Large collections of paraffin-embedded tissue represent a rich resource to test hypotheses based on gene expression patterns; however, measurement of genome-wide expression is cost-prohibitive on a large scale. Using the known expression correlation structure within a given disease type , we sought to identify reduced sets of directly measured (DM) genes which could accurately predict the expression of a maximized number of unmeasured genes.P|) and minimum number of DM genes that must be correlated to an unmeasured gene in order to infer the value of the unmeasured gene (redundancy). We evaluated GGS in the Cancer Genome Atlas (TCGA) HGSC data across 144 combinations of DM size, redundancy (1–3), and |rP| . Across the parameter sweep, GGS allows on average 9 times more gene expression information to be captured compared to the DM set alone. GGS successfully augments prognostic HGSC gene sets; the addition of 20 GGS selected genes more than doubles the number of genes whose expression is predictable. Moreover, the expression prediction is highly accurate. After training regression models for the predictable gene set using 2/3 of the TCGA data, the average accuracy  in the 1/3 testing partition and four independent populations is above 0.65 and approaches 0.8 for conservative parameter sets. We observe similar accuracies in the TCGA HGSC RNA-sequencing data. Specifically, the prediction accuracy increases with increasing redundancy and increasing |rP|.We developed a greedy gene set selection (GGS) algorithm which returns a DM set of user specified size based on a specific correlation threshold  contains supplementary material, which is available to authorized users. Gene expression studies can reveal genes and pathways critical for specific disease phenotypes , 2 and cConceptually, this problem is similar to genotype imputation. Loci physically located near each other on a chromosome tend to be inherited together, and sets of highly associated loci can be identified using linkage disequilibrium (LD) which is a measure of co-occurrence of alleles. Representative or ‘tag’ single nucleotide polymorphisms (SNPs) from these sets can be selected to be assayed and the remaining values inferred based on LD , 14.In an analogous manner, we propose to use the organism-, disease-, and tissue-specific gene expression correlation structure to identify genes which indirectly provide information about the expression of other genes in that tissue. The correlation of gene expression values is well studied and has been used to help inform molecular pathway definitions , 16, disP) to identify sets of correlated genes, and within those sets selects genes to directly measure and genes to attempt to infer using the directly measured genes. We applied this algorithm to the Cancer Genome Atlas (TCGA) HGSC data  algorithm uses pairwise gene expression correlation  of 0.60, 0.65, or 0.70. GGS selects the directly measured genes and the theoretically predictable genes from these eligible genes. The number of genes eligible to be directly measured or predicted varies as a function of the correlation threshold and the minimum number of genes to which an eligible gene must be correlated  respectively. To determine the extent to which the eligible genes represent a wide variety of biological processes, we performed enrichment analysis on the Protein Analysis Through Evolutionary Relationships (PANTHER) GO-slim biological process terms (223 terms) using the 3,695 eligible genes identified using the 0.60 threshold with background frequencies determined by the 8,265 truly expressed genes. After applying a Bonferroni adjustment for the 223 enrichment tests, only 8 processes were underrepresented, and 4 processes were overrepresented at a p-value cutoff of p < 0.05 , leaving 8,265 genes for analysis Fig. . We calcted Fig. –d. More n genes to directly measure by selecting the top n genes correlated with the largest number of genes. In contrast, as GGS constructs the DM and predictable sets, the edges are removed; i.e., correlations associated with those genes are ignored for the remainder of the set construction ; 2. a minimum number of directly measured genes which must be correlated with an unmeasured gene in order to consider that unmeasured gene predictable (redundancy); and 3. the targeted size of the DM set. We used the TCGA HGSC gene by gene correlation matrix and three |rP| values  to create binary matrices, then applied both the ranked-degree and GGS approaches specifying redundancy as 1, 2, or 3 and a targeted DM set size of 400, and calculated the size of the resulting predictable sets , totaling 144 individual GGS runs with resulting DM sets. For each of the DM sets, the size of the corresponding predictable set was calculated  compared to redundancy of 3 (752 genes). The correlation threshold (|rP|) also has a strong effect; when redundancy is 1 and the DM set size is 250, the number of predictable genes is 2.3-fold higher for |rP| of 0.60  compared to |rP| of 0.70 (868 genes). The increase in number of predictable genes as the number of directly measured genes increases is expected. However, a plateau is reached as the size of the DM set increases. This plateau is caused by GGS exhausting the larger sets of correlated genes, and subsequently adding genes to the DM set with a smaller return in increased predictable set size. A network representation of the eligible genes when |rP| is 0.70 and redundancy is 1 is presented in Additional file We performed a parameter sweep across |rWe performed parallel analyses of breast cancer gene expression data from TCGA. We observed results similar to the HGSC datasets, with increasing predictable set size as a function of increasing DM size, decreasing redundancy, and decreasing correlation threshold. Predictable set sizes were consistently larger than those found in HGSC. For example, with a set of 400 DM genes and a correlation threshold and redundancy of 0.70 and 3 respectively, 1,566 predictable genes were identified in the breast cancer data, versus 337 predictable genes observed in the HGSC data. These results are provided in our source code repository .P| was 0.60 and redundancy was 1. These candidate gene sets were created to capture specific biological signals and are not optimized to predict unmeasured gene expression . Once GGS augmented the Yoshihara et al. .Once GGS identified the DM and predictable gene sets, for each predictable gene, we tested how well the DM set inferred unmeasured expression. First, all gene expression vectors were scaled to the range . Using 2/3 of the TCGA Affymetrix data (omForest ) was usea et al. , Tothillata n = 13 we preda et al. ). To asshttps://github.com/lwaldron/doppelgangR), and ggplot2 [https://github.com/greenelab/greedy-geneset-selection).The creation of the correlation and binary matrices as well as the expression scaling and prediction model creation and testing was performed using R 3.0.1 . The fol ggplot2 . GGS was ggplot2  on githuhttps://github.com/greenelab/greedy-geneset-selection) and has a DOI assigned via zenodo: 10.5281/zenodo.32087.While for convenience, we accessed most of our data through the R package “curatedOvarianData” , all dat


Alces americanus ) vehicle collisions (MVCs) are an issue throughout the distribution of moose. Many mitigation strategies have been tested and implemented to reduce the number of MVCs, but there have been few empirical analyses of the effectiveness of roadside vegetation cutting. The goal of this study was to determine if roadside vegetation cutting attracted moose into roadside areas to browse on the vegetation regrowth. We hypothesized that moose would be attracted to roadside areas with cut vegetation. Consequently, we predicted that there would be higher levels of browsing in cut areas compared to uncut areas. To determine if moose were browsing more in cut or uncut areas, we measured the number of plants browsed by moose in paired treatment (cut on or after 2008) and control (not cut since at least 2008) sites, along with a suite of potential environmental covariates. Using a model selection approach, we fit generalized linear mixed-effects models to determine the most parsimonious set of environmental variables to explain variation in the proportion of moose browse among sites. In contrast to our hypothesis, our results show that the proportion of moose browse in the uncut control areas was significantly higher than in the cut treatment areas. The results of this study suggest that recently cut roadside areas (7 years or less based on our work) may create a less attractive foraging habitat for moose. The majority of the variance in the proportion of moose browse among sites was explained by treatment type and nested plot number within site identification (34.16%), with additional variance explained by traffic region (5.00%) and moose density (4.35%). Based on our study, we recommend that vegetation cutting be continued in roadside areas in Newfoundland as recently cut areas may be less attractive browsing sites for moose.Moose ( Wildlife-vehicle collisions are a significant problem in many areas of the world, including the United States, Canada, and Europe –3. As moThere are many different mitigation strategies that have been designed and implemented in an attempt to reduce the number of wildlife-vehicle collisions around the world . CommonRoadside vegetation is being cut in Newfoundland and in many other areas across Canada, allowing drivers an opportunity to see wildlife in the roadside areas and adjust their driving to avoid collisions . HoweverThe goal of this study is to determine if roadside vegetation cutting attracts moose into roadside areas to browse on the vegetation regrowth. Hughes and Fahey  indicateth to July 23rd 2014 along roadsides in two ecoregions in Newfoundland, Canada: maritime barrens and central Newfoundland forest. The sites in the maritime barrens region, on the Avalon Peninsula, were La Manche Provincial Park (MAN), Renews-Cappahayden (REN), and Spaniard’s Bay (SPA) (Picea mariana), balsam fir , tamarack (Larix laricina) and many shrub and lichen species, with a mean annual precipitation of 1,400 mm and temperature of 5.5°C [Betula papyrifera), trembling aspen (Populus tremuloides), and sheep laurel , with a mean annual precipitation of 1,150 mm and temperature of 4.5°C [Newfoundland is an island in the North Atlantic Ocean that falls within the boreal forest region. The study was conducted from June 17ay (SPA) . The regof 5.5°C . The sitof 4.5°C .All vegetation adjacent to the road in Newfoundland is cut back approximately 20 m along main roads, such as the Trans-Canada Highway (TCH). We paired treatment sites with nearby control sites that had similar biophysical traits , but differed in the age of cut vegetation  Tables. 2 quadrat placed in the center of each of the 9 plots per site.We used a stratified random sampling grid to measure the number of plants browsed by moose per plot in roadside areas with cut vegetation and in nearby control areas . A 45 m 2 quadrat. An overall percent ground cover was visually determined for each site. Woody plants within the 9-m2 quadrats were identified to species. Evidence of moose browse is readily detectable on woody plants [To determine the amount of moose browse, we measured a series of plant traits in each 9-my plants ; allowinth to July 1st 2014 indicated that sites on the Avalon Peninsula experienced much higher traffic volumes  than sites in central Newfoundland (mean ±SD number of vehicles per day: 670 ±242).The response variable in this analysis was the proportion of moose browsed plants per plot measured as  on an annual scale. We considered plants that were browsed at least once by moose in the entire study as browsable. We measured a series of discrete and continuous variables that may influence moose browse along roadsides. The discrete explanatory variables were: treatment type, presence or absence of water bodies, and traffic region . TreatmeWe included 6 continuous explanatory variables for variation in the proportion of browsed plants along roadsides: the width of the site, gradient up to the roadside, gradient up to the tree-side, road speed limit, moose density, and plant preference index . Width oWe attempted to control for differences in plant “quality” or preference across sites by selecting control areas close to the treatment areas. While many plants may be only occasionally browsed, preferred species are consumed in a larger proportion than their availability in the environment . Most otrho = −0.30, S = 272145.8, P = 0.002)  . Becausec) to determine the most parsimonious model out of all of the competing models. We considered any model with a ΔAICc<2 as a parsimonious model [2 value when these additional variables were added to the basic model. Width was a potential variable that was included in the original model set, but was a pretending variable  and theAnderson .Rubus idaeus) is abundant (n = 2954) but rarely selected when present, while trembling aspen is scarce (n = 13) but often selected when present  and 14 species showed relatively high frequency of moose browse  . Our seg present .c values of <2 and ωAICc between 0.26 and 0.38 (c = 1.00) was a more parsimonious explanatory variable for variation in moose browse along roadsides than preferred species (ωAICc = 0.00)  . Treatme = 0.00) .The proportion of moose browse in the control areas was 5.67 times higher than the proportion of moose browse in the 2008–2010 cut treatment areas and it was considerably higher than proportion of browse in the 2011–2013 cut treatment areas . The proRoadside cutting is often used as a method to improve visibility of moose on the sides of roadways, but it may have unintended consequences if cut areas act as an attractant for moose. Even though our study only contained six paired treatment-control sites, the effect size of roadside vegetation cutting on moose browsing was very large. Specifically, the proportion of plants browsed by moose in the control sites was on average 1.5 to 23.2 times higher than in the two treatment areas—this despite the sites having similar vegetation communities. These results, which are in contrast to our initial hypothesis, thereby suggest that recently cut areas may not act as attractants for moose browse.Child, Barry  suggesteThe species and availability of plants in roadside areas will be a key determinant of moose browse potential . Many stMoose density also explained a small amount of the variance in moose browse along roadsides . We inclOur correlational study has low inferential strength, but it does provide a new line of evidence about roadside vegetation cutting and its effect on moose browsing. Our study could be improved by having true control areas that had never been cut and by having more treatment and control sites overall to increase the strength of our inference. Our data, however, does clearly indicate that roadside vegetation cutting of secondary roads in Newfoundland does not attract moose to roadside areas to browse on plant regrowth as previously suggested , 38. FutWe provide the first line of evidence that recently cut roadside areas may not be attractive browse areas for moose. Based on our study we recommend that vegetation cutting be continued in roadside areas to both increase driver visibility and to reduce the attractiveness of the area for moose to browse. In our case, sites cut between 1 to 7 years of our sampling had lower moose browse than control areas which suggests that a regime of frequent roadside cutting may help mitigate moose browse along roadsides. Additionally, our surrogate technique for determining preferred forage species will save considerable time in the field and can be applied to studies of ungulate browsing conducted outside of Newfoundland. The main issue of reduction of MVCs in Newfoundland will not, however, be achieved through the implementation of one mitigation strategy. We do not have data to speak beyond roadside clearing as a mitigation strategy but based on other work , 47, a cS1 FigThe proportion of moose browsed plants (dark gray) and the proportion of preferred plants (light gray) for each treatment (trt1 & trt2) and control (ctrl) site. Preferred plants are the 14 forage species that had significantly higher browse frequency as identified with our segmented regression analysis (see text for details). The locations; BAD: Badger, GFW: Grand Falls-Windsor, GAN: Gander Bay, MAN: La Manche Provincial Park, REN: Renews-Cappahayden, and SPA: Spaniards Bay.(DOCX)Click here for additional data file.S2 Figrho = −0.30, S = 272145.8, P = 0.002). Since we were testing for the effect that roadside vegetation cutting had on the proportion of moose browse in roadside areas , treatment type was used as the main explanatory variable rather than preferred plants. For the control and treatment groups: control sites: not cut since at least 2008, treatment 1 sites: cut from 2008–2010, and treatment 2 sites: cut from 2011–2013. For the locations; BAD: Badger, GFW: Grand Falls-Windsor, GAN: Gander Bay, MAN: La Manche Provincial Park, REN: Renews-Cappahayden, and SPA: Spaniards Bay.Displaying the correlation between the proportion of preferred plants per plot and the 3 treatment types. There were higher quality plants present in the control areas than in the treatment areas ((DOCX)Click here for additional data file.S1 TableSite description including; GPS locations, road speed limit, width, presence of water bodies, moose density and the gradient for both the road and tree sides of the site for the cut treatment (TRT) and uncut control (CRL) sites collected from June 17 –July 23 2014 in Newfoundland, Canada. For the locations; BAD: Badger, GFW: Grand Falls-Windsor, GAN: Gander Bay, MAN: La Manche Provincial Park, REN: Renews-Cappahayden, and SPA: Spaniards Bay.(DOCX)Click here for additional data file.S2 TablePictures of one of our control, treatment 1, and treatment 2 sampling sites, showing the width of the cut (in the treatment areas) and the height of the vegetation. A black arrow indicates the location of a person (height: 5’6” or 1.68 m) as a reference for the height of the vegetation.(DOCX)Click here for additional data file.S3 TablePotential hypotheses for the effect that each explanatory variable would have individually on the proportion of moose browse in the roadside areas in Newfoundland, Canada.(DOCX)Click here for additional data file.S4 TablePearson’s and Spearman’s correlation analyses were performed to determine which explanatory variables to include as fixed effects in the models. Pearson’s correlation was performed when both variables were continuous and Spearman’s correlation was performed when either one or both variables were discrete. The tolerance for Type 1 error was set at α = 0.05, therefore variables were considered correlated if the p-value was <0.05.(DOCX)Click here for additional data file.S5 TableTwo generalized linear mixed-effects models used to determine if treatment type or preferred plants were a better predictor of the proportion of browsed plants. The variables plot number and nested within site id were included as random effects in both models.(DOCX)Click here for additional data file.S6 TableDescriptive statistics of plant height in the sampling sites  in Newfoundland. The chart provides an overview of the structure of the plant community, including the proportion of plants in 3 height categories, and 1 combined category. The distinction at 30 cm was made because moose rarely browse below this height .(DOCX)Click here for additional data file.


Saccharomyces cerevisiae strains isolated from different fermented foods of Western Himalayas have been studied for strain level and functional diversity in our department. Among these 23 strains, 10 S. cerevisiae strains on the basis of variation in their brewing traits were selected to study their organoleptic effect at gene level by targeting ATF1 gene, which is responsible for ester synthesis during fermentation. Significant variation was observed in ATF1 gene sequences, suggesting differences in aroma and flavor of their brewing products. Apple is a predominant fruit in Himachal Pradesh and apple cider is one of the most popular drinks all around the world hence, it was chosen for sensory evaluation of six selected yeast strains. Organoleptic studies and sensory analysis suggested Sc21 and Sc01 as best indigenous strains for soft and hard cider, respectively, indicating their potential in enriching the local products with enhanced quality.Twenty-three  Saccharomyces cerevisiae is the most common species associated with fermentation processes , Inter Simple Sequence Repeats (ISSR), Universal Rice Primers (URP), and Delta markers  that wine yeasts of Western Himalayas exhibit, and how this knowledge can been used to develop novel flavor-active yeasts or to incorporate these wild yeasts with great fermentation (flavor) potential in industrial sector for better utilization at commercial level.Western Himalayan region is a rich repository of microbial genetic diversity. Forty-three indigenous isolates of yeasts had already been characterized in the Department of Microbiology, Himachal Pradesh Agricultural University, Palampur from various fermented foods of Western Himalayas. Twenty-three of them were identified as strains of  markers . These s markers . FurtherF2 genes . VerstreS. cerevisiae available in the Department of Microbiology, HPAU, Palampur, India, 10 strains were used in the present investigation on the basis of variation in their brewing traits (Table 1) and were maintained on potato dextrose agar at 4°C and in 50% (v/v) glycerol at –80°C.Out of 23 strains of ATF1 gene sequence, 293bp of upstream related to promoter and TATA box followed by 1578 bp of ORF and 217 bp of 3′UTR was used. For amplifi cation and sequencing, this 2088 bp region was divided into three overlapping sequences. Three separate primer pairs were used to amplify these three overlapping sequences, i.e., ATF1FL (TGCACTCGATGGTCTTCTCA) and ATF1FR (GACAAATTAGCCGCCAACTC) for the first contig, ATF1SL (TGCAATGTTCTGCACGTTATT) and ATF1SR (TAGTTGTGAGCGGCAATCTG) for the second contig and ATF1TL (GAACTTCGAATGGCTTACGG) and ATF1TR (TGCAATGTTCTGCACGTTATT) for the third contig. Polimerase chain reaction (PCR) amplification was carried out in the thermal cycler  with an initial denaturation at 95°C for 2 min, followed by 30 cycles of 94°C for 30 s, 51°C for 30 s, and 72°C for 90 s with a final elongation step at 72°C for 10 min. The PCR product was analyzed on 1.2% agarose gel. For DNA sequencing, purified PCR products were freeze dried (CHRIST ALPHA I-2LD) and custom sequenced (ABI 3730xl automated sequencer) with both forward and reverse primers . The overlapping regions of DNA sequences were aligned for retrieving complete gene sequence. The homology search for ATF1 gene was carried out using NCBI BLASTN program http://www.ncbi.nih.gov/blast and phylogenetic analyses were conducted in MEGA 5.1 software program.For DNA isolation, Yeast DNA isolation Kit was used (Biobasic Inc.). The DNA stock samples were quantified using Nanodrop. Quality and purity of DNA were checked by 0.8% agarose gel electrophoresis. For S. cerevisiae strains, viz., Sc01, Sc02, Sc05, Sc12, Sc21, and Sc24 was prepared by inoculating 2% of seed inoculum to pasteurized apple juice and incubated at 28°C for 24 h under shaking conditions. Pasteurized apple juice was inoculated by 1% inoculum supplemented with di-Ammonium hydrogen phosphate (DAHP) (300 mg w/v) and incubated at room temperature for fermentation. The periodic samples were taken, spun at 6000 rpm for 5 min and analyzed for TSS, pH and ethanol content till no further decrease in °Brix was noticed. After completion of fermentation, analysis of the final product was carried out for various parameters, i.e., Estimation of pH, total soluble solids, titrable acidity , pH, titrable acidity, brix acid ratio, total sugars, reducing sugars, and ascorbic acid. Starter culture of six selected  acidity , brix-ac acidity , ascorbi acidity , reducin acidity , and tot acidity .The organoleptic evaluation of cider was done on the basis of appearance, color, flavor, mouthfeel and overall acceptability by a panel of five judges. Consumer acceptance for the products was evaluated on a nine point “Hedonic scale” .All experiments were performed in triplicate and the results were analyzed statistically by one-way ANOVA and are presented as mean values with the standard error calculated at the 95% confidence level.6–C10 medium-chain fatty acid ethyl esters such as ethyl hexanoate (ethyl caproate) and ethyl octanoate (ethyl caprylate), which have “sour apple” aromas, are also important for the overall bouquet . These enzymes catalyze the formation of acetate esters from the two substrates: alcohol and acetyl-CoA. It was shown that during fermentation, acetate ester production rates followed a pattern corresponding to the AATase activity  based on nucleotide and amino acid sequence analysis, the ATF1 sequence of a strain, KF429732 (Sc01), was found to be highly dissimilar to other strains used in the study. This strain also had most desired organoleptic properties as evident from studies conducted with hard apple cider (Table 5). The phylogenetic tree obtained after amino acid sequence analysis of the ATF1 gene  was almost similar to that obtained after analysis of nucleotide sequences. As evident from the results, ATF1 gene can be used to reveal differences in ester formation among these indigenous yeast strains at genetic level.In selected revisiae . The seqTable 2.Cider is one of the most popular drinks all around the world. In apple producing countries, the apple crop and its subsequent transformation in order to obtain derivatives , is of enormous commercial, economic as well as social relevance. Many different strains of yeast and methods of fermentation are used for producing cider. The interest for locally produced food is increasing due to consumer concern about the environment, distrust of industrial foods and a demand for high quality products. Apple is the predominant fruit crop of Himachal Pradesh and processing of apples into cider could significantly contribute towards the development of the market. The choice of yeast strain as starter culture can have a high impact on the flavor profile of fermented beverages . During Table 3).The fermentation conditions such as initial sugar concentration and temperature have been found to exert both positive and negative influence on the quality of beverage. The interaction between temperature and sugar concentration can determine the final quality of the beverage . Hence tS. cerevisiae strain was found to be best among all other cider preparations (Table 4) and hard cider prepared by Sc01 strain was found to be of standard quality (Table 5) having 5.43% alcohol (v/v) and 7 °Brix of sugar.The apple cider samples were put to sensory analysis to find out the acceptability among the tasters. The soft and hard apple cider was subjected to evaluation by a panel of five judges on a 9 point ‘Hedonic scale’. The soft cider prepared from Sc21 ATF1 gene studies revealed wide variation within the 10 indigenous yeast strains, suggesting great variation in aroma and flavor of the brewing products. These findings signify that this gene can play role in revealing the differences in ester formation among indigenous S. cerevisiae strains. However, other gene groups associated with this trait are further needed to be studied as they are also important factors in deciding the aroma and flavor of brewing products. The ATF1 gene sequence of Sc01 was found to be dissimilar to other strains used in the study and the organoleptic properties of this strain were most desirable among all the indigenous yeast strains. Sensory analysis suggested Sc21 and Sc01 as best strains for soft and hard apple cider, respectively, indicating their role in enhancing the quality of apple products.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lignocellulosic biomass is a complex biopolymer that is primary composed of cellulose, hemicellulose, and lignin. The presence of cellulose in biomass is able to depolymerise into nanodimension biomaterial, with exceptional mechanical properties for biocomposites, pharmaceutical carriers, and electronic substrate's application. However, the entangled biomass ultrastructure consists of inherent properties, such as strong lignin layers, low cellulose accessibility to chemicals, and high cellulose crystallinity, which inhibit the digestibility of the biomass for cellulose extraction. This situation offers both challenges and promises for the biomass biorefinery development to utilize the cellulose from lignocellulosic biomass. Thus, multistep biorefinery processes are necessary to ensure the deconstruction of noncellulosic content in lignocellulosic biomass, while maintaining cellulose product for further hydrolysis into nanocellulose material. In this review, we discuss the molecular structure basis for biomass recalcitrance, reengineering process of lignocellulosic biomass into nanocellulose via chemical, and novel catalytic approaches. Furthermore, review on catalyst design to overcome key barriers regarding the natural resistance of biomass will be presented herein. ILsyl chain , 89.An ideal ILs for lignocellulosic biomass pretreatment process should possess the following: (i) high dissolution capacity for different component by varying organic cation; (ii) low melting point; (iii) low viscosity; (iv) low/no toxicity; and (v) high stability. Considering the fact that fractionation of cellulose from noncellulosic matrix is a complicated process, chemistry of ILs is (anion and cation compositions) needed to be adjusted in order to solubilise hemicellulose and lignin, to ease cellulose fractionation from the biomatrix , 91. ForOther than biomass degradation to isolate cellulose product, ILs pretreatment could reduce crystallinity of cellulose to amorphous nature. Cellulose in the form of fibrils poorly hydrolyse or depolymerise under mild condition due to its intermolecular hydrogen linkages between polysaccharide chains. ILs is capable of disrupting the hydrogen bonds by forming another hydrogen bond between anion of IL with cellulose (sugar hydroxyl protons) in a 1 : 1 ratio. This will break up the cellulose hydrogen bonded structure, thus decreasing the compactness of cellulose and making it more amorphous and susceptible to depolymerisation process .In summary, the chemical pretreatment for fractionation of lignin and carbohydrates (cellulose and hemicellulose) can be achieved through the use of acids, alkalies, solvents, and ionic liquids, which promote selective solubilisation for each biocomponent . The preAlthough chemical process may not be as selective as biological treatment (enzyme), it consisted of many advantages related to operation period, scalability, and process control. Cellulose microfibril contains crystalline and amorphous regions that are randomly distributed along their length. The former, cellulose chain, is packed closely, whereas the latter is in disorder manner, which easily breaks under harsh treatment . NanocelRecently, preparation of nanoscale cellulosic particles under mild conditions is getting attention from the researchers. The top-down destructuring of cellulosic fibers can be conducted by a mechanical reaction  , 94, 95,Among the cellulose depolymerisation treatments, oxidation pretreatment is one of the common techniques used to disintegrate cellulose into nanocellulose by applying 2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPO) radicals. TEMPO-mediated oxidation method generates sinter-fibrillar repulsive forces between fibrils by modifying surface of native cellulose. This led to conversion of primary hydroxyls in cellulose into carboxylate groups, which later become negatively charged and resulted in repulsion of the nanofiber, thus contributing to an easy and fast fibrillation , 98.β-1,4 glycosidic bonds in cellulose. It is well known that acid hydrolysis is the most effective process currently to produce nanocellulose with possibility in lowering energy consumption [2SO4) for depolymerisation of cellulose. The low density of amorphous regions in native cellulose is more accessible to acid and more susceptible to hydrolytic action than the crystalline domains. These amorphous regions will break up, releasing the individual crystallites cellulose when subjected to acid treatment.Acid hydrolysis is a process related to the breakage of sumption –97, 99. 2SO4 and hydroxyl groups to yield “cellulose sulfate” which resulted in negatively charged surface of the cellulose crystallites. The anionic stabilization via the attraction/repulsion forces of electrical double layers is the reason for the stability of colloidal suspensions of crystallites  and technical (rate limiting step of cellulose degradation with long processing period) obstacles. The slow rate of enzymatic hydrolysis is easily influenced by several reasons: structural features resulting from pretreatment and enzyme mechanism –104.  Ta2SO4 and HCl) is not economically and environmentally sustainable.Acid hydrolysis can happen in homogeneous or heterogeneous catalyze reaction with first order kinetic rate. Typically, the acid hydrolysis of cellulose that uses homogeneous catalysis is more feasible in terms of mass transfer and reaction efficiency. However, these methods have major drawbacks such as (i) low chemical recovery, (ii) reactor and equipment corrosion, (iii) extra cost for waste effluents treatment, and (iv) uncontrollable selectivity of nanocellulose production. This can conclude that commercial scale of nanocellulose production by using liquid acid  is a good choice to improve the accessibility of catalyst to cellulose. Other than acting as mass transfer medium, water can take part as catalyst for autohydrolysis process. The hydronium ions (H3O+) formed on the surface of catalyst lead to promotion of cellulose hydrolysis.The main challenge of solid catalysts is the contact between catalyst active sites with solid macromolecule of cellulose. As both reactant and catalyst are present in solid phase, it is difficult for catalytic acid sites to have a close contact with It must be a water-tolerant acid catalyst.It must have strong acidity and high accessibility of acid active sites for reaction.It shall ease in separation between solid catalyst and final nanocellulose product in the powder or gel form.The critical characteristics for ideal acid catalysts for hydrolysis are as follows.Nanoparticle hydrolysis catalysts: they disperse in water solution capability, resulting in facile interaction with cellulose and overcome the difficulty of solid-solid reaction. The monodispersed nanoparticles in form of fluid solution are able to promote large number of surface active site for more access to the oxygen atom in the ether linkage of cellulose.Magnetic properties acid catalyst: magnetization attraction with external magnetic field will facilitate the separation process and catalyst recovery. Presence of repulsion and attraction effect from magnetic nanoparticle is able to reduce the reaction barrier due to adsorption, agglomeration, dispersion, and viscous effects of the reaction mixture.Catalyst with homogeneous, biphasic, and heterogeneous system: catalyst will form liquid-solid phase during hydrolysis reaction through polar or nonpolar solvent and heating. It can be soluble in water and, therefore, more efficient for hydrolysis of solid cellulose. Furthermore, this catalyst can be recovered in solid form after reaction by distilling out the products and solvent.Based on these ideal criteria, several possible green acid catalysts are highly potential to be designed for cellulose hydrolysis .Nanopart3 and Fe2(SO4)3) had been selected as catalyst to accelerate the degradation of hemicellulose in corn stover. FeCl3 significantly increased the hemicellulose degradation in aqueous solutions heated between 140 and 200°C with high xylose recovery and low cellulose removal, amounting to ~90% and <10%, respectively. In this study, hemicellulose removal increased 11-fold when the corn stover was pretreated with 0.1 M FeCl3 compared to pretreatment with hot water under the same conditions, which was also 6-fold greater than pretreatment with dilute sulfuric acid at the same pH [3 significantly increases the hemicellulose (xylose and xylotriose) degradation at 180°C. The result showed that 0.8% of FeCl3 with acid pH of 1.86 rendered 3-fold and 7-fold greater of xylose and xylotriose degradation, respectively, than that for treatment with dilute sulfuric acid with similar pH value. This suggested that different mechanism may exist for the reaction; first, the presence of FeCl3 may induce the formation of H+ ion from water for hydrolysis; second, FeCl3 may catalyze the dehydration of carbohydrates. FeSO4 catalyst also showed strong effects on the pretreatment of corn stover, where hemicellulose significantly degrades to xylose (60.3%) with high recover of cellulose (89.8%) under temperature of 180°C within 20 min. According to Zhao et al. [4 pretreatment is capable of disrupting the ester linkages between cellulose and hemicellulose. It is a potential pretreatment that is able to enhance further hydrolysis reaction of lignocellulosic biomass by destructing chemical composition and altering structural features. In acid hydrolysis, proton (H+ from HCl or H2SO4) plays an important role to weaken the glycosidic bond energy by attracting electron, making it easier for bone rupture. Metal ions also play same function as proton, which consist of more positive charge to pair with more electrons, but proton can only pair with one electron. This fact was in agreement with the studies above, where FeCl3  rendered better hydrolysis effect than FeSO4 , while acid solution performed less effect as compared to inorganic salts [Iron metal-based catalyst with its Lewis acid site can perform as catalyst for acid hydrolysis. Several studies reported that transition metal salts could increase hydrolysis rate for biomass fractionation and nanocellulose synthesis. Iron-based inorganic salts  for preparing cellulose nanocrystal (CNC) via hydrolysis of cellulose. The results show that 22% of CNC was produced under 10% FeCl3, temperature of 110°C for 60 min, and ultrasonic time of 180 min. CNC produced are shaped rod-like with the diameter of 20–50 nm, the length of 200–300 nm, and crystallinity of 76.2% [Recently, researchers started to use transition metal-based catalyst , capable to tune by adjusting the composition of Fe content, resulted in high accessibility of catalytic active sites to hydrolyse glycosidic bond in cellulose . With th3O4, although it consists of potential magnetization properties suitable for separation process for most of the catalytic systems; however, it is so far applied in glucose synthesis [3O4 nanoparticles will incorporate with acid carrier such as carbonaceous support (Fe3O4@C–SO3H) [3O4-SBA-SO3H) [3O4@C–SO3H nanoparticle is composed of magnetic Fe3O4 that doped in a sulphonated carbon shell, which render good characteristic in terms of magnetization separation effect, high stability, and good reusability. The acid catalyst showed 48.6% cellulose conversion with 52.1% glucose selectivity under the moderate conditions of 140°C after 12 h reaction. Experiments on the hydrolysis of cellobiose with Fe3O4-SBA-SO3H showed that the magnetic solid acid gave an even better performance than sulfuric acid as well [3O4 nanocatalyst provides good access of macrocellulose to SO3H group with its functional characteristic which allows it to be separated and regenerated after the process.Same case goes to Feynthesis . General@C–SO3H)  and mesoBA-SO3H)  as an acBA-SO3H)  reported as well . Thus, s3PWO12O40) with its unique physicochemical properties, Bronsted acidity, stability, and high proton mobility, have potential to be used for cellulose hydrolysis. It can be tuned to homogeneous, biphasic, and heterogeneous system at different conditions. HPAs are soluble in water and possess acidic strength similar to sulfuric acid. In theory, HPA acted as H2SO4 by donating H+ ions to oxygen atom in the glycosidic ether bonds of cellulose for depolymerisation process. HPA performed as homogeneous catalyst during hydrothermal hydrolysis, and it can be recovered to solid phase by employing organic solvent such as diethyl ether [Heteropoly acids, HPA  sur3PW12O40 under reaction conditions of 180°C and 2 h of reaction time [xHx3−PW12O40 (Cs-doped HPA) has gained much attention for selective hydrolysis of microcrystalline cellulose (MCC) to sugars in the aqueous phase. Partial substitution of proton by Cs+ improves the characteristic of HPA, where the surface acidity increases with higher surface area, controlled shape selectivity and good hydrophobility effect (insoluble in water). According to Tian's study, Cs1H2PW12O40, with the strongest protonic acid site, showed the best catalytic performance in the conversion of MCC for acid hydrolysis, where highest total reducing sugar (TRS) and glucose yields were 30.1 and 27.2%, respectively [Jiang's study showed that a remarkably high yield of glucose (50.5%) was achieved via acid hydrolysis of Hion time . Furtherectively .Most of the studies used HPA-based catalyst for cellulose hydrolysis to glucose, which was further processed to bioethanol for biofuel application. A control mechanism has to be explored to degrade macromolecule of cellulose to nanomolecular size by using HPA catalyst. It has been suggested that lower reaction temperature and shorter reaction time are the best choice to avoid overdegradation of cellulose to glucose monomer in HPA catalyzed reaction.Ion-exchange resin commercially acted as solid acid catalysts in reactions such as alkylation with olefins, alkyl halides, alkyl esters, isomerization, transalkylation, nitration, and hydrolysis. Compared with homogeneous liquid, ion-exchange resin is easily separated after reaction, less water washing, low equipment corrosion, and high reusability. Huang Biao's team uses cation-exchange resin as an alternative to liquid acid for the hydrolysis of microcrystalline cellulose (MCC) into nanocrystalline cellulose (NCC) with the aid of ultrasonification treatment. This novel hydrolysis showed that 50.04% of NCC (2–24 nm) was achieved at a ratio of resin: MCC (w/w) of 10, temperature of 48°C, and reaction time of 189 min. The crystallinity of hydrolysed MCC was found to be increased from 75.2% to 84.26%, which indicated the removal of amorphous region and realignment of cellulose molecules .Lignocellulosic biomass is the most abundant and biorenewable polymer on earth with great potential for sustainable nanocellulose production. The efficient and controlled breakdown of natural cellulose would produce nanocellulose, which is a mother compound for the synthesis of a large number of chemicals for food, energy, advance material, health, and environmental applications. Nanocellulose can be used as an immobilization support for chemical, microbial, and enzyme-catalysts. Synthesis of synthetic rubbers, bioplastic, pharmaceuticals materials, methyltetrahydrofurans, butanediol, and lactones from nanocellulose would be a quantum leap in nanocellulose industry. Nanocellulose and its derivatives could be further used to synthesize conducting polymers which could be used in biosensor appliances as well as molecular sieves. The complex hierarchy structure of lignocellulose is the main obstacle for major components separation. Overcoming the recalcitrance of lignocellulosic biomass is a key step in separating the biopolymer. Existence of lignin and the stability induced by inter- and intramolecular hydrogen bonding of cellulosic materials makes it a challenge for catalyst design. The current methods to convert cellulose into nanocellulose are based on acid, alkali, supercritical water, and thermal hydrolysis which often destroy the hierarchical structure of cellulose microfibrils and subsequently introduce impurities to the final products and produces unwanted by-products. The current methods also consume a lot of energy during and after the process and thus are deemed to be unprofitable and nonenvironmentally friendly. The typically used liquid-based catalysts are mineral acids and alkali. Introduction of novel catalysts such as iron-based catalyst, heteropoly acids, and ion exchange resin offer an integrated approach combining physical-chemical catalysts for the controlled structured degradation of natural cellulose into nanocellulose which promote more reliable methods in degradation of cellulose into nanocellulose. This shall further encourage more researches towards nanocellulosic field as more nanocellulose is readily available for utilization as a result of more efficient cellulose degradation process.


Pissodes strobi, Peck.) is a native forest insect pest in the Pacific Northwest of North America that attacks species of spruce (Picea spp.) and pine (Pinus spp.). Young Sitka spruce [Picea sitchensis (Bong.) Carr.] trees are particularly susceptible to weevil attack. Pockets of naturally occurring Sitka spruce resistance have been identified in high weevil hazard areas in coastal British Columbia. In this study, we characterize behavioral, physiological and reproductive responses of weevils to an extremely resistant Sitka spruce genotype (H898) in comparison to a highly susceptible genotype (Q903). The experiments relied on a large number of three-year-old clonally propagated trees and were therefore restricted to two contrasting Sitka spruce genotypes. When exposed to resistant trees, both male and female weevils were deterred during host selection and mating, females showed delayed or reduced ovary development, and successful reproduction of weevils was prevented on resistant trees.White pine weevil ( Pissodes strobi, Peck.) attacks several ecologically and economically important conifer hosts [Picea sitchensis (Bong.) Carr.] trees are particularly susceptible to feeding adult insects and their larvae [White pine weevil , and resistant genotypes have been successfully confirmed in replicated clonal field trials as part of the Sitka spruce breeding program of the BC Ministry of Forests and Range (BCMFR) . From thThe Sitka spruce genotypes H898 and Q903 have been propagated in the form of young grafted sapling trees (3 years of growth since the grafting of one-year-old shoots) and provided a unique resource for experiments with weevils that required a large number of clonally propagated trees. In a pair-wise comparative study using the highly resistant H898 genotype and the highly susceptible Q903 genotype, we characterized the behavioral and reproductive response of weevils, including feeding patterns, host choice, ovary development, egg laying behavior, and larval development. The results of this study showed that weevils respond to the highly resistant Sitka spruce genotype H898, but not the susceptible Q903 genotype, with deterrence of both male and female weevils during host selection and mating, delayed ovary development in females, and disruption of egg development, hatching, or development of larvae.The BCMFR  supplied three-year-old grafted Sitka spruce clones of the highly resistant (H898) genotype originating from the Haney area east of Vancouver (49°14’N: 122°36’W) and the highly susceptible (Q903) genotype originating from an area with no history of weevil attack, Haida Gwaii  on BC’s west coast (53°55’N: 132°05’W) . Leader et al. [Weevils were collected by harvesting apical shoot leaders of infested Sitka spruce trees before insect emergence in the fall. Leaders were collected from two BCMFR research plantations in the Campbell River area on Vancouver Island . Adult weevils were left to emerge in 16 L ventilated plastic buckets placed in a controlled environment chamber  set to 22 °C with an 18h:6h light-dark cycle. Every second day, emergent adult weevils were removed from the buckets and transferred to fresh Sitka spruce cuttings of uncharacterized mixed origin. The weevils remained for 5 weeks in the emergence chamber before being transferred to a controlled environment chamber (Conviron) set for a 6 °C day and a 4 °C night regime. Fresh spruce clippings were added as food source at 10–14 day intervals. For all experiments, weevils were separated into males and females according to Harman and Kulman [We characterized constitutive and weevil-induced defenses in the resistant and susceptible genotypes when exposed to weevil feeding and oviposition by assessing the constitutive resin canals , the development of traumatic resin ducts  in response to weevil feeding, and by characterizing the constitutive and weevil-induced monoterpene profiles for each genotype versus untreated control trees.Following earlier work on Sitka spruce terpenoid defenses ,7,10, weet al. [et al. [tert-butyl methyl ether , containing 100 µg/mL isobutyl benzene (Fluka) as an internal standard, and shaken overnight at room temperature. The next day, the extract was washed with 0.3 mL of 0.1 M (NH4)2CO3 (pH 8.0). The extracted tree tissue was removed, dried at room temperature in the fumehood for one week, and then weighed.Extraction and analysis of monoterpene compounds present in the leader tissue was conducted for 3 replicates (destructively sampled at each timepoint) of each Sitka spruce genotype at 2, 5, 10, 15 and 22 days of continuous weevil exposure. Two adult female weevils and two adult male weevils were caged on the leader and first interwhorl (previous year’s leader growth) of an individual tree. Both the presence and amount of monoterpene (μg/g dry weight tissue) was analysed using gas chromatography (GC) (Agilent 6890A series). The extraction method used is based on Lewinsohn et al.  and cond [et al.  with the−1 to 110 °C, then increased at 10 °C min−1 to 180 °C, then finally increased by 15 °C min−1 to 260 °C held for 15 minutes . The initial injection temperature was set at 250 °C, and the initial flow rate was 1 mL He min−1. Stereochemistry of the compounds was determined where authentic standards were available on a Cylcodex-B capillary column  using the following temperature program: the 55 °C initial temperature was increased by 1 °C min−1 to 100 °C, then increased at 10 °C min−1 to 230 °C held for 10 minutes . The initial injection temperature was set at 230 °C, and the initial flow rate was 1mL He min−1. Response factors were calculated for each compound and the compounds were quantified using a known concentration of internal standard, isobutyl benzene. The amount of compound (µg) per gram dry weight of the tissue extracted was calculated and used as the final value for comparisons.Identification of monoterpene compounds was achieved through the comparison of compound retention time with the retention time of commercially available authentic standards. Identities of the compounds were confirmed using GC coupled with mass spectrometry (MS) . The following program was used to separate monoterpenes on a SGE Solgel-Wax capillary column : the 40 °C initial temperature was increased by 3 °C minWe conducted a series of choice and no-choice experiments to assess the responses of weevils to resistant and susceptible tree genotypes. In the no-choice assays, weevils were caged on either individual resistant or susceptible trees. In the choice experiments weevils were free to move between the resistant and susceptible genotypes. We tracked individual weevil movement over time and assessed the number of feeding holes, ovary development, oviposition, and larval numbers surviving on resistant versus susceptible trees.Sitophilus oryzae (rice weevil) ovarian physiology was obtained from Perez-Medonza et al. [Ovary development was recorded based on Pernal and Currie  who devea et al.  and Khana et al. .Ovary development was scored in dissected insects using a dissecting microscope  as one of three categories: low development, moderate development, or mature. Low development includes undeveloped ovaries  as well as the early initiation of oogenesis (cells beginning to swell at the germarian and follicles moving into the vitellarium); moderate development includes follicles that are still in the vitellarium, they are round or bean-shaped and follicular epithelium is still visible; and mature ovaries were identified by highly developed (mature) sausage-shaped eggs in the lower vitellarium, in the lateral oviduct or the common oviduct. The ovary development category was assigned based on the highest level of development noted during examination of the ovaries as the previous stages are usually visible if the later stages are present.This experiment was designed to characterize weevil feeding patterns and oviposition on the resistant versus susceptible trees when given no choice. For the experiment, weevils were removed from 4 °C, placed in petri dishes lined with moistened paper towel, and starved for 48 h prior to placing them on trees. Two adult female weevils and two adult male weevils were caged on the leader and first interwhorl of each tree. Three trees per genotype were analysed for the number of feeding and oviposition holes after 2, 5, 10, 15 and 22 days of continuous weevil exposure.The experimental design above was repeated but with an extended timecourse  and a larger number of weevils per tree  in order to better assess ovary development, and the number of eggs and surviving larvae for adults caged on the resistant versus the susceptible tree genotype. For the weevils recovered at each timepoint, weevil weight and ovary development were recorded. At the beginning of the experiment, 15 female weevils were dissected to ensure that no ovary development had occurred prior to the experiment after 48 hours starvation and before feeding on the experimental tree tissue.The objective of this experiment was to determine whether adult weevils will occupy, feed, and oviposit preferentially when given a choice between trees of the highly susceptible genotype and the highly resistant genotype. Six trees (3 resistant and 3 susceptible) were placed in a 1 m width × 1.5 m length × 1.5 m height mesh cage. The trees were placed in two rows with 3 trees in each row. The genotypes were altered systematically. Weevils were removed from 4 °C, placed in petri dishes lined with moistened paper towel, and starved for 48 hours prior to placing them on trees. The experiment was conducted in two variations, one with a mix of adult males and females, and one with adult male weevils only.In the experiment with males and females mixed, 3 female weevils and 2 male weevils were placed on each tree in the mesh cage at the beginning of the experiment . Individual female weevils were identified with a color code of two colored spots of oil-based paint (Testor Model Paint) applied to the elytra. The location of each female weevil was recorded at 2–3 day intervals for a total of 23 days . The number of males observed on each tree was also recorded at each timepoint. On final day (day 23) of the timecourse, the number of feeding and oviposition holes was counted on the leader and the first interwhorl below the leader. The leader and the interwhorl tissue was dissected in order to record the number of eggs and larvae beneath the bark.In the experiment with males only, the experimental conditions were identical as described above except that 5 adult male weevils were placed on each tree at the beginning of the experiment. Each male was marked with one spot of yellow paint to increase visibility. Again, the number of males on each genotype was recorded at each timepoint  and the number of feeding holes was recorded for each genotype at the end of the experiment on Day 23.2-squared analysis (independence of cases) [Tests were conducted using SYSTAT 11.0 (Systat Software Inc. 2004). Where reported, the analyses satisfied the assumptions for both ANOVA  and Pearson χf cases) .F1, 7 = 102.8, p < 0.001; treatment: F1, 7 = 197.1, p < 0.001), whereas the susceptible genotype showed almost no traumatic resin duct formation. The number and area of constitutive cortical resin ducts were not significantly different between the two genotypes . However, there was no difference in bark thickness of the leaders of the two genotypes  .As bark thickness can play a role in weevil oviposition behavior, we measured both the diameter and average bark thickness of the apical shoot. The leader diameter of resistant trees (average ± 1SE: 4.44 mm ± 0.14) was consistently smaller than that of the susceptible trees (6.15 mm ± 0.18)  and day 22  in the resistant genotype exposed to weevil feeding. Importantly, the monoterpene (+)-3-carene was not detected in Q903 consistent with recent observations of (+)-3-carene as an indicator for Sitka spruce resistance against white pine weevil [The monoterpene profiles over 22 days of continuous weevil feeding were variable but largely unchanged in either resistant or susceptible trees for the compounds we measured, specifically (+)-α-pinene, (−)-limonene, (+)-sabinene and (+)-3-carene . Most ofF1, 4 = 7.0, p = 0.057)   A. The op= 0.013) B. These Ovary development was also different for weevils caged on either resistant or susceptible trees . Two dayA set of choice experiments were performed in order to assess male and female host selection preferences and to assess oviposition choices and larval development on resistant versus susceptible trees. Overall, weevils were highly mobile. On average, a given female weevil was observed on 3.7 different trees. The average number of consecutive sampling days that a weevil was found on the same tree was 2.7 days. No weevils were found on the same tree throughout the complete period of 23 days.Over the entire timecourse of the choice experiment, a higher number of female weevils were observed on the susceptible tree genotype A. Over 7F1, 4 = 7.084, p = 0.056 over the whole tree)  . Successet al. [de novo biosynthesis. In contrast, more abundant compounds may be relatively less affected by loss from feeding holes or the loss may be compensated for by differential de novo formation [In a series of experiments we showed that weevils responded at multiple levels of behavior and reproductive activity differently to resistant and susceptible Sitka spruce sapling trees. Weevils were able to distinguish between the resistant Sitka spruce genotype H898 and susceptible genotype Q903 and when given a choice, showed preference for the susceptible host. When exposed to resistant trees, compared to exposure to susceptible trees, both male and female weevils were deterred during host selection and mating, females showed delayed or reduced ovary development, and no successful reproduction was found on the resistant genotype. We measured a number of parameters of the constitutive and weevil-induced defense response of the resistant and susceptible host genotype that might influence weevil behavior and physiology. Histological analysis showed that although leader diameter may have played a role in the weevil’s original choice not to feed on the resistant leader, bark thickness is not significantly different between the two genotypes at any of the timepoints and thus was likely not a factor in the observed feeding patterns. This is in contrast to the suggestion by Manville et al.  that femet al. , histoloet al. , was sigormation ,21.et al. [Weevils avoided feeding initially on the apical shoot of the resistant genotype. This behavior could be a result of avoiding leader-specific feeding deterrents or toxins that were not present (or are present to a lesser extent) in the lower stem sections of resistant, and in leaders of susceptible genotype, respectively . The amoet al.  suggesteWhen given a choice between the resistant and susceptible genotypes, female weevils were mobile and showed a clear preference for the susceptible genotype. This result suggests that female weevils may test a number of different potential hosts and may oviposit on more than one. In addition, male weevils also distinguished, but more transiently, between resistant and susceptible genotypes, both in the presence or absence of females. The majority of females remained on susceptible trees for the entire period of our experiments (23 days) whereas males showed a preference for susceptible trees only until day 15. Since males showed similar behavior with or without females, it is unlikely that males were simply following female cues when choosing susceptible trees. It is possible that females were more responsive to induced defense in the resistant trees than the males. Induced defense responses are well developed as early as one week after real or simulated insect attack (methyl jasmonate treatment) in Sitka spruce and Norway spruce ,12,21, aet al. [et al. [In spite of a strong induced defense response in resistant trees, our results show that weevils were able to feed, develop ovaries and lay eggs on resistant trees, but ovary development was delayed and reduced, and no surviving offspring (larvae) developed on the resistant trees. Sahota et al.  also sug [et al.  propose  [et al. . Thus, iEven though silvicultural treatments such as shading and planting of non-host species can reduce productivity losses associated with weevils, these interventions are not sufficient to enable commercial-scale regeneration of Sitka spruce. Two areas on the southern mainland, Haney (the origin of the H898 genotype) and Squamish, were noted in particular to have fewer than average attacks per tree even though these areas are rated as high weevil hazard areas . AlthougIn summary, female and male weevils show differential responses to resistant (H898) and susceptible trees (Q903) at multiple levels of host selection, feeding activity, and reproduction. Along with further identification of host resistance traits e.g., ,5,6), in,6, in5,6


Mycobacteria pose a threat to the world health today, with pathogenic and opportunistic bacteria causing tuberculosis and non-tuberculous disease in large parts of the population. Much is still unknown about the interplay between bacteria and host during infection and disease, and more research is needed to meet the challenge of drug resistance and inefficient vaccines. This work establishes a reliable and reproducible method for performing correlative imaging of human macrophages infected with mycobacteria at an ultra-high resolution and in 3D. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) tomography is applied, together with confocal fluorescence microscopy for localization of appropriately infected cells. The method is based on an Aclar poly(chloro-tri-fluoro)ethylene substrate, micropatterned into an advantageous geometry by a simple thermomoulding process. The platform increases the throughput and quality of FIB/SEM tomography analyses, and was successfully applied to detail the intracellular environment of a whole mycobacterium-infected macrophage in 3D. Mycobacterium tuberculosis is the causative agent of tuberculosis, which is the second most deadly infectious disease in the world today [M. avium cause serious infections in immunocompromised, and sometimes healthy, individuals [ld today . Nontubeividuals , 3. Inefividuals , 5.Upon host entry, mycobacteria are phagocytosed by macrophages and dendritic cells. Within macrophages, pathogenic mycobacteria are able to avoid being killed by blocking phagosomal maturation and fusion with the lysosomes , 7, yet High-resolution 3D imaging is required to fully detail the intracellular habitat of the mycobacteria, but many cellular structures of interest are beyond the resolution of light microscopes. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) tomography is a valuable and little explored alternative, with significantly reduced manual labor compared to non-automated 3D electron microscopy (EM) techniques. During FIB/SEM tomography, a dualbeam instrument comprising a FIB and SEM is used to successively mill away thin slices of material  from a sample block, and image the appearing sample surface with the scanning electron beam. This results in an ultra-high resolution image stack representing the cell or region of interest in three dimensions.en bloc technique, and thus requires a different approach, especially when it comes to LM and EM imaging and correlation between the two. Due to the geometry of the FIB/SEM instrument, the throughput of FIB/SEM tomography experiments can be increased if regions of interest are located close to a sample edge rather than on a flat surface, avoiding the need for trench milling around the region of interest [To increase the specificity of FIB/SEM studies, initial selection of a region of interest and imaging of labeled structures can be performed in an optical fluorescent microscope. If the very same region can be relocated and imaged at the EM level, this is called correlative imaging. Correlative imaging studies with light microscopy (LM) and FIB/SEM have been performed –20, but interest . This woTo meet the need for a 3D CLEM platform optimized for LM and FIB/SEM tomography, we developed a reproducible system allowing targeted high throughput and high quality FIB/SEM tomography studies of adherent cells such as macrophages. The presented system is based on a micropatterned Aclar substrate, on which adherent cells are cultured within microwells of a predefined size. The cells can be studied with light microscopy within the wells, and an integrated reference system ensures proper cell localization. After contrasting for EM and plastic embedding, the aclar substrate is removed, leaving cells embedded in small protruding epoxy blocks. We show the successful use of this system for investigation of mycobacterium-infected primary human macrophages. Cells imaged using confocal microscopy were easily relocated for 3D imaging with FIB/SEM, allowing identification of the labeled structures with significantly enhanced resolution. The sample geometry allowed for straightforward overlay between data sets from confocal and FIB/SEM, with minimum manual adjustments. Important cellular structures such as mycobacterium-containing phagosomes were accurately resolved during FIB/SEM imaging. The presented system is simple to produce and use, and increases the quality, specificity and throughput of correlative 3D confocal fluorescence microscopy and FIB/SEM tomography studies.Biological samples are fixed, dehydrated, stained and embedded in a plastic block (e.g. epoxy) before imaging is performed in a FIB/SEM instrument. A system for correlative imaging of adherent cells must therefore consist of a platform where light microscopy imaging and localization of cells can be performed, and where both imaged cells and the reference system used for localization can be transferred to an epoxy block. The following sections describe the production and performance of such a substrate for 3D CLEM, where the optimal geometry for FIB/SEM tomography has been taken into account . The sub× 100 μm × 20 μm microblocks superimposed on a 3μm high reference grid was produced by a combination of UV- and electron-beam lithography and two-step etching (see 2 was used in this work). The pattern was transferred with high fidelity, preserving features down to sub micrometer scale × 100 μm er scale . The tra× 100 μm was suitable to accommodate macrophages, which routinely stretched out more than 50 μm. The repeating pattern of closely spaced microwells assured that a large number of cells grew close to or attached to one of the well edges. This resulted in a large number of cells located on straight sample edges after epoxy embedding, all of which are particularly well suited for FIB/SEM tomography analysis , to address viable mycobacteria having escaped the initial destructive phase. Infected cells could be fixed and stained for cellular components, without detachment from the aclar substrate. For confocal imaging, aclar substrates were placed upside down in a glass-bottomed dish suitable for confocal imaging. Light microscopy was performed on fixed cells in buffer, and was usually best with a water objective due to a thin liquid layer present between the cells and the glass bottom. Both microwells and the reference grid were clearly visible with wide field light microscopy, and z-stacks of stained cells and CFP-expressing bacteria could be gathered by confocal fluorescence imaging  of structures of interest were manually traced on each image of the stack. A surface was generated from the traced contours by interpolating over the known distance between each image (i.e. 35 nm in this case) throughout the whole stack  for EM, compatibility with EM-chemicals, and convenient separation of the substrate from the epoxy-embedded cells after resin curing. To create a substrate of the desired geometry, we exploited a well-known but little used characteristic of aclar, namely its thermoformability. Bunge et al. used this method to create custom shaped cell culture dishes already in 1973 . Here, wM. avium. Here the infected cells were imaged with a confocal microscope after chemical fixation, but as other groups have shown, aclar is also compatible with live cell imaging and subsequent high-pressure freezing and freeze substitution [× 125 μm) to scan a large number of cells for the presence of bacteria. Although not the focus of this work, fixation and imaging procedures could be optimized for higher resolution fluorescence imaging of single cells if desired, which would presumably enhance the detail of the resulting overlay data.The aclar substrates supported differentiation and growth of primary human macrophages, and long-term infection with titution , 32, wittitution . It shouM. avium bacteria in macrophages by providing further details of protein localization and potential interactions. However, it remains to be investigated if the aclar substrates developed here are suitable for superresolution imaging.In particular, modern developments in superresolution fluorescence imaging has pushed the resolution of light microscopy towards the realm of electron microscopy. Single molecule localization techniques such as PALM and STORM have demonstrated 10–20 nm lateral and axial resolution of labelled proteins . LocalizAfter EM sample preparation and embedding in epoxy, the convenient reference grid allowed localization of cells imaged by confocal microscopy in the FIB/SEM instrument. This allowed both initial screening of interesting cells and later correlation of specific confocal data with ultrastructural FIB/SEM data. Volume correlation between the datasets collected by confocal fluorescence microscopy and FIB/SEM tomography was performed based only on the geometrical features present in the moulded aclar substrate. In contrast to methods involving correlation based on biological features or added reference particles  the methIn our system, the accuracy of the correlation procedure was limited by the accuracy of the overlay between ion images of the well replica acquired during the tomography experiment, and DIC images of the edges and base of the aclar well collected in parallel with the confocal data set. The overlay accuracy could thus be further improved by using smaller, easily identified patterns at the bottom of the wells or the rest of the substrate, that could be included in the initial silicon stamp. Optimally, the accuracy of the volume correlation is in the order of the voxel size of the DIC/confocal data set.There are also certain inherent limits to the overlay accuracy, imposed by possible changes to the sample after LM imaging, the limited resolution obtainable by LM and the subjective interpretation of the limits of a fluorescent signal (e.g. the threshold chosen for 3D reconstruction). Dehydration has been reported in the literature to cause tissue shrinkage of various degree –39. HereM. tuberculosis into the cytosol [FIB/SEM tomography has been demonstrated on several biological systems , 40–46,  cytosol , 15. TEM cytosol . Correla cytosol . Similar cytosol .M. avium bacteria localized by fluorescence microscopy, as well as characterization of their intracellular surroundings within host macrophages with nanometer resolution. En bloc staining of macrophage monolayers on aclar gave sufficient contrast to observe the detailed cellular ultrastructure during FIB/SEM tomography experiments, at a resolution comparable to TEM. In one representative cell, no bacteria were observed without a surrounding phagosomal membrane, and single phagosomes often contained several bacteria. Phagosomal membranes were closely spaced around bacteria, corresponding well to descriptions of M. avium-containing phagosomes observed with TEM [M. tuberculosis and M. marinum [In our investigations, FIB/SEM tomography allowed identification of with TEM , and dif marinum , 15. In  marinum , however marinum , 30. TheM. avium, and the method could be similarly applied to detail the intracellular milieu of any adherent cell in high-resolution 3D.In summary, the system developed in the present study constitutes an accessible and easy to use-platform, suitable for streamlined high resolution FIB/SEM investigation of adherent cells. The platform also enables reproducible correlative imaging by high resolution, high numerical aperture confocal fluorescence microscopy and FIB/SEM tomography. Throughout the process, cells are maintained in their adherent state, without resorting to potentially destructive techniques such as scraping or pelleting. Cell growth is performed according to standard protocols, and confocal imaging can be carried out without any modifications to the light microscope. The use of aclar as a growth substrate allows for simple EM sample preparation, including reliable and quick separation of the substrate from cured epoxy. Not only do the wells largely simplify the localization of the exact same cell with several microscopy techniques, but they also constitute a platform that is very advantageous to work with in a FIB/SEM. Straight edges assure good accessibility to many cells for FIB/SEM tomography, and decrease the time required for milling before starting the automatic image acquisition. The correlative imaging platform allowed volume correlation based only on features of the substrate, without bias from cellular components. It was shown to be very useful for inspection of primary human macrophages infected with The master for molding Aclar poly(chloro-tri-fluoro)ethylene microwells was made using silicon microfabrication processes. The hard mask for etching the wells was patterned using photolithography, while the reference grid was patterned with electron beam lithography, before a two-step cryoetching process was performed to transfer the patterns into the silicon wafer.2 into the chamber for reactive DC-sputtering of 15 nm of Al2 O3. The sputtering was performed in an AJA Custom ATC-2200V sputter-coater at 300W and 3 mtorr. Lift-off was performed by sonicating for 5 minutes in Remover PG (Microchem).A p-type <110> silicon wafer was cleaved into 2x2 cm square pieces. SPR700.1 photoresist (Microchem) was patterned using photolithography with an inversed 100/200 μm well-pattern in a Carl Suss MA6 mask aligner. A hard mask for etching the wells was deposited by first DC-sputtering 15 nm aluminum, and then allowing 10% O−2, leading to a total patterning time of only 20 minutes despite the large area. After development, the exposed areas of Omnicoat were removed by exposure to a 50 W 0.6 mbar oxygen plasma for 1 minute in a plasma cleaner (Diener Femto).For producing the reference grid, a double-layer of Omnicoat (Microchem) was applied prior to the SU-8 for simple removal of SU-8 later. A 1 μm SU-8 2 film was patterned by electron beam lithography using a Hitachi S-4300 FEG-SEM modified with a Raith Quantum patterning system, with a voltage of 30 kV, a beam current of 500 pA and a dose of 1 μC cm2 cryogenic etch in an Oxford PlasmaLab 180 ICP-RIE. The etch was performed at −120°C, with 90 sccm SF6, 11.5 sccm O2, an RF platen power of 4W, an ICP power of 750 W and a process pressure of 7.5 mtorr, which gave an etch rate of about 1.45 μm min−1. After 2 minutes of etching, the sample was removed and the remaining SU-8 resist was removed by sonication for 2 minutes in Remover PG at 80°C. The sample was then etched for a further 13 minutes with only the alumina hard mask, defining the wells. The deep silicon etching process ensured straight vertical well edges, which is optimal for FIB/SEM milling and imagingThe reference grid was transferred into the silicon substrate by a highly selective and anisotropic SF6 /OFinally, the aluminum/alumina mask was removed by soaking in MF-26A developer (Microchem) for 2 minutes. The surface was cleaned with a 50 W oxygen plasma for 18s at 0.6 mbar, before the surface was treated with an anti-stick layer of 1H,1H,2H,2H-Perfluorooctyltriethoxysilane (Sigma Aldrich) by vapor silanization for 15 minutes.°C on a hot plate, and the aclar film was carefully placed on top and allowed to conform to the surface. A 2x2 cm piece of a glass microscopy slide was briefly heated on the hot-plate, and then placed on top. Force was applied on top of the glass with the blunt end of a pair of tweezers until no air bubbles in the aclar could be observed. The master, film and glass stack was removed from the hot-plate and allowed to cool down for 5 minutes, before carefully separating the pieces using a scalpel at the edge. The maximum durability of the master was not tested, but it still performed consistently after patterning at least 50 aclar substrates.Aclar poly(chloro-tri-fluoro)ethylene films  were cleaned by rinsing in ethanol. The silicon master was heated to 230Moulded aclar films of size 1x1 cm were sterilized in ethanol, air dried in a sterile bench and placed in 24 well plate flat bottomed culture dishes (Corning Costar) for cell culture.Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats obtained from the Blood Bank, St Olavs Hospital, Trondheim by density gradient centrifugation . The Regional Committees for Medical and Health Research Ethics at NTNU approved use of PBMCs from healthy adult blood donors after written informed consent (identification number 2009/2245-2). 2.5 million PBMCs were seeded per well in a 24-well plate, about 10% of which are monocytes. Monocyte-derived macrophages were generated from those by plastic adherence to the aclar substrates and maintenance in RPMI1640 (GIBCO) supplemented with 30% for 6 days (before infection) or 5% (after infection) of pooled human serum (The Blood Bank).M. avium clone 104 expressing CFP were used for all infection experiments, see [Transformants of the virulent nts, see  for deta°C was added 1:1 directly to the growth medium of the macrophages. After 20 minutes, the solution was exchanged with normal strength fixative (2% GA) and fixation continued for 2 hours. After fixation the cells were washed in 0.3M HEPES. Subsequently fluorescence staining of DNA for nuclear visualization was performed by adding 2.5 μM Draq5 (AH diagnostics) for 10 minutes at room temperature. Confocal imaging of fixed cells was performed with a Zeiss LSM 510 Meta, by putting the film upside down in a glass bottomed dish of thickness 170μm. Cells of interest were protected from being crushed towards the confocal dish because they grew in wells and were hence lifted up 20 μm from the bottom glass. Using a 63x water objective with numerical aperture 1.2, all cells were within the accepted working distance of the objective.Two days post infection, macrophages were fixed using glutaraldehyde  in 0.15M HEPES (Sigma-Aldrich) at pH 7,2. Fixation was performed in two steps. First double strength fixative (4% GA) preheated to 37°C over night. The subsequent day, samples were dehydrated in a graded series of 50%, 70% and 90% ethanol (15min per step on shaker), before they were en bloc stained in 2% uranyl acetate (UA) in 75% ethanol for 1 hour and 20 minutes. After staining, the samples were washed three times in 90% ethanol and further dehydrated 10 min in 90%, 4x15min in 100% ethanol and 2x15min in acetone. The epoxy resin used for infiltration was a mixture of 51% LX-112 epoxy resin, 27% DDSA (Dodecenyl succinic anhydride) and 22% NMA (Nadic methyl anydride), all from LADD Research Industries. 0.15ml DMP-30  phenol, Chemi-Teknik AS) was added to 10ml of mixed epoxy resin just before use. Resin infiltration was performed in three steps of 45min, 1:2, 1:1 and 2:1 resin:acetone respectively. The samples were then left in fresh epoxy on a rotator over night. The following day, monolayer embedding was carried out using epoxy newly mixed with DMP-30 and TEM embedding moulds. The moulds were filled with resin, and the aclar substrates were placed wells-down on top, resting on the edges of the embedding moulds. The resin was polymerized at 60°C for 2 days, before the aclar was peeled off using tweezers. Empty epoxy extending outside the monolayer was cut off using a razor blade. The epoxy blocks were mounted on aluminum sample stubs using a drop of epoxy that was cured over night at 60°C. Total curing time thus became 3 days for FIB/SEM samples. Before imaging, samples were sputter coated twice at different angles with a Cressington sputter coater model 208 HR, each time with a 20 nm thick layer of platinum/palladium.After confocal imaging, samples were postfixed and stained for 4 hours in 1,5% potassiumferrocyanide and 2% osmium tetroxide in 0,1M cacodylate buffer. The samples were then washed 3 times in Milli-Q (MQ) water and left in MQ-water at 4−2. The front face of the relevant well was then milled with the ion beam at 2.7 nA beam current. To avoid shadow effects and redeposition of material during imaging, trenches of about 8 μm width were milled on each side of the cell of interest with an ion beam current of 2.7 nA. The desired position of the trenches could be accurately localized by measuring distances on the corresponding confocal image of the well of interest. Finally the front face of the volume of interest was polished with an ion beam current of 0.9 nA.All FIB/SEM experiments were performed with a Helios NanoLab DualBeam FIB/SEM instrument from FEI Company. Cells of interest were localized using the gridded reference system on the epoxy block, and a protective platinum (Pt) layer of thickness 1 μm was deposited on the top face of the volume of interest using the gas injection system (GIS) in the FIB/SEM instrument. The ion beam was used to induce Pt deposition, at 30kV acceleration voltage and a current corresponding to a current density of 2.55 pA μm× 1768 pixels and the dwell time to 10 μs. Images were collected in immersion mode, using the in-lens detector set to secondary electron detection. A magnification of x6500 was used for electron imaging. The number of slices was set so that the slice thickness became 35 nm, and the ion beam current was set to 0.9 nA for milling during the experiment. The milling depth was set to 3.5 μm and the milling material to Si. A protective pad of Pt with a thickness of 0.6 μm was deposited, before a fiduciary marker was etched with the ion beam. Ion beam drift correction images were collected with a resolution of 1024 × 884 and a dwell time of 3μs, using the Everhart-Thornley detector in secondary electron mode.FIB/SEM tomography was carried out using the Slice and View G2 software from FEI. The electron beam acceleration voltage was set to 3kV, the beam current to 0.69 nA, the resolution to 2048 One sample that was prepared for FIB/SEM as described above, was further processed for TEM imaging after epoxy embedding. Thin sections of 60 nm were cut using a Leica UC6 Microtome with a Diatome diamond knife. Sections were picked up on formvar coated Gilder copper slot grids. Some sections were stained in 4% UA in 50% ethanol for 12 minutes, rinsed in distilled water then counterstained in 4% lead citrate for 4 minutes, while some were imaged without further processing. TEM imaging was performed using a Philips Tecnai 12 Microscope.The images collected during the FIB/SEM tomography experiment were first aligned in Fiji using StackReg or linear stack alignment with SIFT, with the transformation set to translation –53. ThenThe z stack images collected with confocal fluorescence microscopy (with DIC overlay) were adjusted for brightness and contrast in all channels, and a Gaussian blur filter of radius 1 was applied in Fiji to reduce noise. Following this the stack was imported into Avizo, with a scale factor of 7.25 in z direction . Bacteria and nucleus were then surface rendered using Isosurface Rendering in Avizo, with a threshold of 43.2 and 60.7 respectively.Images from FIB/SEM tomography and confocal fluorescence microscopy of the same cell were correlated in Avizo, based on features of the aclar substrate. All relevant images and stacks  were first imported into Avizo with the correct nm/pixel values. The ion images were aligned using the linear stack alignment with SIFT in Fiji , 52 befoS1 FigIn the Dualbeam instrument from FEI used in this study, the electron beam and the ion beam are positioned at 52° tilt compared to each other. During FIB/SEM tomography experiments, the ion beam mills off successive slices at a gracing incidence, while the electron beam images each appearing surface of interest at an angle of 52° compared to the image plane. a) Situation where the region of interest (red) is at a sample edge, and milling and imaging can be started immediately. b) The region of interest is in the middle of a flat surface, and a lot of material needs to be removed before the electron beam can access the whole surface to be imaged. Even more material needs to be removed to avoid shadow effects, because secondary electrons emitted close to a trench wall can be absorbed and will never reach the detector. Trench milling is often done with high ion beam currents to reduce milling time, but this is potentially harmful for the sample.(TIF)Click here for additional data file.S2 FigThe master, aclar and final epoxy block were characterized by SEM imaging. SEM imaging was performed at a 52° tilt angle in order to better observe edge profiles. a) SEM micrograph of a corner of a protruding square on the silicon master. b) The corresponding well on a patterned aclar substrate. c) SEM micrograph displaying the final small epoxy block after embedding. All features are conserved throughout the moulding process, exemplified by the small notch indicated by the arrow on all images. After epoxy embedding, the microwell geometry enabled access to a large number of cells at these straight, vertical sample edges, which greatly facilitated FIB/SEM tomography when compared to flat monolayers. Scale bar 20 μm.(TIF)Click here for additional data file.S3 Figa) and b) are SEM micrographs collected from the surface of the same epoxy block containing embedded macrophages. a) SEM micrograph collected with an acceleration voltage of 3kV. b) SEM micrograph collected with an acceleration voltage of 15kV. The high acceleration voltage used in in b) allows observing the shape of cells just below the surface, while 3kV imaging (a) only shows the surface topography of the block. Scale bar 100μm.(TIF)Click here for additional data file.S4 FigTwo samples with primary human macrophages grown on aclar substrates were prepared in parallel, i.e. with identical conditions for cell growth and sample preparation. One was imaged with SEM during a FIB/SEM tomography experiment (column 1), while the other was sectioned and imaged with TEM, both without (column 2) and with additional staining on the thin sections (column 3). Mitochondria and Golgi are shown as a comparison of the level of detail caught by the two imaging techniques. SEM images all originate from the same tomography data, meaning detail images are cropped out from a larger image of 20 μm x 20 μm. The magnification for the TEM images varies. Scale bars row 1: 5μm, row 2–3: 1μm(TIF)Click here for additional data file.S1 VideoClearly resolved membranes were used to determine boundaries of the nucleus (blue), a vesicle (yellow) and bacteria (red).(MP4)Click here for additional data file.