An anti-tumor protein PFAP specifically interacts with cholesterol-enriched membrane domains of A549 cells and induces paraptosis and endoplasmic reticulum stress.

Nowadays, non-small cell lung cancer (NSCLC) is still one of the most life-threatening diseases in the world. In previous studies, a fungal protein PFAP with anti-NSCLC properties was isolated and identified from Pleurotus ferulae lanzi. In this study, the amino acid sequence of PFAP was analyzed and found to be highly homologous to the aegerolysin family. PFAP, like other members of the aegerolysin family, specifically recognizes lipid raft domains rich in cholesterol and sphingomyelin, which is probably its specific anti-tumor mechanism. Previous studies have shown that PFAP can induce AMPK-mediated autophagy and G1-phase cell cycle arrest in A549 lung cancer cells. This study further revealed that PFAP can also induce paraptosis and endoplasmic reticulum stress (ERS) in A549 cells in vitro by targeting AMPK. PFAP induces multi-pathway death of A549 cells, and thus demonstrates its potential value for developing new drugs for NSCLC.

The high-quality sequencing of the Brassica rapa 'XiangQingCai' genome and exploration of genome evolution and genes related to volatile aroma.

'Vanilla' (XQC, brassica variety chinensis) is an important vegetable crop in the Brassica family, named for its strong volatile fragrance. In this study, we report the high-quality chromosome-level genome sequence of XQC. The assembled genome length was determined as 466.11 Mb, with an N50 scaffold of 46.20 Mb. A total of 59.50% repetitive sequences were detected in the XQC genome, including 47 570 genes. Among all examined Brassicaceae species, XQC had the closest relationship with B. rapa QGC ('QingGengCai') and B. rapa Pakchoi. Two whole-genome duplication (WGD) events and one recent whole-genome triplication (WGT) event occurred in the XQC genome in addition to an ancient WGT event. The recent WGT was observed to occur during 21.59-24.40 Mya (after evolution rate corrections). Our findings indicate that XQC experienced gene losses and chromosome rearrangements during the genome evolution of XQC. The results of the integrated genomic and transcriptomic analyses revealed critical genes involved in the terpenoid biosynthesis pathway and terpene synthase (TPS) family genes. In summary, we determined a chromosome-level genome of B. rapa XQC and identified the key candidate genes involved in volatile fragrance synthesis. This work can act as a basis for the comparative and functional genomic analysis and molecular breeding of B. rapa in the future.

New fungal protein from Pleurotus ferulae lanzi induces AMPK-mediated autophagy and G1-phase cell cycle arrest in A549 lung cancer cells.

A new protein, designated PFAP, with activity against non-small cell lung cancer (NSCLC), was isolated from Pleurotus ferulae lanzi, a medicinal and edible mushroom. The purification method involved hydrophobic interaction chromatography on a HiTrap Octyl FF column and gel filtration on a Superdex 75 column. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single band with a molecular weight of 14.68 kDa. Following de novo sequencing and liquid chromatography-tandem mass spectrometry, PFAP was identified as a protein consisting of 135 amino acid residues, with a theoretical molecular weight of 14.81 kDa. Tandem mass tag (TMT)™-based quantitative proteomic analysis and western blotting revealed that AMP-activated protein kinase (AMPK) was significantly upregulated in NSCLC A549 cells, following PFAP treatment. The downstream regulatory factor mammalian target of rapamycin (mTOR) was suppressed, resulting in the activation of autophagy and upregulated expressions of P62, LC3 II/I, and other related proteins. PFAP blocked NSCLC A549 cells in the G1 phase of the cell cycle via upregulating P53 and P21, while subsequently downregulating the expression of cyclin-dependent kinases. PFAP suppresses tumour growth via the same mechanism in a xenograft mouse model in vivo. These results demonstrate that PFAP is a multifunctional protein with anti-NSCLC properties.

Fungal antitumor protein D1 is internalized via endocytosis and inhibits non-small cell lung cancer proliferation through MAPK signaling pathway.

Lung cancer has the highest mortality among cancer-related deaths worldwide. Among lung cancers, non-small cell lung cancer (NSCLC) is the most common histological type. In the previous research, we isolated a protein (D1) from Boletus bicolor that inhibits the proliferation of NSCLC cell lines. In this study, we elucidated the internalization mechanism and antitumor mechanism of protein D1 in A549 cells. Protein D1 has a strong inhibitory effect on A549 cells. It binds to secretory carrier membrane protein 3 on the A549 cell membrane and enters A549 cells by clathrin-mediated endocytosis. In vitro, protein D1 activates mitogen-activated protein kinase (MAPK) signaling pathway. JNK and p38MAPK are the biological targets for protein D1. In vivo, protein D1 inhibits the tumor growth of NSCLC xenografts by inducing apoptosis and inhibiting cell proliferation. Protein D1 alters the expression of genes related to apoptosis, cell cycle, and MAPK signaling pathway in tumor cells.

Simultaneous high p-nitrophenol concentration and nitrogen removal by two-stage membrane biofilm reactor.

P-nitrophenol (PNP) is highly toxic and difficult to degrade, causing great harm to the ecological environment and human health. A two-stage bench-scale membrane biofilm reactor (MBfR) was constructed to treat wastewater containing high concentration of PNP and the generated nitrogen without external organic carbon sources. The two reactors were supplied with oxygen and methane, respectively. O2-MBfR was used for the degradation of PNP and the improvement of wastewater biodegradability. CH4-MBfR was used for the total nitrogen (TN) removal treatment from O2-MBfR effluent. In this experiment, the performance of the two-stage MBfR process was evaluated and optimized by adjusting operational parameters (aeration pressure, HRT, and pH). Under the optimal operation parameters, the removal efficiencies of PNP (100 mg/L) and TN attained 89.70% and 69.24%, respectively, and the removal loads were 0.930 g·m-2·d-1 and 241.42 mg·m-2·d-1, respectively. The reactor was able to accommodate the concentrations of PNP up to 200-400 mg/L, and the reactor reached maximum efficiency throughout the process when the concentration of PNP in the wastewater was 250 mg/L. The removal rates of PNP and TN reached 95.0% and 69.48%, respectively, and the removal loads were 2.37 g·m-2·d-1 and 96.22 mg·m-2·d-1, respectively. This research provides a better solution for multi-MBfR to treat toxic industrial wastewater containing phenol, nitrophenol, and further TN removal, which would not release any air pollutants into the atmosphere.

RNAi-mediated silencing of Trichinella spiralis serpin-type serine protease inhibitors results in a reduction in larval infectivity.

Trichinella spiralis serpin-type serine protease inhibitors (TsSPIs) are expressed in adult worms (AW), newborn larvae (NBL) and muscle larvae (ML) of T. spiralis, with the ML stage demonstrating the highest expression level. This study aims to determine TsSPI functions in larval viability and invasion of intestinal epithelial cells in vitro, as well as their development, survival, and fecundity in vivo via RNAi. TsSPI-specific siRNAs and dsRNA were transfected into ML by incubation. The silencing effect of TsSPI transcription and expression was determined using qPCR and western blot, respectively. After incubation in 60 ng/µL dsRNA-TsSPI for 3 days, larval TsSPI mRNA and protein expression levels were reduced by 68.7% and 68.4% (P < 0.05), respectively. dsRNA-mediated silencing of TsSPI significantly impacted larval invasion into intestinal epithelial cells in vitro but did not affect the survival rate of larvae. After challenge with dsRNA-TsSPI-treated ML, mice exhibited a 56.0% reduction in intestinal AW burden and 56.9% reduction in ML burden (P < 0.05), but NBL production of female AW remained the same (P > 0.05). Our results revealed that RNAi-mediated silencing of TsSPI expression in T. spiralis significantly reduced larval infectivity and survival in the host but had no effect on the survival rate and fecundity. Furthermore, TsSPIs have no effect on the growth and reproduction of parasites but may be directly involved in regulating the interaction of T. spiralis and the host. Therefore, TsSPIs are crucial in the process of T. spiralis larval invasion and parasite survival in the host.

Experimental Investigation of Hydrophobically Modified α-ZrP Nanosheets for Enhancing Oil Recovery in Low-Permeability Sandstone Cores.

Highly crystalline α-zirconium phosphate (α-ZrP) nanoparticles were synthesized and exfoliated into nanosheets, and then the hydrophilic nanosheets were modified into hydrophobic nanosheets with octadecyltrichlorosilane (OTS). Scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and thermogravimetric analysis were applied to confirm the morphology and chemical structure of the nanosheets. Contact angle measurement was conducted to explore the wettability alteration of the hydrophobically modified α-ZrP nanosheets, and the result showed that the wettability of the core was changed into hydrophobicity. When ZrP-OTS nanosheets were injected during water-flooding, a Pickering emulsion will be formed. The droplet diameters and viscosities of the Pickering emulsions were measured. The hydrophobically modified α-ZrP nanosheets were applied in low-permeability sandstone cores and various concentrations were tested. The injectivity of the hydrophobically modified α-ZrP nanosheets was also studied and the result indicated that the nanosheets exhibit good injectivity. The mechanisms for enhancing oil recovery by utilizing hydrophobic α-ZrP nanosheets were analyzed: forming Pickering emulsions and increasing the viscosity of the displacing phase. Forming emulsions and increasing the viscosity of the flooding phase can enhance the microdisplacement efficiency, while good injectivity can also enhance the macrodisplacement efficiency. The result indicated the possibility of using hydrophobically modified α-ZrP nanosheets for enhancing oil recovery in a low-permeability reservoir.

The universally-conserved transcription factor RfaH is recruited to a hairpin structure of the non-template DNA strand.

RfaH, a transcription regulator of the universally conserved NusG/Spt5 family, utilizes a unique mode of recruitment to elongating RNA polymerase to activate virulence genes. RfaH function depends critically on an ops sequence, an exemplar of a consensus pause, in the non-template DNA strand of the transcription bubble. We used structural and functional analyses to elucidate the role of ops in RfaH recruitment. Our results demonstrate that ops induces pausing to facilitate RfaH binding and establishes direct contacts with RfaH. Strikingly, the non-template DNA forms a hairpin in the RfaH:ops complex structure, flipping out a conserved T residue that is specifically recognized by RfaH. Molecular modeling and genetic evidence support the notion that ops hairpin is required for RfaH recruitment. We argue that both the sequence and the structure of the non-template strand are read out by transcription factors, expanding the repertoire of transcriptional regulators in all domains of life.

Mapping the Escherichia coli transcription elongation complex with exonuclease III.

RNA polymerase interactions with the nucleic acids control every step of the transcription cycle. These contacts mediate RNA polymerase recruitment to promoters, induce pausing during RNA chain synthesis, and control transcription termination. These interactions are dissected using footprinting assays, in which a bound protein protects nucleic acids from the digestion by nucleases or modification by chemical probes. Exonuclease III is frequently employed to study protein-DNA interactions owing to relatively simple procedures and low background. Exonuclease III has been used to determine RNA polymerase position in transcription initiation and elongation complexes and to infer the translocation register of the enzyme. In this chapter, we describe probing the location and the conformation of transcription elongation complexes formed by walking of the RNA polymerase along an immobilized template.

Antihyperglycaemic mechanisms of an aceteoside polymer from rose flowers and a polysaccharide-protein complex from abalone mushroom.

Oral administration of an aceteoside polymer from rose Rosa rugosa (P1-b) and a polysaccharide-peptide complex from abalone mushroom Pleurotus abalonus (LB-1b), both with antioxidant activity, produced antihyperglycaemic effects in alloxan-induced diabetic mice. The expression of insulin, superoxide dismutase and pancreas duodenum homeobox factor-1 essential for pancreatic islet function as estimated by real-time PCR was augmented. The reactive oxygen species-scavenging ability of the rose constituent was notably stronger than the mushroom constituent. Thus, the two biomolecules protected the pancreas from oxidative stress, elevated pancreatic insulin expression and lowered circulating glucose level.

A study of effects of peptide fragments of bovine and human lactoferrins on activities of three key HIV-1 enzymes.

The intent of this study was to examine human and bovine lactoferrin fragments including lactoferrin (1-11), lactoferricin and lactoferrampin, all of which did not demonstrate hemolytic activity toward rabbit erythrocytes at 1 mM concentration, for possible inhibitory effects on the activities of HIV-1 reverse transcriptase, protease and integrase. The data showed that human lactoferricin was the most potent in inhibiting HIV-1 reverse transcriptase (IC50 =2 µM). Bovine lactoferricin (IC50 = 10 µM) and bovine lactoferrampin (IC50 = 150 µM) were less potent. Human lactoferrampin and human and bovine lactoferrin (1-11) at 1 mM concentration did not exhibit any inhibitory effect on HIV-1 reverse transcriptase. All peptides showed only a slight inhibitory effect (from slightly below 2% to 6% inhibition) on HIV-1 protease. Human lactoferrampin and bovine lactoferrampin showed obvious inhibitory effect on HIV-1 integrase at 37 µM and 18.5 µM, respectively. The HIV-1 integrase inhibitory activity of human lactoferrampin and bovine lactoferrampin was dose-dependent. The other peptides were devoid of HIV-1 integrase inhibitory activity. Thus, it is concluded that some lactoferrin fragments exert an inhibitory action on HIV-1 reverse transcriptase and HIV-1 integrase.

Purification and characterization of a novel antitumor protein with antioxidant and deoxyribonuclease activity from edible mushroom Pholiota nameko.

A novel antitumor protein from the edible mushroom Pholiota nameko (PNAP) was purified through a two-step chromatographic procedure including SP cation exchange chromatogram and Superdex gel filtration. The approximate molecular weight was demonstrated to be 18.5 kDa by SDS-PAGE and ultracentrifugation analysis and N-terminal sequence was detected as AGRTFIGYNG by Edman degradation. Biochemical characterization showed that it exhibited significant antioxidant activity by effectively scavenging hydroxyl and 1,1-diphenyl-2-picrylhydrazyl radicals compared to standard antioxidant butylated hydroxy anisole. PNAP had deoxyribonuclease activity with the optimum pH and temperature were 5.0 and 60 °C respectively, as well as it can act on both double-stranded and single-stranded DNA, but preferentially on double-stranded DNA. PNAP displayed antitumor activity against cancer cell lines such as MCF7 and Hela cells. Human breast cancer MCF7 cells treated with PNAP produced typical apoptotic morphological changes including chromatin condensation, accumulation of sub-G1 cells and alternation of mitochondrial permeability. The PNAP induced apoptosis of MCF7 cells entailed loss of mitochondrial membrane potential resulting in release of cytochrome c into cytosol, activation of caspase-9 and caspase-3, which are responsible for the execution of apoptosis, implying intrinsic signal pathway is involved in PNAP induced apoptosis.

Immunoregulatory and anti-HIV-1 enzyme activities of antioxidant components from lotus (Nelumbo nucifera Gaertn.) rhizome.

In the present study, two antioxidant micromolecular components (L2f-2 and L2f-3) and an antioxidant macromolecular component LB2 were extracted from lotus (Nelumbo nucifera Gaertn.) rhizomes. MS, FTIR (Fourier-transform IR) spectroscopy and NMR were used to identify these compounds. L2f-2 was (+/-)-gallocatechin, L2f-3 was (-)-catechin and LB2 was a polysaccharide-protein complex with a molecular mass of 18.8 kDa. LB2 was identified as a polysaccharide sulfate containing α/ß-pyranose and α-furanose according to its FTIR spectrogram. It was composed of mannose, rhamnose, glucose, galactose and xylose with a molar ratio 2:8:7:8:1. The antioxidant components L2f-2, L2f-3 and LB2 strongly inhibited HIV-1 RT (reverse transcriptase) and IN (integrase). LB2 inhibited RT with an IC50 value of 33.7 µM. It also exhibited the highest HIV-1 3'-processing inhibitory activity with an IC50 value of 5.28 µM. Both L2f-2 and L2f-3 up-regulated the expression of IL-2 (interleukin-2) and down-regulated IL-10, while LB2 exhibited positive regulation on IL-2, IL-4 and IL-10. Moreover, L2f-3 and LB2 might inhibit HIV-1 directly by down-regulating TNFα (tumour necrosis factor α). These natural antioxidant components with antiviral and immunoregulatory activities could be potentially important for anti HIV-1 drug development and application to HIV-1 therapy.