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.

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.

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.

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 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.

Review on design strategies and considerations of polysaccharide-based smart drug delivery systems for cancer therapy.

The unique natural advantages of polysaccharide materials have attracted attention in biomedical applications. The abundant modifiable functional groups on the polysaccharide materials surface can facilitate the synthesis of various multifunctional drug delivery carriers. Especially in tumor therapy, the designs of polysaccharide-based drug delivery carriers are diverse. Therefore, this review summarized several latest types of polysaccharide-based drug carriers designs, and focused on the latest design strategies and considerations of drug carriers with polysaccharides as the main structure. It is expected to provide some design ideas and inspiration for subsequent polysaccharide-based drug delivery systems.

Physicochemical properties, content, composition and partial least squares models of A. trifoliata seeds oil.

Physicochemical properties, oil content, and fatty acids (FAs) composition are key for determining the value of oil crops. The aim of this study was to illustrate the potential of exploiting A. trifoliata as an edible oil crop, and establish a rapid measurement model for the A. trifoliata seeds oil (ASO) content and composition. In 130 A. trifoliata germplasms, the highest content of ASO was 51.27%, and unsaturated fatty acids (UFAs) mainly accounted for 74-78% of ASO. The partial least squares (PLS) model based on GC-MS and near-infrared spectroscopy was well-suited for the determination of ASO and UFA content; however, the PLS model for oleic acid (OA) and linoleic acid (LA) was not effective. The acid values and peroxide values for ASO also conformed to the Chinese food safety standards. Our findings will provide new insights and guidance for the use of A. trifoliata as oil crops..

Genetic fate-mapping reveals surface accumulation but not deep organ invasion of pleural and peritoneal cavity macrophages following injury.

During injury, monocytes are recruited from the circulation to inflamed tissues and differentiate locally into mature macrophages, with prior reports showing that cavity macrophages of the peritoneum and pericardium invade deeply into the respective organs to promote repair. Here we report a dual recombinase-mediated genetic system designed to trace cavity macrophages in vivo by intersectional detection of two characteristic markers. Lineage tracing with this method shows accumulation of cavity macrophages during lung and liver injury on the surface of visceral organs without penetration into the parenchyma. Additional data suggest that these peritoneal or pleural cavity macrophages do not contribute to tissue repair and regeneration. Our in vivo genetic targeting approach thus provides a reliable method to identify and characterize cavity macrophages during their development and in tissue repair and regeneration, and distinguishes these cells from other lineages.

Genetic Fate Mapping of Transient Cell Fate Reveals N-Cadherin Activity and Function in Tumor Metastasis.

Genetic lineage tracing unravels cell fate and plasticity in development, tissue homeostasis, and diseases. However, it remains technically challenging to trace temporary or transient cell fate, such as epithelial-to-mesenchymal transition (EMT) in tumor metastasis. Here, we generated a genetic fate-mapping system for temporally seamless tracing of transient cell fate. Highlighting its immediate application, we used it to study EMT gene activity from the local primary tumor to a distant metastatic site in vivo. In a spontaneous breast-to-lung metastasis model, we found that primary tumor cells activated vimentin and N-cadherin in situ, but only N-cadherin was activated and functionally required during metastasis. Tumor cells that have ever expressed N-cadherin constituted the majority of metastases in lungs, and functional deletion of N-cad significantly reduced metastasis. The seamless genetic recording system described here provides an alternative way for understanding transient cell fate and plasticity in biological processes.

A Lipid-Anchored NAC Transcription Factor Is Translocated into the Nucleus and Activates Glyoxalase I Expression during Drought Stress.

The plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) play a vital role in the response to drought stress. Here, we report a lipid-anchored NACsa TF in Medicago falcata MfNACsa is an essential regulator of plant tolerance to drought stress, resulting in the differential expression of genes involved in oxidation reduction and lipid transport and localization. MfNACsa is associated with membranes under unstressed conditions and, more specifically, is targeted to the plasma membrane through S-palmitoylation. However, a Cys26-to-Ser mutation or inhibition of S-palmitoylation results in MfNACsa retention in the endoplasmic reticulum/Golgi. Under drought stress, MfNACsa translocates to the nucleus through de-S-palmitoylation mediated by the thioesterase MtAPT1, as coexpression of APT1 results in the nuclear translocation of MfNACsa, whereas mutation of the catalytic site of APT1 results in colocalization with MfNACsa and membrane retention of MfNACsa. Specifically, the nuclear MfNACsa binds the glyoxalase I (MtGlyl) promoter under drought stress, resulting in drought tolerance by maintaining the glutathione pool in a reduced state, and the process is dependent on the APT1-NACsa regulatory module. Our findings reveal a novel mechanism for the nuclear translocation of an S-palmitoylated NAC in response to stress.

Interaction between PVY HC-Pro and the NtCF1ß-subunit reduces the amount of chloroplast ATP synthase in virus-infected tobacco.

The photosynthetic rate of virus-infected plants is always reduced. However, the molecular mechanism underlying this phenomenon remains unclear. The helper component-proteinase (HC-Pro) of Potato virus Y (PVY) was found in the chloroplasts of PVY-infected tobacco, indicating some new function of HC-Pro in the chloroplasts. We generated HC-Pro transgenic plants with a transit peptide to target the protein to chloroplast. The HC-Pro transgenic tobacco showed a decreased photosynthetic rate by 25% at the light intensity of 600 µmol m(-2) s(-1). Using a yeast two-hybrid screening assay to search for chloroplast proteins interacting with HC-Pro, we identified that PVY HC-Pro can interact with the chloroplast ATP synthase NtCF1ß-subunit. This interaction was confirmed by GST pull-down and co-immunoprecipitation assays. HC-Pro didn't interfere with the activity of assembled ATP synthase in vitro. The HC-Pro/NtCF1ß-subunit interaction might affect the assembly of ATP synthase complex. Quantitative western blot and immunogold labeling of the ATP synthase indicated that the amount of ATP synthase complex was decreased in both the HC-Pro transgenic and the PVY-infected tobacco. These results demonstrate that HC-Pro plays an important role in reducing the photosynthetic rate of PVY-infected plants, which is a completely new role of HC-Pro besides its multiple known functions.

Potato virus Y HC-Pro Reduces the ATPase Activity of NtMinD, Which Results in Enlarged Chloroplasts in HC-Pro Transgenic Tobacco.

Potato virus Y (PVY) is an important plant virus and causes great losses every year. Viral infection often leads to abnormal chloroplasts. The first step of chloroplast division is the formation of FtsZ ring (Z-ring), and the placement of Z-ring is coordinated by the Min system in both bacteria and plants. In our lab, the helper-component proteinase (HC-Pro) of PVY was previously found to interact with the chloroplast division protein NtMinD through a yeast two-hybrid screening assay and a bimolecular fluorescence complementation (BiFC) assay in vivo. Here, we further investigated the biological significance of the NtMinD/HC-Pro interaction. We purified the NtMinD and HC-Pro proteins using a prokaryotic protein purification system and tested the effect of HC-Pro on the ATPase activity of NtMinD in vitro. We found that the ATPase activity of NtMinD was reduced in the presence of HC-Pro. In addition, another important chloroplast division related protein, NtMinE, was cloned from the cDNA of Nicotiana tabacum. And the NtMinD/NtMinE interaction site was mapped to the C-terminus of NtMinD, which overlaps the NtMinD/HC-Pro interaction site. Yeast three-hybrid assay demonstrated that HC-Pro competes with NtMinE for binding to NtMinD. HC-Pro was previously reported to accumulate in the chloroplasts of PVY-infected tobacco and we confirmed this result in our present work. The NtMinD/NtMinE interaction is very important in the regulation of chloroplast division. To demonstrate the influence of HC-Pro on chloroplast division, we generated HC-Pro transgenic tobacco with a transit peptide to retarget HC-Pro to the chloroplasts. The HC-Pro transgenic plants showed enlarged chloroplasts. Our present study demonstrated that the interaction between HC-Pro and NtMinD interfered with the function of NtMinD in chloroplast division, which results in enlarged chloroplasts in HC-Pro transgenic tobacco. The HC-Pro/NtMinD interaction may cause the formation of abnormal chloroplasts in PVY-infected plants.