Discovery and characterization of natural product luteolin as an effective inhibitor of human pyridoxal kinase.

Pyridoxal kinase (PDXK) is an essential enzyme in the synthesis of pyridoxal 5-phosphate (PLP), the active form of vitamin B6, which plays a pivotal role in maintaining the enzyme activity necessary for cell metabolism. Thus, PDXK has garnered attention as a potential target for metabolism regulation and tumor therapy. Despite this interest, existing PDXK inhibitors have faced limitations, including weak suppressive activity, unclear mechanisms of action, and associated toxic side effects. In this study, we present the discovery of a novel PDXK inhibitor, luteolin, through a high-throughput screening approach based on enzyme activity. Luteolin, a natural product, exhibits micromolar-level affinity for PDXK and effectively inhibits the enzyme's activity in vitro. Our crystal structures reveal that luteolin occupies the ATP binding pocket through hydrophobic interactions and a weak hydrogen bonding pattern, displaying reversible characteristics as confirmed by biochemical assays. Moreover, luteolin disrupts vitamin B6 metabolism by targeting PDXK, thereby inhibiting the proliferation of leukemia cells. This research introduces a novel screening method for identifying high-affinity and potent PDXK inhibitors and sheds light on clarification of the structural mechanism of PDXK-luteolin for subsequent structure optimization of inhibitors.

The antipsychotic drug penfluridol inhibits N-linked glycoprotein processing and enhances T-cell-mediated tumor immunity.

Aberrant N-linked glycosylation is a prominent feature of cancers. Perturbance of oligosaccharide structure on cell surfaces directly affects key processes in tumor development and progression. In spite of the critical role played by N-linked glycans in tumor biology, the discovery of small molecules that specifically disturbs the N-linked glycans is still under investigation. To identify more saccharide-structure-perturbing compounds, a repurposed drug screen by using a library consisting of 1530 FDA-approved drugs was performed. Interestingly, an antipsychotic drug, penfluridol, was identified as being able to decrease cell surface Wheat germ agglutinin (WGA) staining. In the presence of penfluridol, cell membrane glycoproteins PD-L1 shifted to a lower molecular weight. Further studies demonstrated that penfluridol treatment caused an accumulation of high-mannose oligosaccharides, especially Man5-7GlcNAc2 glycan structures. Mechanistically, this effect is due to direct targeting of MAN1A1 mannosidase, a Golgi enzyme involved in N-glycan maturation. Moreover, we found that altered glycosylation of PD-L1 caused by penfluridol disrupted interactions between PD-1 and PD-L1, resulting in activation of T-cell tumor immunity. In a mouse xenograft and glioma model, penfluridol enhanced the anti-tumor effect of the anti-PD-L1 antibody in vivo. Overall, these findings revealed an important biological activity of the antipsychotic drug penfluridol as an inhibitor of glycan processing and proposed a repurposed use of penfluridol in anti-tumor therapy through activation of T-cell immunity.

miR-452-5p suppressed the metastasis of Non-small cell lung cancer through regulating Moesin.

Background: Non-small cell lung cancer (NSCLC) is a common malignant tumor, and it is characterized by high mortality. MicroRNA-452-5p (miR-452-5p) and Moesin (MSN) have been proved to be related with regulation of tumors. If miR-452-5p could regulate NSCLC through targeting MSN remain unclear. Methods: TargetScan data and GEPIA databases were used to predict binding site and analyze gene expression, respectively. EdU staining, wound healing, and Transwell assays were performed to measure cell proliferation, migration, and invasion, respectively. Results: The binding site between miR-452-5p and MSN was predicted and validated. Overexpression of miR-452-5p cell lines were constructed, and miR-452-5p mimics markedly inhibited the migration, invasion, and proliferation ability of both H322 and A549 cells, but these effects of miR-452-5p were reversed by pcDNA-MSN. pcDNA-MSN significantly reversed the influence of miR-452-5p mimics on the EMT related proteins expression in H322 and A549 cell lines by decreasing E-cadherin and increasing N-cadherin. Significant higher expression of MSN in lung adenocarcinoma and lung squamous cell carcinoma was observed through GEPIA and TCGA data base analysis. Higher expression of MSN is positively correlated with advanced lung cancer and suggests poor prognosis. Conclusions: We demonstrated that miR-452-5p modulated the cell proliferation, migration, invasion, and EMT process of H322 and A549 cell lines through targeting MSN. This research might provide a novel prevention and treatment target for NSCLC.

Marine-Derived Natural Product HDYL-GQQ-495 Targets P62 to Inhibit Autophagy.

Autophagy is widely implicated in pathophysiological processes such as tumors and metabolic and neurodegenerative disorders, making it an attractive target for drug discovery. Several chemical screening approaches have been developed to uncover autophagy-modulating compounds. However, the modulation capacity of marine compounds with significant pharmacological activities is largely unknown. We constructed an EGFPKI-LC3B cell line using the CRISPR/Cas9 knock-in strategy in which green fluorescence indicated endogenous autophagy regulation. Using this cell line, we screened a compound library of approximately 500 marine natural products and analogues to investigate molecules that altered the EGFP fluorescence. We identified eight potential candidates that enhanced EGFP fluorescence, and HDYL-GQQ-495 was the leading one. Further validation with immunoblotting demonstrated that cleaved LC3 was increased in dose- and time-dependent manners, and the autophagy adaptor P62 showed oligomerization after HDYL-GQQ-495 treatment. We also demonstrated that HDYL-GQQ-495 treatment caused autophagy substrate aggregation, which indicated that HDYL-GQQ-495 serves as an autophagy inhibitor. Furthermore, HDYL-GQQ-495 induced Gasdermin E (GSDME) cleavage and promoted pyroptosis. Moreover, HDYL-GQQ-495 directly combined with P62 to induce P62 polymerization. In P62 knockout cells, the cleavage of LC3 or GSDME was blocked after HDYL-GQQ-495 treatment. The EGFPKI-LC3B cell line was an effective tool for autophagy modulator screening. Using this tool, we found a novel marine-derived compound, HDYL-GQQ-495, targeting P62 to inhibit autophagy and promote pyroptosis.

Effects of Pellet Basicity on the Simulated Deposit Formation in Coal-Fired Rotary Kilns for Iron Ore Pellet Production.

Deposit formation in the coal-fired rotary kiln is frequently found in the production of fluxed iron ore pellets by the grate-kiln process and affects normal production. In this paper, the effects of pellet basicity (CaO/SiO2 mass ratio) on the simulated deposit formation were investigated. The results show that the porosity of deposits samples increases from 30.8 to 41.5% as the pellet basicity increases from 0.6 to 1.2, and most of the holes are irregular in shape. The contents of CaO and Fe2O3 in the silicates of the deposit samples increased with increasing basicity. The primary phase of the deposit samples changed from the M2O3 phase region to the clinopyroxene phase region with a lower melting point. As the basicity increased, the calculated proportions of the liquid phases in the deposit samples had an increasing trend. Moreover, the deposit sample adhesion to the refractory brick increases with the increase in pellet basicity.

Crystal structures of LeuT reveal conformational dynamics in the outward-facing states.

The neurotransmitter:sodium symporter (NSS) homolog LeuT from Aquifex aeolicus has proven to be a valuable model for studying the transport mechanism of the NSS family. Crystal structures have captured LeuT in key conformations visited during the transport cycle, allowing for the construction of a nearly complete model of transport, with much of the conformational dynamics studied by computational simulations. Here, we report crystal structures of LeuT representing new intermediate conformations between the outward-facing open and occluded states. These structures, combined with binding and accessibility studies, reveal details of conformational dynamics that can follow substrate binding at the central substrate binding site (S1) of LeuT in outward-facing states, suggesting a potential competition for direction between the outward-open and outward-occluded states at this stage during substrate transport. Our structures further support an intimate interplay between the protonation state of Glu290 and binding of Na1 that may ultimately regulate the outward-open-to-occluded transition.

The global state of research in pain management of osteoarthritis (2000-2019): A 20-year visualized analysis.

ABSTRACT: There has been a highly active area in the pain management of osteoarthritis (OA) over the past 2 decades. The study aims to unmask the global status and trends in this field.Publications on pain management of OA from 2000 to 2019 were retrieved from the Web of Science (WOS) database. The data were analyzed using bibliometric statistical methodology. The software VOS viewer was used for bibliographic coupling, co-authorship, co-citation, co-occurrence analysis and to investigate the publication trends in pain management of OA.A total of 8207 researches in amount were included. The relative research interests and number of publications indicated a rising trend. The USA made the greatest contribution to this field, with the most publications, total citations and the highest H-index, while Sweden had the highest average citation per publication. The most contributive organization was Boston University. The journal OA and Cartilage published the most relative articles. Researches could be grouped into 5 clusters based on co-occurrence network map: Health and Epidemiology; Sport Medicine; Clinical Study; Mechanism Research and Medical Technology and Science. Medical Technology and Science was predicted as the next research topic in this field.The number of publications about pain management of OA would be increasing based on current global trends. The USA made the largest contribution to this field. The development of Medical Technology and Science may be the next popular topics in the pain management of OA research.

In vitro and in vivo biocompatibility and osteogenesis of graphene-reinforced nanohydroxyapatite polyamide66 ternary biocomposite as orthopedic implant material.

Graphene and its derivatives have been receiving increasing attention regarding their application in bone tissue engineering because of their excellent characteristics, such as a vast specific surface area and excellent mechanical properties. In this study, graphene-reinforced nanohydroxyapatite/polyamide66 (nHA/PA66) bone screws were prepared. The results of scanning electron microscopy observation and X-ray diffraction data showed that both graphene and nHA had good dispersion in the PA66 matrix. In addition, the tensile strength and elastic modulus of the composites were significantly improved by 49.14% and 21.2%, respectively. The murine bone marrow mesenchymal stem cell line C3H10T1/2 exhibited better adhesion and proliferation in graphene reinforced nHA/PA66 composite material compared to the nHA/PA66 composites. The cells developed more pseudopods, with greater cell density and a more distinguishable cytoskeletal structure. These results were confirmed by fluorescent staining and cell viability assays. After C3H10T1/2 cells were cultured in osteogenic differentiation medium for 7 and 14 days, the bone differentiation-related gene expression, alkaline phosphatase, and osteocalcin were significantly increased in the cells cocultured with graphene reinforced nHA/PA66. This result demonstrated the bone-inducing characteristics of this composite material, a finding that was further supported by alizarin red staining results. In addition, graphene reinforced nHA/PA66 bone screws were implanted in canine femoral condyles, and postoperative histology revealed no obvious damage to the liver, spleen, kidneys, brain, or other major organs. The bone tissue around the implant grew well and was directly connected to the implant. The soft tissues showed no obvious inflammatory reaction, which demonstrated the good biocompatibility of the screws. These observations indicate that graphene-reinforced nHA/PA66 composites have great potential for application in bone tissue engineering.

ALEX1 may be a novel biomarker for human cervical squamous cell carcinoma.

UNLABELLED: The armadillo repeat proteins were first found in armadillo gene of Drosophila. Since then a number of proteins containing armadillo repeats have been noticed and studied. These proteins that consist of 6 to 13 armadillo repeat domains are classified as family of armadillo repeat proteins. Recently, several studies indicated that armadillo repeat family of proteins play an important role in the tumorigenesis and maintenance of tissue integrity. ALEX1 (Arm protein lost in epithelial cancers, on chromosome X), contains two armadillo repeats domains, is expressed different in normal and carcinomas tissues. Several studies have found that ALEX1 protein lost in tumors that originated in epithelial tissues. We evaluated the ALEX1 protein expression in 53 cervical cancers and in 53 non-cancerous cervical tissues from patients and adjacent non-cancerous tissues using immunohistochemistry RESULTS: ALEX1 protein expression is significantly increased in 53 cervical cancers tissues compared with non-cancerous tissues. We found, for the first time, that ALEX1 protein expression in cervical cancers tissues is higher than non-cancerous tissues. It is suggested that the ALEX1 protein is associated with tumorigenesis in cervical cancer and we speculate that the ALEX1 may plays a role as an oncogene in cervical cancer. Moreover, ALEX1 may serve as a novel potential diagnostic biomarker in identifying cervical cancer.

Hepatotoxicity and nephrotoxicity of organic contaminants in wastewater-irrigated soil.

The objective of this study is to investigate the hepatotoxicity and nephrotoxicity of organic contaminants in wastewater-irrigated soil using in vivo and in vitro experiments on mice and rat. Soil samples were collected from a wastewater-irrigated area and groundwater-irrigated area, i.e. clean water-irrigated area as control group. The organic contaminants were extracted using an ultrasonic oscillator. In vivo experiment was performed by contamination of hepatocytes of rat using the organic extract, and comet assay was used to analyse the DNA damage of hepatocytes. For in vitro experiment, mice were first gavaged with extracts, and then the indicators for kidney functions, liver functions and oxidative damage of tissues were investigated. The result shows, for in vitro experiments, compared with clean water-irrigated area groups, the average DNA tailing length for the wastewater-irrigated area group is larger, and for the wastewater-irrigated area groups with extract concentration 0.6 g/ml and 0.9 g/ml, the tailing rate increases significantly (P < 0.05). For in vivo experiments, the change of weight across each group shows no significant difference (P < 0.05). Compared with clean water-irrigated groups, the liver indices have decreased for all groups of the wastewater-irrigated area, while both kidney and liver indices decreased for wastewater-irrigated area high-dose group (P < 0.05 or P < 0.01). The total proteins for wastewater-irrigated low-dose group and Gamma-glutamyl transpeptidase, creatinine for high-dose group all increased (P < 0.01). Compared with the reagent control group, total superoxide dismutase activity of liver for wastewater-irrigated groups and glutathione peroxidase activity for high-dose group, malondialdehyde content all decreased (P < 0.05 or P < 0.01); glutathione peroxidase activity of kidney tissue for wastewater-irrigated high-dose group decreased (P < 0.01). The result shows that the joint toxicity in extracts of wastewater-irrigated soil is able to cause DNA damage of hepatocytes in rats, changes of liver functions in mice and lead to oxidative damage of liver and kidney.

MiR-335 inhibits migration of breast cancer cells through targeting oncoprotein c-Met.

Metastasis is the leading cause of death in patients with breast cancer and aberrantly expressed microRNAs (miRNAs) are highly associated with this process. A previous study has shown that miR-335 is downregulated in breast cancer and can suppress tumor invasion and metastasis. Emerging evidences indicate that c-Met is implicated in cell scattering, migration, and invasion. However, little is known about the relationship between miR-335 expression and c-Met alteration in breast cancer. In the present study, we found that miR-335 expression was downregulated and c-Met protein expression was upregulated in two human breast cell lines. MiR-335 was found to negatively regulate c-Met protein level by directly targeting its 3' untranslated region (UTR). Forced expression of miR-335 decreased c-Met expression at protein levels and consequently diminished hepatocyte growth factor (HGF)-induced phosphorylation of c-Met and subsequently inhibited HGF promotion of breast cancer cell migration in a c-Met-dependent manner. MiR-335 expression was increased after 5-aza-2'-deoxycytidine (5-AZA-CdR) treatment, and 5-AZA-CdR treatment resulted in the same phenotype as the effect of miR-335 overexpression. Taken together, these results demonstrate that miR-335 suppresses breast cancer cell migration by negatively regulating the HGF/c-Met pathway.

Bmi-1 regulates epithelial-to-mesenchymal transition to promote migration and invasion of breast cancer cells.

Breast cancer is a highly invasive and metastatic disease. Recent studies report that breast cancer cells that have undergo epithelial-to-mesenchymal transition (EMT) obtain malignant characteristic, however, the molecular mechanism underlying this transition are poorly understood. Here, we found that over-expression associated with the process of breast cancer and that high B-cell-specific moloney murine leukemia virus insertion site 1 (Bmi-1) levels predict shorter survival of breast cancer patients. We demonstrate that Bmi-1 regulates EMT and the migration of breast cancer cells. RNA interference-mediated knockdown Bmi-1 expression restored E-cadherin expression and cell-cell junction formation in breast cancer cells, suppressing cell migration and invasion. In contrast, the over-expression of Bmi-1 decreased the expression of the epithelial mark (E-cadherin) but increased the mesenchymal makers (N-cadherin and vimentin) in breast cancer cells.

MCPH1 Protein Expression in Normal and Neoplastic Lung Tissues.

Lung cancer is the most common cause of cancer-related death in the world. The main types are small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC), the latter including squamous cell carcinoma (SCC), adenocarcinoma and large cell carcinoma. NSCLCs account for about 80% of all lung cancer cases. Microcephalin (MCPH1), also called BRIT1 (BRCT-repeat inhibitor of hTERT expression), plays an important role in the maintenance of genomic stability. Recently, several studies have provided evidence that the expression of MCPH1 gene is decreased in several different types of human cancers. We evaluated the expression of protein MCPH1 in 188 lung cancer and 20 normal lung tissues by immunohistochemistry. Positive MCPH1 staining was found in all normal lung samples and only some cancerous tissues. MCPH1-positive cells were significantly lower in lung carcinoma compared with normal tissues. Furthermore, we firstly found that MCPH1 expression in lung adenocarcinoma is higher than its expression in squamous cell carcinoma. Change in MCPH1 protein expression may be associated with lung tumorigenesis and may be a useful biomarker for identification of pathological types of lung cancer.

[Experimental study on Seprafilm anti-adhesion membrane composed of hyaluronic acid derivative on prevention of tendon adhesion in rabbit].

OBJECTIVE: To evaluate the feasibility of Seprafilm anti-adhesion membrane, a hyaluronic acid (HA) derivative, on prevention of adhesion in acute injured tendon. METHODS: Eighteen 4-month-old Chinese white rabbits (half males and half females, weighing 2.0-2.5 kg) were made the laceration models of the bilateral second and third toes of hindpaw. According to different treatments, the rabbits were randomly divided into 4 groups (n = 18). The second toe of right hindpaw was wrapped with Seprafilm anti-adhesion membrane (group A); the third toe of right hindpaw was wrapped with polylactic acid membrane (group B); the second toe of left hindpaw was coated with sodium hyaluronate gel (group C); and the third toe of left hindpaw did not treated, as control group (group D). The general condition was observed; the range of motion (ROM) of distal interphalangeal joint was measured; the gross observation and histological observation were performed at 1, 2, and 4 weeks, then the degree of adhesion was graded. RESULTS: All rabbits survived to the end of the experiment. There was no significant difference in ROM of distal interphalangeal joint between groups A and B at 1, 2, and 4 weeks (P > 0.05). ROM of group A was superior to that of groups C and D at 2 and 4 weeks (P < 0.05). The gross and histological observations showed the same result in the grading of adhesion. At 1 week, there was no significant difference in the grading of adhesion among 4 groups (P > 0.05); at 2 and 4 weeks, the grading of adhesion in group A was similar to that in group B (P > 0.05), and the grading of adhesion in group A was significantly slighter than that in groups C and D (P < 0.05). CONCLUSION: Seprafilm anti-adhesion membrane composed of HA derivative can prevent tendon adhesion and improve the joint function in acute tendon injury of rabbits.

Porphyrin and galactosyl conjugated micelles for targeting photodynamic therapy.

PURPOSE: To study the targeting and photodynamic therapy efficiency of porphyrin and galactosyl conjugated micelles based on amphiphilic copolymer galactosyl and mono-aminoporphyrin (APP) incoporated poly(2-aminoethyl methacrylate)-polycaprolactone (Gal-APP-PAEMA-PCL). METHODS: Poly(2-aminoethyl methacrylate)-polycaprolactone (PAEMA-PCL) was synthesized by the combination of ring opening polymerization and reversible addition-fragmentation chain transfer (RAFT) polymerization, and then Gal-APP-PAEMA-PCL was obtained after conjugation of lactobionic acid and 5-(4-aminophenyl)-10,15,20-triphenylporphyrin (APP) to PAEMA-PCL. The chemical structures of the copolymers were characterized, and their biological properties were evaluated in human laryngeal carcinoma (HEp2) and human hepatocellular liver carcinoma (HepG2) cells. RESULTS: Both APP-PAEMA-PCL and Gal-APP-PAEMA-PCL did not exhibit dark cytotoxicity to HEp2 cells and HepG2 cells. However, Gal-APP-PAEMA-PCL was taken up selectively by HepG2 cells and had the higher phototoxicity effect. Both polymers preferentially localized within cellular vesicles that correlated to the lysosomes. CONCLUSIONS: The results indicated that porphyrin and galactosyl conjugated polymer micelles exhibited higher targeting and photodynamic therapy efficacy in HepG2 cells than in HEp2 cells.

Assessment of bone metabolism in cadmium-induced renal tubular dysfunction by measurements of biochemical markers.

Bone metabolism related to the severity of cadmium (Cd)-induced renal tubular dysfunction (RTD) was assessed by measuring several bone biochemical markers. Fifty-three female subjects with RTD aged 65-76 years (mean 70.0+/-3.3 years) and who lived in the Cd-polluted Jinzu River basin in Toyama, Japan were studied. Bone alkaline phosphatase (bone-ALP), intact bone Gla-protein (intact-BGP) and carboxy-terminal propeptide of type I collagen (PICP) in serum as bone formation markers and pyridinoline (Pyr) and deoxypyridinoline (Dpyr) in urine as bone resorption markers were measured. All markers of bone turnover were increased and significantly correlated with each other, suggesting that bone formation and resorption were coupled and increased in Cd-induced RTD. Fractional excretion of beta(2)-microglobulin (beta(2)-m, FE(beta 2-m)) as an index of severity of Cd-induced RTD was extremely varied ranging from 0.45 to 53%. There were no significant correlations between FE(beta 2-m) and each of the five bone biochemical markers. The bone turnover in Cd-induced RTD appeared to be determined by the glomerular filtration rate (GFR): in subjects with GFRs above 50 ml/min, the levels of bone-ALP or intact-BGP tended to be inversely related to the GFRs, whereas in subjects with GFRs below 40 ml/min, those levels tended to decrease. These results suggest that the bone turnover, in particular the bone formation, was influenced by renal tubular function as assessed by the levels of GFR in Cd-induced RTD.