Vitis vinifera L.) is considered one of the most environmentally sensitive crops and is characterized by broad phenotypic plasticity, offering important advantages such as the large range of different wines that can be produced from the same cultivar, and the adaptation of existing cultivars to diverse growing regions. The uniqueness of berry quality traits reflects complex interactions between the grapevine plant and the combination of natural factors and human cultural practices which leads to the expression of wine typicity. Despite the scientific and commercial importance of genotype interactions with growing conditions, few studies have characterized the genes and metabolites directly involved in this phenomenon. Here, we used two large-scale analytical approaches to explore the metabolomic and transcriptomic basis of the broad phenotypic plasticity of Garganega, a white berry variety grown at four sites characterized by different pedoclimatic conditions . These conditions determine berry ripening dynamics in terms of sugar accumulation and the abundance of phenolic compounds. Multivariate analysis unraveled a highly plastic metabolomic response to different environments, especially the accumulation of hydroxycinnamic and hydroxybenzoic acids and flavonols. Principal component analysis (PCA) revealed that the four sites strongly affected the berry transcriptome allowing the identification of environmentally-modulated genes and the plasticity of commonly-modulated transcripts at different sites. Many genes that control transcription, translation, transport, and carbohydrate metabolism showed different expression depending on the environmental conditions, indicating a key role in the observed transcriptomic plasticity of Garganega berries. Interestingly, genes representing the phenylpropanoid/flavonoid pathway showed plastic responses to the environment mirroring the accumulation of the corresponding metabolites. The comparison of Garganega and Corvina berries showed that the metabolism of phenolic compounds is more plastic in ripening Garganega berries under different pedoclimatic conditions.Grapevine ( Vitis vinifera L) berries for wine production reflect the outcome of complex interactions between the plant and its environment. In viticulture, the latter is defined as terroir, and it represents a combination of natural factors, such as climate, altitude, exposure, the geological characteristics of the soil, and the microbial community, together with human cultural and production practices, which influence the expression of the berry traits and wine quality  samples were collected from four vineyards during the 2013 growing season at the same time of day (~10.30 a.m.). The parral training system rows were north-south oriented, and SO4 was used as the rootstock. For all vineyards, the planting density was 3000–4000 vines/ha, with vines 10–15 years old. The vineyards were located in different areas of the Soave production region featuring diverse growing conditions, such as altitude and soil composition  and finishing at harvest . For three vineyards , berries were harvested at the so-called “perfect ripening” stage, corresponding to a total soluble solids (TSS) content of 18.5° Brix and pH 3.3. This was never achieved by the VH1 berries. The °Brix of the berry juice was determined using a digital DBR35 refractometer . A single biological replicate was created by pooling about 30 berries collected from clusters of different vines, along one central vineyard row of an average of 70 vines. As the ripening variability within one single cluster is very large, we payed attention on collecting berries also from different position in the clusters. We repeated the same procedure for the other two biological replicates, but each time we collected berries from different clusters of different vines within the same row. This strategy allowed the collection of three biological replicates that represent almost the entire variability of the vineyard. The same sampling collection procedure was applied at each sampling time point for each of the four vineyards, thus the experiment entailed the collection and analysis of 48 berry samples . We removed the seeds from the berries of each biological replicates and the obtained pericarps were powdered with an automatic mill grinder . The obtained frozen powder was used for both transcriptomic and metabolomic analyses.http://ddlab.sci.univr.it/FunctionalGenomics/) representing ~98.6% of the genes predicted in the V1 annotation of the 12x grapevine genome (http://srs.ebi.ac.uk/), as well as 19,091 random probes used as negative controls. Arrays that meet the recommended quality metrics exhibit a background (averaged fluorescence intensity level of empty cells and random probes) of 450–500. Therefore, a fluorescence intensity value of 500 was used as the threshold to define gene expression, and averaged values across the entire dataset lower than 500 were considered to represent minimal/absent expression and were excluded from our analysis.Total RNA was extracted from ~200 mg frozen berry powder using the Spectrum™ Plant Total RNA kit  as previously described  of Turbo DNase (TURBO DNA-free kit—Ambion) according to the instructions provided with the commercial kit. DNase-treated RNA was then used for cDNA synthesis using the SuperScript III Reverse Transcriptase kit (Invitrogen) following the producer's indications. In order to assess if the cDNA had been properly produced, an amplification with primers designed on the 3′ UTR of an Ubiquitin coding gene  was performed.SE)-values were calculated according to Pfaffl et al.  to amplify a specific region of target genes . Reaction mix was composed of 1x GoTaq® Green Master Mix, 200 nM of each primer and 20 ng of cDNA in a final volume of 25 μl. The reaction was carried out on a StepOnePlus™ Real Time PCR System (Applied Biosystems) using the following cycling conditions: 95°C hold for 2 min followed by 40 cycles at 95°C for 15 s, 55°C for 30 s, 60° for 30 s, and 95°C for 15 s. Non-specific PCR products were identified by the dissociation curves. Amplification efficiency was calculated from raw data using LingRegPCR software  with a false discovery rate (FDR) of 0.01%, to extract genes that were significantly modulated during ripening in all four vineyards. The differentially expressed genes were then filtered by applying a fold-change thresholds of ≥2 or ≤ −2. To identify shared and specific transcriptomic ripening programs in the four vineyards, the selected differentially expressed genes  were represented in a Venn diagram . The differentially expressed genes were screened by calculating the coefficients of variation (CV) at the four developmental stages in each vineyard, and then the standard deviation (SD) among the four calculated CVs. This allowed to rank the shared genes by a quantitative measure of the intra-vineyard and inter-vineyard variability of expression during ripening. Transcripts scoring the highest standard deviations (the top 50 genes) were defined as the most plastic genes under our experimental conditions.Principal component analysis (PCA) was applied to the entire transcriptome dataset using SIMCA P+ v13 . Loadings of the first and second principal components were ordered, and genes within the first and last percentiles were extracted to investigate their expression profiles over time in the different vineyards. Multiclass significance analysis of microarrays (SAM) was carried out using TMeV v4.8 (http://www.cytoscape.org/) with PlantGOslim categories. Overrepresented PlantGOslim categories were identified using a hypergeometric test with a significance threshold of 0.05, after Benjamini and Hochberg correction with a FDR of 0.01 , the AROS V1.0 Oligo Set , and the Gene-Chip® Vitis vinifera Genome Array  coupled to electrospray ionization mass spectrometry (RP-HPLC-ESI-MS) or a diode array detector (RP-HPLC-DAD) after dilution  in LC-MS-grade water and passage through a 0.2-μm filter.For each sample, 300 mg of frozen berry powder was extracted in three volumes of cold methanol acidified with 0.1% formic acid. After mixing, the samples were sonicated for 15 min at 4°C and then centrifuged at 16,000 × −1. The HPLC instrument was coupled on-line with an Esquire 6000 ion trap mass spectrometer equipped with an ESI source . MS data were collected using the Esquire Control v5.2 software, and processed using the Esquire Data Analysis v3.2 software . The instrument was set to induce positive and negative ionization in alternating mode. Mass spectra were recorded in the range 50–3000 m/z with a target mass of 400 m/z. MS/MS and MS3 mass spectra were recorded in positive and negative ionization modes in the range 50–3000 m/z with a fragmentation amplitude of 1 V. Nitrogen was used as the nebulizing gas  and drying gas (10 L min−1). Helium was used as the collision gas. The vacuum pressure was 1.4 × 10−8 bar. Additional parameters: capillary source 4500 V; end plate offset –500 V; skimmer: 40 V; cap exit 121 V.RP-HPLC analysis was carried out using a Beckman Coulter  Gold 127 HPLC System equipped with a C18 guard column  and an Alltech  RP C18 column . Two solvents were used: 0.5% formic acid and 5% acetonitrile in water (solvent A) and acetonitrile (solvent B). The gradient was set as follows: 0–10% B in 2 min, 10–20% B in 10 min, 20–25% B in 2 min, 25–70% B in 7 min, isocratic for 5 min, 70–90% B in 1 min, isocratic for 4 min, 90–0% B in 1 min, and 20 min equilibration. For each sample, 20 μL was injected at a flow rate of 0.2 mL minRP-HPLC-DAD analysis was carried out using a Beckman Coulter Gold 126 Solvent Module and Gold 168 Diode Array Detector. HPLC was carried out as above, with a larger injection volume of 100 μL. The wavelength range was 190–600 nm. Data were collected and analyzed using 32 Karat v7.0 (Beckman Coulter). Specific wavelengths were considered to represent the following classes of compounds: 280 nm (flavan-3-ols and their oligomers), 320 nm (hydroxycinnamic acid derivatives), and 350 nm (flavonols).n fragmentation patterns with those of commercial standards in our in-house library. UV/vis spectra recorded by RP-HPLC-DAD were also used to support the LC-MS identification. Fragmentation patterns collected in online databases such as MassBank (http://www.massbank.jp) or reported in the literature were also considered, especially when no authentic standard compounds were available. Neutral losses of 132, 146, and 162 Da were considered diagnostic of the loss of pentose, deoxyhexose, and hexose sugar, respectively.Metabolites were identified by comparing their retention times, m/z-values and MShttp://mzmine.sourceforge.net/). The resulting matrix was analyzed using SIMCA v.13.0 . Pareto scaling was applied to all analytical methods. Unsupervised PCA was used to identify the major clusters defined by the samples prior to supervised partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA and O2PLS-DA) setting the classes according to the ripening stage for each vineyard location. PLS-DA models were validated using a permutation test (200 permutations) and the corresponding OPLS-DA/O2PLS-DA models were cross-validated by analysis of variance (ANOVA) with a threshold of p < 0.01.LC-MS chromatograms were converted to netCDf files for peak alignment and area extraction using MZmine (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE75565). The datasets supporting the Garganega metabolome analysis are included in this article and its supplementary files. The Corvina metabolomics data are available in the Metabolights database under the series entry MTBLS39.Grape berry microarray expression data are available in the Gene Expression Omnibus under the series entry GSE75565  at four ripening stages, focusing on moderately polar metabolites such as phenolic compounds. Data processing revealed 267 signals in negative ionization mode Figure .Another large group of metabolites corresponded to the hydroxycinnamic acids. Within this group, caffeoyltartaric acid was the most abundant, followed by coumaric and ferulic acid glycosides and tartaric acid esters (coutaric and fertaric acid). Minor signals were also assigned to the hexose esters of three benzoic acids .Among the flavonols, the glycosides of quercetin, kaempferol, isorhamnetin, and myricetin were detected, as well as those of the dihydroflavonols, dihydroquercertin, and dihydrokaempferol. Other minor compounds were putatively identified, including two isomers of resveratrol glucoside, the phenylethanoid hydroxytyrosol glucoside, and an N-conjugated glycoside of tryptophan.The accumulation of metabolites among the four vineyards during ripening was initially compared by inspecting the entire data matrix by unsupervised multivariate PCA. Two partially overlapping clusters were observed for the VP and AP samples, whereas the VH1 and VH2 samples formed two independent clusters Figure . This clIn order to find metabolites that characterized the various groups of samples, we applied a supervised O2PLS-DA approach Figure . The plaFollowing microarray hybridization, the pericarp transcriptome dataset of the ripening Garganega berries was inspected by PCA, confirming the consistency of the biological replicates Figure . PC1 expDifferences among the vineyards were highlighted by visualizing the average trend of the first and last percentiles of the PC1 loadings Figure . As expePC2 explained 16.5% of the total variability Figure  and mainThe functional categories of the PC2 first percentile genes indicated that Carbohydrate metabolism, DNA, and RNA metabolic processes  and Transport were key processes  within each group of vineyard samples. A total of 12,931 transcripts were significantly modulated  have already been reported as markers of the veraison phenological phase , mid-maturity (stage 2), and in fully-ripe berries , which regulates the stilbene synthase gene family  in Corvina berries, and VvLAR2  in Garganega berries, but did not correlate with the expression of VvLAR1 , which remained high at the third ripening stage in the Corvina vineyard PM and the Garganega vineyards VP and VH1. Interestingly, the expression profiles of the two MYB transcription factors regulating this branch of the flavonoid pathway differed in the two varieties, i.e., VvMYBPA1 was expressed more strongly in Garganega berries, whereas VvMYBPA2 was expressed more strongly in Corvina berries.The flavan-3-ols and their oligomers declined marginally in both cultivars and in the different vineyards, especially VH1 which featured the strongest reduction among the Garganega vineyards. This correlated with the pronounced decline in the expression of V. vinifera white berry variety. In order to understand the molecular basis of the environmental impact on berry ripening, four vineyards were selected to maximize differences in environmental conditions and to minimize differences due to agricultural practices such as the training system, orientation of the rows, planting layout, vineyard age, and rootstock.We used two large-scale analytical approaches to explore metabolomic and transcriptomic changes during the ripening of Garganega berries, a The four selected growing sites belong to the same production area and were chosen to ensure diverse environmental parameters such as soil origin, texture, and composition. The soils in three of the vineyards were characterized by a low percentage of sand and a low concentration of calcium carbonate, as expected given their volcanic origin, and the fourth was alluvial in origin with a high proportion of clay. Vineyards at different altitudes were chosen to maximize the environmental variability. However, despite the distance of 400 m between the lowest (32 m above sea level) and highest (437 m above sea level) vineyards, the meteorological parameters recorded at the four sites revealed little difference in heat unit accumulation and rainfall during the 2013 growing season. Even so, the number of rainy days during the pre-blooming period  was greater at the VH2 site and there was a higher temperature during July, August, and September. This may have contributed to the specific ripening behavior we observed. Indeed, the pedoclimatic conditions and other uncontrolled variables strongly influenced the ripening dynamics at each site in terms of sugar accumulation and the synthesis of phenolic compounds. The most divergent ripening dynamics were characterized by an early decline in sugar accumulation (VH1) and a consistent high sugar accumulation rate (VH2).V. vinifera white grapes  and the Biotechnology Department of University of Verona. The INNOVINE European Project FP7-311775 “Combining innovation in vineyard management and genetic diversity for a sustainable European viticulture” also supported the present work which benefited from the networking activities coordinated under the EU-funded COST ACTION FA1106 “An integrated systems approach to determine the developmental mechanisms controlling fleshy fruit quality in tomato and grapevine.” SDS was financially supported by the Italian Ministry of University and Research FIRB RBFR13GHC5 project “The epigenomic plasticity of grapevine in genotype per environment interactions.”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. The reviewer CR and handling Editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review.


We aimed to compare the treatment outcomes and the occurrence rates of adverse events associated with different steroid regimens in geriatric patients (aged 65 years or older) with unilateral idiopathic sudden sensorineural hearing loss (ISSNHL). After thorough medical chart reviews of 109 patients with ISSNHL between May 2006 and December 2013, we performed a propensity score-matched analysis using previously known prognostic factors, steroid regimens, and other cointerventions. Patients were divided based on their steroid regimens into group I  and group II . We compared final hearing and the occurrence of adverse events between the two groups. As a result, 20 pairs of propensity score-matched patients (n = 40) were enrolled. Group I patients showed better final hearing levels compared with group II patients , although this difference was marginally significant (p = 0.058). Based on the comparative analysis of each of the frequencies in the final audiograms, lower hearing thresholds at 2 KHz were observed in group I (p = 0.049). There was no significant difference in the occurrence of adverse effects between the two groups (p>0.05). In conclusion, conventional steroid regimens produced adverse event occurrence rates that were similar to those of low-dose treatment but may also have produced superior hearing recovery. The use of steroid dose reduction in geriatric patients with ISSNHL is not preferable to conventional steroid regimens. According to recent guidelines, initial corticosteroid treatment can be administered to patients with idiopathic sudden sensorineural hearing loss (ISSNHL) A study of 18226 patients with diabetes revealed that patients with diabetes and chronic obstructive pulmonary disease who used high-dose corticosteroids were at a greater risk of diabetes-related hospitalization and suggested that the minimally effective corticosteroid dose should be used Prednisone at a dose of 60 mg daily or methylprednisolone (MPD) at a dose of 48 mg for 7 to 14 days is frequently used as an initial medication in the treatment of ISSNHL, and the doses are subsequently tapered Based on these findings, we assumed that, if treatment outcomes following low-dose steroid treatments were as effective as those following higher-dose steroid treatments, the low-dose treatments would be accompanied by a reduced risk of adverse effects and that the use of low-dose steroid treatments for ISSNHL in geriatric patients would be more rational.In this study, we aimed to compare the treatment outcomes and the occurrence rates of adverse events according to different steroid regimens in ISSNHL patients aged 65 years or older by using a propensity score-matched (PSM) analysis.This study was approved by the Institutional Review Board of the Eulji University (No. 2014-02-010). The Board granted a waiver of written informed consent for this retrospective study.Based on retrospective medical chart reviews, we enrolled patients who were aged 65 years or older and had been diagnosed with ISSNHL and admitted to the university hospital between May 2006 and December 2013. The exclusion criteria were as follows: 1) concomitant meningitis, myelitis, vasculopathy, or neuropsychiatric disease; 2) a clinical observation period less than 3 months; 3) previous histories of sudden hearing loss and/or the possibility of Meniere's disease; and 4) uncontrolled hypertension or uncontrolled diabetes mellitus.Age, sex, comorbid diabetes and hypertension, the presence of dizziness and/or tinnitus, the period of time from onset to treatment, the initial hearing levels of both ears, and the final hearing level of the affected side 3 months after the onset of treatment were documented.All patients were hospitalized for 1 week, and one of two types of treatment was provided using the time-variant differential approach: either oral MPD treatment or “low-dose” oral MPD treatment. Oral MPD treatment was administered between 2006 and 2008. Subsequently, oral “low-dose” MPD treatment became the mainstream treatment and was commonly used until 2010. From early 2011, an additional intratympanic dexamethasone injection (IT-DEX) administered as a salvage therapy following oral MPD treatment has been the main treatment for sudden deafness.Patients who received the oral MPD treatment were treated with steroids for 10 days using the same recommended dosage protocol for MPD  Moreover, for patients who required an additional cointervention, a continuous infusion of 10 µg/d of alprostadil over 7 days or daily intravenous infusions of 88 mg of zinc sulfate hydrate were provided.Hearing levels were calculated using the arithmetic mean of the hearing levels at 500 Hz, 1 kHz, 2 kHz, and 4 kHz. Hearing improvement rates were calculated as the hearing gain divided by the initial hearing difference between the lesion side and the healthy side and then multiplied by 100 From the medical chart reviews, insomnia, abdominal discomfort, high blood pressure that occurred more than twice per day , and hyperglycemia for which insulin had been newly prescribed or increased were documented. Additionally, major complications such as myocardial infarction, gastrointestinal bleeding, and death were also documented.The details of the estimation of the propensity scores were as follows: (1) age, sex, accompanying hypertension and diabetes, presence of tinnitus or dizziness, initial hearing levels of the lesion and healthy sides, IT-DEX treatment, and other cointerventions  were selected as covariates based on the results of the previous studies A total of 109 patients were enrolled in this study, including 44 men (40.4%) and 65 women (59.6%), with a mean age of 71.72 years  and mean ISSNHL duration (from the onset to treatment) of 3.94±2.93 days. Diabetes was reported in 26 patients (23.9%) and hypertension in 56 (51.4%). Of accompanying symptoms, 76 patients (69.7%) had tinnitus and 16 (14.7%) had dizziness. The mean initial hearing level was 68.95±23.26 dB and the initial contralateral hearing level was 43.39±25.00 dB. The baseline characteristics revealed marginally significant differences in age (p = 0.045), accompanying hypertension (p = 0.054), and initial hearing levels (p = 0.071) between the steroid regimen groups.After the PSM analysis, 20 pairs of patients were allocated either to group I or II , and theThe hearing improvement rates did not differ between groups I and II . Regarding hearing recovery, group I showed a tendency for better recovery compared with group II, but the difference was not significant , p>0.05.Regarding severe adverse events during treatment, one patient in group II experienced pulmonary edema and upper gastrointestinal bleeding. However, other major complications, such as myocardial infarction and death, did not occur in any of the groups.Regarding minor adverse events, the PSM analysis revealed that patients in group I complained of insomnia (10% of the patients), abdominal discomfort (15%), high blood pressure (35%), and hyperglycemia (35%). However, the occurrence rates of these adverse events were not significantly different between the two groups .Our study showed that patients receiving conventional steroid treatment had slightly better hearing recovery than patients who received reduced steroid doses, although the difference was marginally significant. Similar trends were found when the final audiograms at all frequencies were compared, with the exception of the 4 kHz frequency. No significant differences in the occurrence rates of adverse effects were found between the two groups.These findings may suggest that steroid dose reduction is not preferable to the conventional steroid regimen in geriatric patients with ISSNHL.In contrast, a prospective randomized trial reported that therapeutic outcomes did not differ between 7-day prednisolone and 300 mg dexamethasone pulse therapies With aging, liver function, which is primarily responsible for the metabolism of steroids, is mostly maintained, but phase I metabolism catalyzed by cytochrome P450 tends to decrease, and an increase in interindividual variability is distinctive Apart from systemic steroid treatment, intratympanic steroid injection is currently recommended after the failure of the initial treatment To reduce the confounding effects between diverse treatment options and the observed baseline characteristics, we performed the PSM analysis Our study has several limitations. It seems unnatural to include covariates such as IT-DEX, alprostadil, and zinc, which may act as confounding factors to the outcome, in the PSM analysis. However, the exclusion of all patients who were treated with cointerventions could result in insignificant conclusions owing to a small sample size. Therefore, we controlled cointerventions as covariates to be able to evaluate the sole effect of steroid dose by minimizing the between-group difference.In conclusion, conventional steroid regimens produced the occurrence rates of adverse events that were similar to those of low-dose treatment but may also have produced better recoveries. The use of steroid dose reduction in geriatric patients with ISSNHL is not preferable to conventional steroid regimen.


We further considered the total energy available from each sex and age class of each animal prey over the period they would overlap land-bound polar bears and calculated the maximum number of starving adult males that could be sustained on each food during the ice-free season. Our results suggest that the net energy from land-based food, after subtracting costs of limited movement to obtain it, could eliminate all projected nutritional deficits of starving adult male polar bears and likely other demographic groups as well. The hunting tactics employed, success rates as well as behavior and abundance of each prey will determine the realized energetic values for individual polar bears. Although climate change may cause a phenological mismatch between polar bears and their historical ice-based prey, it may simultaneously yield a new match with certain land-based foods. If polar bears can transition their foraging behavior to effectively exploit these resources, predictions for starvation-related mortality may be overestimated for western Hudson Bay. We also discuss potential complications with stable-carbon isotope studies to evaluate utilization of land-based foods by polar bears including metabolic effects of capture-related stress and consuming a mixed diet.Climate change is predicted to expand the ice-free season in western Hudson Bay and when it grows to 180 days, 28–48% of adult male polar bears are projected to starve unless nutritional deficits can be offset by foods consumed on land. We updated a dynamic energy budget model developed by Molnar et al. to allow influx of additional energy from novel terrestrial foods  that polar bears currently consume as part of a mixed diet while on land. We calculated the units of each prey, alone and in combination, needed to alleviate these lethal energy deficits under conditions of resting or limited movement (2 km d Phoca hispida) )hispida) . The beahispida) . In westhispida) ,6. Assumhispida) ,7,8).-1) but does not allow for energy influx into the system from consuming additional food on land.Molnár et al.  used a m2 of polar bears captured on land. Because energy utilization pathways can change under conditions of extreme stress  and sincaptured ,14,15 thPolar bears are opportunists e.g., ,17) and  and 17])Rangifer tarandus, eggs and Lesser snow geese, hereafter snow geese or LSGO, Chen caerulescens caerulescens) that have more recently been identified in the land-based diet [In this paper, we reconstruct Molnár et al.’s  model tosed diet . The recsed diet . Arrivinsed diet ) as wellsed diet . FurtherHow many individual or combined units of each animal sex and age class  would need to be consumed to prevent starvation in each adult male polar bear?What is the total energy potentially available to polar bears each day from snow geese, eggs and caribou?How many starving adult male polar bears could be supported by each food source?We discuss the limitations of our derived energy calculations in light of the absence of rigorous data on certain aspects of polar bear foraging behavior such as locomotive costs associated with different foraging techniques.To evaluate the potential effectiveness of each food toward fulfilling daily energy requirements of adult males projected to starve while on land for 180 days according to Molnár et al. [We used a 2 component, dynamic energy budget model  developeLBM) and the metabolic rate (m) is the energy required to maintain a unit mass of lean tissue [M [aMb) describes metabolic costs associated with standing and the second, cMbv, describes how energy consumption increases linearly as a function of velocity, v [Application of this model is limited to adult male polar bears (≥ 7 years old) during the ice-free season, so other draws on storage energy, such as thermoregulation, structural growth and reproduction are not accounted for. Because Molnár et al.  assume tn tissue . Movemenissue [M . The firocity, v ,29.α represents the energy density of storage, φ is the proportion of storage mass that is fat and ρSTRk is a constant to estimate structural mass from straight-line body length, L. Storage energy, E, can be expressed as a function of total body mass and straight-line body length  ollowing , body co = 0.684 . Paramet-1 have been reported [v = 0  and v = 2 km d-1 for calculations of energy costs. Also, they observed little variation in straight-line body length among the adult males sampled, so a mean length (L = 2.34 m) was used in all calculations. With initial energy stores, E0, the time to death by starvation was computed by numerically integrating T when E(T) = 0 [-1. As that period expands to 180 days, 28% and 48% would die of starvation if resting or walking, respectively. For sake of reference, adult males comprise approximately 25% (234 polar bears) of the western Hudson Bay population (N = 935 in 2004) based on proportions captured during darting operations once surveys were expanded to include all age and sex classes [Most adult males are reported to be inactive on land during the ice-free season , howeverreported  in westereported  considerE(T) = 0 . Two iceE(T) = 0  estimate classes .E/LBM) for sequential mass values  using Eqs v = 2) for the mass that the bear was on the day before energy stores reached zero. Movement costs were added to the new daily energy requirements regardless of whether these bears had been “moving” prior to starving because movement would be necessary to obtain food from that point forward. The daily energy requirements were summed across all remaining days within the 180-day span for each starving bear  and ranked by total value. These data are illustrated by listing the number of starving polar bears with total energy deficits in sequential 50,000 kcal groupings.To reproduce their results, we computed energy density values , when an ice-free period of 120 days was typical [Although sea ice concentration and extent have delayed freeze-up in parts of Hudson Bay , expansi-1 yr-1 advance from 2008 (21 June) [To estimate snow goose arrival, breeding and molt during the 180-day ice-free season, we projected the mean hatch date in 2068 based on a 0.16 d21 June) . Caribou21 June) . We, theTranslating energy available into energy required to prevent starvation is difficult in a species for which there is little information available on actual terrestrial foraging behaviors or the energetic costs and dynamics associated with those behaviors. In dealing with this uncertainty, we make an initial attempt at integrating the energy available with energy needed by examining maximum potentials and then computing the foraging efficiency that would be required for the translation.th (highest energy requirements), 25th, 50th, 75th and 95th (lowest energy requirements) percentiles with each computed from the average of all energy values falling within 2.5 percentage points (above or below) each of the aforementioned percentiles. We then calculated how many units of each food item  could maximally compensate for the total energy deficits of starving bears in each of the 5 energy condition percentiles for bears that were either resting or walking 2 km d-1 prior to starvation assuming only the added 2 km d-1 movement costs  to procure each food item. Also, because polar bears often consume different foods together [We tabulated the total energy that would be available from each food in 2068 as the ice-free season expands to 180 days. We then compared these energy estimates to different deficit levels that are projected for polar bears that will be susceptible to starvation  according to Molnár et al.’s [together , we prov-1  at the mass the day before they would starve (E(t) = 0). Because most of the starving bears depleted their energy reserves at approximately the same mass (SD = 0.2512) the daily energy requirements (including both somatic and movement costs) did not differ much between individuals so we used the mean value  in calculations. We divided the total energy value of each food item, summed across days, by the mean daily energy deficit  multiplied by the maximum number of days that a starving polar bear would need daily energy supplementation (122 days) to obtain the minimum number of adult male polar bears coming ashore susceptible to starvation that could be supported by each food. This is a conservative estimate of supported bears because individual bears depleted their energy reserves at various points within the 180-day span depending on their arrival mass. 122 days represents the longest period over which males in the worst condition would need to supplementation (days 59 through 180); all others would require food for shorter periods of time. In accordance with Molnar et al. [-1, in calculations of daily energy requirements may lead to overestimation of the number of bears supported by each food item so these estimates should be considered the maximum limits.Adult male polar bears have been observed pursuing and consuming each of the food items discussed, which suggests that the behavior could become widespread through social learning and energetic need ,25. For Recreation of energy profiles for individual polar bears and other computations were completed using R 3.0.1 .7 = 71,068 pairs of nesting geese.In 2006, the nesting population of snow geese on the Cape Churchill Peninsula (CCP) was estimated to be 48,885 pairs . In resp(1/23 days)) [We used the fat and protein content of newly laid snow goose eggs estimated by Badzinski et al.  and desc3 days)) .Puccinellia phryganodes) that goslings forage upon [s = 0.795, computed from the decline in the proportion of goslings between hatch and banding operations 30 days later when the proportion of goslings is again estimated). Years when hatch precedes graminoid emergence (hereafter “bad” years) result in lower survival during the same period . The number of goslings on day 1  was computed from the proportion of successful nests multiplied by 4 . Gosling numbers from day 2 to 30 were computed using daily survival estimates for good (0.9921 = 0.795(1/29 days)), bad (0.9807 = 0.525(1/29 days)) and an average year (0.9864 = 0.660(1/29 days)), using the midpoint of good and bad.Post-hatch gosling survival varies annually, depending, in great part, on the degree to which hatch coincides with peak emergence of wetland grasses  in kilograms and t is time in days between the measurements. Between days 1 and 31 daily increases in protein and mass were 1.1164 and 1.0988 grams, respectively, and between days 32 and 43, growth slowed to 1.0175 and 1.0128 grams of protein and mass, respectively.Fat and protein values were available for neonates , however0.7766; R2 = 0.99). We then multiplied ratios of protein to body mass calculated from the Akimiski Island gosling data [To relate the proportion of protein to mass of the goslings observed on Akimiski Island to the larger ones on the CCP, we first used regression to establish a general relationship of how body mass of CCP goslings changes through the growth period. Using body mass values of neonates from the McConnell River in Nunavut   and thosing data  to the m-0.992; R2 = 0.74) that predicts daily fat content and suggests a drastic drop after day 3 when the remaining yolk is exhausted by the gosling. Grams of fat and protein were converted to gross energy using standard coefficients of 9.39 and 4.30 kcal g-1, respectively [To estimate fat content of goslings, we used a lipid index model from Table 4b in Aubin et al.  that desectively ,48. We fectively .Somateria mollissima) have been observed being attacked by polar bears while guarding their nests [-7.31; R2 = 0.99) to the calculated available energy  modeled with time for the laying, early and late incubation periods and predicted daily energy values from day 1 of incubation through hatch for 35,534 females.Both adult males and females are present during incubation, however, females of both snow geese and common eiders  and used to predict energy values from the beginning of molt (2 July) to flying, approximately 4 weeks later (2 August). Gross energy from protein was scaled by Best’s [Approximately 18 days after hatch, adults begin molting their flight feathers ,53 and bUnlike the case for snow geese, there is no long-term database available for the caribou of the CCP. In generating our estimates of available calories, we have relied on all information there is on this herd and information from studies of mostly nearby herds. Rigorous population surveys are lacking for the Churchill caribou herd, however recent counts (in 2005 and 2012) suggest a stable minimum population size of approximately 3000 animals . UsiCalves were most often first sighted on or around 1 June in the CCP , so this date was chosen as day 1 to determine calf composition based on daily growth with age. This date seems reasonable since it occurs midway between peak calving in the Pen Islands herd to the south (17–28 May) ,60 and t(1/30 days)), we estimated that neonate calves  comprised 29.6% of the population. We used an estimate of over-winter calf survival (14.7%) based on average calf to adult ratios (17.2:100) from late winter surveys of the Churchill herd conducted in 2012 and 2013  to calculate the survival rate (0.6967) from 1 July to 1 March based on the average change in proportion of calves in the population during this period. The average daily survival rate during this period was calculated in a similar fashion as above (0.9985 = 0.6967(1/242 days)).Calf survival was based on observed proportions of calves in the population and estimates of mortality during different times of the year. We used an estimate of 21.1% calves during the post-calving period (~ 1 July) based on the average of counts from 2008 (23.1%) and 2009 (16.1%) in the Pen Islands Herd . We estiWe also used the proportion of calves in the population that survived the winter (14.7%) to estimate the proportion of yearlings available during summer. Given that 12–15% recruitment is generally considered to be the threshold for population stability in caribou populations  and the -1, to convert predicted fat and protein, respectively, to gross energy. Available energy was calculated using digestibilities of fat and protein  provided by Best [The fat and protein content (kg) of adult females and calves at different stages of growth were taken from Gerhart et al.  based on by Best . Energy  by Best  and Sval by Best ,66 at vaDaily changes in fat and protein composition for adults, yearlings and calves were calculated using Of the 97 adult male polar bears sampled, Molnár et al.  predicteThe energetic value of all stages of LSGO  calculated for the 2013 population size was approximately 11,702, 10,959 or 10,334 million kcals depending on whether it was a good, average or bad gosling survival year, respectively. During an average gosling survival year, eggs, pre-hatch adult females and flightless adults comprised 11.8%, 31.2% and 16.4% of the total kcals available to polar bears. Goslings comprised 47.4%, 40.6% and 34.9% of the total available kcals in good, average and bad survival years, respectively. The number of clutches and their respective caloric values both dropped over the course of the 24-day incubation period. For example, on day 1 approximately 71,068 clutches were each worth 840.05 kcal, whereas on day 24 the number of clutches drops to 65,027 and were each worth 493.18 kcal. Goslings, available for 43 days, grew rapidly and range in value from 118.68 kcal at hatch to 1128.23 kcal shortly before flight  needed to prevent starvation for the entire 180-day ice-free period under scenarios of resting or walking. Because of the high caloric value of each caribou and the incidences of multiple polar bears feeding off a single caribou carcass , the exath percentile would require consuming ~5 egg clutches every 3 days, ~1 incubating female off the nest every 3 days, ~2 goslings every 3 days, 1 flightless adult each day and 1 adult male caribou. Different food combinations for each condition percentile for walking and resting starving polar bears are presented in Fig Given the opportunistic nature of polar bears ,21, combAssuming a polar bear population size similar to the last estimate (935)  and the As the ice-free season expands with earlier spring breakup, polar bears are expected to come ashore in western Hudson Bay with smaller energy stores  causing Although local populations of both snow geese and caribou have grown substantially since the 1960s , future The resiliency of caribou in the face of progressive environmental change is less certain . AlthougWith the paucity of knowledge regarding energy consumption rates at varying speeds of travel, especially for adult males i.e., > 235 kg), it is difficult to fully evaluate the feasibility of the foraging scenarios suggested. However, based on past and current behavior, it is clear that polar bears are capable of successfully capturing land-based ungulates, such as caribou  and mus5 kg, it Consumption rates of snow goose eggs and goslings that would compensate starving walking bears ranged from 3 to 26 clutches of eggs and 2 to 19 goslings per day depending on daily energy deficits. High daily depredation rates of goose nests have been reported in populations around the Arctic   but can -1, for example) are quite reasonable. Further, anecdotal observations of flightless goslings and adult snow geese being consumed consecutively by the same bear [Capture rates of goslings and adults are rarely reported ,86 but oame bear  and rema-1 movement cost above somatic maintenance. Similar to lions (Panthera leo), polar bears are considered inefficient walkers so extended pursuits could reduce energetic returns [The large numbers of starving polar bears that can be supported by each food resource suggest that surpluses would be available for other age and sex classes coming ashore with energy deficits. It is important to note, however, that the actual number of bears that will come ashore with energy deficits is unknown and may increase over time as marine food resources become limited. Further, the only costs associated with procurement of prey are a 2 km d returns . Pursuit returns ), howeve returns ,56) suggWilliams and Yeates  calculatPrevious studies have questioned the use of land-based foods to satisfy daily energy requirements while polar bears are on land ,91. HobsIt is possible, however, that the metabolic states of the bears in the Hobson et al.  study weLaminaria spp. and Fucus spp.), typical C4 plants that polar bears commonly consume from land, are more enriched with carbon and have higher δ13C values (-24 to -12‰) compared to most C3  plants, although values range widely depending on plant part and time period sampled [Furthermore, certain foods that polar bears consume on land can complicate results of biochemical studies to distinguish ‘marine’ versus ‘terrestrial’ sources of expended energy using stable carbon isotopes (δ13C) ,95,96. F sampled ,95.Branta canadensis) and common eiders, summering on land in western Hudson Bay can exhibit ‘marine’ signatures from foraging on plants and animals in brackish marshes and marine habitats [Similarly, waterfowl, such as snow geese, Canada geese (habitats ,98. Muschabitats ,97,98, whabitats ,97 and chabitats , especiahabitats .Earlier arriving bears may come ashore with greater nutritional deficits from lost seal hunting opportunities as the ice-free season expands  but calo


Pinus rigida) wood sawdust waste was used as biomass feedstock and the process parameters optimized in this study were the buffering media, the milling time, the enzyme quantity, and the incubation time. Glucose yields were improved when acetate buffer was used rather than citrate buffer. Initially, with each process variable tests, the optimal values were 100 minutes of milling, an enzyme concentration of 16 FPU/g-biomass, and a 12-hour enzymatic hydrolysis. Typically, interactions between these experimental conditions and their effects on glucose production were next investigated using RSM. Glucose yields from the Pinus rigida waste exceeded 80% with several of the conditions tested, demonstrating that milling can be used to obtain high levels of glucose bioconversion from woody biomass for biorefinery purposes.This paper describes an optimization of planetary mill pretreatment and saccharification processes for improving biosugar production. Pitch pine ( Biofuel production is one of major emerging issues in environmental biotechnologies. Producing biofuels from biomass involve a series of processes: producing sugar from biomass through saccharification and biofuels from the sugar through fermentation in order of precedence. However, pretreatment of biomass, typically lignocellulosic, is a currently essential process to obtain high conversion of biomass prior to saccharification and fermentation because of the recalcitrance of biomass. Lignocellulosic biomass consists of cellulose, hemicelluloses, and lignin. Cellulose and hemicelluloses can be hydrolyzed to glucose and xylose, respectively, and subsequently converted to biofuels such as ethanol or butanol through fermentation. The purpose of pretreatment is to make the cellulose within the biomass more readily available for hydrolysis.Previous study showed the potential of using planetary milling to pretreat biomass for downstream fermentation . This mePinus rigida), as the feedstock for pretreatment and glucose production. Softwoods are dominant in the northern hemisphere but they have high lignin content and recalcitrant nature to enzymatic hydrolysis [For an improved sugar production using planetary milling, several factors should be taken into consideration, including the milling time, the amount of enzyme added, and the time given for hydrolysis, as well as the type of buffer used during milling. Hence, we investigated the optimal conditions of pretreatment and enzymatic hydrolysis processes. Glucose yield is a result of combinational interaction between several experimental variables. Therefore, we investigated interactional effects of variable parameters during pretreatment and enzyme hydrolysis in this study, not only single parameter effects. We employed a softwood, pitch pine  after forestry manufacturing process was obtained from Suncheon Forestry Cooperative, Republic Korea. Compositional analysis of the sawdust wood waste was measured according to National Renewable Energy Laboratory protocols [The sawdust wood waste of pitch pine (rotocols .Pinus rigida) was carried out using a Pulverisette 5  in zirconia jar container with 3 mm diameter zirconia balls. The jar was loaded with 7.2 g of biomass and 180 mL of 50 mM sodium acetate (pH 4.8) or 100 mM sodium citrate (pH 4.8). Planetary milling was performed at room temperature by changing the conditions of experimental factors such as milling time and types of buffer solution. The jar was rotated at a velocity of 300 rpm.The planetary mill of sawdust wood waste of pitch pine  was added into the buffer containing milled biomass. Enzymatic hydrolysis was performed at 50°C in a shaking incubator  at 200 rpm. The glucose yield was calculated using the following equation:us study .For investigating the morphology, pretreated pitch pine samples were incubated in dry oven. The samples were coated with Pt on a Cressington Scientific Instruments 108 Auto Sputter Coater . A scanning electron microscope  was used to obtain microscopic images.κα radiation (λ = 1.542 Å) was used for investigating the crystallinity of the samples. Samples were scanned at rate of 2°/min.X-ray diffraction  with Cu The quantities of glucose produced were measured by test strips . The color changes were analyzed using a Reflectometer . Test strips  were sufficiently immersed into test solutions and placed in strip adapter to measure colorimetric changes. Finally, the software installed in the Reflectometer calculated the quantities of glucose contained in the sample by using colorimetric change.All experiments were performed in triplicate for error analysis. Minitab 14  was used for 3D surface plots. Pinus rigida [ P. rigida, however, was similar in both studies with values slightly higher than 40%. The pitch pine sawdust waste used in study had a crystallinity index of 52.5% /(Iθ=22.5°2) × 100, planetary milling was very effective in reducing the crystallinity of the P. rigida cellulose. The crystallinity index of the untreated pitch pine sawdust was 52.5% but was reduced to 36.5 and 31.7% only after 20 minutes of planetary milling when citrate or acetate buffers were used, respectively . To investigate interactional effects of variable parameters, a combinatorial chart was prepared using the conditions listed above as base values along with higher and lower parameter values for analysis . The standard values for the locked parameters were milling for 8 hours, 42.5 FPU/g-biomass and a enzymatic hydrolysis time of 72 hours.After planetary milling, the sizes of biomass were reduced. However, there was no significant difference of biomass size depending on milling time. When it compared with non-pretreated sample, it was clear that planetary mill disrupted the rigid surface and its intact phytoliths . Figure S2 shows scanning electron micrographs of before (a) and after 40 (b), 60 (c) and 120min (d) planetary milled pitch pine wood wastes. Small pieces of non-milling sample were selected to obtain SEM image. Even though the size of biomass does not homogenous, planetary mill was effective to reduce biomass size.


There are limited data on the efficacy of early fluid resuscitation with third-generation hydroxyethyl starch (HES 130) in burn injury. Adverse effects of HES on survival and organ function have been reported.In this randomized, controlled, double-blind trial, 48 patients with severe burn injury were assigned to receive either lactated Ringer’s solution plus 6% HES 130/0.4 in a ratio of 2:1 or lactated Ringer’s solution with no colloid supplement for the first 72 hours. Primary outcome parameter was the group difference of administered total fluid from intensive care unit (ICU) admission up to day 3. Secondary outcomes included kidney and lung injury and failure, length of stay, and mortality.P = 0.39). Creatinine levels from day 1 to 3  −18.7 to 19.5; P = 0.97) and urinary outputs from day 1 to 3  were not different. Six patients in each group developed acute respiratory distress syndrome (ARDS) . Length of ICU stay  and length of hospital stay  were similar. Twenty-eight-day mortality was 4 patients in each group , and in-hospital mortality was 8 in the HES group vs. 5 patients in the lactated Ringer’s group .Three-day totals of administered resuscitation fluid (medians) were 21,190 mL in the lactated Ringer’s group and 19,535 mL in the HES group  in addition to lactated Ringer’s solution would lead to a volume-sparing effect in severe burn injury. Together with the findings that early renal function, incidence of ARDS, length of stay, and mortality were not negatively influenced by HES in this setting, balanced HES 130/0.4 (6%) plus lactated Ringer’s solution could not be considered superior to lactated Ringer’s solution alone.NCT01012648ClinicalTrials.gov  There is an ongoing debate on fluid resuscitation in severe burn injury, especially for the first 24 hours after trauma. Aggressive intravenous fluid therapy according to the Baxter formula is a mainstay of initial therapy. Multiple pathophysiological changes characterize the early post-traumatic phase. Massive systemic inflammation comparable to severe sepsis leads by the release of numerous mediators such as leukotrienes, prostaglandins, and particularly histamine, in combination with complement activation products, to an extensive capillary leak ,2. Intra.This study was an investigator-initiated, prospective, randomized, controlled, double-blind single-center trial. The study protocol was approved by the local ethical committee  and the Swiss Agency for Therapeutic Products (Swissmedic). Power calculation and planning of the statistical analysis were done at the Horten Centre for patient-oriented research at University Hospital of Zurich. Reporting of the study was done according to the CONSORT  guidelines.All adults (age ≥16 years) who had 2nd- or 3rd-degree acute burn injury and more than 15% of body surface area burned and who were admitted to the University Hospital of Zurich burn unit between 1 November 2009 and 31 January 2013 were eligible for the study. All necessary written informed consent  was obtained from the patient or their legal surrogate within 24 hours after inclusion. In the case of written consent of the legal surrogate, all survivors gave written informed consent after recovery, which is in line with the local ethics committee regulations. Patients were excluded when they were expected to succumb within the next 24 to 36 hours  or in situations of palliative care, pregnancy, lack of informed consent, known allergy to HES, contraindications for balanced 6% HES 130/0.4, intracerebral bleeding, acute renal failure, severe hypernatremia and other severe electrolyte disorders, severe von Willebrand Syndrome, and acute liver failure.The study was performed in a tertiary burn unit at the University Hospital of Zurich, Switzerland. The center is the larger of the two national burn units in Switzerland and runs six acute care beds. About 80 severe burn victims are admitted to the university hospital per year.The primary study medication was balanced 6% HES 130/0.4. After patient enrollment and randomization, fluid resuscitation was done as follows. Each patient first received two bags of unblinded lactated Ringer’s solution (500 mL each bag), followed by one bag (500 mL) of blinded study solution, the latter being either again lactated Ringer’s solution or balanced 6% HES 130/0.4. After each bag of study solution, all patients again received two bags of unblinded lactated Ringer’s solution, before a next bag of study solution from the blinded box was infused. This fluid regimen alternating unblinded lactated Ringer’s fluid with blinded study solution ensured an overall ratio of crystalloids versus colloids of 2:1 in the HES patients. The patients not receiving HES but blinded lactated Ringer’s study solution instead were exposed solely to crystalloids during the entire course of the study. Fluid resuscitation was guided by predefined target variables as listed in Figure Primary outcome was the group difference of administered resuscitation fluid within the first 72 hours after admission. Secondary endpoints were creatinine level at day 1 and the difference over day 1 to 3, urine output at day 1 and the difference over day 1 to 3, incidence of acute respiratory distress syndrome (ARDS)  during hpost hoc analysis was performed for 90-day mortality and incidence of renal replacement therapy during hospital stay.A We based our sample size calculations on the primary outcome, which is the total volume of fluids given within the first 72 hours of treatment. We used data from our previous study with HES 200/0.5  to estimA third party not involved in the conduction  performed randomization and prepared the study solution, either balanced 6% HES 130/0.4 or lactated Ringer’s solution, by sealing the identical 500-mL bags in black plastic foil concealing the product label and content. Thus, there was no possibility to recognize the fluid used. Bags were packed into boxes. Three boxes of the same content labelled in consecutive order were assigned to each patient, one box for each 24-hour period up to 72 hours. For randomization, minimization was used with stratification for age (< or ≥50 years). Since minimization does not have a pre-specified randomization list, concealment of random allocation was ensured. Thus, all patients were randomly assigned double-blind either to the lactated Ringer’s plus balanced 6% HES 130/0.4 group or to the lactated Ringer’s-only group, and study medication was assigned to the patients. To make sure that there was no overload of 6% HES 130/0.4 study medication, the maximum amount  was calculated on the basis of the estimated body weight at study enrollment. A body weight of 80 kg, for example, led to 4,000 mL of study solution, which resulted in eight bags of 500 mL each of blinded solution. Not more than that was brought to the patient.We included all patients in the analysis according to the group they were randomly assigned to. To compare the outcomes between the two groups, we used linear regression analysis for continuous outcomes , logistic regression for binary outcomes , and Cox proportional hazard regression for in-hospital mortality, always with group allocation as an independent variable. To compare the total volume of fluids given within the first three days after randomization, creatinine values, and urinary output over the first 72 hours, we used a random effects model that took the auto-correlated structure of repeated measurements  into consideration (xtreg command of STATA). All analyses were conducted by using STATA .From 1 November 2009 through 31 January 2013, 159 patients were assessed for inclusion in the study, and 111 patients were not eligible. From the enrolled 48 patients, 24 patients were assigned to receive lactated Ringer’s solution plus balanced 6% HES 130/0.4 (HES group) and 24 to receive lactated Ringer’s solution (lactated Ringer’s group). Two patients had to be excluded from the study because they retrospectively did not fulfill inclusion criteria. . One patient was lost for analysis in the lactated Ringer’s group because of early discharge within less than 72 hours from the ICU. This patient was formally not excluded, but no study data were available for incorporation in the statistics. Participant flow is shown in Figure During the pre-hospital phase, the lactated Ringer’s group received a median of 1,800 mL and the HES group 2,000 mL of fluid (difference not significant); no colloids were administered. Calculated fluid requirement for the first 24 hours based on the Baxter formula was not different between the two groups . The median 3-day total of effectively administered fluid was 21,190 mL in the lactated Ringer’s group versus 19,535 mL in the HES group. A median amount of 5,650 mL of HES was administered in the colloid group, and no HES was administered in the lactated Ringer’s group (Table P = 0.39). With regard to secondary outcomes, there was no difference over the first 72 hours in creatinine levels  or in urinary output . The incidence of ARDS was 6 patients in each group , and again there was no difference in length of ICU stay and hospital stay , respectively. Twenty-eight-day mortality was 4 patients in each group , and in-hospital mortality was 8 in the HES group versus 5 in the lactated Ringer’s group   −3,975 to 1,549; post hoc analysis of 90-day mortality and incidence of renal replacement therapy showed no difference between the groups. Data are depicted in Table The results of the In this randomized controlled trial, no fluid saving effect was detected by the use of balanced 6% HES 130/0.4 as compared with lactated Ringer’s solution alone in patients with severe burn injury. Furthermore, early renal function as determined by serum creatinine levels, development of ARDS, length of ICU and hospital stay, and in-hospital and 28-day mortality were not different between treatment groups.In severe burn injury with massive systemic inflammation comparable to severe sepsis, aggressive fluid resuscitation to maintain hemodynamic stability and stable kidney function is pivotal. The most widely accepted formula to estimate fluid requirements in burns is the Baxter formula, which, however, rather underestimates the volume needed in about half of the patients -17. The The role of HES in various clinical settings remains controversial. A possible volume-sparing effect, assigned to colloids in general, is the main indication for its widespread use, although the extent of fluid load reduction may be overestimated. There are only few data about the use of HES in patients with burn injury. In a recent randomized controlled trial in burned patients, Vlachou and colleagues  showed aVery limited data are available on modern third-generation HESs such as HES 130 in burns. Only one small randomized open-label study reported more favorable parameters related to the extent of tissue edema and a reduced mortality with HES 130/0.4  just recThe current study has several limitations: firstly, the application of resuscitation fluids was algorithm-based and conducted by different members of our ICU staff. Nevertheless, over two years of study duration, this effect has probably been levelled out over time. Secondly, the used volume resuscitation algorithm was very traditional and did not include any hemodynamic measurement tools such as pulmonary artery catheter or pulse-induced contour cardiac output. Only clinical signs and various hemodynamic and surrogate parameters  were used to guide volume therapy. The reason for this simplified approach comes on the one hand from our clinical experience with fluid therapy in burned patients suggesting that this approach is reliable and on the other hand from the lack of clear evidence for better fluid therapy by the use of advanced hemodynamic guidance tools. Our data show that estimated fluid requirements for the first 24 hours  are comparable to the effectively infused amount of resuscitation fluid in both the HES and the crystalloid group. And as known from the literature, the Baxter formula rather underestimates the necessary amount of infusion fluid -17, whicThe strength of the study is its randomized, double-blinded, and controlled design, making the findings reliable and of clinical relevance. An implication for further research would be the initiation of randomized controlled trials with large sample sizes to strengthen the current evidence of a missing volume-sparing effect of modern HESs in burn injury. With regard to safety concerns that have arisen after the latest meta-analysis reporting a significant risk for mortality and acute kidney injury in various patient groups , furtherThis randomized, controlled, double-blind study did not provide any evidence that early fluid resuscitation with balanced HES 130/0.4 (6%) as an add-on fluid to lactated Ringer’s solution during the first 72 hours after burn injury would lead to a volume-sparing effect. Together with the findings that early renal function, incidence of ARDS, length of stay, and mortality were not negatively influenced by HES in this setting, balanced HES 130/0.4 (6%) plus lactated Ringer’s solution could not be considered superior to lactated Ringer’s solution alone.● All patients received more resuscitation fluid than Baxter formula suggested.● The use of balanced 6% hydroxyethyl starch (HES) 130/0.4 did not result in reduced fluid requirement in severe burn victims.● There is no advantage to using 6% HES 130/0.4 in severe burn injury.ARDS: Acute respiratory distress syndrome; CI: Confidence interval; HES: Hydroxyethyl starch; ICU: Intensive care unit.TAN and RS have occasionally been participants of Fresenius Kabi  and B. Braun  nutrition and fluid therapy advisory board meetings and have received travel and accommodation support and also limited honoraria. MB has received travel and accommodation support and also limited honoraria (Fresenius Kabi and B. Braun). MF, MAP, and RAS declare that they have no competing interests.RAS and MF collected the majority of the data and drafted parts of the manuscript. MAP performed statistical analysis. RS helped to analyze and interpret the data and drafted parts of the manuscript. MB and TAN led the project, collected parts of the data, performed additional statistical analysis, and drafted major parts of the manuscript. All authors read and approved the final manuscript.


Lactobacillus. Subsequently, based on Lactobacillus abundance of an early stage, a model was constructed to predict final wine quality. In addition, three batches of 20 representative wine samples selected from a pool of 110 samples were further analyzed in metagenomics. The results revealed that wine spoilage was due to rapid growth of Lactobacillus brevis at the early stage of fermentation. Gene functional analysis indicated the importance of some pathways such as synthesis of biotin, malolactic fermentation and production of short-chain fatty acid. These results led to a conclusion that metabolisms of microbes influence the wine quality. Thus, nurturing of beneficial microbes and inhibition of undesired ones are both important for the mechanized brewery.Chinese Rice Wine (CRW) is a common alcoholic beverage in China. To investigate the influence of microbial composition on the quality of CRW, high throughput sequencing was performed for 110 wine samples on bacterial 16S rRNA gene and fungal Internal Transcribed Spacer II (ITS2). Bioinformatic analyses demonstrated that the quality of yeast starter and final wine correlated with microbial taxonomic composition, which was exemplified by our finding that wine spoilage resulted from a high proportion of genus  Aspergillus, Rhizopus, Mucor, Absidia, Lactobacillus, Bacillus, and Acetobacteria3Saccharomyces cerevisiae and a few bacterial species, of which S. cerevisiae is the main source of alcohol production. When yeast starter is mixed with wheat Qu under appropriate temperature and humidity conditions, fermentation is initiated that ultimately culminates in producing of specially flavored beverage. As such, many microorganisms play indispensable roles in CRW production and studies on the relevant microbial composition may help to produce high-quality wines.Chinese Rice Wine (CRW), including some acclaimed local varieties produced in Shaoxing, Zhejiang Province, is an alcoholic beverage fermented using rice and wheat. As a traditional alcoholic drink, CRW has been popular in China for centuries and honored as a national banquet wineSaccharomyces, Aspergillus, Bacillus, and Lactobacillus. Another option to evaluate microbial composition is denaturing gradient gel electrophoresis (PCR-DGGE), which however is expensive and time-consuming and has limited ability to detect rare or non-cultivable microbes7Most studies on the influence of microbial composition on CRW quality depended on plate cultivation methods5Since its emergence, the technology of next generation sequencing (NGS) has been used to elucidate the structure of the microbial community of many kinds of biotopes, such as soil, seawater and food910The Illumina Miseq platform was employed to sequence the V3–V4 regions of 16S rRNA gene of 109 samples (14 samples from yeast starter and 95 from 14 batches of wines in fermentation), thereby obtaining 8.04 million clean tags  after concatenation and quality control. Of all test samples, 108 ones produced 3.89 million clean ITS2 tags . One sample failed in sequencing library construction in 16S, as did two in ITS2. Sample statistic information was listed in the To elucidate whether there was a connection between microbial composition and CRW quality, principal coordinate analysis (PCoA) was performed for 12 batches of traditional manual wine samples labeled with quality information , based oLactobacillus appeared antagonist to all of the rest genera. Indeed, Lactobacillus was almost always the most abundant genus over the course of fermentation (LactobacillusLactobacillus are lactic acid bacteria (LAB), which convert carbohydrates to lactic acid in homofermentation or to lactic acid and acetic acid/ethanol in heterofermentation. Production of the two acids acidifies the environment and inhibits the growth of other microbes. Moreover, acetic acid interferes with and slows the fermentation of SaccharomycesLactobacillus percentage over the course of fermentation.As the PCoA analyses revealed differences of taxonomic composition between wines of good and poor qualities , we wereentation  and its Lactobacillus abundance along with Thermoactinomyces abundance which also revealed some weak antagonism with some microbes. The deviation of the abundance of these two genera occurred as early as on the 7th day of the fermentation, which therefore was the earliest time-point to predict the final wine quality. On this day, a low ratio (≤0.8578) of Lactobacillus was always associated with good fermentation, whereas relatively high ratios of Lactobacillus and Thermoactinomyces inevitably resulted in failed fermentation.As illustrated in To identify the genetic elements and species affecting wine quality, 20 wine samples, which were in different fermentation stages and belonged to three batches of wines, were interrogated by analyses of metagenomics. DNA of eukaryotic and prokaryotic origins was extracted, fragmented and sequenced by Illumina Hiseq 2500, which generated a total of 136 Gbp of raw reads of PE125 S. cerevisiae and Saccharopolyspora rectivirgula, consistent with a previous finding that red wine fermentation requires a dominance of S. cerevisiaeAspergillus Oryzae. A. Oryzae has strong secretion of amylases including alpha-amylase, which will certainly accelerate the degradation of grains and provide more nutrients for microbes in fermentation. Li, H. et al.The results of metagenomic taxonomic composition on species level were shown in L. brevis might cause such a problem. The wine quality is mainly evaluated based on taste, which is influenced by acids as they turn the wine sour. Over the course of a good fermentation, the acid level was the highest in the middle phase of fermentation  as well as inter-bacterial competition. For example, bacteria excrete bacteriocins to inhibit other microbes or detect virulence and respond accordingly to survive. Many of these activities are under the control of quorum sensing (QS) systems. Metagenomic proteins were annotated to QS-related proteins. The statistic results of gene number and gene abundance were illustrated in As mentioned above, many microbes coexisted in zymotic fluid, which aggravated the competition for nutrients. In order to compete, some species might produce a variety of antibiotics or bacteriocins to inhibit the growth of other microbes. Analyzing AR genes could indicate the expression level and the strength of antibiotics.etc.) decreased on this day. However, the AR gene abundances were relatively stable over the course of fermentation in the batch “manual3.1”. This supported our previous model using the 7th day for the earliest quality prediction and also agreed with our hypothesis that L. brevis caused a sluggish fermentation. AR gene analysis therefore suggested that regulating the microbial composition might suppress the growth of undesirable microbes during fermentation.S. cerevisiaeMarkers genes that were significantly enriched in the samples of “manual10.1.7” or “manual3.1.7” were annotated on KEGG pathway . Starch,Lactobacillus  in the early stage of fermentation. As such, final wine quality is predictable by samples as early as on the 7th day of fermentation using the relative abundance of Lactobacillus. An early prediction of wine quality will help the wine factory to save time and reduce the loss. Identification of undesired species can remind the factory to take measures to augment the growth of S. cerevisiae. For example, chemical antimicrobial agents, such as SO2, could be employed to inhibit undesired microbes.This study revealed that microbial taxonomic composition apparently differed between high and low quality wines. The spoilage of CRW arose from dominant growth of some species of S. cerevisiae, can promote desirable fermentation. The fast rate in mechanized fermentation could suggest that under such environment, growth of the brewery-beneficial species was encouraged. Improving the fermentation condition to expedite the process is always an important task.Even though the taxonomic compositions were different between samples of “wine.machine2” and “wine.manual10.1”, they were both good quality wines. This indicated that only a few species, such as th day in good fermentation, while AR genes in poor fermentation relatively remained stable. Whether the stability resulted from a good QS-related communication and therefore strong adaptation needs to be studied. Inoculation of some species on 7th day may help the fermentation to enter next stage.Abundances of QS and AR genes were much different between good and poor quality wines. QS analysis revealed that microbes in poor quality wines might have greater level of communications than these in good wines. The result of AR genes displayed a transition phase at around 7KEGG results were consistent with previous reports of other types of wines. “manual10.1” samples harbored relatively high abundance of genes promoting wine fermentation and flavors; on the contrary, “manual3.1” had more genes associated with acetate production that suppresses fermentation. In the zymotic fluid of “manual10.1”, there were more vitamins, such as biotin. These metabolism differences and especially consequent compounds possibly influence fermentation and thus help us to upgrade the fermentation process.L. brevis caused wine spoilage that was possible to be a factor for fermentation delay. Subsequently, we succeeded in establishing a model to predict final wine quality using the bacterial composition on 7th day. The functional analyses indicated some potentially important aspects for CRW fermentation which helps brewery to improve the mechanized processes.In summary, we confirmed the dependence of CRW quality on microbial composition over the course of fermentation after taxonomic analysis. Exceptionally high ratio of Aspergillus oryzae were used as the saccharification agent.At the beginning of the fermentation, a same amount of yeast starters or wheat Qu was added into each fermentation sample. The compositions of the yeast starters and wheat Qu were different for the wine manual and the wine machine. For the manual wine, a naturally cultured yeast starter and wheat Qu were used as the starter inoculum and the saccharification agent respectively. For the mechanized wine, the purely cultured yeast, same as the above, was used as the starter inoculum, while a mixture of the naturally cultured wheat Qu and the wheat Qu inoculated with Samples were collected between February and May, 2011, during the process of manufacturing in a wine-making factory of Shaoxing, Zhejiang Province, China. For the mechanized wine, the supernatant samples and the turbid liquid samples were collected as the follows. At the end of the fermentation, the distillers’ grains were precipitated, and the resulting solution was used as the supernatant. In addition, the distillers’ grains were fully mixed with the supernatant and the mixture used was as the turbid liquid.After the samples were collected, they were transferred to sterile bottles, sealed, and stored at −80 °C. Samples for bacterial and fungal composition analysis were selected after 8-month storage. The alcohol degree and the acidity assay, measured according to the standard of GB/T13662-2008, were employed to evaluate wine quality. The physiochemical parameters of each CRW sample were showed in the Following the manufacturer’s instructions, genomic DNA was extracted using a commercial Fast DNA SPIN Kit for Soil , before the DNA was used to construct a library.Variable regions V3–V4 on 16S rRNA gene of bacteria and the ITS2 region of fungi were amplified using PCR. Tags of ~420 bp of V3–V4 on 16S rRNA gene were amplified with forward primer 5′-ACTCCTACGGGAGGCAGCAG-3′ and reverse primer 5′-GGACTACHVGGGTWTCTAAT-3′After preparation of library, these tags were sequenced on Miseq platform for paired end reads of 300 bp, which were overlapped on their 3′ ends for concatenation into original longer tags. Tags, trimmed of barcodes and primers, were further checked on their rest lengths and average base quality. 16S tag was restricted between 350 bp and 500 bp such that the average Phred score of bases was no worse than 20 (Q20) and no more than 3 ambiguous N. The lengths of ITS tags were between 300 and 420 bp.De novo chimeric sequences were checked and the entire OTU was abandoned if the representative tag was chimeric. All the initial tags of each sample were mapped to non-chimeric representative tag of OTUs with 0.97 identities, augmenting the number of tags in each OTU. The mapped tags of each sample were randomly downsized to the same number to reduce the difference of depth of sequencing. New representative tags with the most abundance were picked. All the above-mentioned steps were mainly performed by usearch (v7.0.1090_i86linux32) and combined with python script of Qiime and perl scripts.We enumerated the copy number of tags and removed redundancy of repeated tagsEach representative tags was assigned to a taxa by RDP classifierThe metagenomic DNA libraries were constructed with 2 μg genome DNA according to the Illumina TruSeq DNA Sample Prep v2 Guide, with an average of 350 bp insert size. The quality of all libraries was evaluated using an Agilent bioanalyser with a DNA LabChip 1000 kit. Sequencing was performed by Illumina Hiseq2500 at WuXi AppTec of China.Oryza sativa, Beijing Rice indica and Triticum aestivum) by SOAPaligner (version 2.21), any hit associated with the reads and their mated reads were removed. Such filtered reads were admitted for the next steps of the analysis.Illumina raw reads were subject to the following treatments: (1) reads with more than 3 ambiguous N bases were removed; (2) reads with less than 60% of high quality bases (Phred score ≥ 20) were deleted; (3) 3′ end of reads were trimmed to the first high quality base. The subsequent high quality reads were further mapped to some cereal genomes  were employed to assemble short reads with parameters ‘-M 3 -u -L 100 -d 1 -F’. k-mers, varying from 39 to 59 by 4, were tested for each sample. The resulting scaffolds were cut into contigs at ambiguous Ns and only contigs longer than 500 bp were saved. N50 were calculated for contigs of different k-mers and only the contigs of largest N50 assembly were attributed to a sample. In order to utilize as many reads as possible, the unassembled reads from the same batch of rice wine fermentation were merged for a second assembly using only one k-mer 59Newly assembled contigs were compared with cereal genomes by blat (v. 35 × 1). The contigs, having ≥90% coverage and ≥95% identity, were considered to belong to cereal genomes and removed.Since a contig might result from either eukaryotes or prokaryotes, which involved different software for gene prediction, it was necessary to classify contigs into either of the two domains. August (version 2.5.5) and MetaGeneMark (version 3.25) were employed for gene prediction of fungi and bacteria, respectively. Because the prediction of August needs a species genome as model, several species were tested, the predicted results with the best probability were chosen. Three steps were performed to separate fungal contigs from bacterial ones, as detailed in the follows: (1) Contigs that were gene-predictable by only one software were classified into the software associated domain. (2) Contigs, predicted by both the software, were compared with bacterial and fungal genomes using blat, contigs were assigned to a tax if (a) the coverage ≥90% and identity ≥95% or (b) the overlap between assembled contigs and genome contigs was larger than 100 bp with the identity ≥95%. If there were several assignments, the best one was preferred. (3) As for the rest predictable contigs, their correspondent protein sequences were annotated according to the NR database, a protein was assigned if E value was smaller than 1e-10. As long as at least 40% of genes on an assembled contig could be assigned to the same domain, that contig was thought to belong to that domain. In case of a contig still having ambiguous origin, it was distributed into a domain that generates a better average score in the output format of blast m8.Removing redundancy was performed respectively on both bacterial and fungal redundant CDS catalogues. Sequences with ≥95% identity and 90% coverage were thought to be redundant and the longer one was left. Therefore, two CDS catalogues were obtained respectively for bacteria and fungi. These two CDS catalogue were merged into a catalogue note C{cds}. Furthermore, We got two others catalogues: (1) catalogue, consisting of non-redundant bacterial CDS catalogue and non-redundant fungal CDS-correspondent transcript catalogue, noted C{trans} and (2) catalogue, comprised of both non- redundant CDS-correspondent protein sequences, noted C{P}.et al.Clean paired reads were mapped to C{trans}, the formula of calculation of gene abundance was related to Qin A local QS database was constructed by searching keywords about QS on UniProtKBC{P} was compared with ARDB (Antibiotic Resistant Database) using ARDB’s own pipeline and 80% identity thresholdC{P} was also annotated by KEGG (Kyoto Encyclopedia Genes and Genomes), using the same methods as QS. Gene abundances were compared between samples. A gene was thought significantly more abundant in one sample than in the other if the gene in one sample is tenfold as much as in the other. These markedly different genes were functionally marked on KEGG pathway.How to cite this article: Hong, X. et al. Metagenomic sequencing reveals the relationship between microbiota composition and quality of Chinese Rice Wine. Sci. Rep.6, 26621; doi: 10.1038/srep26621 (2016).


The roles of mitochondria in various physiological functions of vascular endothelial cells have been investigated extensively. Morphological studies in relation to physiological functions have been performed. However, there have been few reports of morphological investigations related to stem cell differentiation. This was the first morphological study of mitochondria in relation to endothelial differentiation and focused on quantitative analysis of changes in mitochondrial morphology, number, area, and length during differentiation of human mesenchymal stem cells (hMSCs) into endothelial-like cells. To induce differentiation, we engaged vascular endothelial growth factors and flow-induced shear stress. Cells were classified according to the expression of von Willebrand factor as hMSCs, differentiating cells, and almost fully differentiated cells. Based on imaging analysis, we investigated changes in mitochondrial number, area, and length. In addition, mitochondrial networks were quantified on a single-mitochondrion basis by introducing a branch form factor. The data indicated that the mitochondrial number, area per cell, and length were decreased with differentiation. The mitochondrial morphology became simpler with progression of differentiation. These findings could be explained in view of energy level during differentiation; a higher level of energy is needed during differentiation, with larger numbers of mitochondria with branches. Application of this method to differentiation into other lineages will explain the energy levels required to control stem cell differentiation. Mitochondria, the major energy producers in the cell, are known to be involved in various cellular activities and/or functions, including proliferation, aging, and apoptosis –2. RecenAlthough mitochondria in vascular endothelial cells do not occupy a larger volume compared with other closely related cell types and play important roles in cellular processes—such as biogenesis, cellular dynamics, mitophagy, ROS production, and calcium homeostasis –15—there4 cells/cm2 on fibronectin-coated cover glasses on a miniature fluid-chip and cultured for 24 hours to allow stabilization. Endothelial differentiation of hMSCs was induced in Dulbecco’s modified Eagle’s medium with low glucose  containing vascular endothelial growth factor , 5% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. Shear stress was applied as described below for 24 hours after stabilization.Human mesenchymal stem cells (hMSCs) were purchased from Lonza . The cells were cultured according to the manufacturer’s protocol up to passage #4 and seeded at 1×102) were applied for 24 hours after stabilization based on the formula:Mitochondria were stained with 200 nM MitoTracker Deep Red FM  at 37°C for 30 minutes. After staining, the cells were fixed with 4% paraformaldehyde and treated cold acetone to retain mitochondrial staining according to the manufacturer’s guideline. Then, anti-von Willebrand factor (vWF) antibody  and Alexa Fluor 488  were used for immunocytochemistry to confirm endothelial differentiation. Also, nucleus was visualized with 0.5ug/ml of Hoechst 33358 (Invitrogen). Mitochondrial networking and endothelial differentiation were examined by confocal laser scanning microscopy .The number of cells selected per group was 90 on average. The measurement/calculation of the number and area of mitochondria within a cell was performed based on binary images through the thresholding process. The average mitochondrial length was calculated by skeletonizing from the binary images and normalized to the number of mitochondria. MATLAB 2012a  was used to analyze images of single cells acquired within the region of interest. Specifically, the functions of im2bw and bwmorph were used for binarization and skeletonization, respectively.To quantify the networking of a mitochondrion, we defined a branch form factor (BFF) as shown in One-way analysis of variance (ANOVA) was performed using PASW Statistics 18 . When ANOVA indicated a significant difference among groups, the difference was evaluated using the least-significant difference (LSD) test. All data are presented as means ± standard deviation (SD). In all analyses, p < 0.05 was taken to indicate statistical significance.2) and VEGF. This continued even up to 72 hours (day 3). However, fewer cells were found to express vWF under conditions of lower shear stress (2.5 dyne/cm2) with VEGF on day 2. Some of the cells under lower shear stress began to show vWF expression on day 3. No expression of vWF was detected in the group treated with VEGF alone even up to 72 hours. Other extra figures were included for more understanding. 2) < group with VEGF and higher shear stress (10.0 dyne/cm2).vWF is generated only by vascular endothelial cells, and it is therefore widely used as a typical endothelial differentiation marker . Some hMSpecifically, vWF was expressed by larger numbers of cells when exposed to higher shear stress with VEGF. These observations based on vWF staining enabled us to distinguish differentiated from differentiating cells, and therefore we could acquire images to determine the morphological changes occurring in mitochondria during differentiation. In addition, these results reconfirmed that shear stress with VEGF can promote the differentiation of hMSCs into endothelial cells.As we cannot expect all stem cells to differentiate into the desired lineage even under the appropriate conditions for differentiation, we distinguished differentiated from undifferentiated cells by detecting a typical epithelial differentiation marker, vWF.2 or 10.0 dyne/cm2) tended to increase during the early stages of differentiation when compared with that in the cell without shear stresses. Then the number was to decrease as the cell enters the late stage of differentiation. There were fewer mitochondria in cells positive for vWF compared to those without this marker in either the undifferentiated or differentiating state. However, there was no significant difference in the number of mitochondria between groups under lower shear stress (~2.5 dyne/cm2) regardless of vWF expression on day 3. These observations suggested that the number of mitochondria per cell would decrease with differentiation as the higher level of shear stress (~10.0 dyne/cm2) accelerated differentiation compared to the lower level (~2.5 dyne/cm2).The average number of mitochondria in a cell during differentiation is shown in The average total area of mitochondria per cell, which was calculated based on the area measured, was found to decrease with progression of differentiation in the direction of the endothelial lineage  for the statistical support. Also, the stipulation proposed through this study may not be applicable to differentiation to other specific lineages. Further study by accompaniment of such image-based studies with morphologically related protein studies would result in the findings regarding mitochondrial morphology and its role in relation to cellular activities, especially in differentiation, being more physiologically and biologically relevant. In addition, any other image-based analysis techniques such as fractal and textural  are recommended to be adopted as potential tools in detecting cell and/or organelles morphology changes in investigating various cellular activities.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.