Fragaria x ananassa) are valued for their characteristic red color, juicy texture, distinct aroma, and sweet fruity flavor. In this study, genetic and environmentally induced variation is exploited to capture biochemically diverse strawberry fruit for metabolite profiling and consumer rating. Analyses identify fruit attributes influencing hedonics and sensory perception of strawberry fruit using a psychophysics approach. Sweetness intensity, flavor intensity, and texture liking are dependent on sugar concentrations, specific volatile compounds, and fruit firmness, respectively. Overall liking is most greatly influenced by sweetness and strawberry flavor intensity, which are undermined by environmental pressures that reduce sucrose and total volatile content. The volatile profiles among commercial strawberry varieties are complex and distinct, but a list of perceptually impactful compounds from the larger mixture is better defined. Particular esters, terpenes, and furans have the most significant fits to strawberry flavor intensity. In total, thirty-one volatile compounds are found to be significantly correlated to strawberry flavor intensity, only one of them negatively. Further analysis identifies individual volatile compounds that have an enhancing effect on perceived sweetness intensity of fruit independent of sugar content. These findings allow for consumer influence in the breeding of more desirable fruits and vegetables. Also, this approach garners insights into fruit metabolomics, flavor chemistry, and a paradigm for enhancing liking of natural or processed products.Fresh strawberries ( Fragaria x ananassa) fruit is characterized by its large size Modern fully ripe strawberry , contributes to fruit softening The dynamics of fruit development are genetically driven. Microarray analysis determined nearly 15% of probed expressed genes exhibit significant differential expression  in red compared to green fruit FragariaMetabolic profiling indicates an accumulation of sugars, organic acids, and fatty acids as well as the consumption of amino acids during fruit development. Subsequently alkanes, alcohols, aldehydes, anthocyanins, ketones, esters, and furanones increase during fruit ripening H)-furanone, 4-methoxy-2,5-dimethyl-, the current consensus of integral strawberry aroma compounds. Comparisons of consumer preference among a variety of fresh strawberries and their volatile profiles describes less preferable varieties as possessing less esters, more decalactones and hexanoic acid A cross comparison of five previous studies which analyze strawberry volatiles depicts the lack of agreement in defining chemical constituents of strawberry aroma. Each source considers a highly variable subset of volatiles, which are determined by signal intensity and/or human perception of separated compounds Florida strawberry production is concentrated on ten thousand acres near the Tampa Bay. Mild winters allow for annual horticulture which requires continual harvest of ripe fruit from late November through March. Environmental effects on fruit quality are partially attributed to gradually increasing temperatures beginning in mid-January. One result is a late season decline of soluble solids content (SSC) Here we exploit the genetic and within-season variability of fruit to provide as many unique strawberry experiences as possible to a large sample of consumers. To enhance the range and diversity of flavors and chemical constituents 35 genetic backgrounds were included: public and private cultivars representing a large proportion of commercial strawberry acreage in North America, University of Florida advanced breeding selections, and European cultivars . ParalleAll human consumer panels are conducted at the Food Science and Human Nutrition Department at the University of Florida in Gainesville, FL. The University of Florida Institutional Review Board 2 (IRB2) chaired by Ira S. Fischler approved the protocol and written consent form (case 2003-U-0491), which participants are required to complete.http://fawn.ifas.ufl.edu/data/reports) for date ranges January 3, 2011 through February 28, 2011 and December 26, 2011 through March 13, 2012. Daily maximum and minimum temperature recording height is 60 cm, and daily average relative humidity, rainfall, and solar radiation are recorded at 2 m.Thirty-five strawberry cultivars and selections were grown at or in the near vicinity of the Gulf Coast Research and Education Center  during the 2010–2011 (season 1) and 2011–2012 (season 2) winter seasons. Fruit are cultivated according to current commercial practices for annual strawberry plasticulture in Florida 2 (l) and stored at –80°C for later sugar and acid quantification. A two hour collection in a dynamic headspace volatile collection system At least 100 g or seven berries of each sample are removed from 4°C dark overnight storage prior to volatile collection. Samples are homogenized in a blender prior to splitting into three 15 g replicates for immediate capturing of volatile emissions. The remainder is frozen in N−1; splitless injector, temperature 220°C, injection volume 2 µl) equipped with a DB-5 column ((5%-Phenyl)-methylpolysiloxane, 30 m length ×250 µm i.d. × 1 µm film thickness; Agilent Technologies, Santa Clara, CA, USA). Oven temperature is programmed from 40°C (0.5 min hold) at 5°C min−1 to 250°C (4 min hold). Signals are captured with a flame ionization detector (FID) at 280°C. Peaks from FID signal are integrated manually with Chemstation B.04.01 software . Volatile emissions (ng1 gFW−1 h−1) are calculated based on individual peak area relative to sample elution standard peak area. GC-mass spectrometry (MS) analysis of elutions are performed on an Agilent 6890N GC in tandem with an Agilent 5975 MS  and retention times are compared with authentic standards  for volatile identification Quantification of volatiles in an elution is performed on an Agilent 7890A Series gas chromatograph (GC)  is analyzed using biochemical kits (per manufacturer’s instructions) for quantification of citric acid, L-malic acid, D-glucose, D-fructose, and sucrose  with absorbance measured at 365 nm on an Epoch Microplate Spectrophotometer . Metabolite average concentration (mg1 100 gFW−1) is determined from two to six technical replicates per pooled sample. Derived sucrose concentrations via D-glucose and D-fructose are mathematically pooled.Titratable acidity, pH, and soluble solids content 1 sec−1; a flap cut off the opposite provides stability. Maximum force in kg for eight fruit is averaged and reported as a measure of firmness.Firmness of the strawberries is determined as the resistance of the fruit to penetration at its equator with a TA.XTPlus Texture Analyzer . The Texture Analyzer is equipped with a 50 kg load cell and an 8 mm diameter convex tip probe. Whole fruit is penetrated on the side to 7 mm down from the epidermis at a test speed of 2 mmi.e. least to most pleasurable experience i.e. none to most intense sensory stimulus Over the course of two annual seasons, 166 recruited strawberry consumers  evaluate strawberry cultivars. Ages of panelist ranged from 18 to 71, with a median age of 24. Panelists self-classified themselves as 98 White or Caucasian, 11 Black or African-American, 1 Native American, Alaska Native or Aleutian, 41 Asian/Pacific Islander, and 15 Other. An average of 106 (range of 98–113) panelists evaluated between three and five cultivars per session p-values ≤ 0.05, the volatile makes a contribution to perceived sweetness that is independent of the sugar tested.Means and standard errors for consumer, physical, and metabolite measurements are determined from all replicates using JMP . One-way analysis of early and late season fruit quality and consumer response measures was subjected to mean comparison using Tukey’s HSD (α = 0.05). Bivariate analysis among individual measurements of samples allows for linear fit, which includes summary of fit, analysis of variance, t-test, and correlation analysis for density ellipse. Two-way Ward hierarchical cluster analysis of all quantified metabolite and strawberry samples is accomplished in JMP. Amounts of individual volatile compounds are regressed using the “enter” method in SPSS . This is done individually for each of the three sugars: glucose, fructose or sucrose to identify which compounds have an effect on sweetness intensity The inventory of 54 fully ripe  unique si.e. least to most pleasurable experience i.e. none to most intense sensory stimulus Overall liking is a measure of pleasure derived from consuming a strawberry sample. The two samples with the greatest overall liking ratings are of cultivar ‘Festival’. Fruit harvested early in the season, week 2 of season 1 and week 1 of season 2, elicit overall likings of 36.1 and 36.6, respectively . Five we2 = 0.305*) (2 = 0.005) (data not shown) or fructose (R2 = 0.001) (2 = 0.422*)   but not = 0.001) . A harve 0.422*)  through 2 = 0.490*) (2 = 0.742*) (2 = 0.604*) (2 = 0.008) (2 = 0.358*) (2 = 0.488*) (2 = 0.099*) (2 = 0.179*)  , sweetne 0.742*) , and str 0.604*) . However= 0.008) . Increas 0.358*) , and tex 0.488*)  demonstr 0.099*) . Total v 0.179*) . This is 0.179*) .The upper limit for hedonics of texture is comparable to that of overall liking and is observed in ‘Festival’  with an average of 35.7, however, the low texture liking value of 5.8 for ‘Mara Des Bois’  indicates a more drastic disliking of “off” textures than the overall liking of even the lowest rating fruit . FirmnesPerceived sweetness intensity is the greatest predictor of overall liking. In fact, the same samples scoring the highest and lowest for overall liking, ‘Festival’  and ‘Red Merlin’ , elicit the greatest (36.2) and least (14.59) intense sensations of sweetness . The ear2 = 0.984*) (data not shown) within a sample. However, the concentration of glucose or fructose is not predictive of sucrose concentration  (data not shown). Sucrose demonstrated a more dynamic state as its concentration dips as low as 0.16% and up to 2.84%, nearly a seventeen-fold difference among all samples.In the 54 samples assayed, the total sugar concentration ranged from 2.29 – 7.93%, a 3.5-fold difference . Glucose2 = 0.442*) (2 = 0.445*) (2 = 0.337*) (2 = 0.300*) (2 = 0.687*)  . Individ 0.445*) , glucose 0.337*) , and fru 0.300*)  all sign 0.687*)  likely a 0.687*) .2 = 0.314*) (2 = 0.118*), malic acid (R2 = 0.189*) (2 = 0.146*) (2 = 0.49*) (data not shown). There is no correlation of malic acid to TA (R2 = 0.01) (data not shown). The lack of relationship among sourness intensity and overall liking  elicited the most intense sourness response at 24.6 . This sa 0.314*) , when co 0.189*) , or citr 0.146*)  concentrl liking  is shadol liking  and flavl liking  to overa2 = 0.167*) , does not result in the greatest flavor intensity (30.5) and the minimum, 8.5 µg1 gFW−1 hr−1 from ‘Sweet Anne’ , does not rate as the least flavorful (25.8)  , but it l (25.8) .1 gFW−1 hr−1) in at least one sample. Therefore, 38 volatiles are measured in all samples; appearing to be constant in the genetic resources analyzed  and has a significant correlation to flavor (R2 = 0.097*) (1 gFW−1 hr−1 in ‘Sweet Charlie’ is not detected in ‘Red Merlin’. The nerolidol rich ‘Sweet Charlie’ garners greater flavor intensity at 32.2 than deficient ‘Red Merlin’ at 23.95. The impact on flavor intensity by nerolidol (R2 = 0.112*) (1 gFW−1 hr−1(2 = 0.016) (2 = 0.239*) (2 = 0.079*) (1 gFW−1 hr−1 in ‘Treasure’ (sn 1 wk 3). The level of this characteristic strawberry furan is significantly impactful on perceived flavor intensity (R2 = 0.108*) is measured at over 7 µg 0.097*) . A terpe 0.112*)  is greatFW−1 hr−1, and doe= 0.016) . Hexanoi= 0.016) . Convers 0.239*) , while p 0.079*) . The sig 0.079*) . In ‘Cha 0.108*) . In totaH)-furanone, dihydro-5-octyl- (2305-05-7) ; butanoic acid, pentyl ester (540-18-1); butanoic acid, hexyl ester (2639-63-6); acetic acid, hexyl ester (142-92-7); and butanoic acid, 1-methylbutyl ester. Only three compounds are found to be negatively related to sweetness independent of at least one of the sugars: octanoic acid, ethyl ester (106-32-1) exclusively independent of glucose; 2-pentanone, 4-methyl- (108-10-1) mutually independent of glucose and fructose; and 2-buten-1-ol, 3-methyl-, 1-acetate (1191-16-8) exclusively independent of sucrose.Multiple regression of individual volatile compounds against perceived intensity of sweetness is performed independent of glucose, fructose, or sucrose concentration . Twenty Exploitation of genetic diversity and environmental variation allows for a wide range of consumer hedonic and sensory responses. The cultivars in this study represent a large proportion of commercial strawberry acreage in North America, advanced breeding selections, and European cultivars. A genetic collection aimed at enhancing the diversity of physical and chemical constituents, as well as consumer experiences. Despite the perennial life cycle of strawberry much commercial production uses annual methods, which in sub-tropical Florida allows for continual harvest of ripe fruit from late November through March. A nearly three-fold difference in overall liking of strawberry is observable within all samples. The highest and lowest rating samples are ‘Festival’ of the first week in the second season and ‘Red Merlin’ of the sixth week in the first season. These two cultivars are the product of separate breeding programs, have distinct genetic backgrounds, and therefore distinct biochemical inventories. Harvested at opposite ends of the seasons the early and late season fruit are subjected to different environmental conditions, further attenuating genetic differences. The diversity of strawberries samples assayed and range of consumer liking captured  indicatee.g. sugars, acids, and volatile chemical compounds. These elicitors attenuate the perception and hedonics of food Elevated texture liking, sweetness intensity, and strawberry flavor intensity significantly increases overall liking, while sourness intensity alone has no detectable relationship to overall liking . IntegraMuch work has been done to measure sugars and volatile compounds in strawberry fruit in an attempt to understand sweetness and flavor, and these aims are in line with consumer demand. A consumer survey using 36 attributes of strawberry determined “sweetness” and “complex flavor” as consistent favorable characteristics of the ideal strawberry experience The overall liking of strawberry fruit is significantly related to texture liking , and incDespite a moderate range of intensity, perceived sourness has little to no bearing on overall liking . Just ovThe consumer rating of sweetness intensity is the primary factor contributing to overall liking, and sweetness is the component of taste perception facilitating the detection of sugars. Sugars are simple carbohydrates, a readily available form of energy, and the degree of correlation among sweetness and overall liking is due to hedonic effect Drastic fruit quality differences between early and late season fruit result in lower consumer response  Fig. 3EFig. 3E, i.e. a larger imported reserve, facilitating greater flux through primary and secondary metabolism. Generation of glucose and fructose initiates a complex network of primary and secondary metabolism specific to ripening strawberry fruit, in which sucrose is principal and limiting to the strawberry fruit biosynthetic pathways Although total sugar decreases between early and late fruit, a disproportionate amount of the decrease is attributed to sucrose , which iStrawberry flavor intensity is the second greatest determinant of overall liking  and accoFragaria x ananassa analyzed in this study 81 compounds are reproducibly detected, but not one cultivar has detectable amounts of all compounds. The amount of individual volatile compounds within fruit can have a significant influence on flavor intensity, but which volatiles are determinant of flavor has a lack of consensus. Previous determination of flavor relevance relied on approaches in which importance of volatiles is at least initially based on abundance. Determination of flavor descriptors or thresholds of isolated compounds were determined using human perception via orthonasal olfaction Within the genetic resources of H)-furanone, 4-methoxy-2,5-dimethyl-, all of which are quantified in this report. These compounds exhibit adequate variability in fruit samples to discern dose dependent effect on flavor intensity. However, only linalool; butanoic acid, ethyl ester; butanoic acid, methyl ester; and 3(2H)-furanone, 4-methoxy-2,5-dimethyl- show significant positive correlation with flavor intensity -furanone, dihydro-5-octyl- ; butanoic acid, pentyl ester; butanoic acid, hexyl ester; acetic acid, hexyl ester; and butanoic acid, 1-methylbutyl ester , daily average solar radiation (C and D), daily average relative humidity (E and F), and daily total rain fall (G and H) during the 2011  and 2012  seasons. Data for Balm, FL obtained from Florida Automated Weather Network (http://fawn.ifas.ufl.edu/data/reports). Data spans three weeks prior to first harvest through last harvest of each season with individual harvests indicated by dotted vertical line and harvest week number. Dashed horizontal lines represent means of environmental measures. Solid lines are the bivariate fit of environmental measure across season. Coefficients of determination (R2) and p-value of fit is listed above individual scatterplots and are calculated using bivariate fit in JMP 8.(TIF)Click here for additional data file.Figure S2Individual sugars and total volatiles regressed against season progression. Regression of sucrose (A), glucose (B), fructose (C), and total volatiles (D) by harvest week during the seasons. Total volatile content is regressed against sucrose (E) and fructose (F). Sucrose (A) and total volatiles (D) demonstrate a significant negative fit to harvest week, unlike glucose (B) and fructose (C). A strong relationship between total volatile emmission and sucrose concentration is found (E) that is not observed between total volatiles and glucose (data not shown) and fructose (F). Coefficient of determination (R2) and p-value of fit is listed above individual scatterplots and is calculated using bivariate fit in JMP 8. Dashed line represents mean of independent variable, solid line represents linear fit, dashed/dotted ellipse indicates 95% confidence range of data, and asterisk denotes significant fit (α = 0.05).(TIF)Click here for additional data file.Figure S3Chemical structure of volatile compounds. Chemical structure of volatile compounds quantified in strawberry. Sorted by increasing retention time , identified by CAS Registry Number.(TIF)Click here for additional data file.Table S1CAS registry number, chemical name, and formula index. Chemical Abstract Services (CAS) registry numbers were used to query SciFinder® substances database for associated chemical name and molecular formula.(DOCX)Click here for additional data file.Table S2Full data table. Means and standard errors of replicates for all measures for each sample assayed. Includes consumer panel measures, internal and external color, puncture force, organic acids, sugars, SSC, pH, TA, and volatile compounds. High and low value, median, and fold difference for each column displayed above means table.(XLSX)Click here for additional data file.Table S3Fruit attributes bivariate fit during season. Regression of harvest week during season (X) on panel responses and metabolite concentration (Y). Coefficient of determination (R2), correlation coefficient, p-value, sample size (n), mean and standard deviation of X and Y derived from bivariate fit in JMP 8.(DOCX)Click here for additional data file.Table S4Fruit quality bivariate fit. Regression of chemical and physical measures of fruit (X) to panel responses (Y). Coefficient of determination (R2), correlation coefficient, p-value, sample size (n), mean and standard deviation of X and Y derived from bivariate fit in JMP 8.(DOCX)Click here for additional data file.Table S5Multiple regression for identification of sweetness enhancing volatiles. Individual volatile compound concentrations are regressed against perceived sweetness intensity independent of effect from glucose, fructose, or sucrose, separately. Thirty compounds (*) (α = 0.05) were found to enhance intensity of sweetness independent of at least one of the three sugars. Six compounds (bold) were found to significantly enhance intensity of sweetness independent of all three sugars.(DOCX)Click here for additional data file.


Xanthomonas axonopodis pv. citri (X. a. pv. citri) causes citrus canker that can result in defoliation and premature fruit drop with significant production losses worldwide. Biofilm formation is an important process in bacterial pathogens and several lines of evidence suggest that in X. a. pv. citri this process is a requirement to achieve maximal virulence since it has a major role in host interactions. In this study, proteomics was used to gain further insights into the functions of biofilms.X. a. pv. citri mature biofilm and planktonic cells. The biofilm proteome showed major variations in the composition of outer membrane proteins and receptor or transport proteins. Among them, several porins and TonB-dependent receptor were differentially regulated in the biofilm compared to the planktonic cells, indicating that these proteins may serve in maintaining specific membrane-associated functions including signaling and cellular homeostasis. In biofilms, UDP-glucose dehydrogenase with a major role in exopolysaccharide production and the non-fimbrial adhesin YapH involved in adherence were over-expressed, while a polynucleotide phosphorylase that was demonstrated to negatively control biofilm formation in E. coli was down-regulated. In addition, several proteins involved in protein synthesis, folding and stabilization were up-regulated in biofilms. Interestingly, some proteins related to energy production, such as ATP-synthase were down-regulated in biofilms. Moreover, a number of enzymes of the tricarboxylic acid cycle were differentially expressed. In addition, X. a. pv. citri biofilms also showed down-regulation of several antioxidant enzymes. The respective gene expression patterns of several identified proteins in both X. a. pv. citri mature biofilm and planktonic cells were evaluated by quantitative real-time PCR and shown to consistently correlate with those deduced from the proteomic study.In order to identify differentially expressed proteins, a comparative proteomic study using 2D difference gel electrophoresis was carried out on X. a. pv. citri biofilms are enriched for the gene ontology (GO) terms ‘generation of precursor metabolites and energy’ and secondly, the biofilm proteome mainly changes in ‘outer membrane and receptor or transport’. We argue that the differentially expressed proteins have a critical role in maintaining a functional external structure as well as enabling appropriate flow of nutrients and signals specific to the biofilm lifestyle.Differentially expressed proteins are enriched in functional categories. Firstly, proteins that are down-regulated in  Xanthomonas axonopodis pv. citri (X. a. pv. citri) is a gram-negative plant pathogenic bacteria that causes citrus canker -1-propanesulfonate (CHAPS)) with vigorous vortexing at room temperature. Concentration of total protein extracts was estimated by Bradford assay using the Quick Start Bradford protein assay (Bio-Rad). Protein extracts were prepared from three different flasks for both growth conditions.Cellular pellets of X. a. pv. citri planktonic and X. a. pv. citri forming biofilm samples were labeled with Cy3 and Cy5, respectively. Dye swap between samples was carried out to avoid artifacts due to preferential labeling. Three biological replicates and two technical replicates were carried out, giving rise to a total of six gel images per growth conditions. All reactions were carried out on ice and in the dark to limit signal quenching. Labeling was performed for 30 min and terminated by incubation with 10 nmol lysine for 10 min. Equal volumes of urea lysis buffer containing 20 mg/ml DTT and 2% (v/v) IPG buffer, pH range 4–7  were added to each sample and incubated for 15 min. After pooling the samples, the volume was adjusted to 125 μl with rehydration buffer  CHAPS, 2 mg/ml DTT and 1% (v/v) IPG buffer pH 4–7, GE Healthcare) and separated by 2D-DIGE.Prior to 2D-PAGE, protein samples were labeled using the fluorescent cyanine three-dye strategy , according to manufacturer’s instructions. Briefly, proteins (50 μg) of an internal standard containing an equal amount of the control and treated samples were incubated with 400 pmol of Cy2, freshly dissolved in dimethyl formamide (DMF), while Labeled protein samples in urea lysis buffer were used to rehydrate 7 cm-long linear IPG strips, pH range 4–7 . Following overnight rehydration at room temperature, strips were focused for a total of 8,750 Vhrs 50 μA at 20°C, as follows: step, 500 V for 250 Vhrs; step, 1,000 V for 500 Vhrs and step, 8,000 V for 8,000 Vhrs. Prior to SDS-PAGE, strips were equilibrated twice for 15 min in equilibration buffer  glycerol, 6 M urea, 2% (w/v) SDS) first containing 1% (w/v) DTT and then 2.5% (w/v) iodoacetamide with gentle shaking. Strips were loaded on top of 12% SDS-PAGE. Strips were sealed on top of the gel with 1% (w/v) agarose in SDS running buffer  SDS). Gels were run at 50 V for the first 15 min and then at 100 V until the dye reached the bottom of the gels.TM 9410 scanner . Cy2-labeled pool samples were imaged using a 488 nm blue laser and a 520 nm band-pass (BP) 40 emission filter. Cy3 images were obtained using a 532 nm green laser and a 520 nm BP30 emission filter, and the Cy5 images using a 633 nm red laser and a 670 nm BP30 emission filter. Images were analyzed with the Delta2D  software. Spot quantities were calculated by summing pixel intensities within the spot boundaries and used for analyzing gene expression. Normalized expression profile data were used to statistically assess changes in protein spot expression. Differentially expressed protein spots between the two groups were calculated using the Student-T test with a critical p-value ≤ 0.05 and the permutation-based method to avoid biased results that may arise within replicate gels if spot quantities are not normally distributed. The adjusted Bonferroni correction was applied for false discovery rate (FDR) to control the proportion of false positives in the result set. Principal component analysis was performed to determine samples/spots that contributed most to the variance and their relatedness.Gel images were obtained using the Typhoon4HCO3, incubated in 100 μl of 25 mM NH4HCO3 in 50% (v/v) acetonitrile (ACN) for 30 min with gentle shaking, dehydrated with 100 μl of 100% (v/v) ACN for 10 min and then rehydrated with 100 μl of 25 mM NH4HCO3 for 30 min with gentle shaking. Gel pieces were dehydrated again with 100 μl of 100% (v/v) ACN for 10 min and completely evaporated. Proteins were reduced with 50 μl of 10 mM DTT in 100 mM NH4HCO3 at 56°C for 45 min and then alkylated with 50 μl of 50 mM iodoacetamide in 100 mM NH4HCO3 for 30 min at room temperature in the dark. Gel pieces were rinsed with 200 μl of 100 mM NH4HCO3 and then with 200 μl of 100% (v/v) ACN for 10 min each step. These steps were repeated once more. Gel pieces were completely dehydrated and incubated with 200 ng of trypsin  diluted in 50 mM NH4HCO3 overnight at 30°C. Samples were cooled down to room temperature and incubated with 20 μl of 20 mM NH4HCO3 for 10 min. Peptides were extracted twice from the gel pieces with 20 μl of 5% (v/v) formic acid (FA) in 50% (v/v) ACN for 10 min each, collected to separate tubes, evaporated and stored at −20°C prior to mass spectrometry analysis.Differentially expressed protein spots of interest were manually excised and each placed into separate microcentrifuge tubes. Gel pieces were rinsed briefly with 100 μl of 25 mM NHm/z 350 to 1,600. The top 10 precursor ions were selected in the MS scan by Orbitrap with resolution r = 60,000 for fragmentation in the linear ion trap using collision-induced dissociation. The normalized collision-induced dissociation was set to 35.0.Digested peptide mixtures were suspended in 0.1% (v/v) formic acid (FA) in 5% (v/v) ACN, and analyzed with an LTQ Orbitrap mass spectrometer  equipped with an electrospray ion source and coupled to an EASY-nanoLC  for nano-LC-MS/MS analyses. A volume of 5 μl of the peptide mixture was injected onto a 5 μm, 300 Å, 50 mm long × 0.3 mm Magic C18AQ  pre-column and a 3 μm, 100 Å, 100 mm long × 0.1 mm Magic C18AQ  column. A spray voltage of 1,500 V was applied. The mobile phases consisted of 0.1% FA and 5% ACN (A) and 0.1% FA and 90% ACN (B). A three step gradient of 0-40% B in 20 min, then 40-90% B in 5 min and finally 90% B for 20 min with a flow of 500 nl/min over 45 min was applied for peptide elution. The MS scan range was All spectra were converted to mgf using Proteome Discover version 1.2 (Thermo-Scientific) and submitted to a local MASCOT  server and searched against bacteria in the SwissProt (release 57.15) and MSDB databases (release 9.0) with a precursor mass tolerance of 10 ppm, a fragment ion mass tolerance of 0.6 Da and strict trypsin specificity allowing up to one missed cleavage, carbamidomethyl as fixed modification and oxidation of methionine residues as variable modification. Proteins were considered positive if the MASCOT score was over the 95% confidence limit corresponding to a score > 35 for proteobacteria.X. a. pv. citri mature biofilms and planktonic cells was extracted using TRIzol® reagent (Invitrogen), according to the manufacturer’s instructions. After DNAse (Promega) treatment, cDNA was synthesized from 1 μg of total RNA using M-MLV RT (Promega) and the oligonucleotide dN6 was added as follows: 200 U of M-MLV RT , 0.25 μg of primer dN6 and 0.5 mM of deoxynucleoside triphosphates (dNTPs)  and incubated for 1 h at 42°C, and then for 10 min at 94°C. The qRT-PCRs were performed by combining 1 μl of cDNA template, 0.5 U of Go Taq DNA polymerase (Promega), 1 × reaction buffer, 0.2 mM dNTPs and 20 pmol of each primer  in a Mastercycler ep realplex thermal cycler (Eppendorf) using SYBR Green I (Roche) to monitor double-stranded DNA (dsDNA) synthesis. The qRT-PCR conditions were set to 95°C for 1 min, followed by 40 cycles of 95°C for 15 s, 55°C for 30 s and 72°C for 40 s. The primer pairs used for qRT-PCR are provided in Additional file r) according to the equation ΔCt = Ct – Ctr, and quantified as 2–ΔCt. A second normalization using a control (time=0 days) (Ctc), ΔΔCt = Ct – Ctc, produces a relative quantification: 2–ΔΔCt[t-test (p < 0.05).Total RNA from n: 2–ΔΔCt. Values Proteins were considered as differentially expressed when variations between planktonic and biofilm grown cells were at least 1.5-fold and the quantitation p-value of 0.05. The GO enrichment analysis was performed using Blast2GO -66.The authors declare that they have no competing interests.TZ, JO and NG conceived the project and designed the experiments. TZ, LT, CM and BSG designed and performed the experiments. All authors contributed to the analysis and interpretation of the data and LT, CM, BSG, CG, JO and NG wrote the manuscript. All authors read and approved the manuscript.X. a. pv. citri biofilm formation.Complete list of the differentially expressed proteins during Click here for fileOligonucleotides used in qRT-PCR of selected genes.Click here for file


Social anxiety is characterized by fear of evaluative interpersonal situations. Many studies have investigated the perception of emotional faces in socially anxious individuals and have reported biases in the processing of threatening faces. However, faces are not the only stimuli carrying an interpersonal evaluative load. The present study investigated the processing of emotional body postures in social anxiety. Participants with high and low social anxiety completed an attention-shifting paradigm using neutral, angry and happy faces and postures as cues. We investigated early visual processes through the P100 component, attentional fixation on the P2, structural encoding mirrored by the N170, and attentional orientation towards stimuli to detect with the P100 locked on target occurrence. Results showed a global reduction of P100 and P200 responses to faces and postures in socially anxious participants as compared to non-anxious participants, with a direct correlation between self-reported social anxiety levels and P100 and P200 amplitudes. Structural encoding of cues and target processing were not modulated by social anxiety, but socially anxious participants were slower to detect the targets. These results suggest a reduced processing of social postural and facial cues in social anxiety. Social anxiety disorder (SAD) has been characterized by a fear of social situations, often accompanied by their avoidance ,2. CogniElectrophysiology has provided temporal cues of these biases in selective attention processes. First, the P100 component appears enhanced when SAD individuals are confronted with pairs of neutral and angry faces as compared to neutral-happy faces . The P10Interestingly, when the faces are presented one by one and not in pairs, SAD individuals display enhanced P100 compared to that recorded in non-anxious people in response to natural but also schematic and artificial facial stimuli, irrespective of the emotional expressions –37. ThisThe occipital P200 component has also provided evidence of disturbed face processing in social phobia. That component is enhanced during fearful  and angrIn addition to hypervigilance to faces, social phobic individuals may also present attentional biases towards other social cues . Indeed,Faces and bodies also evoke a similar N170 response . This teFor all these reasons, body postures are stimuli of choice in studying biases towards threatening social stimuli in social anxiety. If SAD individuals are constantly scanning their environment for subtle signs of negative evaluation , postureParticipants received details regarding the aims of the study and the procedure to be followed before to give their informed written consent. The study was approved by the Ethics Committee of the Institute of Psychology (Catholic University of Louvain) and was conducted according to the principles described in the Declaration of Helsinki.Thirty-six participants were selected from a group of 250 university students screened using the Liebowitz Social Anxiety Scale . The crt(30)=10.105, p=.000), and that they were more afraid of negative evaluation (t(30)=6.524, p=.000). However, groups did not differ according to trait-anxiety (t(30)=1.366, p=.183). All volunteers were native French speaking, right-handed, between the ages of 18 and 23 years =.673, p=.506), with normal/corrected vision, and no history of psychiatric or neurological disorders.Sample characteristics are reported in The experimental procedure used an ‘attention-shifting paradigm’, similar to the one used by Bar-Haim et al.  see Fig. Each trA practice bloc of 20 trials alternating the different types of cue stimuli was performed before the beginning of the experiment. Participants were then presented with the four experimental blocs. Each bloc comprised one type of cue-stimuli defined by the following variables: type  and gender . Each bloc comprised 96 trials and was offered twice in a randomized order, so the entire experiment comprised 768 trials and lasted 30 minutes.Participants were instructed to focus their gaze on the stimuli presented in the centre of the screen, and then to identify the shape of the target, which could appear in four different locations. Half of the participants had to press “1” in the response box with their left forefinger if the target was a “X”, and “2” with their right forefinger if it was a “O”, and half of participants received the reverse instruction. Participants were informed they could perform the task without ocular movement, and they were asked to refrain from making eye movement. They sat in a chair in a dark room with their head placed 1 m from the screen and restrained in a chin rest. The entire experiment took approximately 50 minutes per participant.The electroencephalogram (EEG) recordings were performed with 32 electrodes mounted in an electrode Quick-Cap with the standard 10-20 International System and intermediate positions. Recordings were made with a linked mastoid physical reference, but were re-referenced by using a “common average” . The EEGF=.000 p=.991, η2=.000), congruently with recent results [Analyses focused on the ERP components elicited by cues and targets, separately see ,74. The F1,=.000 p=. results ,33,56, sStatistical analyses were computed using Statistical Package for Social Sciences, 17th version (SPSS 17.0). Behavioural data see  were anaIn the second step, Pearson correlation coefficients were used to explore the relation between the psychometric factors  and the ERP parameters .F=2.986, p=.094, η2=.091). However, Emotion significantly influenced performance =16.488, p=.000, η2=.355): targets occurring with positive stimuli were less efficiently detected (76.5%) than ones accompanied by neutral (80.2% - p=.000) or threatening stimuli (79.0% - p=.000). The comparison between neutral and threatening stimuli did not reach significance (p=.081).Correct responses: Analyses did not reveal main effect of Cue =4.078, p=.023, η2=.120). To follow up this interaction, separate ANOVAs were conducted for facial and postural Cues. Concerning facial cues, the Emotional effect =15.684, p=.000, η2=.342) revealed significantly better performance for neutral cues (82.3%) as compared to threatening  and positive faces , and higher performance for threatening as compared to positive faces (p=.013). Concerning body postures, the emotional effect =6.588, p=.003, η2=.183) also revealed lower performance for positive postures (75.7%) as compared to neutral  and threatening ones , but without difference between these last emotional categories (p=.832).However, this effect was expressed differently amongst facial and postural cues, as indicated by a significant interaction between Cue and Emotion =1.750, p=.196, η2=.055) and did not interact with Cue =.320, p=.576, η2=.011) or Emotion =.260, p=.748, η2=.009).Group did not influence correct response production =5.306, p=.028, η2=.150), highlighting slower response in HSA as compared to LSA (538.2 vs 503.1ms). No other significant main effect or interaction was found.Response latencies (on correct responses): The 3 x 2 x 2 ANOVA alone displayed a main effect of Group =4.596, p=.040, η2=.133) revealing reduced P100 amplitudes in HSA (.457µV) as compared to LSA (1.756µV)  see .No other significant main effect or interaction was found.F=6.762, p=.014, η2=.184), with enhanced waves recorded in response to faces (-1.324µV) as compared to body postures (-.804µV) (see Cue modulate N170 amplitude (4µV) see .F=7.876, p=.001, η2=.208): Neutral cues evoked reduced amplitudes (-.831µV) as compared to positive  or threatening cues .The N170 was also modulated by Emotion =8.781, p=.001, η2=.226) revealed that the emotion effect was not present for body postures =1.983, p=.146, η2=.062) but well for facial cues =12.976, p=.000, η2=.295): Angry faces (-1.732µV) elicited a more negative N170 than happy  and neutral faces .An interaction between Cue and Emotion =4.289, p=.047, η2=.125) revealing reduced P200 amplitudes in HSA (2.466µV) as compared to LSA (3.533µV)  see .F=10.808, p=.003, η2=.265) showed higher amplitudes on the right hemisphere (3.287 vs. 2.712µV).Second, a main effect of Hemisphere =3.754, p=.029, η2=.111) was decomposed further to show no emotion effect on postures =1.731, p=.186, η2=.050) but a significant influence of Emotion on faces =3.704, p=.030, η2=.110) with higher responses for neutral (3.240 µV) as compared to angry faces .Finally, a interaction between Cue and Emotion =4.806, p=.036, η2=.138) indicated that enhanced amplitudes were produced for targets accompanied by a facial cue, as compared to a body posture =6.777, p=.014, η2=.184) showed higher amplitudes on the right hemisphere.Second, a main effect of Hemisphere =8.166, p=.008, η2=.214), that was decomposed into separate ANOVAs for faces and postures. P100 amplitudes were higher on the right hemisphere for faces =10.988, p=.002, η2=.262) but not for bodies =2.708, p=.110, η2=.080).However, these effects were further qualified by a significant interaction between Cue and Hemisphere  to avoid the problem of comorbidity. However, positive correlation between trait and social anxiety is usual in patients with social phobia  and one In summary, the present study aimed to explore the influence of social anxiety on the processing of social postural and facial cues. Our results show that social anxiety is associated with reduced processing of task-irrelevant postures and faces (diminished P100 and P200 amplitudes). The lack of modulatory effect of cue category or emotional load argues in favour of the automatism of this mechanism, which occurs before the identification of the stimulus and independently of it. The later stages of cognitive processing of targets were comparable between LSA and HSA but SAD modulated task performance by slowing the response latencies. This slowdown appears to be directly associated with the augmentation of evaluation fear symptoms. To conclude, these results argue for a similar processing of postures and faces, but also for the distinct influence of social anxiety in two stages of cognitive processing of social cues. These hypotheses have to be tested in further experiments to better understand the influence of the different facets of social anxiety on cognitive processing.


Escherichia coli. However, product titers and yields remain low. In this work, we demonstrate new methods for improving fatty acid production by modifying central carbon metabolism and storing fatty acids in triacylglycerol. Based on suggestions from a computational model, we deleted seven genes involved in aerobic respiration, mixed-acid fermentation, and glyoxylate bypass  to modify the central carbon metabolic/regulatory networks. These gene deletions led to increased total fatty acids, which were the highest in the mutants containing five or six gene knockouts. Additionally, when two key enzymes in the fatty acid biosynthesis pathway were over-expressed, we observed further increase in strain △cyoA△adhE△nuoA△ndh△pta△dld, leading to 202 mg/g dry cell weight of total fatty acids, ~250% of that in the wild-type strain. Meanwhile, we successfully introduced a triacylglycerol biosynthesis pathway into E. coli through heterologous expression of wax ester synthase/acyl-coenzyme:diacylglycerol acyltransferase (WS/DGAT) enzymes. The added pathway improved both the amount and fuel quality of the fatty acids. These new metabolic engineering strategies are providing promising directions for future investigation.Previous studies have demonstrated the feasibility of producing fatty-acid-derived hydrocarbons in  Escherichia coli. For example, Schirmer et al. identified an alkane biosynthesis pathway from cyanobacteria and transferred it into E. coli, which led to the production and secretion of C13 to C17 alkanes and alkenes [E. coli could be engineered to produce structurally tailored biodiesel, fatty alcohols, and waxes [ Fatty-acid-derived hydrocarbons such as alkanes, alkenes, and biodiesel are promising biofuels. Previous research efforts have demonstrated the feasibility of bio-hydrocarbon production in the well-studied model microorganism nd waxes . HoweverE. coli to improve fatty acid production, as well summarized in a recent review [E. coli to increase medium-chain fatty acids. For example, increases in the levels of medium- and long-chain fatty acids  were achieved by expressing thioesterase (U31813) from Cinnamomum camphorum seeds in an fadD knockout E. coli strain [Umbellularia californica was expressed in E. coli, the predominant fatty acid chain length was changed dramatically from C16 to C12, with up to 75% of the total fatty acid composition consisting of C12 [E. coli was increased by coexpression of fabA, fabB and AtFatA, leading to a 2.3:1 ratio of unsaturated to saturated fatty acids [The past several years have seen increasing efforts in modifying metabolic pathways in t review . Strategt review ,4-8 can t review . In addii strain . Lennen ty acids . ackA-pta and adhE, together with the overexpression of acetyl-CoA synthetase (Acs) [E. coli recombinant strain that showed a 4-fold increase of intracellular malonyl-CoA compared to the wild type [It should be noted that all the research efforts described above focused on local pathways directly related to fatty acids; rerouting metabolic intermediates in distant central carbon pathways was not considered. The biosynthesis of fatty acids requires metabolic precursors and cofactors, which are provided through the concerted action of a large number of interconnected metabolic pathways. Hence, modifications in distant pathways can potentially improve fatty acid synthesis through redistribution of metabolite precursors or indirect global regulatory effects. For example, the level of malonyl-CoA, a precursor for fatty acids, polyketides and flavonoids, was improved 15-fold through the deletion of se (Acs) . The recse (Acs) . Due to se (Acs) , OptReg se (Acs) , OptForcse (Acs) , and Optild type ; Ranganaild type .E. coli, these species capable of TAG and/or WE production exhibit low carbon source flexibilities, require stringent culture conditions, and lack efficient genetic modification tools. Moreover, TAG and WE production with E. coli may offer many advantages over traditional production from plant oils or animal fats by circumventing geographical and seasonal limitations [E. coli [E. coli. One key reason for low bio-hydrocarbon yield and efficiency is that free fatty acids, alkanes, and alcohols are toxic to microorganisms and inhibit their growth. Besides elucidating mechanisms of toxicity and engineering tolerant strains , anotheritations , allowin[E. coli ,23. NeveE. coli strains to produce fatty acids efficiently and to accumulate fatty acids in the form of TAG, similar to oleaginous species. We carried out a series of genetic modifications on the central carbon metabolism of E. coli, suggested by an in silico metabolic and regulatory network model, to maximize fatty acid biosynthesis. In addition, we successfully introduced a TAG biosynthesis pathway into E. coli to serve as storage for fatty acid carbon chains. In this work, our goal is to engineer E. coli BL21 StarTM (DE3) (Invitrogen). Gene knockouts were constructed by P1 phage transduction as previously described [cyoA::kan, ΔadhE::kan, ΔnuoA::kan, Δndh::kan, Δdld::kan, ΔiclR::kan, and ΔdgkA::kan. To knock out simultaneously nuoA and pta, which are in close proximity on the E. coli chromosome, we used strain GNB10760 containing △nuoA::FRT △pta-ackA::kan [TM (DE3) and its derivatives. LB agar with 100 μg/mL kanamycin was used as the selective medium. Transductants were purified from residual P1 phage by isolation streaking on LB agar supplemented with 0.8 mM sodium citrate and 100 μg/mL kanamycin, and verified by colony PCR as per published procedures [TM (DE3) and its derivatives by electroporation transformation. LB agar containing 100 µg/mL ampicillin or 25 µg/mL chloramphenicol was used as the selective medium. Strains and plasmids used in this study are listed in escribed ,25. P1 pescribed ,25, ΔcyockA::kan  as the daccBCDA from E. coli [E. coli thioesterase 'tesA, a ‘leaderless’ version of TesA with cytosolic expression [accBCDA and/or thioesterase.Plasmids pMSD8 and pMSD15 were gifts from Dr. John Cronan. pMSD8 carries  E. coli , while ppression . These tUnless otherwise stated, M9 minimal medium containing 2% glucose was used for fatty acid analysis of knockout strains. LB Lennox broth, and LB Lennox broth supplemented with different concentrations of glucose and sodium bicarbonate as indicated in the text below were used for characterizing TAG-producing strains. Pre-culture in 50 mL test tubes containing 5 mL of medium was performed overnight at 37°C with 250 rpm shaking. Pre-cultures were diluted 1:100 into 100 mL fresh media in 500-mL conical flasks. Ampicillin (100 µg/mL), chloramphenicol (25 µg/mL), and kanamycin (30 µg/mL) were added as needed. Cells were grown at 37 °C to an optical density at 600nm of 0.6, and then incubated with 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) or 0.2% arabinose. After 24 or 48 hrs of culturing, cell cultures were harvested for analysis.E. coli [E. coli [in silico design framework allowed two types of network modifications - removing reactions and re-activating regulatorily inhibited reactions. It was based on a bi-level optimization formulism. The outer level optimization aimed to maximize the synthesis of a product by identifying reactions for removal/activation, through the use of binary decision variables. The inner level optimization served as a constraint of the outer level problem and simulated the network of reactions dictated by the outer level. It considered mass balance of all metabolites along with other constraints such as reversibility of reactions and determined the flux distribution assuming a cellular objective of maximizing growth. The above bi-level mixed integer linear programming (MILP) problem was transformed into a single-level optimization problem [A computational framework developed for maximizing biochemical production in E. coli  was util[E. coli  and inco problem  and solv2SO4 at 0.6 mL/min. Refractive index and UV-vis detectors were utilized. Cell growth status was determined by measuring the optical density of the culture at 600nm using a spectrometer. The concentrations of glucose, acetic acid, and lactic acid were measured through HPLC. Culture samples were first filtered through 0.22-μm sterile filters and loaded to an Aminex HPX-87H ion-exchange column (Bio-Rad) operated at 65°C, then eluted with 5 mM HE. coli cultures were harvested by centrifugation at 3,200 g x 10 minutes.  Cell pellets were washed with Phosphate Buffered Saline (PBS) and dried at 70°C for 24 hrs. The dry cell pellets were subjected to ethanolysis at 90°C for 2 hrs in 5% HCl in anhydrous ethanol. The resulting Fatty Acid Ethyl Esters (FAEEs) were quantified with an Agilent 6890 GC equipped with a 50 m x 0.2 mm x 0.33 mm HPx5 capillary column, flame ionization detector (FID), and autoinjector. 2 µL FAEE samples were analyzed after split injection (1:10). Helium was used as a carrier gas with a constant flow rate of 1.9 mL/min. The temperatures of the injector and detector were 325 and 350°C, respectively. The following temperature program was applied: 5°C for 3 min, increase 10°C/min to 300°C, then 300°C for 10 min. Quantification was done by utilizing authentic standards.Duplicates of 50 mL N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) were added to the GC vials. The vials were stirred for 1 min and then allowed to react for 30 min at room temperature. Samples were diluted in n-heptane (700 µL), stirred for 1 min, and injected into an Agilent 7890 GC-FID with an ASTM6584 column . Injection (1.0 µL) was made to a cool-on-column inlet in oven-track mode with an initial oven temperature of 50°C. After a 1-min hold, the temperature was ramped to 180°C at 15°C/min, 230°C at 7°C/min, and 380°C at 30°C/min. Helium was used as carrier gas at a constant flow rate of 3.0 mL/min. The FID detector temperature was 380°C. Nitrogen served as the makeup gas (30 mL/min). Peaks corresponding to TAGs were identified by retention time and quantified based on internal standard calibration of tricaprin.Triacylglycerols were identified and quantified as described previously . Dry celE. coli. In this work, we employed an in silico modeling and optimization framework that integrates metabolic and regulatory networks to identify targets for genetic manipulations. Similar to OptORF [E. coli [adhE, pta, dld, cyoA, nuoA, and ndh, and activation of the glyoxylate bypass ; ndh encodes the NADH:ubiquinone oxidoreductase II (NDH-2). Removal of these three genes disrupts the oxidation of NADH into NAD+. The increase of NADH in the cell leads to its oxidation into NAD+ and the coupled reduction of NADP+ into NADPH by the membrane-bound pyridine nucleotide transhydrogenase, PntAB [cyoA, nuoA and ndh. On the other hand, the glyoxylate pathway, catalyzed by two enzymes encoded by aceA and aceB, bypasses the conversion of isocitrate to succinate in the TCA cycle, during which one NADPH as well as one NADH is generated. The activation of this glyoxylate bypass leads to more malate, which can be converted back to pyruvate and generate one NADPH when catalyzed by maeB encoded malate dehydrogenase. Hence activation of the glyoxylate bypass might also lead to more NADPH. This can potentially be achieved by deletion of gene iclR, the transcriptional repressor of the glyoxylate bypass operon (aceBAK). Expression of the aceBAK operon is significantly induced by disruption of iclR during growth on glucose [Upon close examination of the corresponding reactions interconnected with others in the network, we hypothesized that this scheme suggested by the model as the optimal design would be promising because it combined two basic strategies: to direct more carbon towards the fatty acid synthesis pathway, by eliminating competing pathways, and to increase the level of cofactor NADPH, which is required by the 3-oxo-acyl-ACP reductase involved in fatty acid biosynthesis . Deletiooduction . The res E. coli . cyoA ene, PntAB . Thus, t glucose . in silico simulations using flux balance analysis [cyoA, adhE, nuoA, ndh, pta, dld, and iclR. P1 transduction was employed to knock out these targeted genes in the above order. The resulting strains were grown using M9 minimal medium supplemented with 2% glucose. Samples were collected after 24 hrs for quantification of glucose and mixed acids by HPLC, and of fatty acids by GC-FID , the total amount of fatty acids was increased from 58.5 mg/g dry cell weight to 66.9 mg/g dry cell weight in this strain. The final lactic acid concentration was increased from 0.28 g/L to 0.94 g/L, while the final acetic acid concentration was reduced slightly, from 2.6 g/L to 2.2g/L. The final OD decreased from 2.9 to 2.3, whereas the amount of glucose consumed was not affected appreciably. We then deleted adhE and created strain △cyoA△adhE. All the properties described above remain similar to those of strain △cyoA. Next we examined strain △cyoA△adhE△nuoA, in which the total amount of fatty acids increased to 72.6 mg/g dry cell weight. The final lactic acid concentration increased substantially to 3.2 g/L; the final acetic acid concentration was reduced slightly to 1.8 g/L. The final OD further decreased to 1.7, whereas glucose consumption remained comparable. Further deletion of ndh caused a slight reduction of fatty acid content in strain △cyoA△adhE△nuoA△ndh. Despite this unexpected result, we continued with the overall scheme and next removed the phosphate acetyltransferase encoded by pta, which is involved in acetate formation from acetyl-CoA. Not surprisingly, the final acetic acid concentration was reduced sharply to 0.45 g/L. This also increased substantially the total amount of fatty acid to 84.9 mg/g dry cell weight, the highest among this set of knockout strains. On the other hand, the final lactic acid concentration was increased to 4.9 g/L. Our second last target was dld, which is responsible for lactate biosynthesis. Contrary to model prediction, its deletion reduced the total amount of fatty acids and led to an increase of lactic acid. Finally, we deleted iclR in order to activate the glyoxylate pathway. Unexpectedly, the total amount of fatty acids was reduced significantly. After further review of literature related to the glyoxylate pathway, we speculate that there are two possible reasons for this reduced fatty acid production. First, the glyoxylate pathway may not have been effectively activated in our strain (see discussion at the end of this section). Second, even though activation of the glyoxylate bypass pathway could potentially benefit fatty acid synthesis by increasing the NADPH level and preventing carbon loss as CO2 in the TCA cycle, this pathway would elevate fatty acid degradation [E. coli growth on acetate or fatty acids as the sole carbon source [We first created strain Δradation  as it isn source . During E. coli exist largely as C12 - C19 molecules, with palmitic acid (C16:0) being the most abundant  and cofactor (i.e. NADPH) from modifications of central carbon metabolism, the capacity of the fatty acid biosynthesis pathway may become a bottleneck. Therefore, combining the rewiring of central metabolic/regulatory networks and modifications of local pathways, as explored in previous studies ,4,7, couE. coli to produce tryacylglycerol, we overexpressed a wax ester synthase/acyl-coenzyme: diacylglycerol acyltransferase (WS/DGAT) enzyme from Streptomyces coelicolor (Sco0958) in BL21 StarTM DE3. No TAG production was observed in the recombinant strain (dgkA), based on a previous report that Sco0958 possessed DGAT activity and its overexpression in a dgkA knockout mutant of E. coli led to the detection of TAG by thin layer chromatogram (TLC) [E. coli, dgkA is involved in the recycle of diacylglycerol to phosphatic acid. Diacylglycerol is produced as a by-product in Membrane-Derived Oligosaccharide (MDO) biosynthesis and serves as the immediate precursor of TAG (dgkA /SCO0958 showed TAG peaks on GC chromatograph (E. coli. To enable t strain . We thenam (TLC) . In E. cr of TAG . The resatograph , demonstE. coli, four other WS/DGAT enzymes from different oleaginous bacteria  was 29.7%, while only 9.3% was unsaturated fatty acids . The cyclopropane fatty acid fraction was reduced to 21.8%, 19.1%, and 9.8% in strains △dgkA/SCO0958, △dgkA/WS1, and dgkA/WS2, respectively; whereas the unsaturated fatty acid fraction was increased to 18.9%, 26.4%, and 34.2%, respectively. The saturated fatty acid fraction  was not changed significantly by overexpressing WS/DGAT enzymes in strain △dgkA. Microbial fuels with a high proportion of unsaturated fatty acids possess favorable low temperature flow properties, improving operation under winter conditions [E. coli enters stationary phase from log phase, unsaturated fatty acids are converted to cyclopropane fatty acids due to modification of the cis double bond by cyclopropane fatty acid synthase [E. coli not only increases the total amount of fatty acids, but also improves the fuel properties of fatty acids.Cellular fatty acid composition was also determined . In BL21nditions . Howeversynthase ,44. It adgkA/WS1 was more effective in producing TAG than strain △dgkA/SCO0958. Hence, enzyme WS1 was used in further TAG production studies, which are presented below. As described above, strain △dgkA/WS1 were compared among five different culture media  in MA/9 minimal medium supplemented with sodium gluconate and glycerol as carbon and energy sources [Next, we tested the effect of media on TAG production. The amounts of TAG and fatty acids produced by strain △re media . These t sources .E. coli by modifying central carbon metabolism. On the other hand, we were able to produce TAG for storing fatty acid carbon chains by introducing a heterlogous TAG biosynthesis pathway. To determine if the combination of these two strategies would further improve fatty acid production, we introduced the TAG biosynthesis pathway into strains △cyoA△adhE△nuoA△ndh△pta△dld and △cyoA△adhE△nuoA△ndh△pta△dld△iclR . The culture medium LB 1-2, which favored TAG production in the previous section, was utilized in characterizing the resulting strains. Results of total fatty acids are shown in dgkA/WS1 and 7△-△dgkA/WS1. All other strains showed comparable or even reduced total fatty acid contents. We also measured the fatty acid contents in these strains in another culture medium LB 5-10 with higher concentrations of glucose and sodium bicarbonate  and in three other random combinations (grey columns).The total amount of fatty acids in recombinant (TIF)Click here for additional data file.Figure S2dgkA/pBAD; 2-∆fadE∆dgkA/pBAD;3-∆cyoA∆adhE∆nuoA∆dgkA/pBAD;4-∆cyoA∆adhE∆nuoA∆fadE∆dgkA/pBAD.TAG production in four additional knockout strains: 1-∆ Cells were cultured in LB and collected at 24 hrs after induction for TAG analysis by GC-FID. (TIF)Click here for additional data file.


T, was determined. The genome comprises a 1,750,259-bp circular chromosome containing single copies of 3 rRNA genes, 43 tRNA genes, and 1,896 protein-coding sequences. In silico genome-genome hybridization suggests the proposal of a novel species, “Thermofilum adornatus” strain 1910bT.The complete genomic sequence of a novel hyperthermophilic crenarchaeon, strain 1910b Thermofilaceae  located near Pauzhetka . The strain grows optimally at 92°C and pH 6.0 to 6.5 in the presence of Fervidicoccus fontis strain 1910a culture broth with glucose and peptone as the substrates.Strain 1910bT and T. pendens strain Hvv3T.A BLAST search  revealedT, we used a combination of fragment and mate-paired library approaches. The fragment library was sequenced with the Illumina MiSeq system, and the mate-paired library, with an average insert length of 2,200 bp, was sequenced with the Life Technologies PGM system. A total of 0.5 million Illumina paired-end reads were trimmed and corrected with the Quake sequencing error correction tool  represent the same species. Based on this evidence, we assume that strain 1910bT represents a novel species “Thermofilum adornatus.” Its complete description will be published elsewhere.The chromosome of strain 1910blgorithm  revealedThermofilum adornatus” strain 1910bT has been deposited in NCBI GenBank under the accession number CP006646.The genome sequence of “


Acetoin and 2,3-butanediol are two important biorefinery platform chemicals. They are currently fermented below 40°C using mesophilic strains, but the processes often suffer from bacterial contamination.Geobacillus strain XT15 capable of producing both of these chemicals under elevated temperatures, thus reducing the risk of bacterial contamination. The optimum growth temperature was found to be between 45 and 55°C and the medium initial pH to be 8.0. In addition to glucose, galactose, mannitol, arabionose, and xylose were all acceptable substrates, enabling the potential use of cellulosic biomass as the feedstock. XT15 preferred organic nitrogen sources including corn steep liquor powder, a cheap by-product from corn wet-milling. At 55°C, 7.7 g/L of acetoin and 14.5 g/L of 2,3-butanediol could be obtained using corn steep liquor powder as a nitrogen source. Thirteen volatile products from the cultivation broth of XT15 were identified by gas chromatography–mass spectrometry. Acetoin, 2,3-butanediol, and their derivatives including a novel metabolite 2,3-dihydroxy-3-methylheptan-4-one, accounted for a total of about 96% of all the volatile products. In contrast, organic acids and other products were minor by-products. α-Acetolactate decarboxylase and acetoin:2,6-dichlorophenolindophenol oxidoreductase in XT15, the two key enzymes in acetoin metabolic pathway, were found to be both moderately thermophilic with the identical optimum temperature of 45°C.This work reports the isolation and identification of a novel aerobic Geobacillus sp. XT15 is the first naturally occurring thermophile excreting acetoin and/or 2,3-butanediol. This work has demonstrated the attractive prospect of developing it as an industrial strain in the thermophilic fermentation of acetoin and 2,3-butanediol with improved anti-contamination performance. The novel metabolites and enzymes identified in XT15 also indicated its strong promise as a precious biological resource. Thermophilic fermentation also offers great prospect for improving its yields and efficiencies. This remains a core aim for future work. Acetoin (3-hydroxybutan-2-one) is widely used in food, flavour, cosmetics, and chemical synthesis. Its redAcetoin and 2,3-butanediol can be produced by bacterial fermentation using renewable biomass. Such platform chemicals are regarded as potential alternatives of non-renewable fossil materials[The scientific understanding and the technological development leading to the production of acetoin and 2,3-butanediol have undergone four periods, evident from the number of the main publications. During the first period 1920s-1981), research work focused on product determination, the basic characterization of microbial properties and development of fermentation processes. Period II (1982–2001) addressed the main biological questions concerning pathways, enzymes, genes, and regulations. This ac1, researTo date, however, almost all these studies have only reported the production of acetoin and 2,3-butanediol from mesophilic fermentation processes or enzymatic reactions, typically at the temperature range of 30-40°C . To avoIn order to resolve these problems, thermophilic fermentation processes, typically operated at 50 – 60°C, have attracted attention over the past few years. RecentlIn this work, we report the isolation and characterization of a novel thermophilic bacterium for the production of acetoin and 2,3-butanediol and the related investigation of its novel metabolites and enzymes, aiming at developing a more efficient and effective fermentation process utilizing the thermophilic strain.Produced water (PW) is the water that is produced along with the crude oil. This isolate XT15 was screened from a PW-based glucose-peptone-yeast extract (PW-GPY) culture medium after 100°C sterilization. The strain was found to grow well on GPY agar, yielding large , white , irregular edged, surface-wrinkled, and opaque colonies. Scanning electron microscopy (SEM) micrographs of XT15 revealed rod-shaped cells of 0.4-0.5 μm × 0.9-1.8 μm in size. Some of the physiological characteristics of XT15 are given in TableGeobacillus derived from the similarity of the 16S rRNA sequences of Geobacillus species , G. thermoleovorans, and G. kaustophilus. Strain XT15 (GenBank accession No. HQ891030.1) is clearly a member of Group I and it is closest to G. kaustophilus and G. bogazici. This strain has been designated as Geobacillus sp. XT15 and deposited in China Center for Type Culture Collection with deposition number M2011022.There are four main branches or groups in the phylogenetic tree of the genus Geobacillus species[Compared with other thermophilic bacteria, the ability of degradation and utilization of hydrocarbons at high temperature is another important feature of  species. After oFor temperature optimization, experiments were performed at 28°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C, respectively. Generation time (doubling time) was calculated according to the specific growth rate. As shown in FigureFor pH optimization, the pH values of the fermentation media were adjusted to 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, respectively, before autoclaving. As shown in FigureThe effect of DO on bacterial growth and product yield was tested by either changing the medium loading volume in shaking flasks or comparing aerobic and anaerobic cultivation. Loading 30 or 20 mL medium per flask did not alter the yield of acetoin. This was because when DO was above a certain level , changes in DO concentration would not affect production any longer. However, under anaerobic condition, no growth or product was detected during a one-week period cultivation of XT15.Geobacillus sp. XT15 could be further developed to utilize cellulosic biomass as the feedstock.Galactose, mannitol, arabionose, and xylose were used as carbon source individually and their influences were compared with glucose (control). As shown in FigureFor nitrogen source screening, peptone, spray-dried corn steep liquor powder (CSLP), malt extract powder, urea, ammonium sulfate, and sodium nitrate were tested Figure. PeptoneAs shown above, glucose was a suitable carbon substrate. The original concentration of glucose in the GPY medium formulation (i.e. 20 g/L) was far from enough to support the maximum acetoin and 2,3-butanediol production. Then glucose concentration in the medium was added up to 220 g/L. Bacterial growth was not influenced by possible substrate inhibition and the high osmotic pressure (data not shown). Therefore, the fermentation medium was changed to: glucose at 220 g/L, CSLP from 50 to 90 g/L, yeast extract at 10 g/L, deionized water, pH 8.0. As shown in FigureDuring the organic solvent extraction process, the addition of a large amount of salt into the cultivation broth was helpful to improve the extraction efficiency. Low pH was effective for the extraction of organic acids. By gas chromatography (GC) analysis, no additional substances (GC peaks) appeared when the aqueous phase was changed from acidic to neutral or alkaline pH. As shown in Figure-, -, and meso-2,3-butanediol. The first two kinds are racemic mixtures and they can only be separated from each other in a chiral column[meso form by ordinary non-chiral GC capillary columns.Peaks 1, 2, 7, and 9 could be easily recognized as diacetyl, acetoin, 2,3-butanediol, and 2,3-butanediol, respectively, in gas chromatography–mass spectrometry (GC-MS) identification. There are 3 stereoisomers of 2,3-butanediol: Ammonia was generated in the medium by the oxidative deamination of amino acids. Nitrogen-containing heterocyclic compounds could be formed from acetoin and ammonia by non-biological processes. ControlAcetylbutanediol , prepared as described before, was useIn addition, 4 organic acids and phenylethyl alcohol were determined in the products of XT15. Therefore, all the main volatile products were identified as shown in Tableα-Acetolactate decarboxylase (ALDC) and acetoin:2,6-dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR or AoDH ES E1) are the key enzymes in acetoin biosynthesis pathway and catabolism pathway, respectively. As shown in FiguresGeobacillus sp. XT15.The spontaneous cleavage (SC) of α-acetolactate should not be neglected as shown in FigureBacillus[Klebsiella[Enterobacter[Serratia[Paenibacillus[By isolation from different environments or genetBacillus,25, Kleblebsiella, Enteroberobacter, Serrati[Serratia, and Paeibacillus,30 have Bacillus strains, which were known as acetion producers, were also isolated from the PW of Shengli Oilfield, China. Geobacillus species were regarded as a branch of the Bacillus genus before Nazina et al. reclassified them in 2001[Geobacillus species, including the ability of hydrocarbon utilization. But XT15 is also capable of acetoin excretion (positive in Voges-Proskauer test), which is unusual in the genus Geobacillus[Geobacillus sp. XT15. This would distinguish XT15 from other Geobacillus species. The substrate conversion rate of XT15 was equivalent to that of Bacillus subtilis CICC 10025[During enhanced oil recovery process, additional water is often injected into reservoirs to help force the crude oil to the surface. Both the injected water and the natural water lying under the hydrocarbons are eventually produced along with the oil. Therefore, the bacteria collected from PW may come from ground or underground. In recent years,  in 2001. Strain obacillus. The recCC 10025.Geobacillus species are chemoorganotrophic, obligately thermophilic, aerobic or facultatively anaerobic, Gram positive, rod-shaped bacteria. Most species are widely distributed in nature. Oxygen is the electron acceptor, replaceable in some species by nitrate. The growth-temperature range is 37-75°C, with an optimum at 55-65°C. Growth occurs in a pH range of 6.0 to 8.5, with an optimum at pH 6.2-7.5. Therefore, the optimum temperature of XT15 is relatively lower, but the pH range is a bit higher.According to Nazina et al., GeobaciOptimization of culture medium components, especially carbon source and nitrogen source, is not only yield-relevant but also cost-effective. Acetoin and 2,3-butanediol are important primary metabolites like ethanol, acetate, and lactate in various microorganisms and the excretion of them has vital physiological implications. Since pThe influences of fermentation ages on acetoin and 2,3-butanediol production were also studied (unpublished data). The maximum yield could usually be achieved 1–3 h after the time point when the carbon source was depleted in the culture medium. But if the carbon source was excessive and could not be used up, the yield usually remained relatively stable after it grew to a certain extent. However, these finding were not always repeatable. The quality of seed culture had great influence on the maturation age of a fermentation process. But generally the time point of the maximum product yield was approximately the same as that for the maximum biomass yield. In FiguresA noticeable observation is that the ratios between acetoin and 2,3-butanediol in the cultivation broth were variable. This could be explained by the physiological state of the bacteria, which would be substantially influenced by the culture medium, especially the content of the carbon source. When the concentration of glucose was low  were identified from the fermentation broth of ic acids. The traic acids,36. As fic acids. ButyrylSulfolobus solfataricus with an optimum temperature of 80°C. This study thus provided an example of a moderately thermophilic AoDH ES. ALDC and AoDH ES E1 were both active in Geobacillus sp. XT15, indicating that the acetoin metabolic pathway was active in this strain. The optimum temperatures and active temperature ranges of the two key enzymes were consistent with those of growth temperatures, respectively, indicating that the acetoin metabolic pathway originally existed in this bacterium. The acetoin metabolic genes in XT15 could have experienced similar evolutionary processes or even affected with each other with Bacillus by horizontal gene transfer, in consideration of the great similarities between Geobacillus sp. XT15 and some Bacillus species.The first step of the acetoin biosynthesis pathway is the reaction from pyruvate to α-acetolactate catalysed by two enzymes: the catabolic α-acetolactate-forming enzyme and the anabolic α-acetolactate-forming enzyme. HoweverGeobacillus bacterium. At its optimum growth temperature range of 45-55°C, XT15 cells would reproduce every 2 h. The optimum initial pH of the culture medium is 8.0. XT15 prefers organic nitrogen sources like CSLP and has the potential to utilize cellulose hydrolysates. 14 products were identified in its cultivation broth, including a novel metabolite. Its two key enzymes in acetoin biosynthesis pathway and catabolism pathway shared identical optimum temperature of 45°C and wide active temperature range. As a novel naturally occurring thermophilic strain for the production of acetoin and/or 2,3-butanediol, there is potential to enhance its yields in further studies.A novel thermophilic acetoin and 2,3-butanediol producer XT15 was isolated. It is an aerobic, gram positive, rod-shaped 2,6-Dichloroindophenol sodium salt hydrate (≥ 97.0%), ethyl 2-acetoxy-2-methylacetoacetate (97%), and thiamine pyrophosphate (≥ 95%) were bought from Sigma-Aldrich. Natural acetoin (GC ≥ 96%), a fermentative product from glucose, was obtained from Shanghai Apple Flavor & Fragrance Co., Ltd. (China). 2,3-Butanediol (GC ≥ 97%), a mixture of racemic and meso forms, was bought from Sinopharm Chemical Reagent Co., Ltd. (China). Other chemicals used in this study were all commonly available analytical reagents. Spray-dried CSLP used in this study was an industrial grade product from Shandong Zouping Hebanshan Tianyuan Chemical Co., Ltd. (China). The composition of CSLP is shown in TableGPY  or modified GPY media were used in this study. Glucose was autoclaved separately. The oil well PW from Shengli Oilfield  was used to replace deionized water for the preparation of screening culture medium (PW-GPY medium). The PW-GPY medium was loaded into Erlenmeyer flasks and heated to 100°C rapidly. Without delay the flasks were then transferred into a shaking bath controlled at 60°C. After 2 d enrichment, the culture was streaked onto GPY agar and incubated at 60°C for strain isolation. Colonies larger in diameters and with deeper red colour in Voges-Proskauer tests were picked up and inoculated into GPY medium for final screening.Physiological characterization was done using a bacterial biochemical identification kit  according to the manufacturer’s instructions. The partial 16S rRNA gene sequence of the isolate was routinely amplified using universal primers 27 F and 1492R. The PCR products were purified and sequenced using an ABI 3730 DNA Analyzer (Applied Biosystems) by Shanghai Sangon (China). Searches of the GenBank database were conducted by using BLAST. 16S rRN2HPO4·3H2O, 0.47 g/L of NaH2PO4·2H2O, 1.32 g/L of (NH4)2SO4, 0.12 g/L of MgSO4, and 1 ml/L of trace element solution . To avoid the entrainment of nutrients from inoculum, the cells in the seed culture were harvested by centrifugation and washed with sterile saline before inoculated into the MS medium. Control experiments were performed using heat-inactivated XT15 cells.Bacterial growth of XT15 on crude oil was tested in a MS medium containing 500 mg/L of crude oil , 2.28 g/L of KCells from slant cultures were inoculated into a pH modified GPY medium  to prepare the seed culture. After 6–8 h incubation at 55°C with agitation, the grown seed culture was inoculated  into appropriate media for optimization experiments including temperature, medium initial pH, DO, carbon source, and nitrogen source. Unless otherwise noted, all the optimization experiments were performed in triplicates using 100 mL Erlenmeyer flasks (loading 30 mL medium per flask) in a reciprocal shaking water bath controlled at 170 rpm and 55°C. For anaerobic cultivation tests, 200 mL serum bottles fully filled with medium were applied and stirrer bars were placed into the serum bottles to keep the bacterial cells well suspended.600) and dry cell weight (DCW) was calculated from OD600 with a linear correlation factor (1 OD600 = 0.39 DCW g/L). The pH values of the cultivation broth were determined using pH 211 (HANNA instruments). Glucose and lactic acid concentrations were enzymatically determined using an SBA-40C biochemical analyser . Acetoin and 2,3-butanediol concentrations in the fermentation broth were analysed periodically with an Agilent 7890A GC equipped with a flame ionization detector and a 30-m HP-5 capillary column (HP Agilent). Prior to GC analysis, the samples were extracted using ethyl acetate with the addition of isoamyl alcohol as the internal standard. The operating conditions were as follows. Nitrogen was used as the carrier gas. The injector temperature and detector temperature were both set at 280°C. The column oven was maintained at 40°C for 2 min and then programmed to increase to 130°C at a rate of 20°C/min.Daily cell observation was performed using a light microscope . Scanning electron microscopy analysis was performed routinely using JSg for 5 min and then 30 g of NaCl was added into the supernatant. Then the supernatant was adjusted to pH 2.0 using 6 M hydrochloric acid before extraction with 20 mL of dichloromethane. The organic phase was concentrated 10 times before GC-MS analysis using Agilent 7890A-5975C equipped with a 30 m HP-INNOWax Polyethylene Glyco column . Helium was used as the carrier gas. The column oven temperature was initially maintained at 40°C for 5 min and then ramped to 200°C at 15°C min-1, maintained at 200°C for 1 min and then ramped to 230°C at 20°C min-1. Mass spectra in the electron impact mode were generated at 70 eV and scan mode in the range of 20–400 amu. The compounds were tentatively identified by comparing their mass spectra with those contained in the MS data system (NIST08.L library). For the further identification of unknown products, high-resolution GC-MS in CI mode was also performed using Waters GCT Premier.In GPY medium fewer kinds of volatile products were detected. In order to make more metabolites detectable, XT15 was cultured in an enhanced GPY medium with elevated carbon substrate and nitrogen substrate concentrations . After 48 h of cultivation at 170 rpm and 55°C, 150 mL of fermentation broth was centrifuged at 8000 g for 20 min at 4°C, and the supernatant was used as the crude cell extract. ALDC activity was assayed by measuring the amount of acetoin generated from α-acetolactate according to Stormer[Strain XT15 was cultured in GPY medium (20 g/L of glucose) and samples were collected at different stages for the analysis of different enzymes. After culturing for 5 h (glucose was not depleted at this time), the cells were harvested by centrifugation for the determination of ALDC activity. About 5 h after glucose was depleted in the culture medium, the cells were harvested for the determination of AoDH ES E1. The harvested cells were resuspended in 50 mM phosphate buffered saline (pH 7.0) and disrupted by sonication. The disrupted cells were centrifuged at 10,000  Stormer. The cre Stormer. All thePW: Produced water; GPY: Glucose-peptone-yeast extract; SEM: Scanning electron microscopy; MS: Mineral-salts; DO: Dissolved oxygen; CSLP: Corn steep liquor powder; GC: Gas chromatography; GC-MS: Gas chromatography–mass spectrometry; TTMP: 2,3,5,6-tetramethylpyrazine; OXZ: 1-ethanol; TMP: 2,3,5-trimethylpyrazine; CI: Chemical ionization; TIC: Total ion chromatogram; ALDC: α-acetolactate decarboxylase; Ao:DCPIP OR or AoDH ES E1: Acetoin:2,6-dichlorophenolindophenol oxidoreductase; SC: Spontaneous cleavage.The authors declare that they have no competing interests.ZX designed the study, performed experiments, analysed the data and drafted the manuscript; XW and YH executed the experimental work and some data interpretation; FH, XZ, and LX participated in the experimental work; JRL coordinated the study and revised the manuscript. All authors read and approved the final manuscript.


A major problem in the formulation of therapeutic proteins is the irreversible protein aggregation. Recombinant human interferon alpha2b (rhIFNα2b) has poor stability and undergoes physical degradation. The aim of this study was to investigate the effect of solution conditions on the heat-induced aggregation of rhIFNα2b. The protein was incubated for 1 h at 40–70 °C and for up to 240 h at 50 °C and its aggregation tendency was then studied using optical density (at 350 nm), SE-HPLC, dynamic light scattering and SDS-PAGE methods. The effect of various pH  and buffer concentrations  on the aggregation of protein following incubation at 50 °C for 72 h was also evaluated. The results obtained for samples incubated at 50 °C for up to 240 h showed that OD350 and the amount of higher molecular weight aggregates (HMW) increased and the monomer content decreased significantly (p<0.05) as the incubation time increased. Following incubation at various temperatures, a significant increase in OD350, drop in monomer content and increase in the amount of HMW aggregates were observed (p<0.05). Data obtained from incubation of samples at 50 °C for 72 h confirmed that regardless of the buffer concentration, the percentage of monomer at pH 6 was significantly higher than that at pH 7 and pH 5 (p<0.05). At constant pH, although not significant, the same trend was observed when the buffer concentration increased to 100 mM. In conclusion, the change in solution conditions can influence the aggregation extent of rhIFNα2b.  During the past three decades, many therapeutic proteins have been developed for a wide range of human disorders . Howevere.g, thermal, acidic, mechanical, and interface adsorption) that proteins may face during the various production and manufacturing processes  have been identified and reported in the literature. In these processes, proteins are exposed to changes of the properties of the solution environment such as pH, temperature, salt concentration, buffer composition, surfactants, preservatives, shear rate and surfaces  are cytokines with immunomodulatory, anti-proliferative and antiviral properties , 11. Spe at risk . The aimMaterials rhIFNα2b was kindly gifted by Pasteur Institute of Iran . Acrylamide/bis acrylamide solution, ammonium peroxodisulfate, bromophenol blue sodium salt, Coomassie brilliant blue, 1,4-dithiothreitol (DTT), glycerol, glycine, iso-butanol, 2-mercaptoethanol, sodium chloride, sodium dodecyl sulfate (SDS), di-sodium hydrogen phosphate anhydrous, sodium dihydrogen phosphate 1-hydrate, tetramethylethylenediamine (TEMED), and tris (hydroxyl methyl) amino methane (Tris) were purchased from Merck . All solutions were prepared with sterile deionized water . Buffers were filtered through 0.2 μm membranes prior to use. Stress studiesThe thermal stability of rhIFNα2b was investigated following heating the protein samples. Protein samples composed of 100 μg/mL in 100 mM sodium phosphate buffer solution pH 7.0) were incubated  for 1 h at various temperatures  and then cooled to room temperature. The level of heat-induced aggregation and monomer content were monitored by both turbidity measurement  at 350 nm and size exclusion high performance liquid chromatography (SE-HPLC). In another experiment, the samples were incubated at 50 °C (up to 240 h) and analyzed by SE-HPLC, optical density determination, dynamic light scattering and electrophoresis techniques. The relationship between thermal stability of protein with pH of sodium phosphate buffer solution  at 350 nm against sodium phosphate buffer as the blank solution to evaluate the aggregate production. Optical density of the protein sample was determined by ScanDropSize exclusion high performance liquid chromatography (SE-HPLC)Size exclusion separation was carried out on a TSKgel G3000 SWXL column . Analysis was performed at 25 °C under isocratic elution at a rate of 0.5 mL/min with a mobile phase consisting of 100 mM sodium phosphate (pH 7.0), 150 mM NaCl and 0.05% (w/v) sodium dodecyl sulfate (SDS).The column was calibrated by protein standard markers.Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)Gels were composed of a separating gel containing 12.5% (w/v) acrylamide and 0.1% (w/v) SDS, and a stacking gel containing 3% (w/v) acrylamide and 0.1% (w/v) SDS. Gels were run under reducing (sample buffer containing 5% v/v β-mercaptoethanol) and non-reducing (sample buffer without β-mercaptoethanol) conditions at 200 V at room temperature, using a MP300 electrophoresis instrument . Dynamic light scattering (DLS)® software for data acquisition and analysis, was used for particle size measurements. A Malvern Zetasizer Nano ZS  laser light scattering system, equipped with a Nano ZSStatistical analysis® SPSS® Statistics, v19, 2010.Results are reported as mean ± SD. Data obtained was compared using one-way ANOVA. Differences between the treatments were assumed to be significant at p < 0.05. Statistical analysis of all data was performed using IBMHeating stress studiesAn increase in optical density signal in the non-absorbing region of a protein UV spectrum 320 nm) is typically considered as an indicator of larger species of aggregate forms , 20.In t nm is ty350, as the incubation time increased  at 58 °C  in 10 mM NaCl solution at room temperature in the pH range of 3-9 by turbidity measurement and found that this process was fastest at pH 5.6 (et al. (Theoretically, a protein has minimum solubility at its pI due to the minimum protein charge-charge repulsions. Nevertheless, the rate and extent of protein aggregation were expected to be maximal around the pI and negatively correlated with the absolute value of charge , 24. Milat 58 °C . Giger et pH 5.6 . However (et al.  found th (et al. . In this (et al. .Buffer concentrations can also influence the aggregation behavior of proteins to a great extent -32. It hDue to the poor stability, proteins are susceptible to physical degradation, specifically aggregation, affecting the efficacy and safety of the biomolecules. To stabilize the protein against aggregation, solution conditions should be carefully chosen and controlled during various processes. The results obtained in this study indicate that thermal stress mediates rhIFNα2b aggregation. Our observations supported a pH and buffer concentration-dependent model for heat-induced aggregation behavior of rhIFNα2b solutions and confirmed that both factors could diminish the aggregation of the protein during thermal stress. Understanding the effects of such factors would play a key role in the protein stabilization and development of therapeutic protein formulations.