Structure characterization and biological activities evaluation of two hetero-polysaccharides from Lepista nuda: Cell antioxidant, anticancer and immune-modulatory activities.

Polysaccharides LNP-1 and LNP-2 were extracted and purified from Lepista nuda, and their structural characteristics and biological activities were evaluated. The molecular weights of LNP-1 and LNP-2 were determined to be 16,263 Da and 17,730 Da, respectively. The monosaccharide composition analysis showed that LNP-1 and LNP-2 were composed of fucose, mannose, glucose, and galactose in a molar ratio of 1.00:2.42:1.09:4.04 and 1.00:2.39:1.61:4.23, respectively. The structure analysis revealed that these two polysaccharides were mainly composed of T-Fuc, T-Man, T-Glc, 1,6-Glc 1,6-Gal, and 1,2,6-Man, 1,2,6-Gal. Additionally, LNP-2 contained an additional 1,4-Glc glycosidic linkage in comparison to LNP-1. Both LNP-1 and LNP-2 exhibited anti-proliferation effects on A375 cells, but not on HepG2 cells. Furthermore, LNP-2 showed better cellular antioxidant activity (CAA) than LNP-1. RT-PCR results indicated that LNP-1 and LNP-2 could induce macrophages to secrete immune-modulatory factors NO, IL-6, and TNF-α by regulating their mRNA expression. Overall, this study provides a theoretical basis for the further development of the structure-function relationship of polysaccharides from L. nuda.

The Impacts of Short-Term NMN Supplementation on Serum Metabolism, Fecal Microbiota, and Telomere Length in Pre-Aging Phase.

Aging is a natural process with concomitant changes in the gut microbiota and associate metabolomes. Beta-nicotinamide mononucleotide, an important NAD+ intermediate, has drawn increasing attention to retard the aging process. We probed the changes in the fecal microbiota and metabolomes of pre-aging male mice (C57BL/6, age: 16 months) following the oral short-term administration of nicotinamide mononucleotide (NMN). Considering the telomere length as a molecular gauge for aging, we measured this in the peripheral blood mononuclear cells (PBMC) of pre-aging mice and human volunteers (age: 45-60 years old). Notably, the NMN administration did not influence the body weight and feed intake significantly during the 40 days in pre-aging mice. Metabolomics suggested 266 upregulated and 58 downregulated serum metabolites. We identified 34 potential biomarkers linked with the nicotinamide, purine, and proline metabolism pathways. Nicotinamide mononucleotide significantly reduced the fecal bacterial diversity (p < 0.05) with the increased abundance of Helicobacter, Mucispirillum, and Faecalibacterium, and lowered Akkermansia abundance associated with nicotinamide metabolism. We propose that this reshaped microbiota considerably lowered the predicated functions of aging with improved immune and cofactors/vitamin metabolism. Most notably, the telomere length of PBMC was significantly elongated in the NMN-administered mice and humans. Taken together, these findings suggest that oral NMN supplementation in the pre-aging stage might be an effective strategy to retard aging. We recommend further studies to unravel the underlying molecular mechanisms and comprehensive clinical trials to validate the effects of NMN on aging.

Effect of COD/N ratio on nitrogen removal and microbial communities of CANON process in membrane bioreactors.

In this study, the effect of COD/N ratio on completely autotrophic nitrogen removal over nitrite (CANON) process was investigated in five identical membrane bioreactors. The five reactors were simultaneously seeded for 1L CANON sludge and be operated for more than two months under same conditions, with influent COD/N ratio of 0, 0.5, 1, 2 and 4, respectively. DGGE was used to analyze the microbial communities of aerobic ammonia-oxidizing bacteria (AOB) and anaerobic ammonia-oxidizing bacteria (AAOB) in five reactors. Results revealed the harmonious work of CANON and denitrification with low COD concentration, whereas too high COD concentration suppressed both AOB and AAOB. AOB and AAOB biodiversity both decreased with COD increasing, which then led to worse nitrogen removal. The suppressing threshold of COD/N ratio for CANON was 1.7. CANON was feasible for treating low COD/N sewage, while the high sewage should be converted by anaerobic biogas producing process in advance.

TGBp3 triggers the unfolded protein response and SKP1-dependent programmed cell death.

The Potato virus X (PVX) triple gene block protein 3 (TGBp3), an 8-kDa membrane binding protein, aids virus movement and induces the unfolded protein response (UPR) during PVX infection. TGBp3 was expressed from the Tobacco mosaic virus (TMV) genome (TMV-p3), and we noted the up-regulation of SKP1 and several endoplasmic reticulum (ER)-resident chaperones, including the ER luminal binding protein (BiP), protein disulphide isomerase (PDI), calreticulin (CRT) and calmodulin (CAM). Local lesions were seen on leaves inoculated with TMV-p3, but not TMV or PVX. Such lesions were the result of TGBp3-elicited programmed cell death (PCD), as shown by an increase in reactive oxygen species, DNA fragmentation and induction of SKP1 expression. UPR-related gene expression occurred within 8 h of TMV-p3 inoculation and declined before the onset of PCD. TGBp3-mediated cell death was suppressed in plants that overexpressed BiP, indicating that UPR induction by TGBp3 is a pro-survival mechanism. Anti-apoptotic genes Bcl-xl, CED-9 and Op-IAP were expressed in transgenic plants and suppressed N gene-mediated resistance to TMV, but failed to alleviate TGBp3-induced PCD. However, TGBp3-mediated cell death was reduced in SKP1-silenced Nicotiana benthamiana plants. The combined data suggest that TGBp3 triggers the UPR and elicits PCD in plants.

Fabrication of macroporous titanium dioxide film using PMMA microspheres as template.

A novel synthetic procedure is described for the fabrication of macroporous titanium dioxide (TiO(2)) films with an ordered, uniform pore framework comprised of nanocrystalline anatase mainly. The synthetic approach involved several processes. First, polymethyl methacrylate (PMMA) microspheres (87 nm) were synthesized by using a dispersion polymerization technique in the presence of Fenton reagent (FeSO(4)/H(2)O(2)) as a novel initiator, which has advantages such as simple and fast polymerization process without deoxygenation. Next, the templates of PMMA microspheres were assembled on clean substrates by dip-drawing technique. Finally, the macroporous TiO(2) films with the average size of pores about 87 nm were obtained by sol-dipping template method and calcination to remove the templates at 550°C. The test results of X-ray diffraction indicate that the nanocrystalline of anatase formed after calcination. The mechanisms of PMMA polymerization and template formation were proposed. Furthermore, both structures and morphologies of the composite films were investigated with field emission scanning electron microscope, and the processes of the thermal decomposition of PMMA and TiO(2) gel were also discussed with thermo gravimetric analysis. This ordered and uniform pore framework could be used as the promising ultrafilter membranes showing active photocatalysis without intensive fouling.

Role of unfolded protein response in plant virus infection.

A new study of Potato virus X (PVX) revealed that a viral movement protein, named TGBp3, triggers the unfolded protein response (UPR) which monitors accumulation of aberrant proteins the endoplasmic reticulum (ER) and targets them for degradation. The PVX TGBp3 resides in ER and activates bZIP60, a transcription factor involved in the UPR pathway. Knockdown of bZIP60 hampers virus infection in protoplasts and whole plants. Preliminary evidence indicates that UPR regulates cellular cytotoxicity that could otherwise lead to cell death if the PVX TGBp3 reaches high levels in the ER. SKP1 expression appears to be linked to bZIP60 and is a component of the SCF-complex mediating proteasomal degradation of cellular substrates. SKP1 expression is induced by PVX TGBp3 and plays a role in regulating PVX spread in whole plants. We propose that SKP1 might be linked to TGBp1-mediated degradation of AGO1. 

The unfolded protein response is triggered by a plant viral movement protein.

Infection with Potato virus X (PVX) in Nicotiana benthamiana plants leads to increased transcript levels of several stress-related host genes, including basic-region leucine zipper 60 (bZIP60), SKP1, ER luminal binding protein (BiP), protein disulfide isomerase (PDI), calreticulin (CRT), and calmodulin (CAM). bZIP60 is a key transcription factor that responds to endoplasmic reticulum (ER) stress and induces the expression of ER-resident chaperones (BiP, PDI, CRT, and CAM). SKP1 is a component of SCF (for SKP1-Cullin-F box protein) ubiquitin ligase complexes that target proteins for proteasomal degradation. Expression of PVX TGBp3 from a heterologous vector induces the same set of genes in N. benthamiana and Arabidopsis (Arabidopsis thaliana) leaves. Virus-induced gene silencing was employed to knock down the expression of bZIP60 and SKP1, and the number of infection foci on inoculated leaves was reduced and systemic PVX accumulation was altered. Silencing bZIP60 led to the suppression of BiP and SKP1 transcript levels, suggesting that bZIP60 might be an upstream signal transducer. Overexpression of TGBp3 led to localized necrosis, but coexpression of TGBp3 with BiP abrogated necrosis, demonstrating that the unfolded protein response alleviates ER stress-related cell death. Steady-state levels of PVX replicase and TGBp2 (which reside in the ER) proteins were unaltered by the presence of TGBp3, suggesting that TGBp3 does not contribute to their turnover. Taken together, PVX TGBp3-induced ER stress leads to up-regulation of bZIP60 and unfolded protein response-related gene expression, which may be important to regulate cellular cytotoxicity that could otherwise lead to cell death if viral proteins reach high levels in the ER.

Conjugated polymer nanoparticles for effective siRNA delivery to tobacco BY-2 protoplasts.

BACKGROUND: Post transcriptional gene silencing (PTGS) is a mechanism harnessed by plant biologists to knock down gene expression. siRNAs contribute to PTGS that are synthesized from mRNAs or viral RNAs and function to guide cellular endoribonucleases to target mRNAs for degradation. Plant biologists have employed electroporation to deliver artificial siRNAs to plant protoplasts to study gene expression mechanisms at the single cell level. One drawback of electroporation is the extensive loss of viable protoplasts that occurs as a result of the transfection technology. RESULTS: We employed fluorescent conjugated polymer nanoparticles (CPNs) to deliver siRNAs and knockdown a target gene in plant protoplasts. CPNs are non toxic to protoplasts, having little impact on viability over a 72 h period. Microscopy and flow cytometry reveal that CPNs can penetrate protoplasts within 2 h of delivery. Cellular uptake of CPNs/siRNA complexes were easily monitored using epifluorescence microscopy. We also demonstrate that CPNs can deliver siRNAs targeting specific genes in the cellulose biosynthesis pathway (NtCesA-1a and NtCesA-1b). CONCLUSIONS: While prior work showed that NtCesA-1 is a factor involved in cell wall synthesis in whole plants, we demonstrate that the same gene plays an essential role in cell wall regeneration in isolated protoplasts. Cell wall biosynthesis is central to cell elongation, plant growth and development. The experiments presented here shows that NtCesA is also a factor in cell viability. We show that CPNs are valuable vehicles for delivering siRNAs to plant protoplasts to study vital cellular pathways at the single cell level.

Mutational analysis of PVX TGBp3 links subcellular accumulation and protein turnover.

Potato virus X (PVX) TGBp3 is required for virus cell-to-cell transport, has an N-terminal transmembrane domain, and a C-terminal cytosolic domain. In the absence of virus infection TGBp3:GFP is seen in the cortical and perinuclear ER. In PVX infected cells the TGBp3:GFP fusion is also seen in the nucleoplasm indicating that events during PVX infection trigger entry into the nucleus. Mutational analysis failed to identify a nuclear targeting domain. Mutations inhibiting TGBp3 association with the ER and inhibiting virus movement did not block TGBp3:GFP in the nucleoplasm. A mutation disrupting the N-terminal transmembrane domain of TGBp3 caused the fusion to accumulate in the nucleus indicating that nuclear import is regulated by ER interactions. Tunicamycin, an ER-stress inducing chemical, caused lower levels of GFP and TGBp3:GFP to accumulate in virus infected protoplasts. MG115 and MG132 were used to demonstrate that wild-type and mutant TGBp3:GFP fusions were degraded by the 26S proteasome. These observations are consistent with an ER-associated protein degradation (ERAD) pathway suggesting that PVX TGBp3, similar to aberrant ER proteins, is translocated to the cytoplasm for degradation. Nuclear accumulation of mutant and wild-type TGBp3:GFP is independent of other PVX proteins and may be another feature of an ERAD pathway.

Subcellular targeting and interactions among the Potato virus X TGB proteins.

Potato virus X (PVX) encodes three proteins named TGBp1, TGBp2, and TGBp3 which are required for virus cell-to-cell movement. To determine whether PVX TGB proteins interact during virus cell-cell movement, GFP was fused to each TGB coding sequence within the viral genome. Confocal microscopy was used to study subcellular accumulation of each protein in virus-infected plants and protoplasts. GFP:TGBp2 and TGBp3:GFP were both seen in the ER, ER-associated granular vesicles, and perinuclear X-bodies suggesting that these proteins interact in the same subdomains of the endomembrane network. When plasmids expressing CFP:TGBp2 and TGBp3:GFP were co-delivered to tobacco leaf epidermal cells, the fluorescent signals overlapped in ER-associated granular vesicles indicating that these proteins colocalize in this subcellular compartment. GFP:TGBp1 was seen in the nucleus, cytoplasm, rod-like inclusion bodies, and in punctate sites embedded in the cell wall. The puncta were reminiscent of previous reports showing viral proteins in plasmodesmata. Experiments using CFP:TGBp1 and YFP:TGBp2 or TGBp3:GFP showed CFP:TGBp1 remained in the cytoplasm surrounding the endomembrane network. There was no evidence that the granular vesicles contained TGBp1. Yeast two hybrid experiments showed TGBp1 self associates but failed to detect interactions between TGBp1 and TGBp2 or TGBp3. These experiments indicate that the PVX TGB proteins have complex subcellular accumulation patterns and likely cooperate across subcellular compartments to promote virus infection.

Molecular biology of potexviruses: recent advances.

Recent advances in potexvirus research have produced new models describing virus replication, cell-to-cell movement, encapsidation, R gene-mediated resistance and gene silencing. Interactions between distant RNA elements are a central theme in potexvirus replication. The 5' non-translated region (NTR) regulates genomic and subgenomic RNA synthesis and encapsidation, as well as virus plasmodesmal transport. The 3' NTR regulates both plus- and minus-strand RNA synthesis. How the triple gene-block proteins interact for virus movement is still elusive. As the potato virus X (PVX) TGBp1 protein gates plasmodesmata, regulates virus translation and is a suppressor of RNA silencing, further research is needed to determine how these properties contribute to propelling virus through the plasmodesmata. Specifically, TGBp1 suppressor activity is required for virus movement, but how the silencing machinery relates to plasmodesmata is not known. The TGBp2 and TGBp3 proteins are endoplasmic reticulum (ER)-associated proteins required for virus movement. TGBp2 associates with ER-derived vesicles that traffic along the actin network. Future research will determine whether the virus-induced vesicles are cytopathic structures regulating events along the ER or are vehicles carrying virus to the plasmodesmata for transfer into neighbouring cells. Efforts to assemble virions in vitro identified a single-tailed particle (STP) comprising RNA, coat protein (CP) and TGBp1. It has been proposed that TGBp1 aids in transport of virions or STP between cells and ensures translation of RNA in the receiving cells. PVX is also a tool for studying Avr-R gene interactions and gene silencing in plants. The PVX CP is the elicitor for the Rx gene. Recent reports of the PVX CP reveal how CP interacts with the Rx gene product.

Mutations in the central domain of potato virus X TGBp2 eliminate granular vesicles and virus cell-to-cell trafficking.

Most RNA viruses remodel the endomembrane network to promote virus replication, maturation, or egress. Rearrangement of cellular membranes is a crucial component of viral pathogenesis. The PVX TGBp2 protein induces vesicles of the granular type to bud from the endoplasmic reticulum network. Green fluorescent protein (GFP) was fused to the PVX TGBp2 coding sequence and inserted into the viral genome and into pRTL2 plasmids to study protein subcellular targeting in the presence and absence of virus infection. Mutations were introduced into the central domain of TGBp2, which contains a stretch of conserved amino acids. Deletion of a 10-amino-acid segment (m2 mutation) overlapping the segment of conserved residues eliminated the granular vesicle and inhibited virus movement. GFP-TGBp2m2 proteins accumulated in enlarged vesicles. Substitution of individual conserved residues in the same region similarly inhibited virus movement and caused the mutant GFP-TGBp2 fusion proteins to accumulate in enlarged vesicles. These results identify a novel element in the PVX TGBp2 protein which determines vesicle morphology. In addition, the data indicate that vesicles of the granular type induced by TGBp2 are necessary for PVX plasmodesmata transport.

[Isolation and activity of bacteria for the biodegradation of microcystins].

The abilities of bacterial communities, which collected from the sediment and surface water of Dianchi Lake, for the biodegradation of microcystins (MCs) were firstly investigated. It was shown that the biodegradation rates of both MC-RR and LR by bacteria in sediment were apparently higher than those by bacteria on surface water. Five strains of bacteria, which have the abilities in the biodegradation of MCs, from the sediment were isolated using the liquid and solid medium containing MC-RR and LR as the carbon and nitrogen sources, which was extracted and purified from the cells of cyanobacterial bloom. Among the bacteria isolated, bacterium D was found to have a strong ability in the biodegradation of MCs. Initial MC-RR and LR of 60.1 mg x L(-1) and 38.7 mg x L(-1) were completely removed in 3 days and the average biodegradation rates per day for MC-RR and LR were 20.0 mg x L(-1) and 12.9 mg x L(-1), respectively.

Photodegradation of acetochlor in water and UV photoproducts identified by mass spectrometry.

The sunlight photodegradation half-lives of 20 mg/L acetochlor were 151, 154 and 169 days in de-ionized water, river water and paddy water, respectively. When exposed to ultraviolet (UV) light, acetochlor in aqueous solution was rapidly degraded. The half-lives were 7.1, 10.1, and 11.5 min in de-ionized water, river water and paddy water, respectively. Photoproducts of acetochlor were identified by gas chromatography/mass spectrometry (GC/MS) and found at least twelve photoproducts resulted from dechlorination with subsequent hydroxylation and cyclization processes. The chemical structures of ten photoproducts were presumed on the basis of mass spectrum interpretation and literature data. Photoproducts are identified as 2-ethyl-6-methylaniline; N,N-diethylaniline; 4,8-dimethyl-2-oxo-1,2,3,4-tetrahydroquinoline; 2-oxo-N-(2-ethyl-6-methylphenyl)-N-(ethoxymethyl) acetamide; N-(ethoxymethyl)-2'-ethyl-6'-methylformanilide; 1-hydroxyacetyl-2-ethoxyl-7-ethylindole; 8-ethyl-1-ethoxymethyl-2-oxo-1,2,3,4-tetrahydroquinoline; 4, 8-dimethyl-1-ethoxymethyl-2-oxo-1,2,3,4-tetrahydroquinoline; 2-hydroxy-2'-ethyl-6'-methyl-N-(ethoxymethyl) acetanilide and a compound related to acetochlor. The other two photoproducts were detected by GC/MS although their chemical structure was unknown.

[Analyses of virus resistance and transgenes for transgenic papaya].

Virus resistance in field and molecular biological characterizations of the transgenes were analyzed for two lines of T(1) generation of transgenic papaya with the replicase mutant gene from papaya ringspot virus (PRV). The transgenic plants showed highly resistant or immune against PRV. Results indicated that the transgenes inherited to and expressed at RNA level in the progenies.

Biodegradation of acetanilide herbicides acetochlor and butachlor in soil.

The biodegradation of two acetanilide herbicides, acetochlor and butachlor in soil after other environmental organic matter addition were measured during 35 days laboratory incubations. The herbicides were applied to soil alone, soil-SDBS (sodium dodecylbenzene sulfonate) mixtures and soil-HA (humic acid) mixtures. Herbicide biodegradation kinetics were compared in the different treatment. Biodegradation products of herbicides in soil alone samples were identified by GC/MS at the end of incubation. Addition of SDBS and HA to soil decreased acetochlor biodegradation, but increased butachlor biodegradation. The biodegradation half-life of acetochlor and butachlor in soil alone, soil-SDBS mixtures and soil-HA mixtures were 4.6 d, 6.1 d and 5.4 d and 5.3 d, 4.9 d and 5.3 d respectively. The biodegradation products were hydroxyacetochlor and 2-methyl-6-ethylaniline for acetochlor, and hydroxybutachlor and 2,6-diethylaniline for butachlor.

Adsorption-desorption behavior of acetochlor to soils in the presence of some environmental substances.

The behavior of herbicide acetochlor adsorption-desorption to soil in the presence of humic acid (HA), anionic surfactant sodium dodecylbenzene sulfonate (SDBS), cationic surfactant hexadecyltrimethyl-ammonium bromide (HDAB) and NH4NO3 as a chemical fertilizer was studied. Observed acetochlor adsorption isotherm were well described using Freundlich isotherm equation, from which the desorption isotherm equation has been deduced. The deduced equation can more directly describe acetochlor desorption process. The results showed that the enhance of acetochlor adsorption capacity by solid HA was greater than by soluble HA. The presence of NH4 NO3 can slightly enhance acetochlor adsorption to soil by comparison with that measured in NH4 NO3-free solution. In soil-water system, surfactant-acetochlor interaction is very complex, and the surfactant adsorptions as well as acetochlor adsorption need to be considered. When acetochlor-soil suspensions contained lower concentration SDBS or HDAB (40 mg/L), Kf for acetochlor adsorption was decreased in comparison to that measured in SDBS- or HDAB-free solution. When acetochlor-soil suspensions contained higher concentration SDBS or HDAB (corresponding 1400 mg/L or 200 mg/L), Kf for acetochlor adsorption was increased in comparison to that measured in SDBS- or HDAB-free solution.