near-infrared spectroscopic method.

Single kernel near-infrared spectroscopy (SKNIRS) could aid in the quality screening of early-generation seeds, to improve the efficiency of seed breeding. However, the application of SKNIRS is limited due to the irregular physical characteristics, the heterogeneous constituent distributions of individual seeds, and the insufficient detection accuracy of the reference method. The reported near-infrared detection results of single seeds are often less accurate than those of dehusked seeds and seed flour. In this paper, a calibration transfer-optimized single kernel near-infrared spectroscopic method is proposed. This method aims to accurately detect the chemical composition of single seeds by using the calibration model of the corresponding dehusked seeds or seed flour. The proposed method was applied to the analysis of the protein content of a single rice kernel. The near-infrared transmission spectra of three forms of rice (single rice kernel (SRK), single brown rice kernel (SBK) and rice flour (RF)) of 201 individual rice seeds and the corresponding protein content values were obtained. By comparing different pretreatment methods and spectral ranges, the spectral range 950-1250 nm, the standard normal variate transformation (SNV) pretreatment, and 9 PLS factors were selected to construct the optimal partial least squares (PLS) regression models. Then, the protein content of single rice kernels were determined through two different methods: (i) the direct method, in which single rice kernels were analyzed using the single rice kernel model directly; and (ii) the proposed method, in which the spectra of single rice kernels were transferred into the spectra of single brown rice kernels and rice flours with a calibration transfer algorithm, spectral space transformation (SST), and were analyzed by the respective calibration models. The external validation coefficient correlation (R) value of the direct method was 0.971, and the R values of the proposed method were 0.962 (SBK) and 0.975 (RF). The root mean square error of prediction (RMSEP) value of the direct method was 0.423, and the RMSEP of the proposed method were 0.480 (SBK) and 0.401 (RF). In addition, the transfer results among the spectra of three forms of rice were compared. By comparison, the results of the proposed method are fairly close to the results of the direct method. The results indicate that the spectra generated from one individual rice seed can be transferred freely among the three forms by means of calibration transfer. The proposed method is a promising way to overcome the challenges associated with analyzing individual seeds and to improve SKNIRS.

Identification of an unknown glycoprotein from whole cell lysate using conA and mass spectrometry.

Medical laboratory technology major was set up to meet rapid development of science and medical research technology in 2013. Students majoring in medical laboratory had learnt a lot of techniques distributed among different specialized courses. But, they did not understand why they had to learn these techniques and how they were applied in a real-world research setting. In a one-month innovation experimental practice described herein, students had learnt to induce, purify and identify an unknown glycoprotein from whole cell lysate using conA-based affinity chromatography and mass spectrometry. Unlike in a traditional cookbook-style experiment, students chose a research subject on their own and did experiment using their selected variables. Over the one-month laboratory periods, students used sterile technique to cultivate cells, induced glycoprotein expression using LPS and IFN-γ, purified glycoprotein from cell lysate using agarose-conA beads, identified a glycoprotein via mass spectrometry, and confirmed the result using western blotting. At end of the practice, students were asked to evaluate their experiences via an anonymous survey. All students declared that this experimental practice was interesting and meaningful to them. The process of completing the project was to apply the learnt techniques to real-world biochemistry research, so they became aware of the importance and significance of techniques. © 2018 by The International Union of Biochemistry and Molecular Biology, 46:373-381, 2018.