Aldehyde group pendant-grafted pectin-based injectable hydrogel.

Periodate oxidation has been the widely accepted route for obtaining aldehyde group-functionalized polysaccharides but significantly influenced the various physicochemical properties due to the ring opening of the backbone of polysaccharides. The present study, for the first time, presents a novel method for the preparation of aldehyde group-functionalized polysaccharides that could retain the ring structure and the consequent rigidity of the backbone. Pectin was collected as the representative of polysaccharides and modified with cyclopropyl formaldehyde to obtain pectin aldehyde (AP), which was further crosslinked by DL-lysine (LYS) via the Schiff base reaction to prepare injectable hydrogel. The feasibility of the functionalization was proved by FT-IR and 1H NMR techniques. The obtained hydrogel showed acceptable mechanical properties, self-healing ability, syringeability, and sustained-release performance. Also, as-prepared injectable hydrogel presented great biocompatibility with a cell proliferation rate of 96 %, and the drug-loaded hydrogel exhibited clear inhibition of cancer cell proliferation. Overall, the present study showed a new method for the preparation of aldehyde group-functionalized polysaccharides, and the drug-loaded hydrogel has potential in drug release applications.

Generation of Allogeneic CAR T Cells through Specific Degradation of the T Cell Antigen Receptor by E3 Ubiquitin Ligase Fusion Proteins.

Receptor downregulation is instrumental for many therapeutic interventions. Receptor knockout through gene-editing technologies is efficient but can introduce off-target mutations and chromothripsis. Regulation of gene expression at the protein level is a promising alternative. Here, we present results showing the targeted T cell antigen receptor (TCR) degradation using chimeric E3 fusion proteins that we call Receptor Targeting Chimeras (ReceptorTAC). We show that TCR degradation is dependent on enzymatically active, membrane-anchored E3 ligase variants. TCR specificity was achieved by direct fusion of an E3 domain to the CD3ζ transmembrane sequence. Jurkat and primary T cells stably expressing the ReceptorTAC constructs showed significantly reduced responses to TCR stimulation. We also used our ReceptorTAC technology to generate TCR-deficient, claudin18.2-specific CAR T cells, where the activity of the CAR was unaffected by the expression of the ReceptorTAC. These data indicate that our ReceptorTAC molecule can be used to generate allogeneic CAR T cells.

Deletion of miR-15a inhibited glioma development via targeting Smad7 and inhibiting EMT pathway.

In the present study, we found the expression of miR-15a-5p (miR-15a) was increased in glioma tissues, and we further explore the underlying mechanism of miR-15a in glioma progression. Microarray analysis used to identify the differentially expressed microRNAs (miRNAs) in glioma tissues. The expression of miR-15a in glioma tissues and cell lines was tested by qRT-PCR. Luciferase assay was used to determine the binding between miR-15a and Smad7. Wound healing and transwell assay were used to examine the role of miR-15a/Smad7 in SHG139 cells. Western blot was used to detect the protein level of Smad7 and epithelial-mesenchymal transition (EMT) markers. A tumor formation model in nude mice was established to measure the role of miR-15a in vivo. MiR-15a was significantly increased in glioma tissues and cells, which indicated a poor prognosis of glioma patients. MiR-15a mimics induced miR-15a level in SHG139 cells, and promoted the malignancy of SHG139 cells, while miR-15a inhibitor showed the opposite effects. Luciferase assay indicated that Smad7 was the direct target of miR-15a, and Smad7 was down-regulated in glioma tissues. Functional experiments revealed that miR-15a inhibitor inhibited the EMT pathway and the migration and invasion of glioma cells, but the silencing of Smad7 reversed the effects of miR-15a inhibitor in EMT pathway and glioma progression. Finally, we performed animal experiments to verify the role of miR-15a in vivo. Present study showed that deletion of miR-15a inhibited the activation of EMT signaling via targeting Smad7, thus suppressed the tumorigenesis and tumor growth of glioma.

Anti-inflammation of epicatechin mediated by TMEM35A and TMPO in bovine mammary epithelial cell line cells and mouse mammary gland.

Epicatechin (EC) has significant antiinflammation, antioxidation, and anticancer activities. It also provides a new alternative treatment for mastitis, which can result in great economic losses in the dairy industry if left untreated. The purpose of this study was to investigate the anti-inflammatory effects of EC on mastitis and the underlying mechanism using in vivo and in vitro systems. The use of ELISA and immunohistochemistry assays showed that EC treatment at 1.5, 7.5, 15, and 30 mg/mL decreased protein expression of inflammatory mediators, including cyclooxygenase-2 and inducible nitric oxide synthase; inflammatory cytokines, which were composed of IL-1ß, TNF-α, and IL-6 in lipopolysaccharide (LPS)-stimulated bovine mammary epithelial cell line (MAC-T); and mouse mammary gland, together with reduced filtration of T lymphocytes in the mouse mammary gland. Furthermore, EC treatment reduced LPS-induced phosphorylation levels of p65 and inhibitor of NF-κB, and blocked nuclear translocation of p65 as revealed by western blot and immunofluorescence test in MAC-T cells and the mouse mammary gland. Epicatechin also attenuated LPS-induced phosphorylation levels of mitogen-activated protein kinase members (i.e., p38, c-Jun N-terminal kinase 1/2 and extracellular regulated protein kinases 1/2). Using RNA-seq and tandem mass tag analyses, upregulation of TMEM35A and TMPO proteins was disclosed in MAC-T cells cotreated with LPS and EC. Although clustered regularly interspaced short palindromic repeats/Cas9-based knockdown of TMEM35A and TMPO attenuated abundance of phosphorylated (p)-p65, p-p38, TNF-α, and iNOS, overexpression of TMEM35A reversed EC-mediated effects in TMPO knockdown cells. Moreover, interaction between TMEM35A and TMPO was detected using the co-immunoprecipitation method. In conclusion, our data demonstrated that EC inhibited LPS-induced inflammatory response in MAC-T cells and the mouse mammary gland. Importantly, TMEM35A mediated the transmembrane transport of EC, and the interaction between TMEM35A and TMPO inhibited MAPK and NF-κB pathways.

[Expression of F0 ATP synthase C subunit in serum of patients with ischemic heart disease and its clinical implications].

OBJECTIVE: To investigate the expression and clinical significance of F0 ATP synthase C subunit (Csub) in patients with ischemic heart disease (IHD). METHODS: The 101 patients with chest pain admitted to the department of emergency of the People's Hospital of Yuhuan from May 2019 to December 2020 were enrolled, including 59 patients with acute myocardial infarction (AMI) and 42 patients with unstable angina pectoris (UAP). At the same time, 50 age-matched healthy subjects in the health examination center were selected as the healthy control (HC). All patients had completed blood sampling before the intervention of drugs or other intervention measures in the emergency room. The content of serum Csub was detected by enzyme linked immunosorbent assay (ELISA), and the relationship between Csub and clinical characteristics was analyzed. At the same time, the contents of hypersensitivity cardiac troponin T (hs-cTnT) and MB isoenzyme of creatine kinase (CK-MB) in blood were detected by electrochemical luminescence. The receiver operator characteristic curve (ROC curve) was drawn to evaluate the value of Csub, hs-cTnT, and CK-MB in the early diagnosis of IHD. RESULTS: The baseline data such as age, gender, and history of the three groups were balanced. There were significant differences in low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), CK-MB, hs-cTnT and B-type natriuretic peptide (BNP), but there were no significant differences in other biochemical indexes. The Csub content in the AMI group and the UAP group were significantly higher than those in the HC group [8.96% (6.37%, 11.53%), 4.27% (3.23%, 6.49%) vs. 1.56% (1.07%, 2.33%), both P < 0.01]. Moreover, the Csub in the AMI group with more severe myocardial ischemia was higher than UAP group [8.96% (6.37%, 11.53%) vs. 4.27% (3.23%, 6.49%), P < 0.01]. A total of 59 patients with AMI were treated with percutaneous coronary intervention (PCI). According to the median of Csub, AMI patients were subdivided into above-median group (29 cases) and below-median group (30 cases). The results showed that there were no significant differences in the number of coronary artery lesion branches, the number of stent implantation and postoperative medication between the two groups. ROC curve analysis showed that the area under the curve (AUC) and 95% confidence interval (95%CI) of Csub, hs-cTnT and CK-MB in the diagnosis of IHD were 0.98 (0.95-1.00), 0.99 (0.99-1.00), 0.94 (0.89-0.99), respectively. The diagnostic efficacy of Csub was slightly lower than that of hs-cTnT but higher than that of CK-MB. When the cut-off value of Csub was 4.74%, the sensitivity and specificity for the diagnosis of IHD were 100% and 87.0%, respectively. CONCLUSIONS: Csub increased significantly in the serum of IHD patients, and further increased with the severity of ischemia. It can be used as a new diagnostic biomarker for the diagnosis and evaluation of the development of myocardial ischemia.

[Construction, Expression of hERα-LBD Prokaryotic Vector and the Activity of Expressed Protein].

OBJECTIVES: To construct the prokaryotic expression system of estrogen receptor α ligand bingding domain (hERα-LBD) and to evaluate the estrogen receptor ligand binding activity of the expressed protein. METHODS: hERα -LBD was amplicated from the plasmid of hERα -LBD by PCR, the identified PCR product was ligated with pGEM-T-easy vector to generate pGM-T-hERα -LBD. After the confirmation, the hERα -LBD fragments were obtained by enzyme digestion and inserted into pET-28a. The expression vectors were expressed in E.Coli to produce hERα-LBD protein. We mixed the hERα-LBD protein and estradiol and bovine serum albumin conjugated antigens (E2-BSA), then evaluated the binding activity of hERα-LBD by electrophoresis. RESULTS: The amplified fragment was about 1.9 kb, which was in agreement with the expected target fragment. Recombinant plasmid of pGM-T-hERα -LBD was confirmed by enzyme digestion and sequencing, then pET-28a(+)-hERα -LBD was constructed successfully. The expressed hERα-LBD protein in E.Coli was observed and the expression amount was 250 mg/L after affinity chromatography purification. hERα-LBD was confirmed to had estrogen binding activity by electrophoresis. CONCLUSIONS: The prokaryotic expression system of pET-28a(+)-hERα -LBD was successfully constructed, and hERα-LBD had the activity of binding.

An overlooked effect of glycine betaine on fermentation: prevents caramelization and increases the L-lysine production.

This article focuses on the effects of glycine betaine on preventing caramelization, and increasing DCW and L-lysine production. The additional glycine betaine not only decreased the browning intensity (decreased 4 times), and the concentrations of 5-hydroxymethylfurfural (decreased 7.8 times) and furfural (decreased 12 times), but also increased the availability of glucose (increased 17.5%) for L-lysine production. The DCW and L-lysine production were increased by adding no more than 20 mM glycine betaine, whereas the DCW and L-lysine production were decreased with the reduction of pH values, although pH had a better response to prevent caramelization than did glycine betaine. For L-lysine production, the highest increase (40%) was observed on the media with 20 mM glycine betaine. The crucial enzymes in glycolysis and L-lysine biosynthesis pathway were investigated. The results indicated that additional glycine betaine increases the activity of enzymes in glycolysis, in contrast to the effect of pH. All the results indicated that glycine betaine can be used to prevent caramelization and increase the L-lysine production. By applying this strategy, glucose would not be have to be separated from the culture media during autoclaving so that factories can save production costs and shorten the fermentation period.

Nickel dichalcogenide hollow spheres: controllable fabrication, structural modification, and magnetic properties.

Magnetize your chemistry! A facile hydrothermal synthetic route was developed for the synthesis of uniform NiS2 hollow spheres, which could be transformed into NiSe2 and NiTe2 hollow spheres through a chemical conversion process. Furthermore, NiS and NiO hollow spheres could be selectively obtained by calcination of NiS2 hollow spheres at different temperatures.

NF-κB p65 and c-Rel subunits promote phagocytosis and cytokine secretion by splenic macrophages in cirrhotic patients with hypersplenism.

Transcription factors of the nuclear factor-kappa B (NF-κB) family play a key role in various biological processes. In this study, we explored the role of NF-κB in the dysfunction of splenic macrophages in hypersplenism due to liver cirrhosis. By using confocal microscopic analysis, Western Blot, TransAM NF-κB ELISA, and chromatin immunoprecipitation (ChIP), we observed that NF-κB p65, p52, and c-Rel were activated in macrophages in patients with hypersplenism (hypersplenic macrophages). Transfection of hypersplenic macrophages with a κB/luciferase reporter plasmid showed that NF-κB complexes were functional. Using co-immunoprecipitation studies, we demonstrated that p65/c-Rel dimers were activated in hypersplenic macrophages. NF-κB activation inhibitor JSH-23 and the small interfering RNA (siRNA)-mediated p65, and c-Rel gene silencing significantly blocked phagocytosis and secretion in hypersplenic macrophages. Using promoter analysis and RNA interference, we found that many phagocytotic and hepatic fibrogenetic regulators, including interleukin (IL)-1α, IL-1ß, interferon-γ (IFN-γ), transforming growth factor-ß1 (TGF-ß1), and tumor necrosis factor-α (TNF-α), were regulated by NF-κB p65 and c-Rel in hypersplenic macrophages. Our findings demonstrate that NF-κB p65 and c-Rel play an important role in phagocytosis and secretion in hypersplenic macrophages. Activation of NF-κB p65 and c-Rel may be considered an important regulator of hypersplenism and liver cirrhosis.