Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques.

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)(1) and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)(2). Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MS(n). Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

A study of the correlation between dengue and weather in Kandy City, Sri Lanka (2003 -2012) and lessons learned.

BACKGROUND: Weather variables affect dengue transmission. This study aimed to identify a dengue weather correlation pattern in Kandy, Sri Lanka, compare the results with results of similar studies, and establish ways for better control and prevention of dengue. METHOD: We collected data on reported dengue cases in Kandy and mid-year population data from 2003 to 2012, and calculated weekly incidences. We obtained daily weather data from two weather stations and converted it into weekly data. We studied correlation patterns between dengue incidence and weather variables using the wavelet time series analysis, and then calculated cross-correlation coefficients to find magnitudes of correlations. RESULTS: We found a positive correlation between dengue incidence and rainfall in millimeters, the number of rainy and wet days, the minimum temperature, and the night and daytime, as well as average, humidity, mostly with a five- to seven-week lag. Additionally, we found correlations between dengue incidence and maximum and average temperatures, hours of sunshine, and wind, with longer lag periods. Dengue incidences showed a negative correlation with wind run. CONCLUSION: Our results showed that rainfall, temperature, humidity, hours of sunshine, and wind are correlated with local dengue incidence. We have suggested ways to improve dengue management routines and to control it in these times of global warming. We also noticed that the results of dengue weather correlation studies can vary depending on the data analysis.

The reducing end sequence of wheat endosperm cell wall arabinoxylans.

Walls from wheat (Triticum aestivum L.) endosperm are composed primarily of hetero-(arabino)xylans (AXs) (70%) and (1→3)(1→4)-ß-D-glucans (20%) with minor amounts of cellulose and heteromannans (2% each). To understand the differential solubility properties of the AXs, as well as aspects of their biosynthesis, we are sequencing the xylan backbone and examining the reducing end (RE) sequence(s) of wheat (monocot) AXs. A previous study of grass AXs (switchgrass, rice, Brachypodium, Miscanthus and foxtail millet) concluded that grasses lacked the comparable RE glycosyl sequence (4-ß-D-Xylp-(1→4)-ß-D-Xylp-(1→3)-α-L-Rhap-(1→2)-α-D-GalpA-(1→4)-D-Xylp) found in dicots and gymnosperms but the actual RE sequence was not determined. Here we report the isolation and structural characterisation of the RE oligosaccharide sequence(s) of wheat endosperm cell wall AXs. Walls were isolated as an alcohol-insoluble residue (AIR) and sequentially extracted with hot water (W-sol Fr) and 1M KOH containing 1% NaBH4 (KOH-sol Fr). Detailed structural analysis of the RE oligosaccharides was performed using a combination of methylation analysis, MALDI-TOF-MS, ESI-QTOF-MS, ESI-MS(n) and enzymic analysis. Analysis of RE oligosaccharides, both 2AB labelled (from W-sol Fr) and glycosyl-alditol (from KOH-sol Fr), revealed that the RE glycosyl sequence of wheat endosperm AX comprises a linear (1→4)-ß-D-Xylp backbone which may be mono-substituted with either an α-L-Araf residue at the reducing end ß-D-Xylp residue and/or penultimate RE ß-D-Xyl residue; ß-D-Xylp-(1→4)-[α-L-Araf-(1→3)](+/-)-ß-D-Xylp-(1→4)-[α-L-Araf-(1→3)](+/-)-ß-D-Xylp and/or an α-D-GlcpA residue at the reducing end ß-D-Xylp residue; ß-D-Xylp-(1→4)-[α-L-Araf-(1→3)](+/-)-ß-D-Xylp-(1→4)-[α-D-GlcAp-(1→2)]-ß-D-Xylp. Thus, wheat endosperm AX backbones lacks the RE sequence found in dicot and gymnosperm xylans; a finding consistent with previous reports from other grass species.

In vitro grown pollen tubes of Nicotiana alata actively synthesise a fucosylated xyloglucan.

Nicotiana alata pollen tubes are a widely used model for studies of polarized tip growth and cell wall synthesis in plants. To better understand these processes, RNA-Seq and de novo assembly methods were used to produce a transcriptome of N. alata pollen grains. Notable in the reconstructed transcriptome were sequences encoding proteins that are involved in the synthesis and remodelling of xyloglucan, a cell wall polysaccharide previously not thought to be deposited in Nicotiana pollen tube walls. Expression of several xyloglucan-related genes in actively growing pollen tubes was confirmed and xyloglucan epitopes were detected in the wall with carbohydrate-specific antibodies: the major xyloglucan oligosaccharides found in N. alata pollen grains and tubes were fucosylated, an unusual structure for the Solanaceae, the family to which Nicotiana belongs. Finally, carbohydrate linkages consistent with xyloglucan were identified chemically in the walls of N. alata pollen grains and pollen tubes grown in culture. The presence of a fucosylated xyloglucan in Nicotiana pollen tube walls was thus confirmed. The consequences of this discovery to models of pollen tube growth dynamics and more generally to polarised tip-growing cells in plants are discussed.

Fructose containing sugars modulate mRNA of lipogenic genes ACC and FAS and protein levels of transcription factors ChREBP and SREBP1c with no effect on body weight or liver fat.

The aim of this study was to determine the effects of high-glucose, high-fructose and high-sucrose diets on weight gain, liver lipid metabolism and gene expression of proteins involved with hepatic fat metabolism. Rats were fed a diet containing either 60% glucose, 60% fructose, 60% sucrose, or a standard chow for 28 days. Results indicated that high-fructose and high-sucrose diets were associated with higher mRNA levels of gene transcripts involved with fat synthesis; ACC, FAS and ChREBP, with no change in SREBP-1C mRNA. The protein level of ChREBP and SREBP1c was similar in liver homogenates from all groups, but were higher in nuclear fractions from the liver of high-fructose and high-sucrose fed rats. The mRNA level of gene transcripts involved with fat oxidation was the same in all three diets, whilst a high-fructose diet was associated with greater amount of mRNA of the fat transporter CD36. Despite the changes in mRNA of lipogenic proteins, the body weight of animals from each group was the same and the livers from rats fed high-fructose and high-sucrose diets did not contain more fat than control diet livers. In conclusion, changing the composition of the principal monosaccharide in the diet to a fructose containing sugar elicits changes in the level of hepatic mRNA of lipogenic and fat transport proteins and protein levels of their transcriptional regulators; however this is not associated with any changes in body weight or liver fat content.

Rapid bioassay-guided screening of toxic substances in vegetable oils that shorten the life of SHRSP rats.

It has been consistently reported that vegetable oils including canola oil have a life shortening effect in Stroke-Prone Spontaneously Hypertensive Rats (SHRSP) and this toxic effect is not due to the fatty acid composition of the oil. Although it is possible that the phytosterol content or type of phytosterol present in vegetable oils may play some role in the life shortening effect observed in SHRSP rats this is still not completely resolved. Furthermore supercritical CO2 fractionation of canola oil with subsequent testing in SHRSP rats identified safe and toxic fractions however, the compounds responsible for life shortening effect were not characterised. The conventional approach to screen toxic substances in oils using rats takes more than six months and involves large number of animals. In this article we describe how rapid bioassay-guided screening could be used to identify toxic substances derived from vegetable oils and/or processed foods fortified with vegetable oils. The technique incorporates sequential fractionation of oils/processed foods and subsequent treatment of human cell lines that can be used in place of animal studies to determine cytotoxicity of the fractions with structural elucidation of compounds of interest determined via HPLC-MS and GC-MS. The rapid bioassay-guided screening proposed would require two weeks to test multiple fractions from oils, compared with six months if animal experiments were used to screen toxic effects. Fractionation of oil before bio-assay enhances the effectiveness of the detection of active compounds as fractionation increases the relative concentration of minor components.