The Nano Spray Dryer B-90 offers a new, simple, and alternative approach for the production of drug nanocrystals. Among attractive drugs, calpain inhibitor that inhibits programmed cell death ‘apoptosis’ is a candidate for curing apoptosis-mediated intractable diseases such as Alzheimer’s disease and Parkinson’s disease. In this study, the preparation of calpain inhibitor nanocrystals using Nano Spray Dryer B-90 was demonstrated. The particle sizes were controlled by means of selecting mesh aperture sizes. The obtained average particle sizes were in the range of around 300 nm to submicron meter. Using drug nanoparticles for a drug delivery system has attracted considerable attentions in the field of nanomedicine. Especially, nanoparticles with a drug filling rate of 100% including their amorphous form are called drug nanocrystals. Additio2CO3) hollow spheres used in lithium batteries[Several approaches for preparing drug nanocrystals are classified as top-down and bottom-up procedures. For theatteries have beeRecently, our interest is focused on preparing calpain inhibitor nanocrystals. Calpain inhibitors inhibit the calpain activity, resulting in reducing the calpain-meditated apoptosis. Severalv/v) were purchased from Wako Pure Chemical Industries . The calpain inhibitor SNJ-1945 was kindly provided by Senju Pharmaceutical Co., Ltd. . Each drug was dissolved in ethanol (5 mg/10 ml). Then, each ethanol-dissolved drug solution was used for preparing each nanocrystal using Nano Spray Dryer B-90 (Büchi®). A schematic image of Nano Spray Dryer B-90 is illustrated in Figure Tin and Tout, respectively. The operating conditions for the experiments were kept constant at Tin = 50°C, Tout = 35°C, feed rates of 25 ml/h, and a drying gas flow rate of 100 L/min. Spray mesh aperture sizes of 4.0, 5.5, and 7.0 μm were used in this experiment. Finally, the resulting nanocrystal powders were collected using a rubber spatula. The morphology and size of collected particles were observed by scanning electron microscopy . The average size and particle size distribution were calculated by means of counting more than 300 particles from the obtained SEM images.The calpain inhibitor I and ethanol  was prepared with the following experimental conditions: concentration = 5 mg/10 ml, Tin = 50°C, gas flow rate = 100 L/min, and mesh aperture size = 4.0 μm  and high gas flow rate (150 L/min) did not significantly affect the particle size compared to the effect of utilizing high concentration of 50 mg/10 ml (cases I to IV in Table Tin = 100°C and gas flow rate = 150 L/min are shown in Figure Tin = 100°C and gas flow rate = 150 L/min were 515 ± 197 and 536 ± 230 nm, respectively (Figure Tin and gas flow rate. More detailed experimental conditions related to the concentration of dissolved drug solution, temperature, and gas flow rate, which will affect particle formation, will be investigated as future works including several kinds of calpain inhibitor nanocrystals. We also investigated the effects of the experimental conditions relating to the concentration of ethanol-dissolved drug solution, We succeeded in preparing size-controlled calpain inhibitor nanocrystals using Nano Spray Dryer B-90. To the best of our knowledge, this is the first time to prepare calpain inhibitor nanocrystals. The particle sizes were associated with the mesh aperture sizes, namely when the mesh aperture sizes decreased, the particle sizes decreased. The concentration of ethanol-dissolved drug solution significantly affected the particle size. Calpain inhibitors will be a promising drug for curing intractable diseases such as Alzheimer’s disease and Parkinson’s disease. Therefore, utilizing the calpain-inhibitor-nanocrystal-based drug delivery system targeting such intractable diseases will be quite attractive topics in the near future, and thus our fundamental findings of preparing calpain inhibitor nanocrystals in this research are noteworthy. We are also investigating the preparation of water dispersion of calpain inhibitor nanocrystals. This nanocrystal water dispersion will act as attractive nanocrystal-based eye drops for the treatment of ophthalmic disorders such as Fuchs’ endothelial dystrophy of the cornea in the near future.The authors declare that they have no competing interests.KB contributed to the conception of the study, carried out all the experiments, and drafted the manuscript. KN contributed to the interpretation of data and revised the manuscript. Both authors read and approved the final manuscript.KB is a specially appointed associate professor and KN is a professor and a medical doctor at the Department of Ophthalmology, Osaka University Graduate School of Medicine, Japan.


Intracellular skeletal muscle water is redistributed into the extracellular compartment during periods of dehydration, suggesting an associated decline in muscle volume. The purpose of this study was to evaluate skeletal muscle volume in active (knee extensors (KE)) and less active  musculature following dehydration induced by exercise in heat.ETBW), which was assessed by changes in body mass, and (2) fluid replacement (FR) where 3% and 5% losses of ETBW were counteracted by intermittent (20 to 30 min) fluid ingestion via a carbohydrate-electrolyte beverage. During both conditions, serum osmolality and skeletal muscle volume (assessed by magnetic resonance imaging) were measured at baseline and at the 3% and 5% ETBW loss measurement points.Twelve participants  cycled in the heat under two conditions: (1) dehydration (DHYD) resulting in 3% and 5% losses of estimated total body water (p = 0.005) and 5% (p < 0.001) ETBW losses, while FR decreased serum osmolality at the 5% loss of ETBW time point (p = 0.009). In DHYD, KE muscle volume declined from 1,464 ± 446 ml to 1,406 ± 425 ml  at 3% ETBW loss and to 1,378 ± 421 ml  at 5% ETBW loss. The largest decline in KE volume in DYHD occurred in the mid-belly  and proximal  regions of the grouped vasti muscles. There were no changes in volume for the biceps/triceps (p = 0.35) or deltoid (p = 0.92) during DHYD. FR prevented the loss of KE muscle volume at 3%  and 5%  ETBW loss time points compared to baseline .In DHYD, serum osmolality increased at 3% (Following exercise in the heat, the actively contracting muscles lost volume, while replacing lost fluids intermittently during exercise in heat prevented this decline. These results support the use of muscle volume as a marker of water loss. Water accounts for 50% to 60% of the total body mass  and apprStorage of glycogen is also associated with water storage, and therefore, the concentration of glycogen will directly influence total muscle water . ApproxiMagnetic resonance imaging (MRI) offers outstanding spatial resolution of muscle anatomy including the ability to noninvasively evaluate the volume of individual muscles and/or muscle groups ,19. The n = 7) and women (n = 5) were recruited for the investigation (mean ± standard deviation (SD): body mass = 69.8 ± 16.4 kg, body fat = 17.2 ± 7.9%, estimated TBW (ETBW) = 40.0 ± 10.1 kg). Prior to participation, all subjects were screened for sickle cell trait  due to the risk of microvascular occlusion during physical exertion [Twelve recreationally active men  + 0.73 kg (DEXA fat − free mass) [Prior to experimental testing, an estimate of stable baseline euhydrated body mass (nearest hundredth of 1 kg) was determined for each subject from mean values obtained over consecutive days (9.3 ± 0.8 days). Each measurements were obtained semi-nude using a calibrated digital scale . These measurements were conducted at the same time of day in the laboratory after voiding and prior to eating. Fat and fat-free body mass were also determined via Dual Energy X-ray Absorptiometry (DEXA) . Using the fat and fat-free mass from DEXA, ee mass) ,21. DuriBoth DHYD and FR testing sessions were counterbalanced and began the morning (0700 hours) following an overnight fast (>8 h). On arrival to the laboratory, subjects voided their bladders for assessment urine specific gravity via a refractometer . Urine specific gravity was only evaluated at baseline to document that subjects were not dehydrated prior to baseline data collection. Body mass was measured using the digital scale previously described (0715 hours), and participants rested in the supine position of 30 min. Blood samples (approximately 5 ml) were obtained via arm venipuncture using Vacutainer®  containing a clot activator and gel for serum separation (0745 hours). All blood samples were immediately transported to SUNY Medical University Hospital for clinical analysis of serum osmolality . Once blood samples were obtained, subjects were transported to the MRI scanner in the supine position using a gurney to avoid fluid shifts . Once coETBW loss. Rectal temperature  and heart rate  were monitored throughout the exercise sessions. During the DHYD exercise in heat session, no fluids were provided. Participants exercised in heat and were weighed every 20 to 30 min until ETBW loss reached 3% as assessed by body mass. At this time, subjects were allowed to void their bladders and walk to the resting area where a final weight was recorded. Subjects rested in the supine position until a blood sample and MRI scan was obtained 45 min and 60 min, respectively, after exercise. After dependent measures were obtained, the participants went back to the climate chamber for additional exercise in heat. No fluids were provided during acquisition of dependent measures; therefore, participants cycled in heat and were weighed until an additional 2% ETBW was lost . Once 5% ETBW loss was achieved, the dependent measures  were obtained as described previously.During the DHYD testing condition, participants cycled at 60 rpm on a Monark cycle ergometer in a climate chamber at 38°C, 32% humidity, and 13 km/h wind. Participants began pedaling at 1 kp but were allowed to change the cycle load if needed to achieve the desired ETBW. This amount was recorded, and fluids were provided. Participants were then re-weighed with the consumed fluid and returned to the thermal chamber for an additional 20 to 30 min of exercise in heat. This process was repeated until the total amount of fluid lost after being removed from the thermal chamber totaled 3% ETBW loss. At this point, our goal was to have adequately replaced the 3%ETBW fluid, so there was no change in body mass. Participants were allowed to void their bladders and walk to the resting area where a final weight was recorded and dependent measures were assessed. Participants then returned to the heat chamber and exercised, and every 20 to 30 min, they were then weighed and given the appropriate amount of fluids. This process continued until an additional 2% ETBW was lost (then replaced); hence, the total amount of fluid lost then replaced was equivalent to 5% ETBW. At this point, the goal was to have no change in body mass from the baseline despite sweating out 5% ETBW during the exercise and heat session. After the 5% ETBW loss, time point was reached in FR, a final body mass was recorded, and dependent measures were reassessed.The FR condition was identical to the DHYD condition except that all fluids lost during exercise and heat were intermittently (20 to 30 min) replaced at 1.5 times the rate they were lost using a carbohydrate-electrolyte beverage  . ReplaceFollowing both DHYD and FR conditions, MRI images were transferred to a computer for tracing, and skeletal muscle volume was calculated using the NIH ImageJ analysis software  . Knee ext tests. Dependent measures of body mass, serum osmolality, and skeletal muscle volume were analyzed using within-subject analysis of variance (ANOVAs) with repeated measures on condition  and time . When significant condition × time interactions were determined, differences within and between conditions were evaluated using Bonferroni corrected Student's t tests. Regional  changes in muscle volume were also evaluated post hoc using ANOVA with repeated measures on the muscle  and time  with follow-up Bonferroni corrected Student's t tests. Associations among dependent variables were further explored using Pearson's correlations. MRI image quality (motion artifact) at one time point prevented the analysis of muscle volume on one male subject for the knee extensors and one female subject for the arm muscles. Therefore, both leg and arm MRI statistical analyses were performed using n = 11. Significance was determined at p < 0.05. All values are mean ± SD.Urine specific gravity, chamber temperature, relative humidity, duration of chamber exposure, exercise workload, exercise heart rate, and core body temperature were compared between DHYD and FR conditions using paired p = 0.42) and were 1.016 ± 0.01 and 1.018 ± 0.01 for DHYD and FR conditions, respectively. Chamber temperature, humidity, exercise workload, and core temperature were closely matched between conditions . Body mass was significantly lower in the DHYD compared with that in the FR at 3% (p = 0.007) and 5% (p < 0.001) ETBW loss measurement time points. Overall, ETBW loss at 3% and 5% measurement points were 3.45 ± 0.73% (p < 0.001) and 5.75 ± 0.86% (p < 0.001) in DHYD and 0.78 ± 0.81% (p < 0.05) and 0.64 ± 0.79% (p < 0.05) in FR, respectively.There was a significant condition × time interaction for body mass . In DHYD, osmolality increased at the 3% (p = 0.005) and 5% (p < 0.001) ETBW loss. In FR, serum osmolality decreased only at the 5% (p = 0.009) ETBW loss measurement time point. Serum osmolality was significantly greater in the DHYD condition compared with that in FR at both the 3% (p < 0.001) and 5% (p < 0.001) ETBW losses.There was a significant condition × time interaction for serum osmolality . In FR, there were no changes in knee extensor volume at 3%  or 5%  ETBW loss compared to that in the baseline. However, during DHYD, knee extensor volume decreased significantly at both 3%  and 5%  ETBW loss measurement points, which represented reductions of 3.96 ± 1.31% and 5.87 ± 1.97%, respectively. Knee extensor volume was lower in the DHYD compared with that in the FR at 3% (p = 0.005) and 5% (p < 0.001) ETBW losses. An example MRI slice from DHYD and FR conditions at each time point is shown in Figure R = 0.57, R2 = 0.39, p = 0.006) between the loss of body mass (fluid loss via sweat) and the loss of knee extensor volume during the DHYD condition. The delta change in body mass relative to the delta change in knee extensor muscle volume is displayed in DHYD ; p < 0.001), mid-belly (p = 0.002), and proximal (p = 0.02) regions . The volume of the vasti muscles also declined significantly from the baseline to the 5% ETBW measurement point in the distal (p < 0.001), mid-belly, (p = 0.001), and proximal (p = 0.001) regions. There were no significant changes from the baseline to the 5% ETBW measurement point in any region of the rectus femoris (Table p = 0.35) or deltoid (p = 0.92) muscle volumes. In addition, no main effects were observed at these sites  were evaluated by region across the distance of the leg in DHYD only, there were significant muscle × time point interactions in the distal . The dehydration-related loss of the knee extensor muscle volume as a result of exercise in heat was counteracted by intermitted fluid replacement. We also show for the first time that less active upper body muscles (biceps/triceps or deltoid) maintain volume when dehydration is induced by exercise in heat.ETBW), the decline in knee extensor muscle volume was 5.89 ±1.9%. The association (R2 = 0.32) between the change in body mass and the change in knee extensor volume suggests that muscle water was sequestered from intracellular stores of the active muscle tissue to the extracellular space. The most notable comparison to the present study is the investigation of Costill et al. [By the end of the dehydration protocol , there was a small but significant decline in body mass from the baseline at both testing time points (approximated average 0.30 kg). However, knee extensor skeletal muscle volume was preserved, suggesting no change in muscle water content when fluids are replaced during exercise in heat. Participants had a greater total exposure time in the heat chamber in FR and performed exercise at the same workload albeit at a lower heart rate. These data are in agreement with previous studies demonstrating the benefits of maintaining adequate hydration during physical exertion -31.Contrary to our hypothesis, there were no changes in muscle volume in less active muscles  during the exercise protocols. Our representation of deltoid muscle volume was small (approximately 17 ml) as it consisted five to six MRI slices superior from its appearance. Therefore, it is plausible that greater muscle volumes are required to accurately detect change via MRI. We speculate that vasoconstriction to less active muscle groups and the absence of any significant metabolic perturbations in these muscles  are likeThe time course of the changes observed during our study is worth noting. MRI scanning occurred 60 min following completion of the exercise in heat sessions to allow for equilibration of fluids across compartments  and to bDehydration reduces physical work capacity, lowers tolerance to heat, and remains a significant health risk . AssessmIn summary, this study evaluated changes in skeletal muscle volume using MRI following exercise in heat, with or without fluid replacement. There was a significant decline in knee extensor (active muscles) volume following dehydration induced by prolonged, submaximal cycling in the heat, wherein the greatest loss of muscle volume occurred in the mid and proximal regions of the knee extensor musculature. This loss of knee extensor volume was prevented through provision of a carbohydrate-electrolyte beverage. No changes in muscle volume in the bicep/triceps brachii or deltoid (less active muscles) were detected with dehydration. The results from this study indicate that skeletal muscle volume assessed by MRI is a useful application to dehydration-related research and warrants further investigation.The authors declare that they have no competing interests.KJH participated in data acquisition, completed MRI and statistical analysis, and drafted the manuscript. SBC assisted with the coordination of the study, participated in data acquisition, completed MRI analysis, and provided edits to drafts of the manuscript. TJF assisted with the coordination of the study, participated in data acquisition, and provided edits to the manuscript. LPS designed the study, secured funding, assisted with the coordination of the study, participated in data acquisition, and provided edits to drafts of the manuscript. All authors read and approved the final manuscript.


Lactobacillus plantarum is a plant-associated bacterial species but it has also been found in human, mouse and porcine gastrointestinal tracts. It can ferment a broad spectrum of plant carbohydrates; it is tolerant of bile salts and low pH, and it has antagonistic potential against intestinal pathogens. However, experiments reporting the use of L. plantarum as a probiotic are limited. In this study, the effects of L. plantarum ZJ316 isolated from infant fecal samples on pig growth and pork quality were investigated.L. plantarum and no antibiotic; and Group 5 was fed a mixture of mequindox and L. plantarum. After a 60 days initial treatment, samples were collected for evaluation. The results showed that, the L. plantarum ZJ316 has probiotic effects on pig growth and that these effects are dose dependent. The effects of a dose of 1 × 109 CFU/d were more pronounced than those of a dose of 5 × 109 CFU/d or 1 × 1010 CFU/d. In Group 2 (1 × 109 CFU/d), the diarrhea (p = 0.000) and mortality rates (p = 0.448) were lower than in antibiotic-treated pigs (Group 1), and the daily weight gain (p = 0.001) and food conversion ratios were better (p = 0.005). Improved pork quality was associated with Lactobacillus treatment. pH , hardness (p = 0.000), stickiness (p = 0.044), chewiness (p = 0.000), gumminess (p = 0.000) and restoring force (p = 0.004) were all significantly improved in Lactobacillus-treated pigs (Group 2). Although we found that L. plantarum exerted probiotic effects on pig growth and pork quality, the mechanisms underlying its action require further study. Polymerase chain reaction-denaturing gradient gel electrophoresis results showed that the gut bacterial communities in Lactobacillus- and antibiotic-treated pigs were very similar and the quantity of L. plantarum ZJ316 was below the detection limits of DGGE-band sequencing. The concentration of short-chain fatty acids in Lactobacillus- and antibiotic-treated fecal samples were not significantly different (p = 0.086). However, the villus height of ilea (p = 0.003), jejuna (p = 0.000) and duodena (p = 0.036) were found to be significantly improved by Lactobacillus treatment.One hundred and fifty newly weaned pigs were selected randomly and divided into five groups. Group 1 was fed a diet supplemented with the antibiotic mequindox; Groups 2, 3 and 4 were fed a diet supplemented with L. plantarum ZJ316 was found to have probiotic effects, improving pig growth and pork quality. The probiotic mechanism might not involve L. plantarum colonization and alteration of the gut bacterial community. Rather, it might be related to the inhibition of the growth of opportunistic pathogens and promotion of increased villus height. Weaning stress can destroy the balance of intestinal microbiota in young mammals. Such periods of stress may allow opportunistic pathogens to multiply and cause gastrointestinal (GI) disorders. To promLactobacillus, which is part of the normal mucosal microbiota of pigs, have been found to be good probiotics.L. plantarum ZJ316 culture supernatants were evaluated on 17 bacteria strains  and lactic acid (15 mM/L) were assessed using the solutions of these two acids with pH 3.5.The inhibitory effects of the raw extraction of At the end of the study (day 95), average daily feed intakes (ADFI), average daily weight gain (ADG), food conversion ratio (FCR), mortality and diarrheal rate were calculated for evaluating the effects of different treatments on pig growth. The quantity of food provided and food left over was recorded every day, and the ADFI was calculated as follows: /(number of pigs × number of days). The ADG was calculated as follows: (average pig weight at the end – average pig weight at the beginning)/(number of days). The FCR was calculated as follows: (ADG/ADFI). The numbers of dead and diarrheal pigs were recorded every day and the ratios were calculated as follows: (number of dead pigs)/ and /. Nine pigs were selected for slaughter at the end of the experiment; and then muscle samples were collected for pork quality evaluation, ileal mucosa samples were collected for morphology observation, and ileal mucosa and cecal contents were collected for bacterial community analysis.Lactobacillus, three pigs from Group 1, six pigs from Group 2  were randomly selected for evaluation of pork quality. After slaughter, muscle samples from the Longissimus thoracis  and fillet were collected for evaluation of the pork quality. A reflectance spectrophotometer  was used to measure the color at the surface of a 2-cm-thick steak of Longissimus thoracis muscle after exposure to air for two hours. The parameters registered were L* (lightness), a* (redness), and b* (yellowness). Each value was the mean of 10 determinations per sample on the same slice, avoiding areas with excess fat.After 60 d of exposure to antibiotics and Muscle pH was measured at 45 min and at 24 hours (starting points from the minute the muscles were removed from the corpse) using a portable pH meter equipped with a glass electrode . To measure the drip loss, samples were placed on a supporting mesh in a sealed plastic container with no contact between sample and container. Three pigs were selected for sample collection and three muscle samples were collected from each pig. After a storage period of 24 hours and 48 hours at 4 °C, the samples were taken out of the container, dabbed lightly onto filter paper, and weighed again. Drip loss was expressed as a percentage of the initial weight based on Honikel. MarblinTexture profile analysis (TPA) was measured using a TA-XT2 Texture Analyser  equipped with a 25 kg load cell. The Texture Expert computer program  was used for data collection and calculations. Before TPA analysis, samples were vacuum-packaged , placed in a cooler  for 7 days, and then frozen on stainless steel trays at -20 °C. At the beginning of TPA analysis, the samples were fast-thawed in tap water (4 h). Then the vacuum was broken and the samples were wrapped in aluminum foil and cooked at 200 °C in a double-plate grill (Sammic GRS-5) until the internal temperature reached 72 °C. After cooking, steaks were placed in a vacuum bag and immediately immersed in an ice bath to stop further cooking. In this study, hardness, stickiness, springiness, chewiness, gumminess, cohesiveness, and restoring force were determined as described by Bourne.After animals were killed, ileal tissues  were harvested and cut into 1.5 cm × 1.5 cm × 0.3 cm pieces for sectioning. The pieces were fixed in 10% formalin after washing with PBS. Then, the formalin-fixed samples were dehydrated in ethanol, cleared with xylene, and embedded in paraffin wax. Sections (6 μm thick) were stained with hematoxylin and eosin and observed using a light microscope .For measurement of villus height and crypt depth, 10 villi and crypts were selected per section using Leica MZ16A software. The villus height was measured from the villus tip to the bottom, not including the intestinal crypt. The crypt depth was measured from the crypt tip to the bottom. An average of 10 villi and crypts per section was expressed as a mean villus height and crypt depth for each pig.TM universal detection system  with 8% polyacrylamide gels  at 60 °C. The gels were electrophoresed at 200 V for 4 h, and then stained with SYBR GREEN І. The bands were visualized and analyzed with Quantity One software  using a match tolerance of 2%.Ileal mucosa  and cecal contents (5-10 g) were collected after slaughter. Fresh fecal samples were collected at the beginning and end of the experiment, corresponding to 35, 95 and 102 days of age. Some gut samples collected from Group 1, Group 2-1 and Group 2-2 were used for bacterial genomic DNA extraction. In this study, a bead-beating method was used as previously described. The conDGGE band sequencing was carried out according to Li et al.. The staTM Organic Acid column (250 mm × 4.6 mm) was used with the detection conditions: temperature, 40 °C; wavelength, 217 nm; pressure, 0.1-4,000 psi.Fecal samples (0.5 g) were dissolved into 10 ml phosphate buffered saline (PBS), vortexed, and then centrifuged at 12,000 × g for 10 minutes at 4 °C. Then the supernatants were filtered through a 0.45 μm membrane filter for HPLC detection. In this study, the levels of acetic, butyric, citric, formic, isovaleric, lactic, malic, propionic, and tartaric acids were investigated. A PrevailAn Oxford cup test was executed as described above, and the inhibitory effects of 50 μg/ml, 100 μg/ml, 250 μg/ml, 500 μg/ml and 750 μg/ml of mequindox were evaluated.To analyze growth, each pen from each group was considered a single replicate. For meat color, pH value, drop loss and marbling score, each pig from each group was considered a single replicate. For meat TPA analysis, three samples were collected from each pig and used as replicates. For gut villus height and crypt depth analysis, 30 values were collected from each group and used as replicates. All of the above data were statistically analyzed using the One-Way ANOVA program included in the statistical software package SPSS 13.0 . Least-significant difference (LSD) was selected for post hoc multiple comparisons. For fatty acid analysis, fecal samples were collected from pigs that had been selected for meat quality evaluation and were considered as replicate. They were then analyzed using the means program included in the statistical software package SPSS 13.0 . All values were presented as mean ± standard error. p < 0.05 was considered significant.CS performed the pig growth experiment and carried out the data analyses; YY designed the experiments, carried out data analyses, and drafted the manuscript; XW performed the pork quality experiment; XL performed the inhibitory experiment of antibiotic; DS isolated the Lactobacillus strain and assisted with the pork quality experiment; XW designed the experiments and revised the manuscript; QG designed the experiments, carried out data analyses, and drafted the manuscript. All authors read and approved the final manuscript. Fermentation pattern of L. plantarum ZJ316*. Supplementary Figure 1. Inhibitory effects of the Lactobacillus plantarum ZJ316 culture supernatants at pH 6.0. Culture supernatants of L. plantarum ZJ316 were collected and adjusted to pH 6.0 using 1 mol/L NaOH. The inhibitory effects were evaluated on Escherichia coli and Salmonella using agar plates. A: Inhibitory effects of the culture supernatants on Escherichia coli. B: Inhibitory effects of the culture supernatants on Salmonella. Supplementary Figure 2. Comparison of the inhibitory effects of culture supernatants of Lactobacillus plantarum ZJ316 with acetic acid and lactic acid at pH 3.5. Salmonella was used as indicator bacteria for comparing the inhibitory effects at pH 3.5. 1, represents culture supernatants of Lactobacillus plantarum ZJ316 was added into the Oxford; 2, represents lactic acid was added into the Oxford and 3, represents acetic acid was added into the Oxford.Click here for file


Side population (SP) refers to a group of cells, which is capable to efflux Hoechst 33342, a DNA-binding dye. SP cells exist both in normal and tumor tissues. Although SP abundance has been used as an indicator for disease prognostic and drug screening in many research projects, few studies have systematically examined the factors influencing SP analysis. In this study we aim to develop a more thorough understanding of the multiple factors involved in SP analysis including Hoechst 33342 staining and cell culture. RPMI-8226, a high SP percentage (SP%) human myeloma cell line was employed here. The results showed that SP% was subject to staining conditions including: viable cell proportion, dye concentration, staining cell density, incubation duration, staining volume, and mix interval. In addition, SP% was highest in day one after passage, while dropped steadily over time. This study shows that both staining conditions and culture duration can significantly affect SP%. In this case, any conclusions based on SP% should be interpreted cautiously. The relation between culture duration and SP% suggests that the incidence of SP cells may be related to cell proliferation and cell cycle phase. Maintaining these technical variables consistently is essential in SP research. Specifically they display a distinct staining pattern, based on the phenomenon of a differential emission of blue (450 nm) versus red (670 nm) emission fluorescence upon UV excitation, such that SP appears as a tiny population on the lower left-hand side of a red (x)-blue (y) flow cytometry scattergram. This differential blue-red emission allows clear identification of a cell population that locates sideways from the diagonal and was thus named “side” population. Recent studies have shown the presence of SP cells in many types of cancer including ovarian cancer, glioblastoma cancer, lung cancer, nasopharyngeal cancer, gastrointestinal cancers hepatocellular carcinoma, mesenchymal tumors, and multiple myeloma [Side population (SP) cells were first described as a subset of adult mouse bone marrow with enriched hematopoietic stem cells (HSCs) , 2. This myeloma –11. SP cAlthough studies indicated that SP% varied among different types of cancer and from sample to sample as well –11, somein vitro, we systematically explored the variables involved with optimization and standardization of Hoechst 33342 staining variables such as dye concentration, cell density, staining duration, staining volume, and mix interval during staining as well as cell viability prior to flow cytometry (FCM) analysis. Importantly, we found that the time after cell subculture is the single most important factor affecting the SP% and this has not been reported before. In summary, this study suggests that both the Hoechst staining and subculture duration affect the proportion of SP. Hence, the conclusions of any other studies based on the change of SP% should be interpreted cautiously. Attempts to maintain these factors in a more consistent manner is instrumental for building reliable platforms for drug screening which target SP. In this study, using the human myeloma cell line RPMI-8226 as a convenient cellular model system μg/mL of streptomycin (Invitrogen), and 10% fetal bovine serum (FBS) . Cells were cultured in T75 or T25 flasks kept in a humidified incubator with 5% CO2 at 37°C. The cells were seeded at the density of 0.2 × 106 cells/mL.The human myeloma cell line RPMI-8226 was obtained from the American Type Culture Collection . RPMI-8226 cells were maintained in RPMI-1640  containing 100 u/mL of penicillin (Invitrogen), 100 + containing 2% FBS and 10 mM HEPES buffer (Invitrogen). Live cell number was counted at least twice and adjusted to a final cell density of 1 × 106 cells/mL by adding appropriate volume of warm staining medium. Hoechst 33342 water solution (1 mg/mL) was then added to make a final concentration of 10 μg/mL followed by incubation in a water bath at 37°C for 90 min with shaking every 30 min . To help gate SP on flow cytometry, samples treated with 1 μg/mL fumitremorgin C (FTC) , an ABCG2 transporter inhibitor, were included during the entire staining procedure as controls. Once incubation finished, samples were immediately put on ice to stop dye efflux. Subsequently, the cells were centrifuged for 5 min at 300 g at 4°C and resuspended in 100 μL of ice-cold Hanks' Balanced Saline Solution (HBSS) (Invitrogen) containing 2% FBS, 100 u/mL of penicillin, 100 μg/mL of streptomycin, and 10 mM HEPES. The samples were kept on ice before flow cytometry analysis. Propidium iodide (PI) (Sigma) solution was added at a final concentration of 2 μg/mL to exclude dead cells just before flow analysis.RPMI-8226 cells were harvested after culture for different periods of time and then stained with Hoechst 33342 dye (Invitrogen). Briefly, after discarding culture medium, cells were suspended in the staining medium RPMI16406 cells/mL under the same conditions. The cells were cultured for up to four days and then harvested at the end of 1, 2, 3, and 4 days, respectively. Cells were counted using trypan blue at least twice to adjust same staining density (1 × 106 cells/mL) across different experiment days. Two batches of samples were prepared from different but close passages of cells. Cells were subsequently stained under the conditions described as below. A subsequent independent experiment was further performed at the laboratory of Hong Kong Polytechnic University with different batches of RPMI-8226 passage and Hoechst dye. To explore how cell culture affected SP%, RPMI-8226 cells were maintained in culture medium for a range of time prior to being harvested for downstream staining. Preparation of the samples in order to examine the effect of culture time course on SP% was as follows: RPMI-8226 cells were seeded to four T-25 flasks at 0.2 × 10μg/mL), cell density (0.25 × 106–2.00 × 106 cells/mL), volume (100–2000 μL), duration (30–180 min), shaking interval (10–90 min), and viable cell proportion (20–80%). Only one factor was varied and examined each time, while the others were kept constant. The standard protocol described before was employed for the staining procedure except for the testing of the effects of cell viability prior to FCM analysis on SP%. For those experiments, two tubes of cell suspensions with a density of 2 × 106 cells/mL were prepared in staining medium, one of which was treated by incubation at 60°C for 20 min until all the cells died. We then mixed 0.1, 0.2, 0.3, 0.4, 0.5 mL treated cells suspension with 0.5 mL cell suspension without heat treatment, respectively. The resulting samples with a range of viable cell percentages were prepared for subsequent staining.We also investigated the SP% associated with an array of different staining conditions: Hoechst 33342 concentration . The Hoechst 33342 dye was excited at 355 nm, and its emission fluorescence was detected using 440/30 nm (Hoechst 33342-Blue) and 670/40 (Hoechst 33342-Red) filter systems. PI was excited at 488 nm, and its fluorescence was detected using a 610/20 nm filter for dead cell exclusion. The flow data was analyzed using FlowJo Version 8 software .This study is based on our previously published results showing a convincing FCM pattern of stained RPMI-8226 cells with Hoechst 33342, where a remarkable SP of about 20% presented with a clear gap existing between SP and non-SP cells . Thus, wPrevious studies suggested that SP and non-SP cells were in different cell cycle phases . We hypoμg/mL and then assessed for (i) cytotoxicity as measured by PI uptake indicating loss of plasma membrane integrity and (ii) change of SP%. RPMI-8226 cell viability was sensitive to Hoechst 33342 in a dose-dependent manner, which indicated that this DNA-binding dye was toxic to cells in higher concentrations (μg/mL (μg/mL (26.3%) to 10 μg/mL (4.4%), which was followed by a much gentle reduction to 12.5 μg/mL (0.16%) and onwards  resist current drug therapies and repair DNA after radiation treatment more efficiently than their differentiated, daughter cells and that these CSCs are responsible for the recurrence of tumours after treatment –20. SimiIt has been reported that the number of companies devoted to research into novel medicines targeting CSCs doubled from 2007 to 2008 . In addiin vitro, we systematically explored multiple factors, namely, Hoechst 33342 dye concentration, cell density, staining duration, staining volume, mix interval during staining, and cell viability prior to enumerating SP cells by FCM analysis. We have highlighted the large variability in SP% which is obtained when different conditions are used and have optimised the technical aspects of staining SP cells. With regards to cell viability, a previous study has also shown that, for consistent SP staining, the cell count before the Hoechst staining should include all cells ranging from dying, dead, to live cells [Few prospective systematic studies have examined the factors influencing SP% in normal tissues or cancer cells. In this study, using the human myeloma cell line RPMI-8226 as a convenient cellular model system ve cells . The samve cells . Importantly, we found that the time after cell subculture is an important factor affecting the SP% which has not been reported previously. While this factor will not affect the staining of SP in primary tissues, we envisage that a drug screening platform targeting SP for various cancer types will utilize stable cell lines. Thus, this new finding should lead to the modification of the preparation of cell samples for drug screening purposes. Furthermore, since RPMI-8226 harbors a rich and constant source of SP cells, it could be used as the positive control for this platform and for the detailed monitoring of the day-to-day variation of Hoechst staining performance.


During a followup of 1364 ± 628 days, 5 cardiac deaths and 20 unscheduled cardiovascular hospitalizations were observed. Among LV diastolic function indices, Tau > 48 ms and e′ < 5.8 cm/s were each significantly associated with lower rate of survival free of cardiovascular hospitalization. Even after adjustment for potential confounders , the predictive value of Tau > 48 ms and e′ < 5.8 cm/s remained significant. No predictive value was observed in EDP, E/e′, or LV ejection fraction. In conclusion, LV diastolic dysfunction, particularly impaired LV relaxation assessed by both cardiac catheterization and Doppler echocardiography, is independently associated with increased risk for cardiac death or cardiovascular hospitalization in patients with known or suspected CAD.We hypothesized that left ventricular (LV) diastolic dysfunction assessed by cardiac catheterization may be associated with increased risk for cardiovascular events. To test the hypothesis, we assessed diastolic function by cardiac catheterization (relaxation time constant (Tau) and end-diastolic pressure (EDP)) as well as Doppler echocardiography (early diastolic mitral annular velocity ( Left ventricular (LV) diastolic dysfunction refers to abnormalities in relaxation, filling, and distensibility . EvidencWe studied 222 consecutive Japanese patients who underwent cardiac catheterization for the evaluation of CAD between January 2004 and August 2006. All the patients had symptoms suggestive of angina and/or clinical signs of CAD . No patients with acute coronary syndrome, congestive heart failure, atrial fibrillation, primary valvular diseases, idiopathic dilated or hypertrophic cardiomyopathy, congenital heart disease, end-stage renal disease on maintenance hemodialysis, or malignant neoplasms were included. Medication status and a history of coronary revascularization were determined by review of medical records. Hypertension was defined as systolic blood pressure >140 mmHg and/or diastolic blood pressure >90 mmHg measured by indirect arm-cuff sphygmomanometry at rest or use of antihypertensive drugs. Diabetes was defined as a fasting blood glucose level >126 mg/dL or treatment with dietary modification, insulin, or oral hypoglycemic agents. Dyslipidemia was defined as low-density lipoprotein level ≥140 mg/dL, high-density lipoprotein level <40 mg/dL, and/or triglyceride level ≥150 mg/dL or treatment with antihyperlipidemic agents. Within a week before the index cardiac catheterization for this study, blood chemistry was obtained for assessment of clinical features. All the patients gave written informed consents to participate in the study, and this study was performed according to the regulations proposed by the Ethical Guidelines Committee of the Nagoya City University Graduate School of Medical Sciences.Before contrast material was injected into the LV or coronary artery, LV and aortic pressure waves were obtained with a catheter-tipped micromanometer  and recorded on a polygraph system  as previously reported . From thE) and atrial contraction (A) at the mitral orifice were obtained with the use of pulsed Doppler echocardiography in the apical 4-chamber view. Deceleration time was measured as the time from the peak early filling velocity to termination of early filling. The peak early diastolic annular velocity (e′) was measured with the use of pulsed Doppler echocardiography at the septal and lateral mitral annular sites. The values of e′ measured at both sites were averaged. The median values of measurements of 3 consecutive beats were used for statistical analyses. LV mass was calculated from M-mode echocardiographic measurements [The day before the index cardiac catheterization, ultrasound examination was performed with the use of a commercially available echocardiographic machine  with a 3-MHz transducer. Patients were examined at rest in the left lateral decubitus position. LV diastolic function was evaluated according to the published guideline . Transmiurements  and LV mFollowup was determined in October 2010 with the use of the medical record and/or at regular visit to determine the patients' vital status and any cardiovascular hospitalizations. The outcome of the present study was cardiac death  or unscheduled admission for cardiovascular causes. Sudden cardiac death was defined as unexpected death within one hour after the onset of a new symptom, or unexpected, unobserved death.t-test and the Fisher exact probability test. The association between continuous variables was determined by Pearson's correlation analysis. Survival curves of patients stratified by LV function indices were calculated by the Kaplan-Meier method and compared using the log-rank test. EDP of 16 mmHg, Tau of 48 ms, and EF of 50% were used as cut-off points, because these values are established thresholds for separating normal and abnormal levels [e′ and E/e′) for the diagnosis of diastolic dysfunction or the prediction of adverse prognosis are variable depending on age [e′ recorded at lateral mitral annular site is usually higher than e′ at septal site [P < 0.05 was considered significant.We used the SAS program package  for statistical analyses. Differences in quantitative and categorical data at baseline between groups were compared by the Student's g on age  and popue′ and were less likely to have hypertension. Based on the findings of the index coronary angiography, 50 percutaneous coronary interventions and 2 coronary artery bypass surgeries were performed. During a followup of 1364 ± 628  (median (range), 1444 (16–2433)) days, 5 cardiac deaths (4 heart failure and 1 sudden cardiac death) and 20 hospitalizations due to cardiovascular causes , 3 heart failure, and 2 arrhythmia) were observed. Clinical, hemodynamic, and echocardiographic features of all patients and patient subgroups are shown in e′ and E/e′ were determined as 5.8 cm/s  and 13.2 , respectively. Among LV function indices, significant univariate predictors for death or cardiovascular hospitalization included Tau > 48 ms  and e′ < 5.8 cm/s . In contrast, no significant predictive value was observed in EDP > 16 mmHg , EF < 50% , or E/e′ > 13.2 . Survival curves of patients stratified by Tau and e′ are shown in P < 0.01). Based on receiver-operating characteristic analyses for the prediction of death or cardiovascular hospitalization, the best cut-off points for e′ are shown in β-blockers, and to undergo coronary revascularization after the index cardiac catheterization. Compared with patients with e′ ≥ 5.8 cm/s, those with e′ < 5.8 cm/s were more likely to be older, to have diabetes and history of myocardial infarction, to be prescribed angiotensin converting enzyme inhibitors/angiotensin receptor blockers, and to undergo coronary revascularization after the index cardiac catheterization and were less likely to be prescribed calcium blockers.Clinical features of patients stratified by Tau and e′ are also shown in Tables e′ ≥ 5.8 cm/s, those with e′ < 5.8 cm/s were more likely to have greater number of coronary arteries narrowed and higher levels of Tau, EDP, and LV volumes and lower levels of EF. Hemodynamic and echocardiographic features of patients stratified by Tau and e′ < 5.8 cm/s (n = 41)) were more likely to be older, to have history of myocardial infarction, to be higher in serum creatinine levels, LV volume, and LV mass index, and to be lower in EF, compared with those who were diagnosed to have abnormal LV relaxation by only one modality (n = 82).Patients who were diagnosed to have abnormal LV relaxation by both cardiac catheterization and Doppler echocardiography , the predictive value of Tau > 48 ms continued to be significant . Similar5.8 cm/s .e′  and E/e′  and EDP correlated with E/e′  but not with e′ .Modest but significant correlations were observed between LV diastolic function indices determined by cardiac catheterization and those by Doppler echocardiography; Tau correlated with e′ < 5.8 cm/s were each associated with an increased risk for cardiac death or subsequent cardiovascular hospitalization in patients undergoing cardiac catheterization for CAD. In contrast, no prognostic value was observed in EDP, E/e′, or EF.In the present study, we found that Tau > 48 ms and E/e′ ≥ 15 were each predictive of future heart failure events. In contrast, in our study, no prognostic value was observed in EDP or E/e′. Compared with a cohort of Liang et al., our patients had lower EDP (18.2 ± 7.3 versus 14.4 ± 5.4 mmHg) and E/e′ (12.5 ± 5.6 versus 9.6 ± 3.3) levels, due probably to that patients with congestive heart failure were not included in our study. Thus, EDP or E/e′ may not be predictive of adverse prognosis in a cohort of patients without congestive heart failure in which the majority have normal or slightly elevated EDP and E/e′.Although studies have reported that LV diastolic abnormalities assessed by Doppler echocardiography are predictive of cardiac mortality and morbidity in patients with myocardial infarction , those we′ on Doppler echocardiography, e′ quantitates the peak velocity of early diastolic longitudinal motion of the mitral annulus [e′ only provides best-available noninvasive assessment of LV relaxation. Our study is significant in showing for the first time the prognostic value of abnormal LV relaxation determined by cardiac catheterization in patients with known or suspected CAD.The strength of the present study is that LV hemodynamic variables were obtained with the use of a micromanometer catheter but not a fluid-filled catheter. Although a fluid-filled catheter accurately measures late diastolic LV pressures, it cannot precisely determine rapidly changing pressures as occur during LV isovolumetric relaxation . Use of  annulus  and measAlthough the present study does not provide direct mechanisms underlying the association between abnormal LV relaxation and adverse prognosis in patients with known or suspected CAD, there are possible explanations. The most common cause of cardiac death or cardiovascular hospitalization in our study was ischemic myocardial events. It is well-established that the presence of myocardial ischemia impairs LV relaxation . In factAnother possible explanation for our observed association of impaired LV relaxation with adverse prognosis is that LV diastolic abnormalities may coexist with other disorders that may adversely impact on prognosis. Specifically, Lee et al. reported that altered LV diastolic function assessed by Doppler echocardiography was independently associated with endothelial dysfunction and hemostatic abnormalities , both ofE/e′. Forth, our study patients were composed of stable known or suspected CAD patients without congestive heart failure who were referred for cardiac catheterization for assessment of CAD. Our findings cannot be extended to other populations. Finally, our study is not large enough to permit us to evaluate the association of LV diastolic dysfunction with specific causes of death or hospitalization.There are several limitations in the present study. First, LV mass was available in the limited number of the patients (68%), which could have influenced our results . Furthere′ by Doppler echocardiography may provide useful information for risk stratification in known or suspected CAD patients without congestive heart failure. The cut-off point of e′ used in the present study, however, is higher than that previously reported. Specifically, Wang et al. reported that e′ < 3 cm/s was a powerful predictor for cardiac death in patients with reduced (<50%) EF [e′ < 3.5 cm/s for cardiac death in hypertensive subjects with LV hypertrophy [e′ for the prediction of adverse prognosis may be variable depending on population studied. Further studies are necessary for determining the optimal cut-off points of e′ for identifying high-risk patients in various populations.Our study suggests that measurement of <50%) EF . Similarertrophy . These rOur findings suggest that patients with impaired LV relaxation may need more aggressive treatment of CAD. A recent study has reported that altered LV relaxation assessed by Doppler echocardiography is improved after coronary revascularization in patients with ischemic cardiomyopathy . The pro


Very high gravity (VHG) fermentation using medium in excess of 250 g/L sugars for more than 15% (v) ethanol can save energy consumption, not only for ethanol distillation, but also for distillage treatment; however, stuck fermentation with prolonged fermentation time and more sugars unfermented is the biggest challenge. Controlling redox potential (ORP) during VHG fermentation benefits biomass accumulation and improvement of yeast cell viability that is affected by osmotic pressure and ethanol inhibition, enhancing ethanol productivity and yield, the most important techno-economic aspect of fuel ethanol production.Batch fermentation was performed under different ORP conditions using the flocculating yeast and media containing glucose of 201 ± 3.1, 252 ± 2.9 and 298 ± 3.8 g/L. Compared with ethanol fermentation by non-flocculating yeast, different ORP profiles were observed with the flocculating yeast due to the morphological change associated with the flocculation of yeast cells. When ORP was controlled at −100 mV, ethanol fermentation with the high gravity (HG) media containing glucose of 201 ± 3.1 and 252 ± 2.9 g/L was completed at 32 and 56 h, respectively, producing 93.0 ± 1.3 and 120.0 ± 1.8 g/L ethanol, correspondingly. In contrast, there were 24.0 ± 0.4 and 17.0 ± 0.3 g/L glucose remained unfermented without ORP control. As high as 131.0 ± 1.8 g/L ethanol was produced at 72 h when ORP was controlled at −150 mV for the VHG fermentation with medium containing 298 ± 3.8 g/L glucose, since yeast cell viability was improved more significantly.No lag phase was observed during ethanol fermentation with the flocculating yeast, and the implementation of ORP control improved ethanol productivity and yield. When ORP was controlled at −150 mV, more reducing power was available for yeast cells to survive, which in turn improved their viability and VHG ethanol fermentation performance. On the other hand, controlling ORP at −100 mV stimulated yeast growth and enhanced ethanol production under the HG conditions. Moreover, the ORP profile detected during ethanol fermentation with the flocculating yeast was less fluctuated, indicating that yeast flocculation could attenuate the ORP fluctuation observed during ethanol fermentation with non-flocculating yeast. Fuel ethanol that is renewable and environmentally friendly has been produced around the world as an alternative to fossil fuels. HoweverApparently, ethanol-tolerant strains are prerequisite for more efficient ethanol production under VHG conditions in order to overcome stuck fermentation, in which significant sugars are present at the end of fermentation, and ethanol yield, the most important techno-economic aspect of fuel ethanol production, is compromised, correspondingly. Stuck fermentation results from severe ethanol inhibition in yeast cells. When yeSaccharomyces cerevisiae subjected to ORP control, high ethanol productivity and yield could be achieved[Redox potential (ORP) reflects electron activities during fermentations, which provides real-time information on the physiological status of cells. Controlachieved-13.In this study, ORP profiles were monitored and analyzed for ethanol fermentation with the flocculating yeast. Furthermore, ORP control strategy was developed by controlling ORP at two different levels in order to improve ethanol productivity and yield for VHG ethanol fermentation with the flocculating yeast.S. cerevisiae and ethanol fermentation under VHG conditions[ORP affected the growth of non-flocculating nditions. Thus, tWhen yeast flocs were inoculated into fresh media, vigorous growth quickly depleted dissolved oxygen in the media and fermentation reducing power outperformed oxidizing power, leading to a drastic decline of ORP from 110 mV to −220 mV in Region I, which was characterized by a logarithmic growth of yeast flocs. Yeast growth was then retarded due to ethanol accumulation and its inhibition effect, and a stationary phase was developed, correspondingly, which was characterized by a slow decrease of ORP till the lowest level of −280 mV in Region II. Near the end of fermentation, severe ethanol inhibition completely inhibited yeast growth, and in the meantime the lysis of yeast cells occurred, resulting in an increase of ORP.S. cerevisiae[Compared with ethanol fermentation with non-flocculating erevisiae, time coFigureBased on ORP profiles recorded in FigureIn reference to ethanol fermentation without ORP control, more ethanol was produced when the ORP control strategy was applied. Since feedstock consumption is the major cost for fuel ethanol production, more etThe highest ethanol concentration of 131.0 ± 1.8 g/L was obtained for ethanol fermentation with medium containing 298 ± 3.8 g/L glucose when ORP was controlled at −150 mV. Whereas, for ethanol fermentation with media containing glucose of 252 ± 2.9 and 201 ± 3.1 g/L, ethanol production was enhanced more significantly when ORP was maintained at −100 mV.Ethanol production is attributed to both yeast cell viability and biomass accumulation. Yeast cell viability was improved more significantly when ORP was controlled at −150 mV, since more reducing power would favor the maintenance metabolism of yeast cells and correspondingly improve their viability,15, whicGlucose was completely consumed for ethanol fermentation with media containing 201 ± 3.1 and 252 ± 2.9 g/L glucose when ORP was controlled at −100 mV. Although ethanol fermentation under 298 ± 3.8 g/L glucose conditions was improved when ORP was controlled at −150 mV, as high as 24.0 ± 1.1 g/L glucose was remained unfermented, which would be not acceptable from the point of view of industrial application. Hence, various fermentation strategies and process configurations such as tanks-in-series fermentation systems have been developed,17.S. cerevisiae[Compared with the bathtub ORP profile previously reported for VHG ethanol fermentation with erevisiae, differeFluctuation in ORP during the stationary phase of non-flocculating yeasts was recorded. In contrast, ORP was relatively stable during ethanol fermentation with the flocculating yeast. We postulated that this difference might be due to the morphological change associated with the flocculation of yeast cells.S. cerevisiae was employed[The flocculating yeast formed flocs during ethanol fermentation and the size of the yeast flocs increased as the fermentation proceeded Figures and2. Aemployed.Compared to non-flocculating yeast, no lag phase was observed for growth and ethanol fermentation of the flocculating yeast under VHG conditions, which resulted in a drastic decline of ORP, and in the meantime stuck fermentation with prolonged fermentation time and significant glucose remained unfermented was observed due to mass transfer limitation associated with the flocculation of yeast cells. The ORP control provided a strategy for improving ethanol productivity and yield for the VHG fermentation system.Ethanol fermentation performance is affected by biomass accumulation as well as yeast cell viability, and thus the ORP control impacts the fermentation system differently. With media containing glucose of 201 ± 3.1 and 252 ± 2.9 g/L, complete fermentation was observed at 32 and 56 h when the ORP was controlled at −100 mV, since yeast cell growth was stimulated by more oxygen supply through aeration. On the other hand, for ethanol fermentation with medium containing 298 ± 3.8 g/L glucose, as high as 131 ± 1.8 g/L ethanol was produced at 72 h when the ORP was controlled at −150 mV, because the lower ORP level with more reducing power available for maintaining metabolism of yeast cells improved their viability more significantly. Although the ORP control improved ethanol fermentation performance, residual glucose was still high for the VHG ethanol fermentation, which would be not acceptable from the view point of industrial application, and other bioprocess engineering strategies such as tanks-in-series systems that can alleviate ethanol inhibition in yeast cells should be developed and integrated with the ORP control.When ORP control was applied, the ORP profile detected during ethanol fermentation with the flocculating yeast was more stable, compared with that observed in ethanol fermentation with non-flocculating yeast, indicating that the morphological change associated with the flocculation of yeast cells and micro-environment created within yeast flocs not only attenuated the flocculation of ORP, but also might prevent yeast lysis, which would benefit process control and optimization.The flocculating yeast strain was developed at Dr. Bai’s lab at Dalian University of Technology. This stAn autoclavable ORP electrode  was inserted into the fermentor to monitor ORP, and the measured voltage signal was processed by a pH/ORP controller . When the measured ORP was lowered than the set point, a proper amount of filter-sterilized (0.2 μm nylon membrane) air was sparged into the fermentor to maintain ORP at designated levels: -100 or −150 mV. The amount of air was determined by the PID algorithm built-in in the pH/ORP controller.2SO4, and the flow rate was set at 0.6 mL/min. The temperature for column and RI detector were set at 50°C. Yeast cell viability was determined as a ratio of viable to total cell numbers counted under microscope by methylene-violet staining procedure[A 4-mL fermentation broth was sampled every 6 or 8 h. The sample was centrifuged at 10000 × g for 5 min. The precipitate was washed twice with deionized water, dried at 85°C for 48 h, and weighted. The supernatant was used to quantify glucose and ethanol by HPLC (Model: Waters 1525) with an RI detector (Model: Waters 2414). An ion exclusion column  was used to separate metabolites. The mobile phase consisted of 10 mM Hrocedure.A focused beam reflectance measurement system was used to monitor yeast flocs in situ during the fermentation, which were characterized by the statistic mean of the yeast floc distribution.HG: High gravity; VHG: Very high gravity; ORP: Oxidation-reduction potential, also known as redox potential.The authors declare that they have no competing interests.CGL, under the supervision of YHL and FWB, developed the research scheme and prepared the draft manuscript. NW carried out the experimental work. CGL, YHL and FWB involved data interpretation, result discussion, and manuscript revision. All authors agreed and approved the manuscript submission.CGL completed his PhD study on very high gravity ethanol fermentation in 2011 at Dalian University of Technology (DUT), China, and is now working as a post-doctoral fellow at DUT. NW is a MSc graduate at DUT, working on ethanol fermentation kinetics of flocculating yeast. YHL is a Professor at the Department of Chemical and Biological Engineering, University of Saskatchewan, Canada. FWB is a Professor at DUT’s School of Life Science and Biotechnology. YHL and FWB co-supervised CGL's PhD study.


Atrial fibrillation (AF) and heart failure (HF) are 2 of the most common cardiovascular conditions nationally and AF frequently complicates HF. We examined how AF has impacts on adverse outcomes in HF‐PEF versus HF‐REF within a large, contemporary cohort.We identified all adults diagnosed with HF‐PEF or HF‐REF based on hospital discharge and ambulatory visit diagnoses and relevant imaging results for 2005–2008 from 4 health plans in the Cardiovascular Research Network. Data on demographic features, diagnoses, procedures, outpatient pharmacy use, and laboratory results were ascertained from health plan databases. Hospitalizations for HF, stroke, and any reason were identified from hospital discharge and billing claims databases. Deaths were ascertained from health plan and state death files. Among 23 644 patients with HF, 11 429 (48.3%) had documented AF . Compared with patients who did not have AF, patients with AF had higher adjusted rates of ischemic stroke , hospitalization for HF , all‐cause hospitalization , and death . The associations of AF with these outcomes were similar for HF‐PEF and HF‐REF, with the exception of ischemic stroke.AF is a potent risk factor for adverse outcomes in patients with HF‐PEF or HF‐REF. Effective interventions are needed to improve the prognosis of these high‐risk patients. The sites were identified on the basis of providing care to an ethnically and socioeconomically diverse population across varying clinical practice settings and geographically diverse areas. Each site also had a Virtual Data Warehouse (VDW),10 which served as the primary data source for identifying and characterizing study subjects. The CVRN VDW is a distributed standardized data resource composed of linked demographic, pharmacy, laboratory test results, and health care utilization  data for health plan members receiving care within participating CVRN sites.10The source population included members from 4 participating health plans within the National Heart, Lung, and Blood Institute–sponsored Cardiovascular Research Network (CVRN).Institutional review boards at participating sites approved the study, and waiver of consent was obtained due to the nature of the study.International Classification of Diseases, Ninth Edition (ICD‐9) codes were used to identify potential HF cases: 398.91, 402.01, 402.11, 402.91, 404.01, 404.03, 404.11, 404.13, 404.91, 404.93, 428.0, 428.1, 428.20, 428.21, 428.22, 428.23, 428.30, 428.31, 428.32, 428.33, 428.40, 428.41, 428.42, 428.43, and 428.9. When compared against medical record abstraction and use of the Framingham Heart Study clinical criteria, use of the primary discharge diagnosis of HF based on these codes showed a positive predictive value of >95%.14 We determined the level of left ventricular systolic function closest to the qualifying HF diagnosis, based on clinically obtained echocardiograms and other relevant imaging modalities. We classified patients into categories of preserved and reduced left ventricular ejection fraction. We defined PEF as left ventricular ejection fraction ≥50% and/or a physician's qualitative assessment of preserved or normal systolic function.15 REF was defined as left ventricular ejection fraction ≤40% and/or a physician's qualitative assessment of moderate, moderate‐to‐severe, or severe systolic dysfunction. To limit misclassification, we excluded participants with ejection fraction >40% and <50% and/or a physician's qualitative assessment of mild systolic dysfunction.We identified all individuals aged ≥21 years with diagnosed HF between January 1, 2005, through December 31, 2008, based on either ≥1 hospitalization with a primary discharge diagnosis of HF and/or ≥3 ambulatory visits coded for HF with ≥1 of the ambulatory diagnoses from a cardiologist to enhance diagnostic specificity. The following 4 We defined preexisting AF as AF documented any time during the 5 years before cohort entry, and incident AF as AF occurring anytime during follow‐up among those patients with HF without AF at baseline.We ascertained AF based on ≥1 primary hospital discharge and/or ≥2 ambulatory diagnoses of AF (ICD‐9 code 427.31) or atrial flutter (ICD‐9 code 427.32) from each site's VDW.16 Occurrence of death was identified using data from member proxy report, state death certificate registries, and Social Security Administration files as available at each site. These approaches have yielded >97% vital status information in our prior studies.17Follow‐up occurred through December 31, 2008, which was the latest date on which complete data on death were available at the time of analysis. Subjects were censored if they disenrolled from the health plan or reached the end of study follow‐up. Hospitalizations for HF were identified from each site's VDW based on a primary discharge diagnosis for HF using the same inclusion criteria ICD‐9 codes. Ischemic strokes were identified from hospital discharge and billing claims databases using previously validated ICD‐9 codes.18 We defined prevalent HF as any hospitalization or ambulatory HF diagnosis before the index date. We collected baseline and follow‐up data on diagnoses of coronary artery disease, acute myocardial infarction, coronary artery revascularization, stroke and transient ischemic attack, peripheral artery disease, diabetes, hypertension, cancer, liver disease, valvular heart disease, lung disease, and ventricular fibrillation/tachycardia based on previously described ICD‐9 codes and Current Procedural Terminology procedure codes.18 For the purposes of this study, “baseline” was defined by the period 5 years before the index date for data regarding comorbidities and laboratory values. For medication use, “baseline” was determined by any use within 120 days before the index date and active use within 30 days of index date.We ascertained information on coexisting illnesses based on diagnoses or procedures using relevant ICD‐9 codes, laboratory results, or filled outpatient prescriptions from health plan hospitalization discharge, ambulatory visit, laboratory, and pharmacy databases, as well as from site‐specific diabetes mellitus and cancer registries.2) based on outpatient serum creatinine results.19 We characterized longitudinal receipt of HF‐related medications including angiotensin‐converting enzyme inhibitors, angiotensin receptor blockers, β‐blockers, digoxin, thiazide and loop diuretics, nitrates, aldosterone receptor antagonists, and statins using previously described methods.12 We also identified receipt of cardiac resynchronization therapy (with or without defibrillator), implantable‐cardioverter defibrillator placement, and pacemaker placement using ICD‐9 procedure and Current Procedural Terminology codes.10Using each site's VDW, we captured ambulatory measurements of systolic and diastolic blood pressure, serum low‐density lipoprotein cholesterol, and blood hemoglobin level on or before the index date and during follow‐up. We also classified baseline and longitudinal kidney function using the Chronic Kidney Disease Epidemiology Collaboration formula for estimating glomerular filtration rate . We compared baseline characteristics by AF status  using the following methods: Kruskal–Wallis test for comparing median values, ANOVA for comparing mean values, and χWe calculated rates (per 100 person‐years) and associated 95% CIs for each outcome according to AF status among patients with HF, overall and stratified by HF‐PEF versus HF‐REF using a time‐to‐event approach. We then conducted multivariable extended Cox regression models to examine the association between AF status and each outcome, overall and separately in those with HF‐PEF versus HF‐REF. Death was treated as a censoring event when analyzing time to event outcomes.). Of note, at baseline, 58% of the overall cohort received an angiotensin‐converting enzyme inhibitor or angiotensin receptor blocker, 63% of participants received a β‐blocker, and 26% received a calcium channel blocker . Overall, 9081 (38.4%) had preexisting AF and 2348 (9.9%) had developed newly diagnosed (incident) AF during the study period . The freTable 1 blocker .Median follow‐up of the overall cohort was 1.8 years (interquartile range 0.8 to 3.1). The rate of death from any cause in the overall cohort was 14.1 per 100 person‐years (95% CI 13.8 to 14.5). The crude rate (per 100 person‐years) of death was higher in those with incident AF compared with those who had preexisting AF or no AF . In the The rate of ischemic stroke was 2.0 per 100 person‐years (95% CI 1.9 to 2.2) in the overall cohort. Crude rates (per 100 person‐years) of ischemic stroke were 1.5, 2.5, and 3.4 for those with no AF, preexisting AF, and incident AF, respectively . In the In the overall cohort, the rate of hospitalization for HF was 16.6 per 100 person‐years (95% CI 16.2 to 17.0). Crude rates (per 100 person‐years) were higher in those with preexisting AF (18.9) or with incident AF (19.6) compared with patients who did not have AF (14.9) . In the The rate of hospitalization from any cause in the overall cohort was 68.2 per 100 person‐years (95% CI 67.1 to 69.2). Crude rates (per 100 person‐years) were higher in those with preexisting AF (75.3) or incident AF (81.0) compared with patients who did not have AF (63.1) . In the Within a multiethnic community‐based cohort of >23 600 adults with HF, we demonstrate that both preexisting and incident AF are common in patients with HF and are associated with major adverse cardiovascular outcomes. Newly diagnosed AF complicating HF was associated with the highest risk for adverse complications, but the higher multivariable adjusted hazards of ischemic stroke, hospitalization for HF, hospitalization for any cause, and death associated with AF did not materially differ between participants with HF‐PEF and those with HF‐REF. These associations persisted even after accounting for a broad spectrum of potential confounders as well as differential longitudinal exposure to relevant medications.23 Although data are more limited for patients with HF‐PEF, the frequency of preexisting AF (43.2%) among patients with HF‐PEF with our study was similar to the proportion of patients with AF in the Irbesartan in heart failure with PRESERVEd systolic function (I‐PRESERVE) study as well as 2 studies involving 2802 (31.8%) and 6072 (41.3%) subjects with HF conducted in 2001.24 We also noted a high and relatively similar rate of new‐onset AF complicating HF‐REF and HF‐PEF, with approximately 1 in 10 participants developing AF during a median 1.8 years of follow‐up. Incidence rates of AF observed in our study are significantly higher than those reported in 2 community‐based investigations involving 708 and 1664 patients with HF, in which 17% and 23% of patients with HF developed new‐onset AF during 4 years of follow‐up, respectively.7 The higher rate of incident AF in our study likely relates to the higher proportion of subjects with HF‐PEF, higher prevalence of AF risk factors, including hypertension and diabetes, and access to ambulatory diagnosis data, compared with other studies.We found that nearly one third of patients with HF‐REF had preexisting AF, which is consistent with previous estimates (15% to 35%).25 As expected, we found that patients with HF with AF in our cohort were more likely to have worse kidney function, documented hypertension, and known valvular heart disease.26 Notably, however, patients with HF and AF were not more likely to have diabetes or coronary artery disease than were patients without AF, which is consistent with other studies.28The epidemiologic similarities between AF and HF, as well as their frequent concurrence, are explained, at least in part, by shared underlying risk factors, including hypertension, diabetes mellitus, chronic kidney disease, ischemic heart disease, and valvular heart disease.7 Among participants in the Candesartan in Heart failure—Assessment of Reduction in Mortality and morbidity (CHARM) study, crude death rates were 24% in those with AF and HF‐PEF and 37% in those with HF‐REF.28 In contrast to CHARM,28 in our study of 23 644 participants with HF, we found that incident AF was associated with a notably higher rate of death that was similar in those with HF‐PEF or HF‐REF. The reasons for the differences between our study and CHARM are not clear but may relate to differences between the selected subjects with HF enrolled in the CHARM clinical trial versus the more representative patients with HF treated in community‐based practice settings in our study.Absolute rates of death in our study were high and consistent with rates seen in previous studies of patients with AF and HF. Among 1470 individuals with HF from the Framingham Heart Study, incident AF was associated with a 60% higher risk for death in men and nearly 3‐fold higher risk for death in women.29 Of note, however, preexisting AF was associated with higher adjusted hazard of ischemic stroke in those with HF‐PEF but not for those with HF‐REF. Although the CHARM investigators reported higher rates of ischemic stroke in patients with known AF and HF‐PEF compared with HF‐REF , this difference did not achieve statistical significance (P=0.32).28 However, the CHARM study was underpowered to evaluate the association between prevalent AF and ischemic stroke due to a low number of fatal or nonfatal strokes in their cohort with AF . The reasons for why preexisting AF was a strong predictor of ischemic stroke in those with HF‐PEF but not HF‐REF are not clear, but patients with preexisting AF may have preferentially experienced stroke or other AF‐related complications and death before the development of HF and therefore led to a more selected subgroup of patients with preexisting AF and HF in our cohort. Although we adjusted for age, the interaction between age and risk for ischemic stroke might have modified the relation between preexisting AF and HF‐PEF in a manner distinct from HF‐REF. Moreover, although we did not observe significant differences in the death rates of patients with HF‐PEF compared with HF‐REF, it is also possible that our findings could also be explained by the fact that patients with HF‐REF may have been more likely than patients with HF‐PEF to die before ischemic stroke develops.As expected, we found that AF was associated with an elevated risk for ischemic stroke in adults with HF. We observed that incident AF conveyed a particularly high hazard of ischemic stroke (≈2.5‐fold higher adjusted rates compared with no AF), which is consistent with previous analyses.30 In contrast to the findings of a Japanese HF registry, which included 319 patients with HF hospitalized in 2006–2007,31 we observed a significant increase in adjusted rates of HF‐related and all‐cause hospitalization among patients with preexisting and incident AF. Our findings are consistent with those of the CHARM substudy, which found a 4‐fold higher risk for cardiovascular‐related rehospitalization in patients with new‐onset AF and HF, regardless of systolic function.28 Even after extensive adjustment for potential confounders and longitudinal use of therapies, we found an approximately 1.5‐fold higher hazard of hospitalization associated with incident AF. The difference in the magnitude of association between these findings may be explained by the greater comorbid disease burden in our community‐based cohort compared with the younger and healthier participants enrolled in the CHARM clinical trial. This hypothesis is supported by the overall higher rates of HF and all‐cause hospitalizations in our study relative to CHARM. Our finding that AF had a similarly negative impact on HF‐specific and all‐cause hospitalizations among patients with HF‐PEF and those with HF‐REF highlight the importance of AF on the high burden and cost to patients and the health care system.Overall rates of hospitalization observed in our study (66.6%) were higher than those reported in a study of 17 448 Medicare beneficiaries hospitalized for HF  during 1991–1994.32 We did not include information about certain AF treatment modalities, including AF ablation, but prior investigations have not consistently shown that AF ablation reduces HF hospitalizations, total death rates, or the risk of stroke.33 Because we relied on clinically obtained assessments of left ventricular systolic function, systematic data on structural aspects of the atria were unavailable. However, we believe that our sample represents a “real world” cohort of adults with clinically recognized HF managed in typical care settings and therefore provides generalizable results to the broader US population.Our study included a large socioeconomically and racially diverse multicenter cohort recruited from multiple geographic areas and varying clinical practice settings. Another strength of our study is the use of a standardized data resource (CVRN VDW) with linked demographic, health care utilization, pharmacy, laboratory, and vital status information. Our study also had several limitations. Our study was conducted in an insured population, so the findings may not be fully generalizable to uninsured persons or other practice settings. The large sample size facilitated many statistically significant findings, but we focused on clinically meaningful effect sizes. Information was unavailable on the type of AF , although previous studies support similar relationships between AF type and the risk of ischemic stroke.In sum, both preexisting and new‐onset AF were frequent complications of HF and increased the rates of ischemic stroke, hospitalization for HF or any cause, and death, overall and similarly in those with HF‐PEF or HF‐REF. Incident AF consistently carried a worse prognosis for each of these outcomes compared with preexisting AF. Our study emphasizes the need to develop novel prevention strategies for AF and its associated complications in patients with HF‐PEF and HF‐REF.


In addition to its antiviral effect, Ritonavir directly inhibits the insulin-regulated glucose transporter GLUT4 and blocks glucose entry into fat and muscle cells. However, the effect of Ritonavir on cardiac GLUT4 inhibition during myocardial necrosis is not investigated. In the present study, we evaluated the role of Ritonavir in isoproterenol-induced myocardial necrosis Isoproterenol (ISO)  was administered to mice to cause myocardial necrosis. Phlorizin (400 mg/kg/day i.p twice daily for 2 days) and Ritonavir (10 mg/kg/day i.p twice daily for 2 days) were administered in two different groups of mice before isoproterenol administration.In vitro study with heart homogenate confirmed no antioxidant effect of ritonavir and phlorizin in the absence and presence of isoproterenol.Isoproterenol (ISO)  administration caused significant (p < 0.05) increase in heart/body weight ratio, and myocardial necrosis as evident by significant (p < 0.05) increase in serum markers i.e. SGOT and CK; and cardiac histopathological changes. Significant (p < 0.05) reduction in myocardial SOD and catalase activities, and GSH level along with a significant (p < 0.05) rise in myocardial TBARS and nitric oxide levels were observed after ISO administration. However, administration of phlorizin, a SGLT1 inhibitor has been found to exhibit partial protection in ISO induced myocardial necrosis, as observed by significant decrease in heart/body weight ratio and myocardial nitric oxide level; significant increase in myocardial SOD and catalase activities along with no histopathological alterations. On the other hand, administration of ritonavir, a nonspecific GLUT inhibitor has been found to exhibit complete protection as observed by normalisation of heart/body weight ratio, serum markers, antioxidant enzymes activities and histopathological alterations. Our study concluded that ritonavir, a nonspecific GLUT inhibitors showed complete protection in catecholamine induced myocardial necrosis. Myocardial infarction (MI) is one of the main causes of death from cardiovascular disease. MI is defined as an acute condition of necrosis of the myocardium that occurs as a result of imbalance between coronary blood supply and myocardial demand . The heaIn mammalian heart, glucose transport is believed to be mediated mainly by two members of the GLUT family, GLUT1 and GLUT4. Increased activity of GLUTs in diseased heart has been reported earlier . SGLT isIn the present study, we inhibited SGLTs and GLUTs transporters in heart by using pharmacological inhibitors, phlorizin (nonselective SGLT blocker) and ritonavir (nonselective GLUTs blocker) respectively, and evaluated the relative importance of these transporters during myocardial necrosis induced by isoproterenol.ad libitum. All animal experiments were undertaken with the approval of Institutional Animal Ethics Committee of Indian Institute of Chemical Technology, Hyderabad, India.Male Swiss albino mice (20-25 gm) were provided by National Institute of Nutrition (NIN), Hyderabad. The animals were housed in BIOSAFE, an animal quarantine facility of Indian Institute of Chemical Technology (IICT) Hyderabad. The animal house was maintained at temperature 22 ± 2°C, relative humidity 50 ± 15% and 12 hour dark/light cycle was maintained throughout the study. Mice had free access to food  and water Weight matched male swiss albino mice were randomly divided into four groups with each group having eight animals. Six and two animals from each group were kept for biochemical and histopathological evaluation, respectively. The doses used in this study were selected on the basis of reports of previous studies ,8.•Control group .•ISO group (SC injection of ISO 150 mg/kg/day for 2 consecutive days).•ISO+Phz group (IP injection of phlorizin 400 mg/kg/day 10 min. prior to ISO dose for 2 days).•ISO+RTV group (IP injection of ritonavir 10 mg/kg/day 10 min. prior to ISO dose for 2 days).ISO is dissolved in PBS while phlorizin and ritonavir were dissolved in vehicle . Control group received phosphate buffer saline (PBS) and vehicle at the time of ISO and ‘phlorizin and ritonavir’ administration, respectively. ISO group received vehicle at the time of phlorizin and ritonavir administration.The animals in all groups were sacrificed 48 hrs after first dose of isoproterenol injection. Cardiac tissues were collected and stored at - 80°C for further biochemical evaluation. At the time of sacrifice, blood was collected by cardiac puncture, serum was separated by centrifugation at 4000 rpm (4°C) for 15 minutes and serum markers (SGOT and CK) were analysed by auto blood analyser (Bayer diagnostic). SGOT and CK were expressed in IU/L.Each heart was homogenized with 20 times volume of heart weight in ice cold 0.05 M potassium phosphate buffer and treated separately as described below for the measurement of different biochemical parameters . 20% homAll cardiac samples after euthenisation were fixed in 10% neutral buffer formalin. Paraffin embedded 5 μm thick sections were obtained and stained with Hematoxylin and Eosin (H&E stain). Prepared sections were examined under light microscope to assess gross myocyte injury and the effects of interventions.-5 M-1 cm-1).Adult male swiss albino mice were euthanized. Heart was excised, washed with 0.9% NaCl solution and homogenised with 20-times volume of heart weight in 0.05 M ice-cold phosphate buffer [pH 7.4] . Heart hAll values were expressed as mean ± SEM. Data were statistically analyzed using one way ANOVA for multiple group comparison, followed by student unpaired ‘t’ test for group wise comparison. Significance was set at P ≤ 0.05. Data were computed for statistical analysis by using Graph Pad Prism Software.A significant p ≤ 0.001) increase in heart weight / body weight ratio was observed in ISO group in comparison to control. However, significant (p ≤ 0.05) decrease in heart weight / body weight ratio was observed both in ISO + Phz and ISO + RTV groups in comparison to ISO group (Table  increasep < 0.05) in ISO group in comparison to control group. Significant (p < 0.05) increase in SGOT level but no change in CK level was observed in ISO + Phz group. However, significant (p < 0.05) decrease in both SGOT and CK levels was observed in ISO + RTV group in comparison to ISO group (Table SGOT and CK levels were significantly increased (up Table .p < 0.01) in ISO group in comparison to control. There was significant (p < 0.05) decrease in myocardial nitric oxide level but no change in myocardial TBARS level in ISO + Phz group in comparison to ISO group. However, there was significant (p < 0.05) decrease in both myocardial TBARS and nitric oxide level in ISO + RTV group in comparison to ISO  in ISO group in comparison to control group. There was significant (p < 0.01) increase in both myocardial catalase and SOD level but no significant change in myocardial GSH level in ISO + Phz group in comparison to ISO. However, significant (p < 0.05) increase in all three parameters was observed in ISO + RTV group in comparison to ISO  and ritonavir (RTV group) treatment in comparison to control. Significant (p < 0.05) increase in TBARS level was observed after addition of isoproterenol (ISO group). However, no significant change in TBARS level was observed in ISO group after phlorizin (ISO + Phz group) and ritonavir (ISO + RTV group) treatment in comparison to ISO group  and SGOT enzymes. These enzymes enter into the blood stream and thus increasing their concentration in the serum, and serve as the diagnostic markers of myocardial tissue damage -23. Isop2 · -] and H2O2, which have damaging effects in cells [Lipid peroxidation and endogenous antioxidants were also measured to confirm the myocardial oxidative stress after administration of isoproterenol. Isoproterenol produce quinones which react with oxygen to generate superoxide anions [Oin cells . ISO indin cells ,22,25. Iin cells . Increasin cells , no antiin-vitro antioxidant assay with heart homogenate. Our study showed no significant change in TBARS level, a marker of lipid peroxidation, after addition of phlorizin and ritonavir in heart homogenate in absence or presence of isoproterenol. This indicates that both phlorizin and ritonavir had no additional antioxidant effect in presence of isoproterenol. Thus the cardioprotective effect of phlorizin and ritonavir might be due to the inhibition of glucose uptake in heart.To further confirm whether phlorizin and ritonavir have any antioxidant property, we did ISO-induced myocardial necrosis is also confirmed by histopathological findings. ISO-treated heart showed the features of severe inflammation, the presence of inflammatory cells between the fibers, vacuolation of myocytes and few focal areas of myocytolysis. These all changes were previously reported in various ISO-induced myocardial necrosis models ,25. HoweCatecholamine or isoproterenol generally increases heart rate and glucose uptake in heart. The reason of myocardial injury by isoproterenol might be through catecholamine induced free radical generation. It is possible that increased glucose uptake in heart might be responsible for more free radical generation and oxidative stress. Our present study showed that inhibition of cardiac glucose uptake through GLUT and SGLT1 blockers were beneficial for ISO-induced necrotic heart. We found partial protection by phlorizin, a SGLT1 inhibitor, in ISO-induced myocardial necrosis in mice. This protection might be possible through inhibition of cardiac glucose uptake through SGLT1 glucose transporter. Most important finding in our study is that ritonavir (GLUTs inhibitor) provides complete protection in ISO induced myocardial necrosis. It attenuated heart/body weight ratio, serum markers and oxidative stress. Hence ritonavir proved to be a better cardioprotective agent and prevents myocardial oxidative stress in ISO induced myocardial necrosis.Thus our study concludes that non-specific GLUT inhibitor, ritonavir showed complete protection during ISO-induced myocardial necrosis. However, further studies should be carried out to find out whether ritonavir possess cardioprotection through additional effect unrelated to glucose transporter inhibition.The authors declare that there are no conflicts of interest.PG, AK, YB, TNK, BS and MK carried out animal experimentation, biochemical estimation and statistical analysis of results. UKP did all histopathology work and its interpretation. SKB conceived the study, and participated in its design, coordination and drafted the manuscript. All authors read and approved the final manuscript.PG, YB and BS are MS in Pharmacology , AK and TNK are Senior Research Fellow from Council of Scientific and Industrial Research, MK (PhD) is Senior Technical Assistant, UKP is a scientist in National Institute of Nutrition, Hyderabad and SKB (PhD) is Principle Investigator in the Division of Medicinal Chemistry and Pharmacology, Indian Institute of Chemical Technology (IICT), Hyderabad-500607, India.