On the Chemical Purity and Oxygen Isotopic Composition of α-Cellulose Extractable from Higher Plants and the Implications for Climate, Metabolic, and Physiological Studies.

The 18O/16O ratio of α-cellulose in land plants has proved of interest for climate, environmental, physiological, and metabolic studies. Reliable application of such a ratio may be compromised by the presence of hemicellulose impurities in the α-cellulose product obtainable with current extraction methods, as the impurities are known to be isotopically different from that of the α-cellulose. We first compared the quality of hydrolysates of "α-cellulose products" obtained with four representative extraction methods (Jayme and Wise; Brendel; Zhou; Loader) and quantified the hemicellulose-derived non-glucose sugars in the α-cellulose products from 40 land grass species using gas chromatography-mass spectrometry (GC/MS). Second, we performed compound-specific isotope analysis of the hydrolysates using GC/Pyrolysis/IRMS. These results were then compared with the bulk isotope analysis using EA/Pyrolysis/IRMS of the α-cellulose products. We found that overall, the Zhou method afforded the highest purity α-cellulose as judged by the minimal presence of lignin and the second-lowest presence of non-glucose sugars. Isotopic analysis then showed that the O-2-O-6 of the α-cellulose glucosyl units were all depleted in 18O by 0.0-4.3 mUr (average, 1.9 mUr) in a species-dependent manner relative to the α-cellulose products. The positive isotopic bias of using the α-cellulose product instead of the glucosyl units stems mainly from the fact that the pentoses that dominate hemicellulose contamination in the α-cellulose product are relatively enriched in 18O (compared to hexoses) as they inherit only the relatively 18O-enriched O-2-O-5 moiety of sucrose, the common precursor of pentoses and hexoses in cellulose, and are further enriched in 18O by the (incomplete) hydrolysis.

Leaf transition from heterotrophy to autotrophy is recorded in the intraleaf C, H and O isotope patterns of leaf organic matter.

RATIONALE: Quantitatively relating 13 C/12 C, 2 H/1 H and 18 O/16 O ratios of plant α-cellulose and 2 H/1 H of n-alkanes to environmental conditions and metabolic status should ideally be based on the leaf, the plant organ most sensitive to environmental change. The fact that leaf organic matter is composed of isotopically different heterotrophic and autotrophic components means that it is imperative that one be able to disentangle the relative heterotrophic and autotrophic contributions to leaf organic matter. METHODS: We tackled this issue by two-dimensional sampling of leaf water and α-cellulose, and specific n-alkanes from greenhouse-grown immature and mature and field-grown mature banana leaves, taking advantage of their large areas and thick waxy layers. Leaf water, α-cellulose and n-alkane isotope ratios were then characterized using elemental analysis isotope ratio mass spectrometry (IRMS) or gas chromatography IRMS. A three-member (heterotrophy, autotrophy and photoheterotrophy) conceptual linear mixing model was then proposed for disentangling the relative contributions of the three trophic modes. RESULTS: We discovered distinct spatial leaf water, α-cellulose and n-alkane isotope ratio patterns that varied with leaf developmental stages. We inferred from the conceptual model that, averaged over the leaf blade, only 20% of α-cellulose in banana leaf is autotrophically laid down in both greenhouse-grown and field-grown banana leaves, while approximately 60% and 100% of n-alkanes are produced autotrophically in greenhouse-grown and field-grown banana leaves, respectively. There exist distinct lateral (edge to midrib) gradients in autotrophic contributions of α-cellulose and n-alkanes. CONCLUSIONS: Efforts to establish quantitative isotope-environment relationships should take into account the fact that the evaporative leaf water 18 O and 2 H enrichment signal recorded in autotrophically laid down α-cellulose is significantly diluted by the heterotrophically formed α-cellulose. The δ2 H value of field-grown mature banana leaf n-alkanes is much more sensitive than α-cellulose as a recorder of the growth environment. Quantitative isotope-environment relationship based on greenhouse-grown n-alkane δ2 H values may not be reliable.

The effect of processing medium on the 2 H/1 H of carbon-bound hydrogen in α-cellulose extracted from higher plants.

RATIONALE: Although the 2 H/1 H ratio of the carbon-bound hydrogens (C-Hs) in α-cellulose extracted from higher plants has long been used successfully for climate, environmental and metabolic studies, the assumption that bleaching with acidified NaClO2 to remove lignin before pure α-cellulose can be obtained does not alter the 2 H/1 H ratio of α-cellulose C-Hs has nonetheless not been tested. METHODS: For reliable application of the 2 H/1 H ratio of α-cellulose C-H, we processed plant materials representing different phytochemistries and photosynthetic carbon assimilation modes in isotopically contrasting bleaching media (with an isotopic difference of 273 mUr). All the isotope ratios were measured by elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). RESULTS: Our results show that H from the bleaching medium does appear in the final pure α-cellulose product, although the isotopic alteration to the C-H in α-cellulose due to the incorporation of processing H from the medium is small if isotopically "natural" water is used to prepare the processing medium. However, under prolonged bleaching such an isotope effect can be significant, implying that standardizing the bleaching process is necessary for reliable 2 H/1 H measurement. CONCLUSIONS: The currently adopted method for removing lignin for α-cellulose extraction from higher plant materials with acidified NaClO2 bleaching is considered acceptable in terms of preserving the isotopic fidelity if isotopically "natural" water is used to prepare the bleaching solution.

Assessment of the hemolysis and endothelial cell cytotoxicity induced by residual linear alkylbenzene sulfonates on pharmaceutical rubber stoppers based on HPLC-ESI-MS.

This study was designed to develop a high-performance liquid chromatographic-electrospray ionization-mass spectrometry (HPLC-ESI-MS) method for quantitative determination of residual surfactant linear alkylbenzene sulfonate (LAS) compounds on pharmaceutical rubber stoppers. An HPLC-ESI-MS method was developed for separation and determination of five LAS homologs (C10-C14) under gradient conditions using methanol and ammonium acetate as mobile phases. Hemolysis activity of residual LAS compounds was analyzed by spectrophotometry. Expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in human umbilical vein endothelial cells (HUVECs) after LAS compound treatment was examined by enzyme-linked sorbent assay. LAS compounds were well separated and determined by the established gradient conditions. The linear range was 0.05-8 µg/mL with correlation coefficients ≥0.997. Recoveries were from 73 to 134% and the relative standard deviation was <13.7%. There was a correlation between hemolysis rate and LAS compounds concentration when it was ≥0.8 µg/cm(2). LAS compounds decreased the viability of HUVECs and promoted the production of IL-6 and TNF-α. The developed analytical method was successful for quantitative determination of residual LAS compounds on pharmaceutical rubber stoppers and it is important to monitor and control the amount of LAS compounds on rubber stoppers.

Biodegradable and flame-retardant cellulose-based wearable triboelectric nanogenerator for mechanical energy harvesting in firefighting clothing.

Integrating flexible triboelectric nanogenerators (TENGs) into firefighting clothing offers exciting opportunities for wearable portable electronics in personal protective technology. However, it is still a grand challenge to produce eco-friendly TENGs from biodegradable and low-cost natural polymers for mechanical-energy harvesting and self-powered sensing. Herein, conductive polypyrrole (PPy) and natural chitosan (CS)/phytic acid (PA) tribonegative materials were employed onto the Lycra fabric (LC) in turn to assemble the biodegradable and flame-retardant single-electrode mode LC/PPy/CS/PA TENG (abbreviated as LPCP-TENG). The resultant LPCP-TENG exhibits truly wearable breathability (1378.6 mm/s), elasticity (breaking elongation 291 %), and shape adaptivity performance that can produce an open circuit voltage of 0.3 V with 2 N contact pressure at a working frequency of 5 Hz with a limiting oxygen index of 35.2 %. Furthermore, facile monitoring for human motion of firefighters on fireground is verified by LPCP-TENG when used as self-powered flexible tactile sensor. In addition, degradation experiments have shown that waste LPCP-TENG can be fully degraded in soil within 120 days. This work broadens the applicational range of wearable TENG to reduce the environmental effects of abandoned TENG, exhibiting prosperous applications prospects in the field of wearable power source and self-powered motion detection sensor for personal protection application on fireground.

High-throughput BCRP inhibitors screening system based on styrene maleic acid polymer membrane protein stabilization strategy and surface plasmon resonance biosensor.

Multidrug resistance (MDR) is a dominant challenge in cancer chemotherapy failure. The over-expression of breast cancer resistance protein (BCRP) in tumorous cells, along with its extensive substrate profile, is a leading cause of tumor MDR. Herein, on the basis of styrene maleic acid (SMA) polymer membrane protein stabilization strategy and surface plasmon resonance (SPR) biosensor, a novel high-throughput screening (HTS) system for BCRP inhibitors has been established. Firstly, LLC-PK1 and LLC-PK1/BCRP cell membranes were co-incubated with SMA polymers to construct SMA lipid particles (SMALPs). PK1-SMALPs were thus immobilized in channel 1 of the L1 chip as the reference channel, and BCRP-SMALPs were immobilized in channel 2 as the detection channel to establish the BCRP-SMALPs-SPR screening system. The methodological investigation demonstrated that the screening system was highly specific and stable. Three active compounds were screened out from 26 natural products and their affinity constants with BCRP were determined. The KD of xanthotoxin, bergapten, and naringenin were 5.14 µM, 4.57 µM, and 3.72 µM, respectively. The in vitro cell verification experiments demonstrated that xanthotoxin, bergapten, and naringenin all significantly increased the sensitivity of LLC-PK1/BCRP cells to mitoxantrone with possessing reversal BCRP-mediated MDR activity. Collectively, the developed BCRP-SMALPs-SPR screening system in this study has the advantages of rapidity, efficiency, and specificity, providing a novel strategy for the in-depth screening of BCRP inhibitors with less side effects and higher efficacy.

Accessing the position-specific 18O/16O ratios of lignin monomeric units from higher plants with highly selective hydrogenolysis followed by GC/Py/IRMS analysis.

The 18O/16O of lignin at bulk, molecular and positional levels can be used to extract valuable information about climate, plant growth environment, plant physiology, and plant metabolism. Access to the individual oxygen isotope compositions (δ18O) in the lignin monomeric units is, however, challenging as depolymerization of lignin to release the monomeric units may cause isotope fractionation. We have developed a novel method to measure the δ18O of the three oxygens (O-3, O-4 and O-5) attached to the aromatic ring of the monomeric units (bearing no oxygen in their side chains) releasable by highly selective W2C/AC (tungsten carbide supported by activated carbon)-catalyzed hydrogenolysis of lignin. O-4 is obtained by measuring the δ18O of H-type monomeric unit, while O-3 and O-5 can be calculated following isotope mass balance between H, G and S-type monomeric units measurable simultaneously with GC/Py/IRMS (gas chromatography-pyrolysis-isotope ratio mass spectrometry). The measurement precisions are better than 1.15 mUr and 4.15 mUr at molecular and positional levels, respectively. It was shown that there were a δ18OH > Î´18OG > Î´18OS isotopic order in the herbaceous plant lignin and an (inclusive) opposite order in woody plant lignin. Such differences in isotopic order is likely to be caused by the fact that both L-tyrosine, which carries an 18O-enriched leaf water signal, and L-phenylalanine, which carries mainly a molecular O2 isotopic signal, serve as the precursors for lignin biosynthesis in herbaceous plants while only the latter serves as precursor for lignin biosynthesis in woody plants. We have highlighted the potential application of such molecular and positional levels isotopic signals in plant physiological, metabolic, lignin biosynthetic and climate studies.

The use of nanofibrillated cellulose to fabricate a homogeneous and flexible graphene-based electric heating membrane.

Nanofibrillated cellulose (NFC) as a natural macromolecule, binder, dispersant, enhancer, was utilized to facilitate the assembly of graphene sheets, imparting a steady stacked structure by the sheets to the electric heating membrane with flexibility and uniform heating performance. Strong interface bonding formed in the membrane, which combined graphene sheets to be a steady conductive network structure for electric heating. The membrane attained an equilibrium temperature rise to 60°C in 3min under 2000Wm-2, which increased linearly with increasing power density and graphene content. Decreased resistance between two electrodes was caused by electric-heat coupling effect which led to a decrease in the membrane's oxygen-containing groups as conducting electrification. The temperature distributing on membrane surface, and that as bent and distorted to different angles even simultaneously at the electric heating status, were all characterized by infrared thermal imaging to indicate the uniform distribution and well bonding performance between NFC and graphene, as well as the great flexibility in the biomass membrane. This study would further broaden the utilization of the natural nanocellulose-graphene biomass composites.

Highly efficient siRNA transfection in macrophages using apoptotic body-mimic Ca-PS lipopolyplex.

BACKGROUND: The discovery and development of RNA interference has made a tremendous contribution to the biochemical and biomedical field. However, liposomal transfection protocols to deliver siRNAs to certain types of cells, eg, immune cells, are not viable due to exceedingly low transfection efficiency. While viral delivery and electroporation are two widely adopted approaches to transfect immune cells, they are associated with certain drawbacks such as complexity of preparation, biosafety issues, and high cytotoxicity. We believe amendments can be made to liposomal formulas and protocols to achieve a highly efficient knockdown of genes by liposome-loaded siRNAs. AIM: The aim of this study was to use the apoptotic-mimic Ca-PS lipopolyplex to achieve highly efficient siRNA knockdown of genes in the hard-to-transfect macrophages with reduced cytotoxicity and more efficient cellular uptake. RESULTS: We devised an anionic liposomal formula containing phosphatidylserine to mimic the apoptotic body, the Ca-PS lipopolyplex. Ca-PS lipopolyplex was proven to be capable of delivering and effecting efficient gene knockdown in multiple cell lines at lowered cytotoxicity. Among the two types of macrophages, namely Ana-1 and bone-marrow derived macrophages, Ca-PS lipopolyplex showed an improvement in knockdown efficiency, as high as 157%, over Lipo2000. Further investigations revealed that Ca-PS promotes increased cellular uptake, lysosomal escape and localization of siRNAs to the perinuclear regions in macrophages. Lastly, transfection by Ca-PS lipopolyplex did not induce spontaneous polarization of macrophages. CONCLUSION: The apoptotic body-mimic Ca-PS lipopolyplex is a stable, non-cytotoxic liposomal delivery system for siRNAs featuring vastly improved potency for macrophages and lowered cytotoxicity. It is speculated that Ca-PS lipopolyplex can be applied to other immune cells such as T cells and DC cells, but further research efforts are required to explore its promising potentials.

Quality improvements of cell membrane chromatographic column.

Cell Membrane Chromatography (CMC) is a biological affinity chromatographic method using a silica stationary phase covered with specific cell membrane. However, its short life span and poor quality control was highlighted in a lot of research articles. In this study, special attention has been paid to the disruption, cell load and packing procedure in order to improve the quality of the CMC columns. Hereto, two newly established CMC models, HSC-T6/CMC and SMMC-7721/CMC have been developed and used in this research project. The optimization of the abovementioned parameters resulted in a better reproducibility of the retention time of the compound GFT (RSD<10%) and improved significantly the quality of the CMC columns. 3.5×10(7)cells were the optimal cell load for the preparation of the CMC columns, the disruption condition was optimized to 5 cycles (400W and 20s interval per cycle) by an ultrasonic processor reducing the total time of cell disruption to 1.5min and the packing flow rate was optimized by applying a linear gradient program. Additionally, 4% paraformaldehyde (PFA) was employed to improve the column quality and prolong the column life span. The results showed that the retention time was longer with PFA treated columns than the ones obtained with the control groups.