Morphological determination of localization and function of Golgi proteins.

In animal cells, the Golgi apparatus serves as the central hub of the endomembrane secretory pathway. It is responsible for the processing, modification, and sorting of proteins and lipids. The unique stacking and ribbon-like architecture of the Golgi apparatus forms the foundation for its precise functionality. Under cellular stress or pathological conditions, the structure of the Golgi and its important glycosylation modification function may change. It is crucial to employ suitable methodologies to study the structure and function of the Golgi apparatus, particularly when assessing the involvement of a target protein in Golgi regulation. This article provides a comprehensive overview of the diverse microscopy techniques used to determine the specific location of the target protein within the Golgi apparatus. Additionally, it outlines methods for assessing changes in the Golgi structure and its glycosylation modification function following the knockout of the target gene.

Study on the underlying molecular mechanism of benzene-induced nervous system damage in mice based on tandem mass tag (TMT) proteomics.

Benzene is known to be a common toxic industrial chemical, and prolonged benzene exposure may cause nervous system damage. At present, there were few studies on benzene-induced neurological damage. This research aimed to identify the protein biomarkers to explore the mechanism of nervous system damage caused by benzene. We established a benzene poisoning model of C57 mice by gavage of benzene-peanut oil suspension and identified differentially expressed proteins (DEPs) in brain tissue using tandem mass tag (TMT) proteomics. The results showed a significant weight loss and decrease in leukocyte and neutrophil counts in benzene poisoning mice compared to the control group. We also observed local cerebral oedema and small vessel occlusion in the cerebral white matter of benzene poisoning mice. TMT proteomic results showed that a total 6,985 proteins were quantified, with a fold change (FC) > 1.2 (or < 1/1.2) and P value <0.05 were considered as DEPs. Compared with the control group, we identified 43 DEPs, comprising 14 upregulated and 29 downregulated proteins. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis results showed that the candidate proteins were mainly involved in cholesterol metabolism, complement and coagulation cascades, african trypanosomiasis, PPAR signaling pathway, and vitamin digestion and absorption. Three proteins, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (UGT8), Apolipoprotein A-I (APOA1) and Complement C3 (C3) were validated using immunoblotting and immunohistochemical. In conclusion, our study preliminarily investigated the mechanism of benzene toxicity to the nervous system by analyzing DEPs changes in the brain.

TaNRAMP3 is essential for manganese transport in Triticum aestivum.

Manganese (Mn) is an essential trace element for almost all living organisms. In plants, Mn deficiency, which is occurs in calcareous soils or alkaline soils, severely limiting crop yields. However, the potential mechanism of Mn transport in Triticum aestivum is still obscure. Here, we found that TaNRAMP3, a member of the naturally resistant macrophage protein (NRAMP) family in Triticum aestivum, is located in the plasma membrane of protoplasts and functions as an influx transporter for Mn in yeast (Δsmf1). The expression of TaNRAMP3 was induced under Mn-deficiency conditions. Furthermore, TaNRAMP3-RNAi plants exhibited a sensitive phenotype, while transgenic plants overexpressing TaNRAMP3 showed a tolerant phenotype. In addition, TaNRAMP3 rescued the sensitive phenotype of Arabidopsis nramp1 mutant under Mn deficiency condition. In summary, our study reveals the key role of TaNRAMP3 in Mn transport in Triticum aestivum, allowing it to adapt to Mn-deficiency stress. These findings provide new insights for the cultivation of Mn-deficiency tolerant wheat varieties.

Preparation of an Ultrahigh-DAR PDL1 monoclonal antibody-polymeric-SN38 conjugate for precise colon cancer therapy.

Antibody-drug conjugates (ADCs) are the most potent active tumor-targeting agents used clinically. However, the preparation of ADCs with high drug-to-antibody ratios (DARs) remains a major challenge. Herein, a Fab-nondestructive SN38-loaded antibody-polymeric-drug conjugate (APDC), aPDL1-NPLG-SN38, was prepared that had a DAR as high as 72 for the first time, by increased numbers of payload binding sites via the carboxyl groups of poly (l-glutamic acid) (PLG). The bonding of Fc-III-4C peptide with PLG-graft-mPEG/SN38 (Fc-NPLG-SN38) was achieved using a click reaction between azide and DBCO groups. The aPDL1-NPLG-SN38 conjugate was then synthesized by the high-affinity interaction between the Fc-III-4C peptide in Fc-NPLG-SN38 and the crystallizable fragment (Fc) of PDL1 monoclonal antibody (aPDL1). This approach avoided the potential deleterious effects on the Fab structure of the monoclonal antibody. The aqueous environment used in its preparation helped maintain monoclonal antibody recognition capability. Through the specific recognition by aPDL1 of PDL1 that is highly expressed on MC38 tumors, the accumulation of aPDL1-NPLG-SN38 in the tumors was 2.8-fold greater than achieved with IgG-NPLG-SN38 that had no active tumor-targeting capability. aPDL1-NPLG-SN38 exhibited excellent therapeutic properties in both medium-sized and large MC38 tumor animal models. The present study provides the details of a novel preparation strategy for SN38-loaded ADCs having a high DAR.

Calcium-dependent protein kinases CPK21 and CPK23 phosphorylate and activate the iron-regulated transporter IRT1 to regulate iron deficiency in Arabidopsis.

Iron (Fe) is an essential micronutrient for all organisms. Fe availability in the soil is usually much lower than that required for plant growth, and Fe deficiencies seriously restrict crop growth and yield. Calcium (Ca2+) is a second messenger in all eukaryotes; however, it remains largely unknown how Ca2+ regulates Fe deficiency. In this study, mutations in CPK21 and CPK23, which are two highly homologous calcium-dependent protein kinases, conferredimpaired growth and rootdevelopment under Fe-deficient conditions, whereas constitutively active CPK21 and CPK23 enhanced plant tolerance to Fe-deficient conditions. Furthermore, we found that CPK21 and CPK23 interacted with and phosphorylated the Fe transporter IRON-REGULATED TRANSPORTER1 (IRT1) at the Ser149 residue. Biochemical analyses and complementation of Fe transport in yeast and plants indicated that IRT1 Ser149 is critical for IRT1 transport activity. Taken together, these findings suggest that the CPK21/23-IRT1 signaling pathway is critical for Fe homeostasis in plants and provides targets for improving Fe-deficient environments and breeding crops resistant to Fe-deficient conditions.

An MMAE-loaded PDL1 active targeting nanomedicine for the precision treatment of colon cancer.

Tumor-active-targeting drugs such as antibody-drug conjugates have emerged as promising accurate therapeutic agents. However, their complex preparations risk compromising the targeting ability of the fragment antigen binding (Fab) region and promote aggregation over long-term storage. Here, we propose a tumor-active-targeting nanomedicine, aPDL1-PLG-MMAE, that effectively targets programmed death-ligand 1 (PDL1) high-expressing tumors and delivers monomethyl auristatin E (MMAE). aPDL1-PLG-MMAE consists of an anti-PDL1 monoclonal antibody (aPDL1) and poly(L-glutamic acid) (PLG) grafted Fc-III-4C peptide/Val-Cit-PAB-MMAE (Fc-PLG-MMAE). Fc-PLG-MMAE was obtained by conjugating the Fc-III-4C peptide and Val-Cit-PAB-MMAE to PLG via amide condensation. The strong affinity between the fragment crystallizable (Fc) region of aPDL1 and the Fc-III-4C peptide enabled aPDL1 and Fc-PLG-MMAE to self-assemble into aPDL1-PLG-MMAE after four hours of coincubation in PBS. As this nanomedicine can be quickly prepared for immediate use, the required antibodies can be stored separately from the Fc-PLG-MMAE portion for extended periods, which also facilitates transport. Moreover, aPDL1-PLG-MMAE demonstrated robust tumor recognition and targeting effects on MC38 colon cancer cells, resulting in potent therapeutic efficacy without significant toxicities.

CBL1/9-CIPK23-NRAMP1 axis regulates manganese toxicity.

Manganese (Mn) is an essential micronutrient in plants. However, excessive Mn absorption in acidic soils can cause Mn toxicity, which adversely affects plant growth and crop yields. At present, acidic soils cover c. 30% of the Earth's surface. However, the mechanism underpinning Mn uptake remains largely unknown. We identified cbl1/9 and cipk23 mutants exhibiting high-Mn-sensitive phenotype through the reverse genetics method. Furthermore, we identified the CIPK23 phosphorylated NRAMP1 through a variety of protein interaction techniques and protein kinase assays. Here, we demonstrated that two calcineurin B-like proteins, CBL1/9, and their interacting kinase CIPK23 positively regulated the tolerance of Mn toxicity in Arabidopsis. The cbl1 cbl9 double mutant and cipk23 mutants exhibited high-Mn-sensitive phenotypes, which manifested as decreased primary root length, biomass, and chlorophyll concentration, and higher accumulation of Mn. In addition, CIPK23 interacted with and phosphorylated the Mn transporter NRAMP1 primarily at Ser20/22 in vitro and in vivo, and thereby induced clathrin-mediated endocytosis of NRAMP1 to reduce its distribution on the plasma membrane and enhance plant tolerance to Mn toxicity. In summary, we found that the CBL1/9-CIPK23-NRAMP1 module regulates the tolerance to high-Mn toxicity and provide insight into a mechanism of the tolerance of plants to Mn toxicity.

The comparison of the Effect of double reverse traction repositor (DRTR) and traction table assisted Anterograde Intramedullary nail in treatment of femoral shaft fractures.

OBJECTIVE: The objective of this study was to compare the clinical efficacy of DRTR (Double Reverse Traction Repositor, DRTR)and traction table in the treatment of femoral shaft fractures with the aid of AN-IMN (Antegrade intramedullary nailing). PATIENTS AND METHODS: In this study, patients with femoral shaft fractures admitted to the Department of Orthopedics at Zhaoqing First People's Hospital from May 2018 to October 2022 were recruited. All patients were treated with anterograde intramedullary nailing, with 23 patients in the DRTR-assisted group and 21 patients in the traction table-assisted group. The demographic characteristics, fracture classification, intraoperative data, postoperative data, and prognostic indicators of the two groups were recorded and analyzed retrospectively. All procedures were performed by the same team of experienced physicians. RESULTS: All the patients in the two groups were followed up for more than 12 months. Both traction methods could provide stable traction for the operator during AN-IMN, and there was no significant difference in demographic characteristics and fracture classification. The intraoperative fluoroscopy times and opening reduction rate of the DRTR group were lower than those of the traction table group (P < 0.05), and the postoperative Harris Hip Score, as well as the Lyshol Lysholm knee function Score of the DRTR group, were significantly higher than the traction table group members (P < 0.05). Postoperative complications such as perineal soft tissue injury and lateral femoral cutaneous nerve injury occurred in the traction table group, but not in the DRTR group. CONCLUSION: DRTR can safely and effectively provide continuous and stable traction in the femoral shaft fractures surgery, and outperforms the traction table in the number of intraoperative fluoroscopy, opening reduction rate, reduction of complications, and postoperative joint function score.

Isolation of Intact Vacuoles from Arabidopsis Root Protoplasts and Elemental Analysis.

The vacuole is one of the most conspicuous organelles in plant cells, participating in a series of physiological processes, such as storage of ions and compartmentalization of heavy metals. Isolation of intact vacuoles and elemental analysis provides a powerful method to investigate the functions and regulatory mechanisms of tonoplast transporters. Here, we present a protocol to isolate intact vacuoles from Arabidopsis root protoplasts and analyze their elemental content by inductively coupled plasma mass spectrometry (ICP-MS). In this protocol, we summarize how to prepare the protoplast, extract the vacuole, and analyze element concentration. This protocol has been applied to explore the function and regulatory mechanisms of tonoplast manganese (Mn) transporter MTP8, which is antagonistically regulated by CPK4/5/6/11 and CBL2/3-CIPK3/9/26. This protocol is not only suitable for exploring the functions and regulatory mechanisms of tonoplast transporters, but also for researching other tonoplast proteins. Graphical abstract.

Bipotent transitional liver progenitor cells contribute to liver regeneration.

Following severe liver injury, when hepatocyte-mediated regeneration is impaired, biliary epithelial cells (BECs) can transdifferentiate into functional hepatocytes. However, the subset of BECs with such facultative tissue stem cell potential, as well as the mechanisms enabling transdifferentiation, remains elusive. Here we identify a transitional liver progenitor cell (TLPC), which originates from BECs and differentiates into hepatocytes during regeneration from severe liver injury. By applying a dual genetic lineage tracing approach, we specifically labeled TLPCs and found that they are bipotent, as they either differentiate into hepatocytes or re-adopt BEC fate. Mechanistically, Notch and Wnt/ß-catenin signaling orchestrate BEC-to-TLPC and TLPC-to-hepatocyte conversions, respectively. Together, our study provides functional and mechanistic insights into transdifferentiation-assisted liver regeneration.

Plasma membrane-associated calcium signaling regulates arsenate tolerance in Arabidopsis.

Arsenate [As(V)] is a metalloid with heavy metal properties and is widespread in many environments. Dietary intake of food derived from arsenate-contaminated plants constitutes a major fraction of the potentially health-threatening human exposure to arsenic. However, the mechanisms underlying how plants respond to arsenate stress and regulate the function of relevant transporters are poorly understood. Here, we observed that As(V) stress induces a significant Ca2+ signal in Arabidopsis (Arabidopsis thaliana) roots. We then identified a calcium-dependent protein kinase, CALCIUM-DEPENDENT PROTEIN KINASE 23 (CPK23), that interacts with the plasma membrane As(V)/Pi transporter PHOSPHATE TRANSPORTER 1;1 (PHT1;1) in vitro and in vivo. cpk23 mutants displayed a sensitive phenotype under As(V) stress, while transgenic Arabidopsis plants with constitutively active CPK23 showed a tolerant phenotype. Furthermore, CPK23 phosphorylated the C-terminal domain of PHT1;1, primarily at Ser514 and Ser520. Multiple experiments on PHT1;1 variants demonstrated that PHT1;1S514 phosphorylation is essential for PHT1;1 function and localization under As(V) stress. In summary, we revealed that plasma-membrane-associated calcium signaling regulates As(V) tolerance. These results provide insight for crop bioengineering to specifically address arsenate pollution in soils.

Brassinosteroid transcription factor BES1 modulates nitrate deficiency by promoting NRT2.1 and NRT2.2 transcription in Arabidopsis.

Nitrogen (N) is one of the most essential mineral elements for plants. Brassinosteroids (BRs) play key roles in plant growth and development. Emerging evidence indicates that BRs participate in the responses to nitrate deficiency. However, the precise molecular mechanism underlying the BR signaling pathway in regulating nitrate deficiency remains largely unknown. The transcription factor BES1 regulates the expression of many genes in response to BRs. Root length, nitrate uptake and N concentration of bes1-D mutants were higher than those of wild-type under nitrate deficiency. BES1 levels strongly increased under low nitrate conditions, especially in the non-phosphorylated (active) form. Furthermore, BES1 directly bound to the promoters of NRT2.1 and NRT2.2 to promote their expression under nitrate deficiency. Taken together, BES1 is a key mediator that links BR signaling under nitrate deficiency by modulating high affinity nitrate transporters in plants.

Overall analyses of the reactions catalyzed by acetohydroxyacid synthase/acetolactate synthase using a precolumn derivatization-HPLC method.

A precolumn derivatization-HPLC method using 2,4-dinitrophenylhydrazine and 4-nitro-o-phenylenediamine as respective labeling reagents for comprehensive analyses of the reactions catalyzed by acetohydroxyacid synthase (AHAS)/acetolactate synthase (ALS) is developed and evaluated in this research. Comparison with the classic Bauerle' UV assay which can analyze the enzymes only through measurement of acetoin production, the HPLC method shows advantages because it can analyze the enzymes not only via determination of consumption of the substrate pyruvate, but also via measurement of formation of the products including acetoin, 2,3-butanedione, and acetaldehyde in the enzymatic reactions. Thus the results deduced from the HPLC method can reflect the trait of each enzyme in a more precise manner. As far as we know, this is the first time that the reactions mediated by AHAS/ALS using pyruvate as a single substrate are globally analyzed and the features of the enzymes are properly discussed.

Plasma membrane-associated calcium signaling modulates cadmium transport.

Cadmium (Cd) is a toxic heavy element for plant growth and development, and plants have evolved many strategies to cope with Cd stress. However, the mechanisms how plants sense Cd stress and regulate the function of transporters remain very rudimentary. Here, we found that Cd stress induces obvious Ca2+ signals in Arabidopsis roots. Furthermore, we identified the calcium-dependent protein kinases CPK21 and CPK23 that interacted with the Cd transporter NRAMP6 through a variety of protein interaction techniques. Then, we confirmed that the cpk21 23 double mutants significantly enhanced the sensitive phenotype of cpk23 single mutant under Cd stress, while the overexpression and continuous activation of CPK21 and CPK23 enhanced plants tolerance to Cd stress. Multiple biochemical and physiological analyses in yeast and plants demonstrated that CPK21/23 phosphorylate NRAMP6 primarily at Ser489 and Thr505 to inhibit the Cd transport activity of NRAMP6, thereby improving the Cd tolerance of plants. Taken together, we found a plasma membrane-associated calcium signaling that modulates Cd tolerance. These results provide new insights into the molecular breeding of crop tolerance to Cd stress.

Functional study of CYP90A1 and ALDH3F1 gene obtained by transcriptome sequencing analysis of Brassica napus seedlings treated with brassinolide.

Sclerotinia disease and weeds of Brassica napus greatly reduce crop yields. However, brassinolides can improve the resistance of plants to sclerotinia diseases and herbicides. In this study, we investigated the effects of brassinolide on the occurrence, physiological indices, yield, and gene expression of Fanming No. 1 seeds under sclerotinia and glufosinate stress. The results showed that soaking of the seeds in 0.015% brassinolide for 6 h reduced the incidence of sclerotinia by 10%. Additionally, in response to glufosinate stress at the seedling stage, the enzyme activities of catalase and superoxide dismutase increased by 9.6 and 19.0 U/gFW/min, respectively, and the soluble sugar content increased by 9.4 mg/g, increasing the stress resistance of plants and yield by 2.4%. LHCB1, fabF, psbW, CYP90A1, ALDH3F1, ACOX1, petF, and ACSL were screened by transcriptome analysis. ALDH3F1 and CYP90A1 were identified as key genes. Following glufosinate treatment, transgenic plants overexpressing ALDH3F1 and CYP90A1 were found to be resistant to glufosinate, and the expression levels of the ALDH3F1 and CYP90A1 were 1.03-2.37-fold as high as those in the control. The expression level of ATG3, which is an antibacterial gene related to sclerotinia disease, in transgenic plants was 2.40-2.37-fold as high as that in the control. Our results indicate that these two key genes promote plant resistance to sclerotinia and glufosinate. Our study provides a foundation for further studies on the molecular mechanisms of rapeseed resistance breeding and selection of new resistant varieties.

Ca2+-dependent phosphorylation of NRAMP1 by CPK21 and CPK23 facilitates manganese uptake and homeostasis in Arabidopsis.

Homeostasis of the essential micronutrient manganese (Mn) is crucially determined through availability and uptake efficiency in all organisms. Mn deficiency of plants especially occurs in alkaline and calcareous soils, seriously restricting crop yield. However, the mechanisms underlying the sensing and signaling of Mn availability and conferring regulation of Mn uptake await elucidation. Here, we uncover that Mn depletion triggers spatiotemporally defined long-lasting Ca2+ oscillations in Arabidopsis roots. These Ca2+ signals initiate in individual cells, expand, and intensify intercellularly to transform into higher-order multicellular oscillations. Furthermore, through an interaction screen we identified the Ca2+-dependent protein kinases CPK21 and CPK23 as Ca2+ signal-decoding components that bring about translation of these signals into regulation of uptake activity of the high-affinity Mn transporter natural resistance associated macrophage proteins 1 (NRAMP1). Accordingly, a cpk21/23 double mutant displays impaired growth and root development under Mn-limiting conditions, while kinase overexpression confers enhanced tolerance to low Mn supply to plants. In addition, we define Thr498 phosphorylation within NRAMP1 as a pivot mechanistically determining NRAMP1 activity, as revealed by biochemical assays and complementation of yeast Mn uptake and Arabidopsis nramp1 mutants. Collectively, these findings delineate the Ca2+-CPK21/23-NRAMP1 axis as key for mounting plant Mn homeostasis.

Generation of Plvap-CreER and Car4-CreER for genetic targeting of distinct lung capillary populations.

It has been reported recently that there are two distinct subpopulations of capillary endothelial cells in the mammalian lungs: gCap (general capillary) and aCap (aerocyte). They are identified by two unique markers, respectively: plasmalemmal vesicle-associated protein (PLVAP) and carbonic anhydrase IV (CAR4). Here, we report two novel knock-in mouse lines Plvap-CreER and Car4-CreER, which genetically target gCap and aCap, respectively. Induced by tamoxifen, the Plvap-CreER and Car4-CreER alleles mediate specific and efficient Cre-loxP recombinations in PLVAP+ gCap and CAR4+ aCap, respectively, in the lungs. These two mouse lines are useful genetic tools to investigate cell fates and functions of PLVAP+ and CAR4+ cells in lung homeostasis, injury and repair.