Supplement intake frequency (6.8 ± 8.4 times/24 hrs) was highest during the night hours and lower at the beginning and end of the race. Elevated food and fluid intake among participants tracked across all race segments (P < 0.001). In conclusion, the nutrition strategy employed by ultra-MTBers was similar to those demonstrated in previous studies of ultra-cyclists with some exceptions among selected individuals.We investigated seventy-four ultra-mountain bikers (MTBers) competing in the solo category in the first descriptive field study to detail nutrition habits and the most common food before during and after the 24 hour race using questionnaires. During the race, bananas (86.5%), energy bars (50.0%), apples (43.2%) and cheese (43.2%) were the most commonly consumed food, followed by bread (44.6%), rice (33.8%) and bananas (33.8%) after the race. Average fluid intake was 0.5 ± 0.2 l/h. The main beverage was isotonic sports drink (82.4%) during and pure water (66.2%) after the race. The most preferred four supplements in the four weeks before, the day before, during and after the race were vitamin C (35.1%), magnesium (44.6%), magnesium (43.2%) and branched-chain amino acids (24.3%), respectively. Total frequency of food intake (30.6 ± 10.5 times/24 hrs) was associated with fluid intake (The online version of this article (doi:10.1186/2193-1801-3-715) contains supplementary material, which is available to authorized users. The continuous physical stress in ultra-endurance performances leads to an unavoidable energy deficit Knechtle . In mostThe nutritional strategy during a 24-hour race and a defined plan for feeding can be a key strategy to finish a race successfully Burke . On the Information about energy intake in ultra-endurance cyclists during a 24-hour race is scarce  and the study was approved by the local institutional ethics committee . Ultra-MTBers were notified approximately three months before the race of the study via an e-mail and were informed about the planned investigation with indication that participation is voluntary. Prior to the race, athletes were informed of the procedures and gave their informed written consent.i.e. 40 in Race 1 and 37 in Race 2).In the current study, we observed the nutritional habits of entrants in two 24-hour MTB races held in the Czech Republic in 2012. The first measurement was performed at the ‘Czech Championship 24-hour MTB’ in Jihlava (Race 1). The ultra-MTBers started on May 19th 2012 at 12:00 and finished on May 20th 2012 at 12:00. The course was primarily held on a 9.5 km single-track trail with an elevation of 220 m. The second measurement was performed at the 24-hour race ‘Bike Race Marathon MTB’ in Liberec (Race 2) taking place from June 9th 2012 to June 10th 2012. The course was comprised of a 12.6 km loop with an elevation of 250 m. Nearby temperature during both races ranged from 6°C to 23°C. We acknowledge that the use of the temperatures from the nearest local weather stations underestimated the actual temperatures through some parts of the course. A single aid station was positioned at the start/finish area, where a variety of food and beverages such as hypotonic sports drinks, tea, soup, caffeinated drinks, water, fruit, vegetables, energy bars, bread, soup, sausages, cheese, bread, chocolate and biscuits were available. The ultra-MTBers could also use their own supplies during pre-determined breaks. In total, 77 racers consented to participate in the present study . The subjects were not told what food or fluid to consume during the race. During the event, the time and race speed taken by each athlete to complete each lap were recorded by a personal lap counter (Sportsoft timing spol. s.r.o. chip technology). After the race, the ultra-MTBers sent their completed questionnaires and reports by fax, postal letter or e-mail to the investigator. Participants were instructed to respond within ten days of the event.Volunteers were asked to complete a training diary in the three-month period before the race. The training records consisted of sex, age, the number of years that they had actively participated in cycling, their best result (measured in km) in a 24-hour race, the number of hours spent by training per week as well as the number of hours of cycling training per week. The MTBers also completed a questionnaire about their pre-race diet and the use of supplements before the race. Prior to the start of the race, a second questionnaire about race and post-race nutrition was administered to the participants by trained personnel. The MTBers and their support teams marked items and noted the amount of fluid and the frequency of food and supplements consumed at the aid station or provided by the support crew. For the purpose of recording, the race was broken into discrete segments (n = 23) were calculated using one-way ANOVA after fulfilling two basic presumptions – homogeneity of variance (Levene test) and the normality of distribution (Shapiro-Wilks test). The association between pre-race characteristics and the amount of fluid intake, the frequency of food or supplement intake was calculated using Spearmans rank correlation coefficient. Spearman correlation coefficient was used to reveal the measure of statistical dependence between race speed and fluid intake, between the frequency of food and/or supplements intake and the amount of fluid intake mutually, between total nutrition intake and intake in all race segments. Results in the text are presented as mean ± SD. For all statistical tests, significance was set at a level of 0.05.The statistical software Statistica  was used for the statistical analysis. Differences between pre-race variables  in the racers from the two races in the subgroup  between the racers from two races in this subgroup (P > 0.05). Pre-race characteristics and an average race speed are presented in Table Seventy-four 96.1%) of the 77 race participants successfully finished the race and returned their completed questionnaires. One MTBer was forced to retire due to an equipment failure, while two had medical complications. Fluid intake and the frequency of food and supplement intake were evaluated in a subgroup of 23  ultra-MTBers (30.1%). Incomplete questionnaires were excluded from the final analysis. We found no significant differences in pre-race variables  followed no special pre-race diet  racers accomplished a carbohydrate-rich and twelve racers (16.2%) a protein-rich diet. Twenty-five (33.8%) ultra-MTBers consumed ergogenic supplements with a preference for BCAA (17.6%) and L-Carnitine (10.8%). Thirty-nine (52.7%) racers ingested vitamins and favoured vitamin C (31.1%), multi-vitamin products (12.2%) and B vitamins (12.2%). Forty-nine (66.2%) ultra-MTBers consumed minerals with a special preference for magnesium (44.6%) and multi-mineral products (12.2%) (Table .8% racer® (54.1%), and tea (51.4%) (Table i.e. 12.00 -18.00 hrs) 3.5 ± 1.5 l, respectively 0.6 ± 0.2 l/h] with an average fluid consumption decreasing during each subsequent segment [S2 (i.e. 18.00 - 24.00 hrs) 3.1 ± 1.4 l equal to 0.5 ± 0.3 l/h, S3 (i.e. 24.00 - 6.00 hrs) 2.7 ± 1.3 l, respectively, 0.5 ± 0.3 l/h, S4 (i.e. 6.00 - 12.00 hrs) 2.6 ± 1.2 l equal to 0.4 ± 0.2 l/h. Racers were assigned to the three groups based on their race speed   , equal to 1.6 ± 0.8 times/h; with a decrease (6.9 ± 3.8 times) during the second segment (i.e. 18.00 - 24.00 hrs), 1.1 ± 0.6 times/h, respectively. A moderate increase appeared during the third segment (i.e. 24.00 - 6.00 hrs) 7.0 ± 3.5 times, equal to 1.2 ± 0.6 times/h and during the last fourth segment (i.e. 6.00 - 12.00 hrs) 7.3 ± 3.6 times, equal to 1.2 ± 0.6 times/h, respectively.Ultra-MTBers ate with an average frequency 30.6 ± 10.5 times/24 hrs, equally to 1.3 ± 0.4 times/h. Bananas (86.5%) were preferred before energy bars (50.0%), apples (43.2%), cheese (43.2%), biscuits (40.5%) and bread (37.8%) (Table i.e. 12.00 - 18.00 hrs), 2.0 ± 2.3 times during the second segment (i.e. 18.00 - 24.00 hrs), 1.9 ± 2.6 times during the third segment (i.e. 24.00 - 6.00 hrs) and 1.7 ± 2.2 times during the final race segment (i.e. 6.00 - 12.00 hrs).BCAA were the preferred ergogenic supplement during the race for twenty-two athletes (29.7%), and L-Carnitine for fifteen racers 20.3%). Thirty-two (43.2%) racers consumed magnesium, nine 12.2%) consumed vitamin C, five (6.8%) B vitamins, and eight (10.8%) multi-mineral products . Also the total supplement frequency  and the food intake frequency  associated with frequencies in all race segments. Fluid intake was related to the frequency of food intake , but not related to the frequency of supplement intake (P > 0.05). Food intake did not associate with the frequency of supplement intake (P > 0.05). We found no significant differences between pre-race variables  and the amount of fluid intake, the frequency of food intake, or the frequency of supplement intake (P > 0.05). The highest correlation appeared between the amount of fluid intake and the participants best result in a 24-hour race (r = 0.24), however, the correlation was not significant (P > 0.05).The total amount of fluid ingested was related to fluid intake in each race segment , from energy beverages (e.g. isotonic sports drinks and Coca Cola®), from energy bars, carbohydrate gels, or from other carbohydrate-rich foods such as biscuits and bread. The most frequently consumed food in the present ultra-MTBers were bananas, which was in an agreement with Knechtle et al. study about the ultra-cycling race‘ RAAM‘  and at the end of the race (the last race segment). In the ultra-MTBers with the highest dietary intake, this characteristic tracked across all four segments. Average fluid intake was in an accordance with the International Marathon medical Directors Association  is widespread in athletes  and minerals (®. Little is known about the intake of ergogenic supplements and their effect on ultra-endurance performance (Singh et al. The third most drank beverage during and after the race in the present study was Coca ColaThe limitation of the study is the use of the self-perception of the athletes to define the type of diet followed in the 3 month period prior to the race.vice versa. Future studies need to use employ a more accurate method of measuring and recording dietary intake in order to estimate macronutrient, micronutrient, mineral and supplement intake during ultra-endurance races.The dietary intake and use of supplements seen in the present study were similar to common recommendations for ultra-cyclists, with exceptions according to each athlete's individual nutrition habits. The ultra-MTBers who drank more, had a higher frequency of food intake. In all participants, increased levels of food, fluid and supplement ingestion tracked across all segments of the race and


Mrakia blollopis exhibited distinct growth characteristics under subzero conditions: SK-4 grew efficiently, whereas TKG1-2 did not. I analysed the metabolite responses elicited by cold stress in these two M. blollopis strains by using capillary electrophoresis–time-of-flight mass spectrometry. M. blollopis SK-4, which grew well under subzero temperatures, accumulated high levels of TCA-cycle metabolites, lactic acid, aromatic amino acids and polyamines in response to cold shock. Polyamines are recognized to function in cell-growth and developmental processes, and aromatic amino acids are also known to improve cell growth at low temperatures. By contrast, in TKG1-2, which did not grow efficiently, cold stress strongly induced the metabolites of the TCA cycle, but other metabolites were not highly accumulated in the cell. Thus, these differences in metabolite responses could contribute to the distinct abilities of SK-4 and TKG1-2 cells to grow under subzero temperature conditions.Microbes growing at subzero temperatures encounter numerous growth constraints. However, fungi that inhabit cold environments can grow and decompose organic compounds under subzero temperatures. Thus, understanding the cold-adaptation strategies of fungi under extreme environments is critical for elucidating polar-region ecosystems. Here, I report that two strains of the Antarctic basidiomycetous yeast  Approximately, 98% of Antarctica is covered by ice and snow, and the temperatures in its coastal areas typically range from −35°C to 5°C; moreover, the temperatures on Antarctic plateaus are considerably more extreme: they range from around −70°C in winter to −25°C in summer [Cold environments cover a large area on the Earth: approximately 85% of the biosphere is continuously exposed to temperatures below 5°C , and aroMost culturable microorganisms are mesophiles and they occupy temperature niches that are not regarded as extreme. Conversely, psychrotrophic or psychrophilic microorganisms are especially adapted to low-temperature environments . MicrobeFungi that inhabit cold environments can grow and decompose organic compounds under subzero temperatures. Thus, cold-adapted fungi work as decomposers and play critical roles in the carbon cycle in polar-region ecosystems . ConsequSaccharomyces cerevisiae, which is considered a ‘conventional’ yeast [S. cerevisiae is categorized as an ascomycetous fungus. Basidiomycetous yeasts differ taxonomically from ascomycetous fungi.Although the response to cold stress has been widely studied in bacteria and plants ,9, littll’ yeast ,12. Basil’ yeast  and basil’ yeast ,15, and Mrakia spp. and Mrakiella spp. have been found in the Arctic, Siberia, Alaska, Alps, Apennines, Patagonia and Antarctica [Mrakia accounts for approximately 24% of the culturable fungi in Antarctic soil. Moreover, I previously reported that Mrakia spp. constitute approximately 35% of the culturable fungi isolated from the lake sediments and soils of East Antarctica [Mrakia spp. are the dominant culturable fungi near Syowa station, East Antarctica. Among this genus, Mrakia blollopis is the most frequently isolated species from the region and is well adapted to the environment.The cold-adapted basidiomycetous yeasts tarctica –23. Di Mtarctica  reportedtarctica . My repoM. blollopis under subzero temperatures. This is the first study to detect the metabolite changes in psychrophilic fungi under cold stress, and the results provide a clear overview of the metabolite responses elicited by cold shock in M. blollopis.In this study, I used capillary electrophoresis–time-of-flight mass spectrometry (CE-TOFMS) to analyse the metabolite responses to cold stress in two strains of the Antarctic basidiomycetous yeast 2.2.1.M. blollopis were used, SK-4 and TKG1-2 [In this study, two strains of d TKG1-2 , both of2.2.Mrakia blollopis SK-4 and TKG1-2 were first cultivated in 20 ml of yeast extract--peptone--dextrose liquid medium  at 120 r.p.m. for 120 h at 10°C, following which the cells from the cultures were collected through centrifugation at 3500g for 5 min at 4°C. The cell pellets were resuspended in distilled water, and the washed cells were inoculated into 50 ml of modified YPD liquid medium . For all cultivation conditions tested, the initial cell density in the YPD medium was adjusted to a 600 nm absorbance (OD600) of 2. Both strains were cultivated at 120 r.p.m. at −3°C and 10°C for 120 h, and 2–10 ml of each sample was collected once every 24 h and the glucose concentration in the supernatants was measured using high-performance liquid chromatography (HPLC). All experiments were conducted independently in four vials.2.3.600 with a BioSpectrometer , and the glucose concentration in the YPD liquid medium was determined by using an HPLC equipped with a refractive index detector  and an Aminex HPX-87H column . The HPLC equipment was operated at 65°C with a mobile phase of 8 mM H2SO4 at a flow rate of 0.6 ml min−1 [Cell density was monitored by measuring the ODml min−1 .2.4.et al. [600 was measured. The culture volume used in the metabolome analysis was calculated using this formula:g for 5 min at 4°C. The supernatant was transferred to an Amicon Ultrafree MC filter unit (Millipore) and centrifuged at 9100g for 120 min at 4°C. Lastly, the four filtrates obtained from the four vials were mixed in one tube, dried and then dissolved in 50 µl of MilliQ water.Intracellular metabolites were extracted according to the method of Matsushika et al.  with min2.5.m/z range. Anionic metabolites were separated in a fused silica capillary filled with an anion buffer solution , and the same sheath liquid mentioned above was delivered. The sample solution was injected at 50 mbar for 25 s and a positive voltage of 30 kV was applied. ESI-MS was conducted in the negative mode and the capillary voltage was set at 3500 V. Exact mass data were acquired over a 50–1000 m/z range. Peaks were extracted using the automatic integration software MasterHands v. 2.16.0.15 [m/z, peak area, and migration time. Principal component analysis (PCA) and hierarchical clustering analysis were performed using SampleStat v. 3.14 (HMT) and PeakStat v. 3.18 (HMT).CE-TOFMS analysis was performed using an Agilent CE-TOFMS system . Cationic metabolites were separated in a fused silica capillary  filled with a cation buffer solution , Turuoka, Yamagata, Japan), and the sheath liquid  was delivered. The sample solution was injected at 50 mbar for 10 s and a positive voltage of 27 kV was applied. Electrospray ionization--mass spectrometry (ESI-MS) was conducted in the positive mode and the capillary voltage was set at 4000 V. Exact mass data were acquired over a 50–1000 .16.0.15 , and eac3.3.1.600 peaked at 48 h and then gradually decreased up to 120 h, whereas at −3°C, glucose was consumed at a steady rate up to 120 h and the OD600 also increased up to 120 h. At 10°C and −3°C, the final OD600 values reached were almost the same (a). By contrast, when TKG1-2 cells were grown at 10°C, the glucose concentration decreased up to 120 h and approximately 14 g l−1 glucose remained in the medium, and the OD600 gradually increased until 96 h; moreover, at −3°C, glucose was consumed more slowly than at 10°C and approximately 25 g l−1 glucose remained in the medium, and the OD600 also increased more slowly than at 10°C (b).When SK-4 cells were grown at 10°C, glucose was completely consumed by 72 h and the ODthe same a. By con at 10°C b.Figure3.2.M. blollopis. The metabolite response elicited by cold stress was determined by extracting and measuring metabolites in SK-4 and TKG1-2 cells at 4 and 3 time points, respectively, during the 120-h cultivation at −3°C and 10°C; sample collection, metabolite extraction and pooling, and CE-TOFMS analysis are detailed in §§2.4 and 2.5. In the CE-TOFMS analysis, 219 metabolites  were detected. Moreover, 88 metabolites, which included amino acids, organic acids, sugar phosphates and nucleotides, were quantified using external standards and targeted metabolite analysis.Next, the effect of cold-shock stress on metabolite accumulation was examined in the two strains of After CE-TOFMS analysis, heatmap analysis and PCA were performed to determine whether or not cold stress induced changes in metabolite accumulation and responses. The results of the heatmap analysis showed that the metabolite levels in SK-4 were drastically altered by cold stress , which aThe PCA results showed that the first two components accounted for approximately 55.5% of the total variance; whereas the first component (PC1) explained 34.4% of the variation, the second component (PC2) explained 21.1%. In the case of SK-4, the metabolite-response pattern for cold shock A in  was clea3.3.d-glycerate , succinyl coenzyme A (Suc-CoA) and oxaloacetic acid were not detected. In SK-4 cells grown at 10°C, the following 11 of 19 metabolites in the central carbon pathway reached their peak concentration at 24–48 h and then decreased rapidly within the next 24 h: glucose 6-phosphate (G6P), fructose 6-phosphate (F6P), fructose 1,6-diphosphate (FBP), 3-phosphoglyceric acid (3-PG), 2-phosphoglyceric acid (2-PG), pyruvic acid, aconitic acid, isocitric acid, 2-oxoglutaric acid (2-OG), fumaric acid (FUM) and malic acid (MAL). By contrast, at −3°C, the FBP concentration was decreased after the first 24 h and then increased slightly; 3-PG gradually increased up to 72 h; and acetyl coenzyme A (AcCoA) and 2-PG markedly accumulated within 48 h. Moreover, lactic acid, AcCoA, CoA, FUM and MAL were accumulated to a greater extent at −3°C than at 10°C.The extent of metabolite changes in the central carbon metabolism pathway, including in glycolysis, the pentose phosphate pathway and the TCA cycle, is shown in In the case of TKG1-2 cells cultivated at 10°C, the results obtained for the metabolites of the central carbon pathway were similar to those obtained for SK-4 cells; F6P, 3-PG, 2-OG, FUM and MAL were all detected, but most metabolites in TKG1-2 were scarcely accumulated as compared to the levels in SK-4 under both temperatures. However, citric acid, FUM and MAL in TKG1-2 cells showed similar accumulation as in SK-4 cells at −3°C.3.4.Glycerol is produced during glycolysis as follows: G6P is converted to FBP, which is converted to dihydroxyacetone phosphate (DHAP) and glycerol 3-phosphate (G3P) by triose phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase, and, finally, G3P is converted by glycerol-3-phosphatase (GPP) into glycerol, which either accumulates in the cell or is ejected into the extracellular medium . In SK-4The trehalose biosynthesis pathway metabolites detected here were glucose 1-phosphate (G1P), uridine diphosphate glucose (UDP-glucose) and trehalose 6-phosphate (T6P); 3.5.In SK-4 cells cultivated at −3°C, the concentration of histidine increased drastically between 24 and 48 h, and finally was 41.5-fold higher than that at 0 h. Phenylalanine, tryptophan and tyrosine increased continuously and reached concentrations that were approximately 6.6-, 4.0- and 8.4-fold higher than those at 0 h, respectively. In TKG1-2 cells grown at −3°C, the concentration of histidine decreased to 0.56-fold and then increased to 2.4-fold relative to that at 0 h, whereas phenylalanine, tryptophan and tyrosine increased continuously and reached concentrations that were approximately 3.0-, 1.2- and 2.0-fold higher than those at 0 h, respectively. At 10°C, histidine concentrations in SK-4 and TKG1-2 cells increased for the first 24 h and finally decreased by 1.69-fold and 0.68-fold relative to the levels at 0 h, respectively. In SK-4 cells, phenylalanine, tryptophan and tyrosine concentrations peaked within 24 h and then dropped to levels that were approximately 1.6-, 1.4- and 1.4-fold higher than control; the concentrations of these amino acids in TKG1-2 also peaked at 24 h and then finally decreased by 0.37-, 0.50- and 0.41-fold relative to the control levels, respectively.3.6.S-adenosylmethionine (SAM) levels increased continuously and finally were 10.6-, 28.6- and 4.7-fold higher than the levels at 0 h. Furthermore, the highest concentrations of other metabolites in this pathway were recorded at 72 h. At 10°C, GLU, ornithine (ORN) and spermidine (SPD) concentrations peaked at 24 h, and ORN and SPD reached concentrations that were 3.5- and 3.1-fold higher than the corresponding maximal concentrations at −3°C. Moreover, 4-aminobutanoic acid  peaked at 48 h and its concentration was 2.0-fold higher than its highest concentration at −3°C. In TKG1-2 cells, the concentrations of polyamine metabolites at −3°C were almost the same as those at 10°C, but the concentration of GLU was 2.4-fold higher than its maximal concentration at 10°C.The changes in metabolite concentrations in the polyamine synthesis pathway are shown in 3.7.Adenylate energy charge (AEC) and guanylate energy charge (GEC) in SK-4 and TKG1-2 cells were calculated using this formula:4.Mrakia blollopis strains SK-4 and TKG1-2 were isolated from, respectively, the lake sediment and soil collected near Syowa station in East Antarctica. These strains show greater than 99.5% similarity in the ITS  region and D1/D2 domain of 26S rDNA with the type strain of M. blollopis, CBS 8921 [CBS 8921 . HoweverCBS 8921 ,27. Here600 of the cultures at −3°C reached almost the same value as that at 10°C; by contrast, in the case of TKG1-2 cells grown at −3°C, more than 25 g l−1 glucose remained after 120 h and the OD600 was approximately half that at 10°C . These results indicate that whereas the strain SK-4 is well adapted to subzero conditions, TKG1-2 does not grow efficiently at subzero temperatures. Accordingly, the metabolite responses induced by cold stress in these two strains were studied using CE-TOFMS. To the best of my knowledge, only a few studies have investigated the metabolite responses elicited by cold shock in fungi. Specifically, changes in the metabolites of the central carbon pathway have been only reported by López-Malo et al. [S. cerevisiae grown in the presence of fermentation inhibitors [SK-4 cells completely consumed the supplied glucose within 120 h at −3°C, and the OD at 10°C a,b. Theso et al. , althoughibitors .S. cerevisiae was cultivated at a low temperature, glycerol was highly induced [S. cerevisiae, but in cryotolerant species Saccharomyces bayanus var. uvarum, T6P was strongly induced by low temperature [Mrakia spp. are known to accumulate trehalose at low levels in the cell at low temperatures, Glaciozyma antartica, another Antarctic basidiomycetous yeast, accumulated approximately 20% (w/w) trehalose in the cell [Mrakia strains studied here are considered to convert UDP-glucose into compounds in the glycogen synthetic pathway. SK-4 markedly induced lactic acid at −3°C, whereas TKG1-2 accumulated lactic acid to approximately one-third the level in SK-4. When S. cerevisiae NBRC 0308, which is known as sake yeast, was frozen in the presence of lactic acid at −20°C for 10 days at pH 5.0, the viability of the yeast cells was decreased to approximately 40%; however, when it was frozen in the absence of lactic acid, cell viability was decreased considerably more, to around 15% [Glycerol and trehalose are recognized as cryoprotectants that prevent the freezing of microbial cells at near-freezing temperatures . In the  induced , but G3P induced . In the  induced . Therefoperature . By contthe cell . The twoound 15% . Based oound 15% , I propoS. cerevisiae under nitrogen- and sulfur-starvation conditions [Polyamines are generally recognized to function in cell-growth and developmental processes . The strnditions , and SAMnditions . TherefoS. cerevisiae, aromatic amino acids are highly accumulated under carbon starvation and in the presence of fermentation inhibitors. Moreover, increased biosynthesis of aromatic amino acids, especially tryptophan, reflects the resistance of microbial cells to transport impairment and an improvement of cell growth at low temperature [Aromatic amino acids, which have been reported to accumulate under cold, heat, acidity and oxidative stresses , are amoperature . Here, hMrakia has been reported from low-temperature areas worldwide, but Mrakia spp. scarcely produce extracellular polysaccharides, accumulate glycerol and trehalose in the cell as cryoprotectants, and do not harbour genes encoding anti-freeze proteins [Mrakia can grow below −10°C [M. blollopis isolated from Antarctica experiences strong cold-shock stress. Consequently, SK-4 cells, which grew efficiently under subzero conditions, accumulated high levels of TCA-cycle metabolites, lactic acid, aromatic amino acids and polyamines as an adaptation to resist cold shock. By contrast, TKG1-2 cells, which did not grow efficiently in subzero temperatures, strongly induced the metabolites of the TCA cycle, but did not accumulate other metabolites at high levels in response to cold-shock stress. These differences in metabolite responses could contribute to the distinct abilities of SK-4 and TKG1-2 cells to grow under subzero temperature conditions.Lastly, the genus proteins ,32. Howeow −10°C  and is aow −10°C . My resu


This study investigated the effects of the selective dopamine D1 receptor agonist SKF81297 on acquisition and retrieval in the novel object recognition task in male Wistar rats. SKF81297 (0.4 and 0.8 mg/kg s.c.) given 15 min before the sampling phase impaired novel object recognition evaluated 10 min or 24 h later. The same treatments also reduced novel object recognition memory tested 24 h after the sampling phase and when given 15 min before the choice session. These data indicate that D1 receptor stimulation modulates both the encoding and retrieval of object recognition memory. Microinfusion of SKF81297 (0.025 or 0.05 μg/side) into the prelimbic sub-region of the medial prefrontal cortex (mPFC) in this case 10 min before the sampling phase also impaired novel object recognition memory, suggesting that the mPFC is one important site mediating the effects of D1 receptor stimulation on visual recognition memory.Previous studies have shown that dopamine D For example, rats given the choice between a novel and familiar object spontaneously spend more time exploring the novel object. To discriminate between novel and familiar stimuli requires the ability to remember previously encountered stimuli . As thisThe neurobiological substrates of recognition memory have been extensively studied  and show3 receptor antagonist, S33084, into the PFC of rats caused a dose-related (0.025 or 0.05 μg/side) improvement, while injection of the preferential D2 receptor antagonist, L741,626, into the same region caused a dose-related (0.025 or 0.05 μg/side) impairment in NOR  neurotransmission disrupts recognition memory. For example, both acute and subchronic injection of the DA releasing agent, methamphetamine reduced NOR . The mPFt in NOR . While ig memory , questiolidation  and retrlidation . However1 receptor full agonist, R/S-(+/−)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine  and a long inter-trial interval delay (24 h). Second microinjection of SKF81297 into the prelimbic (PL) part of the mPFC prior to the acquisition of object memory was performed to determine whether short term recognition memory was dependent on mPFC DA D1 receptor activation.Therefore, the present study used systemic administration of the selective D22.1n=36), 2 (n=35), 3 and 5 (n=36), non-naive rats (mean weight 431 g) were used, in each case counterbalanced for previous experimental experience. In experiments 4 and 6, 11 naive rats  were used. One rat was excluded from the analysis because it escaped from the NOR box during both the sampling and choice phase.Adult male Wistar rats  were caged in group of four on a 12:12 h light/dark cycle with food and water ad libitum. Rats were handled for approximately 10 min per day for 1 week prior to any procedure. All procedures were carried out in accordance with the United Kingdom (UK) Animals Scientific Procedures Act 1986, Project Licence number: PPL 40/3163. In experiments 1 (2.21 agonist SKF81297 (0.4 or 0.8 mg/kg s.c.) was injected 15 min before the sampling phase of the NOR procedure (see below). In experiments 3 and 5, the same doses were administered 15 min before the NOR testing phase and immediately prior to exposure to the locomotor activity boxes respectively. Drug doses were those used in a previous study in our laboratory  were implanted with bilateral infusion guide cannula consisting of a 5 mm plastic pedestal holding two 26 gauge metal tubes 1.2 mm apart and projecting 4.5 mm from the pedestal was implanted through small holes drilled in the skull. The tips of the guide cannula were aimed 0.5 mm above the injection site in the PL part of the prefrontal cortex, at the following coordinates  inserted into the guides such that tips extended 0.5 mm into the mPFC, and the injector ends were connected through polyethylene tubing to 5-µl syringes mounted on a microinfusion pump . A volume of 0.5 μl/side of 0.154 M saline or SKF81297 (0.025 or 0.05 μg/side) was then infused bilaterally over 1 min. The movement of an air bubble, which was included in the tubing, was monitored to verify that liquid was successfully infused into the brain. The injector remained in place for one additional minute to allow for tissue absorption of the infusion bolus. The injectors were then removed and the stylets replaced. The choice phase of novel object discrimination (NOD) commenced 10 min after infusion. Locomotor testing began as soon as possible after the infusion and the time course of activity was followed for 60–90 min to determine the optimal onset of the drug action. In experiments 4 and 5b, SKF81297 used was dissolved in saline at a concentration of 0.05 μg/0.5 μl. This solution was aliquoted and kept frozen until use. On the day of infusion an aliquot was thawed and a part of this solution was diluted to a concentration of 0.025 μg/0.5 μl with saline.2.5All testing was conducted in a 38×40 cm opaque plastic rectangular arena with 54 cm high walls. The stimuli consisted of duplicate copies of bottles and flasks made of glass, metal or plastic of varied shape, colour and size which were too heavy to be displaced by the animal , for mor2.6The NOR task was based on previously established protocols . One day1 receptor agonist SKF81297. The order of the three infusions was counterbalanced using a Latin square design. During the 5 min sampling period the rats were exposed to two identical objects. After a delay of 10 min in which the rats were returned to their home cage, each rat was tested for 3 min in the arena containing a novel object and an identical copy of the object previously seen during the sampling phase. Again, the time spent exploring the familiar and novel object was recorded. In the course of the three sampling/choice sessions, three different object pairs were used, counterbalanced across the infusions conditions.Comparison between the systemic drug studies (experiments 1–3) provides the temporal resolution to distinguish effects at encoding versus retrieval in the NOR procedure. Since both encoding and retrieval were affected, the experiment 4 infusion study used the experiment 1 timeline  which does not distinguish effects on encoding and retrieval. Experiment 4 was run over a 6 day cycle in a within-subjects design to reduce the number of animals subjected to the cannulation procedure. On days 1, 3 and 5 rats were placed individually into the arena for 1 h. On days 2, 4 and 6, rats underwent a re-acclimatisation of 3 min to the arena before receiving one of three bilateral mPFC infusions 10 min before the sampling phase: saline, 0.05 μg (0.025 μg/side), 0.1 μg (0.05 μg/side) of the selective D2.6.1r=0.85, p<0.0001) indicating robust inter-rater reliability.An independent experimenter who was blind to the treatment and object contingencies rescored 20% of all test phases from the original video recording. The rescored results significantly correlated with the original scores ; 2.10.1t-tests were systematically used to test for NOD.In experiments 1–3, choice exploration times were analysed in a mixed design using treatment as the between-subjects factors. Object (novel versus familiar) was a within-subjects factor as was 1-min block (at 3 levels). Thus, this design examines minute-by-minute variation in NOR over the duration of the test session. The results obtained using the Latin Square procedure of experiment 4 were analysed in an entirely within-subjects design, in the same way as experiments 1–3 but in this case treatment was also within-subjects. ANOVAs showed marked variation in NOR over the 3 min of test (as reflected in significant interactions between object and 1-min block). Therefore, based on the results obtained in experiments 1 which consistently indicated that the preferential object exploration was greater in the first min of the choice trial, follow up analyses were restricted to the first min. Where ANOVA showed significant interactions, separate follow-up analyses of the familiar or novel object exploration time were performed using treatment as the factor. Post-hoc pairwise comparisons were performed using Fisher׳s PLSD. On the basis of the pattern of results of the first experiment, planned comparisons requiring a paired comparisons 2.10.2t-tests were used to compare performance measures to the 0.5 value of the ratio which reflects chance level performance.The time spent exploring the novel object divided by the total time spent exploring both objects was calculated for the first min and total three min of exploration. In experiment 1–3 the data were subjected to ANOVA with drug treatment as a between-subjects factor. In experiment 4 drug treatments was a within-subject factor. Effects revealed by the ANOVA were further analysed using Fisher׳s PLSD test. One sample 2.10.3In experiments 5 and 6 the activity levels were examined in a mixed design with doses as the between-subjects factor and 10 min blocks (locomotor activity) as the within-subjects factor.33.1F=15.81; p<0.0001) and choice phases =6.17; p<0.006) of the NOD test when given by systemic administration  and the highest dose reduced exploration during the choice phase (p<0.05)  .F=2.89; p<0.029). Inspection of the means indicated that the effect of SKF81297 was due to a decrease of the time spent exploring the novel object during the first minute of the choice trial =8.49; p<0.002). This interaction was however not significant during the second =0.79; p=0.46) and the third =1.34; p=0.27) 1-min blocks.A minute by minute change in object exploration over the 3 min test period of the choice trial was revealed by a three-way interaction of treatment×object×1-min block =7.55; p<0.002). This arose because of decreased novel object exploration compared to saline after both low  and high doses  of SKF81297. There was no significant effect of drug on familiar object exploration =1.32; p=0.28). Furthermore, planned comparison (by paired t-test) showed that the exploration times during the first 1-min block were significantly higher for the novel object than for the familiar object in the control group (t(11)=15.12; p<0.0001) and after an injection of 0.8 mg/kg of SKF81297 (t(11)=2.85; p<0.05), but not after injection of 0.4 mg/kg (t(11)=1.52; p=0.15)  B.F=3.82; p<0.04). Post-hoc comparisons indicated that the lower (p<0.05), but not the higher dose (p=0.16) of SKF81297, decreased recognition of the novel object compared to saline. This was also confirmed by one sample t-tests comparing the discrimination ratio in each group to chance (0.5). Performance differed from chance in the saline group (t(11)=11.29; p<0.0001) and the group receiving 0.8 mg/kg (t(11)=3.23; p<0.05), but not in the group receiving an injection of 0.4 mg/kg of SKF81297 (t(10)=0.70; p=0.49).It was also clear from the discrimination ratios that during the first 1-min block the drug groups differed markedly in the proportion of time spent exploring the two objects B. ANOVA 1 receptor before encoding impaired the ability of the rats to discriminate novel from familiar object using a trial delay of 10 min.These data indicate that systemic activation of D3.2F=5.10; p<0.03) but not the choice =0.32; p=0.73) phase after systemic administration before object encoding .In this experiment, SKF81297 affected exploration time during the sample =1.53; p=0.20). However, there was a main effect of object =9.80; p<0.004) reflecting a preferential exploration of the novel object and a main effect of 1-min block =47.52; p<0.0002), consistent with greater exploration of the novel object during the first min =8.32; p<0.007) but not during the second =3.57; p=0.06) and third minutes =0.07; p=0.79). Accordingly, and for comparison with experiment 1, planned comparisons of the novel versus familiar object were restricted to the first minute only. These showed that rats in the saline group explored the novel object (t(11)=2.79; p<0.05) more than rats injected with 0.4 mg/kg (t(10)=1.06; p=0.31) and 0.8 mg/kg (t(11)=1.08; p=0.30) SKF81297 confirming that the main effect of SKF81297 was restricted to early exploration =0.57, p=0.56) only the performance of the control group was better than chance (t(11)=3.23; p<0.05).The discrimination ratio during the first 1-min of testing showed SKF81297 treated rats explored both objects equally, whereas the saline group tended to show a preference for the novel object B. Even t1 receptor transmission before the encoding tended to impair the recognition of a novel object after a delay of 24 h.These data indicate that systemic activation of the D3.3F=0.815; p=0.45) but exploration was decreased under SKF81297 during the choice phase =19.19; p<0.0001) .The groups were well matched for exploration time during the sample phase (<0.0001) . Post-hoF=3.38; p<0.02) suggests that the effects on object exploration changed over the 3 min of the choice trial. Examination of the means confirmed that NOR was again more marked during the first minute of exploration =7.40; p<0.003), suggesting that the drug preferentially reduced novel object exploration time. This interaction was not significant during the second =0.84; p=0.44) and the third =0.47; p=0.63) 1-min blocks.A significant interaction between treatment×object×1-min block (loration A and prop<0.0001) and 0.8 mg/kg  of SKF81297 decreased novel object exploration time. While there was also a significant effect of drug on familiar object exploration =5.04; p<0.02), this effect only reached significance after injection of 0.4 mg/kg SKF81297 . Planned comparisons demonstrated that the exploration times were significantly higher for the novel than the familiar object in the control group (t(11)=3.93; p<0.001), but not after 0.4 mg/kg (t(11)=1.63; p=0.13) or 0.8 mg/kg of SKF81297 (t(11)=1.51; p=0.15)  A.F=1.43, p=0.25), However, planned comparison showed that performance in the saline-injected control group was greater than chance (t(11)=4.23; p<0.05), whereas it was not with either 0.4 mg/kg (t(10)=0.4; p=0.69) or 0.8 mg/kg (t(11)=0.11; p=0.91) of SKF81297. Thus these data suggest that the retrieval of NOR is also disrupted by DA D1 receptor stimulation.Both doses of SKF81297 tended to reduce the discrimination ratio compared to saline during the first 1-min of the choice trial B but the3.4F=1.42; p=0.26) but showed a reduction in total exploration during the choice phase =4.86, p<0.03) with injection of SKF81297 into the mPFC =7.37; p<0.03). F=3.50; p=0.05) while novel object preference in the 2-min =1.62; p=0.22) and 3-min blocks =0.49; p=0.95) was unaffected. Analysis of the 1 min novel and familiar object exploration times separately showed a main effect of drug =4.64; p<0.03). Furthermore novel object exploration time was decreased compared to the saline group after mPFC-infusion of SKF81297 at 0.025 μg/side (p<0.05) and 0.05 μg/side (p<0.05). On the contrary, the exploration time of the familiar object was unaffected by this treatment =1.62; p=0.22). Planned comparisons demonstrated that the exploration times between the novel and the familiar object were significantly different in the saline group (t(9)=5.94; p<0.001) and after an infusion of 0.025 μg/side of SKF81297 (t(9)=2.40; p<0.05). However at 0.05 μg/side, there was no difference (t(9)=0.80; p=0.44) (There was a 3 way interaction of treatment×object×1-min block in the linear trend ( p=0.44) A.F=8.87; p<0.002). Post-hoc comparisons revealed a significant decrease in performance after infusion of 0.05 μg/side of SKF81297 compared to the control group  =6.92; p<0.0001; chance vs. 0.025 μg/side t(9)=1.29; p=0.23; chance vs. 0.05 μg/side t(9)=0.18; p=0.86)  C. Additi p=0.86) B. Togeth3.5F=0.55; p=0.56) nor any interaction of treatment×10 min time bin =2.22; p=0.07). Systemic administration of SKF81297 increased activity at all doses compared to saline starting 10 min after injection and lasting for about 50 min. There was both a main effect of treatment =15.9; p<0.0001) and an interaction of treatment×10 min bin =6.41; p<0.0001). Separate ANOVAs and post-hoc comparisons of locomotor activity during the 6 10-min blocks revealed that, compared to saline, 0.4 and 0.8 mg/kg SKF81297 did not increase locomotor activity during the first 10-min bin =1.68; p=0.20) whereas groups differed during the other 10-min bins .SKF81297 markedly increased locomotor activity in the activity boxes . In the 3.6F<1) nor any interaction of treatment×10 min bin: <1). Following infusion there was still no main effect of treatment <1) or interaction of treatment×10 min bin <1).SKF81297 infusion into the mPFC did not affect locomotor activity in the activity boxes . The dif3.7No gross damage was seen after drug infusion, and the morphological structure of the mPFC was preserved 41 receptor agonist SKF81297 before encoding impaired NOR at the lowest dose after a delay of 10 min. Both doses of SKF81297 (s.c.) decreased NOR encoding and retrieval after a delay of 24 h. Both doses of SKF81297 (s.c.) stimulated locomotor activity. In experiment 4, we used a 10 min delay to show that intra-mPFC infusion of SKF81297 induced a dose-related impairment in encoding and/or retrieval. Both overall object exploration and locomotor activity were unaffected by this manipulation. Thus the present study shows that DA D1 receptor activation modulates the formation and expression or retrieval of visual recognition memory and that this effect is, at least in part, mediated in mPFC. In contrast, the effects of DA D1 receptor activation on general levels of activity and exploration are not necessarily mediated in mPFC.Systemic (s.c.) administration of the D4.11 receptor agonist SKF81297 is consistent with previous reports (1 receptor-mediated transmission (Increased locomotor activity after s.c. administration of the D reports . Similar reports . Indepensmission .1 receptor stimulation decreases object exploration (as measured during the sampling phase). It could be argued that the NOR memory deficits observed relate to this reduction in exploratory encoding of the object to be remembered. In experiment 1 however, only the lowest dose of SKF81297 impaired memory, whereas (during the sampling phase) object exploration was decreased at both doses. If reduced sample exploration caused the apparent NOR impairment, both doses should have decreased NOR. In experiment 3, SKF81297 also decreased exploration of both the familiar and the novel object when injected before the test phase, possibly reflecting some locomotor effects. However the difference between the exploration times of the novel and familiar objects so clearly present in the saline group was absent after injection of SKF81297. Moreover, the pattern of results seen in experiment 1 shows that deficits in NOR are dissociable from non-specific locomotor effects.General locomotor activity levels do not necessarily predict object exploration. As can be seen from comparison of the saline groups, there were different baseline levels of exploration, but each experiment controls for these unexplained shifts . Also in both experiments 1 and 2, D4.21 receptor activation. Importantly, consolidation, which refers to a category of processes that stabilise a memory trace after its initial acquisition, has been demonstrated to be dependent on D1 receptor activation (1 receptor transmission may play a key role in NOR is in line with previous studies suggesting the importance of DA D1 receptor activation in different forms of memory (1 receptor induction and maintenance of long-term potentiation (LTP) is well characterised  remain poorly understood. The results of experiments 1 and 2 show that SKF81297 impaired NOR when injected prior to the sampling phase and after a delay of 10 min and 24 h. At a short delay, it is impossible to discriminate whether memory formation or retrieval has been affected, the drug being effectively active in the brain during both processes. However, the persistence of the impairment after a delay of 24 h implies that the formation of a familiar memory is dependent on Dtivation . It is tf memory  and memof memory . Interesf memory  and the cterised . It has asticity ;. Too muasticity . Togethesolation . Our pre4.31 receptor stimulation decreased NOR retrieval after a delay of 15 min. The present study confirmed that a similar dose of SKF81297 decreased performance after a delay of 10 min. However, the above study also reported enhanced retrieval after a long delay  within the PFC . The BBSRC had no further involvement in the study design, in the collection, analysis or interpretation of data; in the writing of the report; nor in the decision to submit the paper for publication.M.A. Pezze, H.J. Marshall, H.J. Cassaday designed and conceived the study. M.A. Pezze, H.J. Marshall, H.J. collected data and conducted statistical analysis. M.A. Pezze wrote the first draft of the manuscript. H.J. Cassaday, K.C.F Fone supervised the work. M.A. Pezze, H.J. Cassaday and K.C.F Fone critically reviewed the manuscript. All the authors contributed to and have approved the final manuscript.The authors declare no competing financial interests.


Solution-processed organic photovoltaics (OPV) offer the attractive prospect of low-cost, light-weight and environmentally benign solar energy production. The highest efficiency OPV at present use low-bandgap donor polymers, many of which suffer from problems with stability and synthetic scalability. They also rely on fullerene-based acceptors, which themselves have issues with cost, stability and limited spectral absorption. Here we present a new non-fullerene acceptor that has been specifically designed to give improved performance alongside the wide bandgap donor poly(3-hexylthiophene), a polymer with significantly better prospects for commercial OPV due to its relative scalability and stability. Thanks to the well-matched optoelectronic and morphological properties of these materials, efficiencies of 6.4% are achieved which is the highest reported for fullerene-free P3HT devices. In addition, dramatically improved air stability is demonstrated relative to other high-efficiency OPV, showing the excellent potential of this new material combination for future technological applications. In organic photovoltaics, the best performing devices usually involve low-bandgap polymers whose limited solubility and stability constrain the scalability of organic solar cells. Here, Holliday et al. develop a new acceptor and pair it with canonical P3HT to obtain 6.4% efficient and stable devices. In addition, the large extent of spectral overlap of FBR with P3HT and lack of long-wavelength absorption reduced the ability to harvest photons across the spectrum, further limiting the generated photocurrent.The efficiency of solution-processed organic photovoltaics (OPV) has been increasing rapidly, with the development of new high-performing benzodithiophene12360BM refs , 9, 10. n-octyl) alkyl chains yielding a more crystalline material with a further red-shifted absorption onset relative to branched (2-ethylhexyl) chains, resulting in higher Jsc and PCE. Power conversion efficiencies of up to 6.4% were achieved, which is, to the best of our knowledge, the highest reported for fullerene-free P3HT solar cells. The oxidative stability of these devices is also found to be superior to the benchmark P3HT:PC60BM devices, as well as devices with several of the high-performance polymers tested alongside, demonstrating this to be a robust and highly promising new materials combination for OPV.We now present a new acceptor derivative that has been designed to address both the spectral overlap and morphological issues with FBR via replacement of the fluorene core with an indacenodithiophene unit. This has the effect of planarizing the molecular structure and thus significantly red-shifting the absorption as well as increasing the tendency to crystallize on length scales commensurate with charge separation and extraction. We show how these properties can be further tuned via side-chain engineering, with linear (n-octyl (O-IDTBR) or branched 2-ethylhexyl (EH-IDTBR) side chains as shown in o-xylene (60 °C), enabling facile solution processing of OPV devices. In the case of FBR, a torsional angle of 33.7° was calculated between the fluorene core and the adjacent benzothiadiazole unit by density functional theory (DFT) methods. By contrast, IDTBR was calculated to be essentially planar (355 M−1 cm−1 (measured in solution) is over twice the value of FBR and demonstrates the potential of these molecules to contribute significantly more to the photocurrent relative to PC60BM for which the maximum extinction coefficient in the visible region (400 nm) was measured alongside to be only 3.9 × 103 M−1 cm−1 in CHCl3  values close to 3.9 eV. The EA of P3HT was measured for comparison to be 3.2 eV, allowing sufficient energetic offset for electron transfer between the donor and acceptor. The ionization potential (IP) of O-IDTBR was measured to be slightly smaller than that of EH-IDTBR, which accounts for the small difference in optical bandgap  in the thin film shows that both EH-IDTBR and O-IDTBR have electron affinity  data for the optimized devices with an active device area of 0.045 cm2, which were measured under simulated AM1.5G illumination at 100 mW cm−2. Both acceptors yielded high open-circuit voltage (Voc) values (0.7–0.8 V) relative to reference devices with PC60BM as the acceptor, which gave 0.58 V  profile of O-IDTBR, which extends beyond 800 nm due to the red-shifted absorption of the acceptor after annealing. Although the Voc and fill factor (FF) are both slightly lower for the linear chain analogue, this significantly larger Jsc leads to an overall increase in average PCE from 6.0% for EH-IDTBR to 6.3% for O-IDTBR, with a maximum PCE of 6.4% for the best performing device. This is among the highest efficiencies for fullerene-free devices as well as being the highest published efficiency for non-fullerene acceptor devices with P3HT. It is also significantly higher than the reference PC60BM:P3HT device efficiency of 3.7%, despite the reduced active layer thickness of 75 nm for the IDTBR devices compared with 150 nm for the fullerene-based device. This difference in active layer thickness can also explain the increased peak EQE in the PC60BM:P3HT devices as shown in J–V properties on active area was analysed for O-IDTBR:P3HT devices, as shown in 2, the PCE is maintained at 6.3% and for areas as large as 1.5 cm2, the PCE is still relatively high as 4.5%, owing to a slight reduction in Jsc and FF. It should be noted that these larger area devices were prepared using procedures optimized for the 0.045 cm2 cells, and that with further optimization of large-area devices their performance may be further improved, demonstrating these materials to be promising candidates for large-area, scalable OPV.Solar cells were fabricated using P3HT as the donor polymer due to the favourable energetic offsets mentioned above, as well as its widespread availability of P3HT and its potential for technological scale-up. An inverted device architecture of glass/ITO/ZnO/P3HT:IDTBR/MoOe 0.58 V  and thisTc of 115 °C. No such thermally induced crystallization occurs during the heating cycle of EH-IDTBR, explaining the different optical response of the acceptors to thermal annealing. DSC measurements were also carried out on drop-cast blends of the acceptors with P3HT to determine the extent of crystallization within the blend. The blend of FBR:P3HT  was used to compare the crystallinity of the acceptors in films that were slightly thicker than those used in device fabrication (280–290 nm) in order to provide enough resolution to observe crystalline reflections by this method. FBR:P3HT  shows onGrazing incidence XRD (GIXRD) was used to further investigate the formation of pure donor and acceptor domains in the thin-film blends. Qz=1.69 Å−1, and several rings in its diffraction pattern. The peak most probably results from a portion of face-on π–π stacking of EH-IDTBR aggregates. The rings indicate that besides the aggregates with face-on orientation, the film also has a considerable amorphous fraction. When EH-IDTBR is blended with P3HT, a new peak at Qz=0.48 Å−1 appears, partly overlapping with the broad P3HT (001) alkyl peak at 0.39 Å−1. This peak does not correspond to any features seen in the diffraction pattern of neat EH-IDTBR, suggesting that in the presence of P3HT, EH-IDTBR crystallizes in a different orientation or a different polymorph than in neat form, although the diffraction data is not complete enough to allow us to distinguish between these two hypotheses. It should also be noted that the diffraction pattern of P3HT in the blends is the same as that of a pure P3HT filmEH-IDTBR has an out-of-plane peak centred at 2 active area (see td) of 1 μs. From the measured photocurrent transients, the charge carrier mobility (μ) is calculated using the following It is well known that charge transport is crucial for efficient OPV devices. Carrier mobility of both donor and acceptor materials can be affected by morphology, field or carrier densities in bulk heterojunction active layers under operating conditions4244area see . Figure d is the active layer thickness, A is the voltage rise speed A=dU/dt, U is the applied voltage, tmax is the time corresponding to the maximum of the extraction peak, and j(0) is the displacement current. The photo-CELIV mobilities for the charge carriers in the O-IDTBR and EH-IDTBR blends with P3HT is found to be 5.4±0.4 × 10−5 and 5.0±0.3 × 10−5 cm2 V−1 s−1 after averaging over various delay times, respectively. The O-IDTBR:P3HT blend shows slightly higher charge-carrier density (which is the integrated area of the photo-CELIV curve at 1 μs delay time) than the branched chain analogue system. In addition to photo-CELIV, the electron mobility of EH-IDTBR:P3HT and O-IDTBR:P3HT blends was determined by space charge-limited current (SCLC) measurements on electron-only devices as well as the hole mobility of EH-IDTBR:P3HT blends on hole-only devices. Both acceptors exhibited electron mobilities ∼3–6 × 10−6 cm2 V−1 s−1, while the hole mobility of EH-IDTBR:P3HT was found to be ∼3–7 × 10−4 cm2 V−1 s−1 (see where  s−1 see . Both men) using charge extraction (CE)4546Voc in O-IDTBR solar cells compared with branched EH-IDTBR cells with P3HT. All samples were operated at Voc, but under different background illumination intensities and then shorted in the dark to enable CE. The measured average n as a function of Voc is depicted in n(Voc) indicates a 40 meV smaller electronic bandgap for O-IDTBR devices, which is consistent with the reduced open-circuit value (0.73 V) for O-IDTBR:P3HT devices compared with EH-IDTBR:P3HT solar cells (0.77 V). This reduced Voc can be explained by the more ordered microstructure of O-IDTBR:P3HT blends, as confirmed with GIXRD measurements, which results in a reduced electronic bandgap in the bulk.Charge-carrier density (Photoluminescence (PL) studies were carried out on the EH-IDTBR:P3HT and O-IDTBR:P3HT blends relative to neat reference films of EH-IDTBR, O-IDTBR and P3HT to compare the PL quenching efficiency (PLQE) as shown in The charge generation process was studied with femtosecond–nanosecond transient absorption spectroscopy (TAS). Transient spectra of EH-IDTBR and O-IDTBR blends, measured with the acceptors excited selectively at 680 nm, are shown in 60BM:P3HT as well as three of the most widely reported high-efficiency polymers PTB7, PCE-10 (PTB7-Th) and PCE-11 (PffBT4T-2OD)548Oxidative stability is an essential consideration for the technological implentation of OPV materials60BM is that large-scale aggregates and crystals emerge from the meta-stable blend morphology over time. This process can be monitored by polarized optical microscopy during accelerated aging of the films upon annealing2960BM:P3HT were prepared on ZnO/ITO substrates and these were subjected to annealing at 140 °C for 1 h. As the micrographs in In addition to oxidative stability, the morphological stability of the O-IDTBR:P3HT blends was investigated. One of the main issues with fullerene-based acceptors like PCJsc and an increase in PCE from 6.0 to 6.4%. CE measurements at the same light intensity reveal a reduced electronic bandgap for O-IDTBR relative to EH-IDTBR, which explains the difference in Voc measured for these devices. As well as affecting the optoelectronic properties, the enhanced intermolecular interactions of the linear alkyl chain also have an effect on the blend morphology. Relative to FBR, both IDTBR acceptors exhibit increased crystallinity and, crucially, formation of pure acceptor domains as evidenced by GIXRD and DSC studies. O-IDTBR in particular shows pronounced crystal packing upon annealing, which is consistent with the reduced optical bandgap. This results in greater phase segregation for the linear analogue which is manifested in reduced PL quenching of the acceptor emission, as well as a delayed polaron generation and slower recombination dynamics in the O-IDTBR:P3HT blend. Interestingly, the charge-carrier mobilities measured for the IDTBR:P3HT blends appear quite low, considering the reasonably high FFs obtained from devices (up to 64%) and the charge recombination dynamics of these systems therefore warrant further investigation to determine whether non-geminate recombination is significantly suppressed. In addition to high efficiencies, IDTBR:P3HT devices demonstrate improved stability in ambient conditions compared with the benchmark PC60BM:P3HT device, as well as several systems with typical low-bandgap, high-performance polymers, which were found to degrade at a dramatic rate when exposed to air. IDTBR devices also showed improved morphological stability to fullerene devices in accelerated aging studies. These results strongly supports the use of P3HT, in conjunction with non-fullerene acceptors such as IDTBR, for high-efficiency, scalable and stable OPV for future technological applications.In this work, we present a new small molecule electron acceptor IDTBR that is based on an indacenodithiophene core with benzothiadiazole and rhodanine flanking groups. IDTBR is designed to give high performance with the donor polymer P3HT, chosen for its commercial scale-up potential both in terms of cost, scalability and stability. In comparison with our previously published acceptor FBR, which had an essentially overlapping absorption profile with P3HT, this new acceptor has a significantly reduced optical bandgap owing to the more planar molecular backbone, delocalized electronic structure and push–pull molecular orbital hybridization, resulting in a UV–vis absorption profile that is now highly complementary to that of P3HT. This gives broader photon harvesting across the incident solar spectrum within the active layer, which is reflected in higher short-circuit currents and power conversion efficiencies relative to FBR:P3HT devices. Furthermore, the absorption onset of this new IDTBR acceptor can be tuned by judicial choice of solubilizing alkyl chains on the IDT unit. Linear (O-IDTBR) chains promote stronger intermolecular packing, which is particularly enhanced by thermal annealing, relative to branched (EH-IDTBR) chains. One effect of this is to further red-shift the absorption of O-IDTBR relative to the branched counterpart, which results in a broader EQE profile, higher 1H and 13C NMR spectra were collected on a Bruker AV-400 spectrometer at 298 K and are reported in p.p.m. UV–vis absorption spectra were recorded on a UV-1601 Shimadzu UV–vis spectrometer. DSC experiments were carried out with a Mettler Toledo DSC822 instrument at a heating rate of 5 °C min−1 under nitrogen. Samples were prepared by drop-casting the materials from CHCl3 solution directly into the DSC pan and allowing the solvent to evaporate under Ar. Specular XRD was carried out on thin films of the acceptors spin-coated from CHCl3 solutions  using a PANalytical X'Pert PRO MRD diffractometer equipped with a nickel-filtered Cu-Kα1 beam and X'Celerator detector, with a current I=40 mA and accelerating voltage U=40 kV. Samples for GIXRD were spin-coated on Si (100) substrates following the same spin-coating and annealing procedures as were used in fabricating solar cells.1a and 1b were prepared according to literature procedure34The compounds 2a. A solution of 1a  in anhydrous tetrahydrofuran (200 ml) was stirred at −78 °C for 30 min. n-BuLi  was added dropwise and the solution was stirred at −78 °C for 30 min followed by −10 °C for 30 min. After cooling again to −78 °C, trimethyltin chloride was added  and the solution was allowed to return to room temperature overnight. The reaction was then poured into water and extracted with hexane, washed successively with acetonitrile to remove excess trimethyltin chloride and dried over MgSO4 to yield 2a as a yellow oil . 1H NMR  δ: 7.25 , 6.97 , 1.97–1.91 , 1.86–1.78 , 1.23–1.05 , 0.83–0.80 , 0.39 ; 13C NMR  δ: 157.15, 153.47, 147.71, 139.24, 135.31, 129.55, 113.42, 53.06, 39.20, 31.87, 30.07, 30.03, 29.31, 24.17, 22.68, 14.14 and −8.02. MS (ES-ToF): m/z calculated for C54H90S2Sn: 1,040.45; m/z found 1,041.40 (M+H)+.3a. A solution of 2a  and 2,1,3-benzothiadiazole-4-carboxaldehyde  in anhydrous toluene (40 ml) was degassed for 45 min before Pd(PPh3)4  was added and this solution was heated at 100 °C overnight. The reaction mixture was then cooled and purified by flash column chromatography on silica mixed with potassium fluoride using CHCl3 as the eluent. Further purification by column chromatography on silica using CH2Cl2/pentane (1:1) followed by precipitation from methanol yielded 3a as a dark purple solid . 1H NMR  δ: 10.72 , 8.27 , 8.25 , 8.06 , 7.45 , 2.05 , 1.05–1.2 , 0.99–0.81 , 0.77 ; 13C NMR  δ: 188.44, 157.04, 154.02, 152.29, 147.00, 140.67, 136.44, 134.14, 132.87, 131.62, 124.87, 124.8, 122.80, 114.12, 54.43, 39.16, 31.79, 29.98, 29.29, 29.20, 24.29, 22.58, 14.04. MS (ES-ToF): m/z calculated for C62H78N4O2S4: 1,038.5; m/z found 1,041.40.O-IDTBR.3a  and 3-ethylrhodanine  were dissolved in tert-butyl alcohol (30 ml). Two drops of piperidine were added and the solution was left to stir at 85 °C overnight. The product was extracted with CHCl3 and dried over MgSO4. The crude product was purified by flash column chromatography on silica in CH2Cl2 and precipitated from methanol. The precipitate was collected and dried by vacuum filtration to afford O-IDTBR a dark blue solid . mp=219–221 °C. 1H NMR  δ: 8.54 , 8.24 , 8.03 , 7.74 , 7.45 , 4.27 , 2.18–1.96 , 1.35 , 1.22–1.12 , 0.99–0.90 , 0.80 . 13C NMR  δ: 193.04, 167.59, 157.05, 154.63, 154.22, 151.77, 146.15, 141.02, 136.41, 131.37, 130.54, 127.29, 124.49, 124.25, 124.08, 123.82, 113.97, 54.38, 39.94, 39.19, 31.82, 30.02, 29.33, 29.24, 24.30, 22.61, 14.08 and 12.35. MS (matrix-assisted laser desorption/ionization–time of flight): m/z calculated for C72H88N6O2S8: 1,324.5; m/z found 1,326.0 (M+H)+.2b. A solution of 1b  in anhydrous tetrahydrofuran (40 ml) was stirred at −78 °C for 30 min. n-BuLi  was added dropwise and the solution was stirred at −78 °C for 1 h. Trimethyltin chloride was then added  and the solution was allowed to return to room temperature overnight. The reaction was then poured into water and extracted with hexane, washed successively with acetonitrile to remove excess trimethyltin chloride and dried over MgSO4 to yield 2b as a yellow oil . 1H NMR  δ: 7.28 , 6.99 , 1.96–1.88 , 1.87–1.82 , 0.99–0.46 , 0.37 . 13C NMR  δ: 157.40, 153.43, 147.51, 140.73, 135.20, 130.04, 113.95, 53.52, 43.59, 34.89, 32.20, 29.75, 28.74, 28.10, 22.67, 14.16 and −8.16.3b. A solution of 2b  and 2,1,3-benzothiadiazole-4-carboxaldehyde  in anhydrous toluene (30 ml) was degassed for 45 min before Pd(PPh3)4  was added and this solution was heated at 110 °C overnight. The reaction mixture was then cooled and purified by flash column chromatography on silica mixed with potassium fluoride using CHCl3 as the eluent. Further purification by column chromatography on silica using CH2Cl2/pentane (1:1) followed by precipitation from methanol yielded 3b as a dark purple solid . 1H NMR  δ: 10.72 , 8.37–8.30 , 8.25 , 8.03 , 7.49 , 2.15–2.05 , 1.05–0.85 , 0.74–0.50 . MS (ES-ToF): m/z calculated for C62H78N4O2S4: 1,038.50; m/z found 1,038.50 (M+).EH-IDTBR.3b  and 3-ethylrhodanine  were dissolved in tert-butyl alcohol (15 ml). 1 drop of piperidine was added and the solution was left to stir at 85 °C overnight. The product was extracted with CHCl3 and dried over MgSO4. The crude product was purified by flash column chromatography on silica with CH2Cl2 as the eluent followed by precipitation from methanol to yield EH-IDTBR as a dark blue solid . mp=218–220 °C. 1H NMR  δ: 8.53 , 8.27 , 7.99 , 7.73 , 7.47 , 4.25 , 2.07 , 1.34 , 0.95–0.90 , 0.69–0.54 . 13C NMR  δ: 193.07, 167.58, 156.76, 154.63, 153.93, 151.80, 146.14, 140.46, 136.38, 131.37, 130.64, 127.31, 125.08, 124.51, 124.30, 123.73, 114.82, 54.19, 39.94, 35.13, 34.16, 28.64, 28.25, 27.26, 22.86, 14.18, 12.33 and 10.60. MS (matrix-assisted laser desorption/ionization–time of flight): m/z calculated for C72H88N6O2S8: 1,324.5; m/z found 1,325.9 (M+H)+.+ reference electrode. Measurements were carried out in anhydrous and deoxygenated acetonitrile with 0.1 M of tetrabutylammonium hexafluorophosphate (TBA PF6) as the supporting electrolyte, and calibrated against ferrocene in solution using a cylindrical Pt working electrode. IP and EA values were calculated from the following equations:CV measurements were performed using an Autolab PGSTAT101 potentiostat. Thin films of the acceptor were spin-coated onto ITO-coated glass substrates to be used as the working electrode, alongside a platinum mesh counter electrode and Ag/AgEred and Eox are taken from the onset of reduction and oxidation, respectively, and EFc is taken as the half-wave potential of ferrocene.where 3/Ag). Glass substrates were used with pre-patterned indium tin oxide (ITO). These were cleaned by sonication in detergent, deionized water, acetone and isopropanol, followed by oxygen plasma treatment. ZnO layers were deposited by spin-coating a zinc acetate dihydrate precursor solution (60 μl monoethanolamine in 2 ml 2-methoxyethanol) followed by annealing at 150 °C for 10–15 min, giving layers of 30 nm. The P3HT:IDTBR (1:1 ratio by mass) active layers were deposited from 24 mg ml−1 solutions in chlorobenzene by spin-coating at 2,000 r.p.m., followed by annealing at 130 °C for 10 min. Active layer thicknesses were 75 nm (averaged over six devices) for both acceptor blends. P3HT:PC60BM (1:1 ratio by mass) layers were spin-coated at 1,500 r.p.m. from 40 mg ml−1 solutions in o-dichlorobenzene, followed by annealing in the glovebox at 130 °C for 20 min, resulting in active layer thicknesses of 148 nm. MoO3 (10 nm) and Ag (100 nm) layers were deposited by evaporation through a shadow mask yielding active areas of 0.045 cm2 in each device. (J–V) characteristics were measured using a Xenon lamp at AM1.5 solar illumination (Oriel Instruments) calibrated to a silicon reference cell with a Keithley 2400 source meter, correcting for spectral mismatch. Incident photon conversion efficiency was measured by a 100 W tungsten halogen lamp (Bentham IL1 with Bentham 605 stabilized current power supply) coupled to a monochromator with computer controlled stepper motor. The photon flux of light incident on the samples was calibrated using a UV-enhanced silicon photodiode. A 590-nm long-pass glass filter was inserted into the beam at illumination wavelengths longer than 580 nm to remove light from second-order diffraction. Measurement duration for a given wavelength was sufficient to ensure the current had stabilized.Bulk heterojunction solar cells were fabricated with an inverted architecture  and PCE-11 (PffBT4T-2OD) used in stability studies were obtained from Ossila, and the active layers for these devices were prepared as follows, with the same architecture a used for the IDTBR:P3HT devices.−1). To completely dissolve the polymer, the active layer solution was stirred on a hot plate at 80 °C for at least 3 h. Active layers were spin-coated from the warm polymer solution on preheated substrates in a nitrogen glove box at 1,500 r.p.m.Active layer solutions (D:A ratio 1:1.5) were prepared in CB with 3 wt% 1,8-diiodooctane . To completely dissolve the polymer, the active layer solution was stirred on a hot plate at 80 °C for at least 3 h. Active layers were spin-coated from the warm polymer solution onto preheated substrates in a nitrogen glove box at 1,500 r.p.m.Active layer solutions (D:A ratio 1:1.5) were prepared in CB with 3 wt% 8-diiodooctane . To completely dissolve the polymer, the active layer solution was stirred on a hot plate at 110 °C for at least 3 h. Active layers were spin-coated from the warm polymer solution onto preheated substrates in a nitrogen glove box at 1,000 r.p.m.Active layer solutions (D:A ratio 1:1.4) were prepared in CB/o-DCB (1:1 volume ratio) with 3 wt% 8-diiodooctane . A fast electrical switch was used to isolate the cell and prevent CE or sweep out during the laser pulse and the delay time. After a variable delay time, a linear extraction ramp was applied via a function generator. The ramp, which was 20 μs long and 2 V in amplitude, was set to start with an offset matching the A is the device area (4 mm2), ɛ=3 and, ɛ0=8.85 × 10−12 F m−1 are the relative and absolute dielectric permittivity, respectively, and d is the active layer thickness (90 nm). C is then calculated as 1 nF. Assuming Rload=50 nm, the RC value is 5.9 × 10−8 s. Assuming the electrical field (E) is 1 × 105 V m−1, the transient time (t) is calculated with the following formula:where et al.In CE measurements, the devices were illuminated in air with a 405 nm laser diode for 200 μs, which was sufficient to reach a constant open-circuit voltage with steady state conditions. At the end of the illumination period, an analogue switch was triggered that switched the solar cell from open-circuit to short-circuit (50 ω) conditions within less than 50 ns. By adjusting the laser intensity, different open-circuit voltages were obtained which allowed a plot to be generated of charge-carrier density over voltage. As described by Shuttle SCLC measurements were performed on electron-only devices of the structure ITO/PEDOT:PSS/Al/P3HT:acceptor/Al and on hole-only devices of the structure ITO/PEDOT:PSS/P3HT:acceptor/Au using a Paios (FLUXiM AG) measurement system. The current–voltage characteristics were fitted by the Mott–Gurney law in the region where the current follows the square of the voltage to extract the carrier mobility.−2 with a TOPAS-Prime (light conversion) optical parametric amplifier. The other laser output was used to generate the probe light in near visible continuum (450–800 nm) by a sapphire crystal. The spectra and decays were obtained by a HELIOS transient absorption spectrometer  and decays to 6 ns. The samples were measured in N2 atmosphere. Deconvolution of the blend spectra was conducted by fitting the singlet EH-IDTBR exciton spectrum (Sexciton) and the P3HT:EH-IDTBR polaron spectrum (Spolaron) at 6 ns to the blend spectra for 20 different time delays using the equation:Samples for TAS and PL spectroscopy were spin-coated onto glass using the same conditions as for solar cells. Spectra were measured using a steady state spectrofluorimeter . The spin-coated films were excited at 680 nm. Sub-picosecond TAS was carried out at 800 nm laser pulse  by using a Solstice (Newport Corporation) Ti:sapphire regenerative amplifier. A part of the laser pulse was used to generate the pump laser at 680 nm, 2 μJ cmA1 and A2 are linear coefficients that estimate the percentage contribution of the spectra to the experimental blend spectra. Sexciton was derived from the transient absorption spectra of the EH-IDTBR, which peaks at 1,150 nm at selected time delays .Supplementary Figures 1-15 and Supplementary Tables 1-4