Xinxiang”, “Jinyu”, “Zimei” and “Yuzishu 263” were treated by baking, boiling and steaming and subsequently analyzed for starch content, amylase activity and sugar contents including glucose, fructose, sucrose and maltose. Results indicated that cooking reduced starch content and final amylase activity and increased reducing sugar content especially maltose content, but did not have significant influence on non-reducing sugar content. These effects were different among the four cultivars and three cooking methods. Baking led to the least starch reduction. Storage roots of “Jinyu” contained the highest amount of sugar content and thus sweetest. Sugar composition analysis suggested that cultivars “Xinxiang” and “Jinyu” belong to high-maltose cultivars. This study may provide useful information for evaluating the cooking quality of sweetpotato cultivars.Sweetpotato has rich nutrition, good ecological adaptability and high yield. There is a lack of knowledge about the effects of cooking methods on starch and sugar components in elite Chinese cultivars. In this study, sweetpotato storage roots from four cultivars “ Ipomoea batatas), a dicotyledonous plant, belongs to the Convolvulaceae family. It is a root vegetable due to its large, starchy, sweet-tasting storage roots. Sweetpotato is native to the tropical regions in America. It is distantly related to common potato (Solanum tuberosum).Sweetpotato  and the purple flesh cultivars (“Zimei” and “Yuzishu263”). “XinXiang” and “Jinyu” were created by Agricultural Academic Science Institution of Zhejiang Province. “XinXiang” storage root is red skin and oval shape. “Jinyu” storage root is red skin and long. “Yuzishu 263” was bred by Sweetpotato Institution of Southwest University in Chongqing City, and the storage root is purple skin and looks long. “Zimei” was introduced from Vietnam, and the storage root is purple skin and oval shape.Sweetpotato cultivars selected in the study were representatives of the yellow flesh cultivars (“All storage roots were freshly harvested within one week before being used in the experiments. The same sizes of storage roots (about 250 g) were used in the study. Chemicals  were purchased from East China Medicine Company.Boiling: sweetpotato storage roots were washed with water, soaked into water in a pan followed by heating them using electric cooker. It took 28 min for boiling in boiled water. Steaming: sweetpotato storage roots were put on steaming box above the water line in the pan. It also took 28 min for steaming since water boiled. Baking: sweetpotato storage roots were settled in a baking box, set temperature at 200°C. It took 45 min for baking at 200°C. We did not peel the storage roots and performed any cutting before cooking. After cooking, samples were cooled to normal temperature, and cut into pieces (1.0 cm*1.0 cm*0.2 cm). Some of them were freezing drought under -45°C for 48 hr before being smashed; others were baked at 80°C until the weight kept consistent. Raw sweetpotatoes (as control treatment) were also cut into pieces (1.0 cm*1.0 cm*0.2 cm) before drought as above.Bake-dried rate and freeze-dried rate were calculated as follows: Bake-dried rate = baked sugar content (g) / bake-dried weight (g)*100%. Freeze-dried rate = freeze-dried sugar content (g) / freeze-dried weight (g)*100%.rpm for 15 min. The supernatant was collected, filtered through a 0.45-μm membrane filter and injected into a high performance liquid chromatography  with a refractive index detector. HPLC employed reverse-phase C-18 column ; sample volume was 10 μL; mobile phase was 70% acetonitrile:H2O (7:3); and flowing rate was 0.5 mL/min. The column was heated to 35°C.Soluble sugars were extracted from the sweetpotato samples based on the published method ,14 with Sweetpotato sweetness was calculated according to the method  using thStarch was prepared and detected based on the method of Cao . It was rpm for 15 min, the supernatant was used to determine amylase activities.α-amylase activities were determined by the starch azure assay with minor modification . The samAll the analyses were done in triplicates. The data were subjected to statistical analysis using the statistical software SPSS13.1 and Microsoft Excel. Variance analysis had been applied, and Duncan’ test was also used.Zimei” as an example, under raw condition, the ratio of Dw/Fw (dry weight to fresh weight) between freeze-dried and bake-dried “Zimei” were 36.5% and 34.9%, while it was 38.2% and 38.5% under baked condition, 27.3% and 27.6% in boiling, and 33.3% and 35.3% in steaming. Every pair of data appeared approximately the same without statistical difference between the freeze-dried and bake-dried sweetpotatoes. Similarly, no significant difference on Dw/Fw was observed between freeze-dried and bake-dried sweetpotatoes for other cultivars under the same cooking method , whereas “Jinyu” had the lowest (124.5 mg/g.Dw)  . In genef starch .Xinxiang” (peaks a-c) and generated extra maltose (peak d) , glucose (peak b) and sucrose (peak c) . Cooked (peak d) .Jinyu”. The total sugar content in sweetpotatoes from “Jinyu” under baking, boiling and steaming were 378, 238 and 250 mg/g of dry weight, respectively. Most of the increased sugars were mainly due to maltose accumulation  followed by “Xinxiang” (117.4 mg/g.Dw). Sucrose was the most abundant sugar in the raw sweetpotatoes of “Yuzishu263”and “Zimei”  contained more sugars than purple flesh cultivars (“Zimei” and “Yuzishu263”). The order of total sugar content in raw sweetpotatoes were “Jinyu” > “Xinxiang” > “Zimei” > “Yuzishu263” (Raw sweetpotatoes from “ “Zimei” . Glucosee sugars . In geneishu263” .Xinxiang” had the highest reducing sugar (210.7 mg/g.Dw) (Zimei” was the highest (163.6 mg/g.Dw) (Jinyu” (294 mg/g.Dw) (Yuzishu263” cultivar had the highest reducing sugar content (136 mg/g.Dw)  . As to bmg/g.Dw) . Under bmg/g.Dw) . Among tmg/g.Dw) . Non-red methods . Except  methods .Jinyu” was the highest either in raw sweetpotatoes (index 0.77) or under baking (index 6.5) and steaming (index 4.4). By baking, boiling and steaming, sweetness of “Jinyu” increased respectively by 8.44-, 4.64- and 5.71-fold, compared to raw state  but boiled “Xinxiang” turned to be the least (index 1.75)  . By baki.50-fold . Meanwhi.37-fold , while “ectively .-amylase activity while those in other treatments approached to be zero  contained more sugars than those from the purple flesh cultivars (“Zimei” and “Yuzishu263”). Cooking increased total sugar content especially reducing sugars. This effect was different among cultivars and cooking methods. Cooking methods did not have any significant effects on non-reducing sugar content, which is in agreement with the results of Bian and Damir [Our study showed that sugar composition variation was related to the color of sweetpotato flesh. In general, sweetpotato storage roots from the yellow flesh cultivars  have been employed to degrade starch, compared to method of Enzymatic hydrolysis , there was little difference. Sweetpotatoes from cultivar “Jinyu” had the highest soluble sugar content, the lowest starch content, the highest amylase activity and highest sweetness. Amylase activity and starch content of sweetpotatoes from other cultivars had less significant correlation with soluble sugar content. It may be related to other factors such as cooking temperature and time, as well as cultivar difference, etc. This observation was similar to that reported previously [We did not cut the storage root into pieces before boiling while Lyimo et al. (2010) sliced the storage root into pieces of 3–5 cm thick then boiled (moist heat) in clean water until ready to eat (about 30–45 minutes). We considered that pieces in hot moist may lose some components into distilled water which will influence the results. For example, as to boiling, our result of reducing sugar was 75–163.6 mg/g.Dw among the four cultivars, compared to 72.02–110.4 mg/100g.Dw in Lyimo et al. (2010). As to cultivars, we selected the typical materials by their color flesh, and we supposed that different color flesh perform significantly different nutritional quality. As to starch, HClOeviously ,26.In our study, cooking decreased starch content, increased sugar content especially reducing sugars and therefore increased sweetness. This is expected since starch is degraded by enzymes and transformed into soluble sugars during cooking processes –29. It wOur report showed that the sweetpotatoes still had some residual amylase activities after 28 min of cooking . The resOur results showed that cooking decreased starch content and increased soluble sugar content, which support the well-known phenomena that cooked sweetpotato usually tastes sweeter than fresh ones. Our experiment demonstrated that different cooking methods produced different sweetness and that steaming produced the highest sweetness. Soluble sugars of cooked sweetpotatoes had higher correlation with sweetness than those of raw sweetpotatoes, suggesting that soluble sugars in cooked sweetpotatoes are more suitable than those in raw sweetpotatoes for evaluating cultivar quality during sweetpotato breeding .We determined the effects of cooking methods on starch, amylase and sugar components in four elite Chinese sweetpotato cultivars. Cooking reduced starch content and amylase activity and increased reducing sugar content especially maltose content as well as sweetness, but did not have significant changes in non-reducing sugar content. These effects were different among the four varieties and three cooking methods. This study provides useful information for quality analysis and cultivar appraisal of sweetpotatoes.


H∞ and passive synchronization problem for Markov jump neural networks. The randomly occurring controller gain fluctuation phenomenon is investigated for non-fragile strategy. Moreover, the mixed time-varying delays composed of discrete and distributed delays are considered. By employing stochastic stability theory, synchronization criteria are developed for the Markov jump neural networks. On the basis of the derived criteria, the non-fragile synchronization controller is designed. Finally, an illustrative example is presented to demonstrate the validity of the control approach.This paper studies the non-fragile mixed  There have been significant attentions on dynamic behaviors of neural networks, since they have various current and future potential applications, i.e., signal processing, optimization problems, pattern recognition and so forth. –9. In paOn another research line, the synchronization problem has become a hot topic in the fields of neural networks , 12. WheH∞ and passive filtering design, which can provide a more flexible design than common H∞ approach T denotes the state of the neuron; f(x(t)) = T is the neuron activation function; A(r(t)) = (aij(r(t)))n×n, B(r(t)) = (bij(r(t)))n×n and D(r(t)) = (dij(r(t)))n×n represent the connection weight matrix, the discretely delayed connection weight matrix and the distributively delayed connection weight matrix, respectively; τ(t) and d(t) denote the discrete delay and distributed delay, respectively, which satisfyμ, and x(s) = ϕ(s), Consider the Markov jump neural networks with mixed time-varying delays in r(t), t ≥ 0} is a right continuous continuous-time Markov process with t > 0, lim(o(Δt)/Δt) = 0 and πij ≥ 0 ) inis continuous and bounded, and satisfieswherefi(0) = 0, α, α ≠ βandandare known real constants.r(t) by i indices. Moreover, it is assumed that the mode of the drive and the response neural networks can be identical all the time  is a known constant.Consequently, The following definitions and lemmas are introduced.Definition 1.  represents the parameter that defines the trade-off betweenH∞and passive performance.ition 1.  System   DefinitiLemma 1. T and y(t) = T, respectively. Under the modes evolution shown in Set the initial values as H∞ and passive synchronization problem of Markov jump neural networks with mixed time-varying delays is addressed. By utilizing the stochastic stability theory, delay-dependent criteria are derived for ensuring that the desired synchronization is achieved and the non-fragile synchronization controller is designed. An interesting further research direction is extending the derived results to the uncertainty cases.The non-fragile mixed S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.


Acidibacillus ferrooxidans” Huett2, a novel strain of an acidophilic, heterotrophic, iron-oxidizing bacterium belonging to the phylum Firmicutes. It was isolated from a water drainage system of a former mine field in Freiberg, Germany.Here, we communicate the draft genome of “ Acidibacillus” within the Firmicutes. All characterized strains of “Acidibacillus ferrooxidans” (mesophilic iron oxidizers) and “Acidibacillus sulfuroxidans” (moderately thermophilic iron- and sulfur oxidizers) are strictly dependent on a source of organic carbon. The region of Freiberg within the ore mountains in Germany is marked by centuries of mining activities. The novel A. ferrooxidans strain Huett2 was isolated from water of the mine drainage ditch “Hüttenrösche” which was streaked out on solid plates supporting the growth of acidophilic bacteria. Hereinafter, the isolate was cultivated in liquid MAC medium . The library was prepared with the NexteraXT DNA library kit  and checked with an Agilent technology bioanalyzer 2100. Paired-end libraries were sequenced using Illumina v3 chemistry. The resulting 8,040,162 reads were uploaded to the web-based genome analysis tool Galaxy  using SPAdes  array were predicted.The genome of burg.de 35). Read g SPAdes . The finMWPS00000000.This whole-genome shotgun project has been deposited at GenBank under the accession number


The calibrations were subsequently applied at a single bean and pixel level, so that the distribution was visualised within and between single seeds . HSI is thus suggested as a promising approach to estimate cocoa bean composition rapidly and non-destructively, thus offering a valid tool for food inspection and quality control.The aim of the current work was to use hyperspectral imaging (HSI) in the spectral range 1000–2500 nm to quantitatively predict fermentation index (FI), total polyphenols (TP) and antioxidant activity (AA) of individual dry fermented cocoa beans scanned on a single seed basis, in a non-destructive manner. Seventeen cocoa bean batches were obtained and 10 cocoa beans were used from each batch. PLS regression models were built on 170 samples. The developed HSI predictive models were able to quantify three quality-related parameters with sufficient performance for screening purposes, with external validation R Non-destructive and rapid techniques for the analysis of granular food commodities are interesting for the food industry as well as for research laboratories. In addition, methods able to predict sample composition at an individual seed level may offer the advantage of understanding the natural variations in bean-to-bean composition.Hyperspectral imaging (HSI) is a relatively novel technology in the field of food science and technology. It can analyse individual grain or bean samples in a rapid, non-destructive and non-contact manner and offers the possibility to scan samples at high throughput, also visualising the spatial distribution. Different configurations exist for data acquisition, with the “push-broom” approach being the most promising one for its practical applicability, as it acquires one spatial line at a time. The resulting image is commonly referred to as a “hypercube”, containing the two spatial dimensions and a third dimension represented by the spectra at each pixel. In food science, HSI has been mostly applied for the study of meat and fish products , and les−1 (dmb) expressed as epicatechin equivalent in the freshly harvested product, with variation further depending on the botanical variety, geographical origin and postharvest processing.Cocoa is the key ingredient for chocolate production. This paper investigated three quality-relevant constituents or properties of cocoa beans, namely polyphenols, antioxidant activity and fermentation index. Cocoa polyphenols are antioxidant compounds with beneficial health properties, and are responsible for the typical bitter taste of raw cocoa seeds . PolyphePolyphenols have received attention because of their physiological effects including their antioxidant activity, i.e. the capacity to retard lipid oxidation. The antioxidant capacity of foods can be assessed by several methods, the most common being 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. Solid foods have complex composition and contain both hydrophilic and lipophilic compounds, found in free or bound states, thus representing an issue for their extraction. In addition, polyphenols have different polarity, which further complicates the measurement of total antioxidant capacity. An approach recently proposed to overcome this issue, named QUENCHER , involves the direct addition of free radicals to the solid food . ABTS orAnother important parameter related to cocoa bean quality is the fermentation level, which can affect the final flavour of cocoa powder and chocolate. Fermentation is often carried out by making a heap of fresh cocoa beans or placing them in wooden boxes for 3–7 days. The beans are then dried. An indication of the fermentation level can be obtained by visual assessment, using the cut test: unfermented beans are grey (slaty), under-fermented beans are violet or purple and fully fermented beans are brown in colour. However, this method is time consuming and subjective. Alternatively, a fermentation index (FI) can be obtained from an analytical determination that involves grinding the cocoa beans, pigment extraction using methanol:HCl and spectrophotometric readings at 520 and 460 nm. Despite the more objective measurement, this method is still time-consuming (overnight extraction) and involves the use of harmful reagents. Thus, rapid methods to predict fermentation index of cocoa, potentially on a single bean basis, would be of great practical interest.Despite no work being published on within-batch cocoa seed variation, a large variability of chemical composition is expected, similar to that of other seeds or fruits. For other tree species, large natural variability was reported even within the same plant . The posRelatively little research has been published for the quantification of cocoa constituents using non-destructive techniques. Examples include the application of traditional NIR spectroscopy for the prediction of fat content in ground cocoa , as well2 value of 0.98, for total procyanidin oligomers.Other properties of cocoa beans have been estimated using FT-IR, in particular pH and fermentation index , with pr2 = 0.84 and RMSECV = 0.93 mg g−1 using vector normalisation pre-treatment. Similarly, good prediction was obtained for fermentation index, with the best model showing R2 = 0.88 and RMSECV = 0.06.Only one research paper used IR methods on whole cocoa beans for quantitative prediction. Other papers reporting on NIR applied to cocoa beans used ground beans or cocoa mass, thus little investigation has been carried out on whole cocoa nibs. No information has been published using HSI or even traditional NIR spectroscopy for single cocoa beans. The hypothesis tested in the present research was that HSI is able to predict cocoa chemical constituents and properties on a single seed basis.Therefore, the aim of the present work was to use hyperspectral imaging (HSI) in the spectral range 1000–2500 nm to predict fermentation index, total polyphenols and antioxidant activity of individual dry fermented cocoa beans. In addition, we report on the distribution of these compounds in a wide range of samples showing single-seed variability.22.1All the reagents used were analytical grade. HPLC-grade methanol and ethanol, and HCl were supplied by VWR . ABTS was supplied by Roche . Folin-Ciocalteu reagent, Ferulic acid (99% purity), (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid  and potassium persulfate (99.99%) were supplied by Sigma-Aldrich .Seventeen batches of fermented, unroasted cocoa beans were obtained from importers representing a range of producing countries: Brazil (1 batch), Cameroon (1), Ecuador (3), Ghana (4), Indonesia (1), Ivory Coast (3), Mexico (1), Nigeria (2) and Venezuela (1). Ten representative samples of sound unbroken cocoa beans were selected from each batch, thus obtaining 170 single beans, each bean representing a sample for the purposes of our work.2.2−1. A lens of focal length 31 mm was used, giving a field of view of 35 mm, with a pixel size of 0.109 mm (320 pixels). Two 500 W incandescent lamps were used as the illumination source, oriented at 45°. A baseline correction was made for each image and a normalisation was made using a white (PTFE) reference to calculate absorbance hypercubes , including a SWIR camera  with a cooled 14 bit 320 × 256 pixel HgCdTe detector. 240 spectral bands were obtained covering the range 1000–2495 nm, with ∼6 nm resolution. Cocoa beans were analysed by HSI as shelled whole beans (cotyledons). Ten seeds at a time were placed on a movable plastic stage and scanned using the push-broom approach, at a frequency of 100 lines spercubes .Image processing was done as reported by 2.3Fermentation index (FI) was measured according to the method of 2.4Total polyphenols (TP) were analysed according to the Folin-Ciocalteu method , as descg using a Mikro 22 centrifuge . 100 µL of the supernatant was taken and 750 µL of Folin-Ciocalteu reagent were added. After exactly 5 min, 750 µL of a 0.45 M sodium hydrogen carbonate solution was added to stop the reaction. After 90 min, the absorbance was read at 725 nm using an Evolution 201 spectrophotometer. A 7-point calibration curve was built using pure ferulic acid in the range 0–0.20 mg mL−1, with R2 = 0.993. The standard error (SE) was calculated as the ratio between the standard deviation of the measurements and the square root of the number of replicates. The SE for total polyphenol reference analysis was 3.23 mg Ferulic acid g−1 cocoa. A second indicator of the repeatability of the reference measurement is given by the coefficient of variation (CV), which was 3.68% for polyphenol content analysis.1 mL of an ethanol:water solution (70:30 v/v) was added to a 100 mg sample of ground cocoa. The dispersion was shaken for 30 s and centrifuged for 5 min at 15002.5g for 2 min. The supernatant was taken and absorbance at 734 nm was read using an Evolution 201 spectrophotometer. The reading was performed at exactly 6 min after mixing the ABTS reagent with the sample. Trolox was used to build a calibration curve to express the antioxidant capacity as Trolox equivalent antioxidant capacity (TEAC), i.e. mmol Trolox equivalent kg−1 cocoa. A 5-point calibration curve was built in the range 20–120 mM Trolox equivalent, showing R2 = 0.978. The SE for this method was 21.02 mmol Trolox equivalent kg−1 cocoa, with CV value of 14.78%.The antioxidant activity (AA) was evaluated using the QUENCHER approach, according to 2.62), the root mean square error of calibration (RMSEC) and prediction (RMSEP), as well as the Ratio of Performance to Deviation (RPD) , 70% of the beans were randomly selected to be part of the calibration set, while the remaining 30% were used as the validation set. The two average spectra belonging to a single cocoa bean were both included either in the calibration or in the validation dataset. The spectral data exported for each cocoa bean were treated in The Unscrambler 10.3 . Several spectral pre-treatment algorithms were tested to remove light scattering interferences and other noise, including area normalisation, Standard Normal Variate (SNV), and first and second derivative treatment using Savitzky-Golay smoothing . Partial Least Squares (PLS) regression was applied to build quantitative models, using the spectral range 1000–2500 nm, corresponding to 240 bands. The optimal number of latent variables (LVs) in the PLS was chosen by leaving the software to suggest the best number of latent variables (based on the amount of explained variance and the prediction error in relation to the number of latent variables), which is commonly carried out to reduce the cross-validation prediction error, while avoiding model overfitting. In particular, models using a full cross-validation on the calibration datasets were built, the prediction error was evaluated as a function of the number of LVs, and the model with the minimum root mean square error of calibration (RMSECV) was selected. Once the optimal number of LVs had been established, further validation was carried outbuilding new models using the external validation dataset. The performance of the prediction models was tested by evaluating the coefficient of correlation (Ron (RPD) . In addi33.1−1 cocoa, mean 186.1 and standard deviation (SD) of 116.1 mmol Trolox equivalent kg−1 sample. Average polyphenol content was 68.4 mg ferulic acid equivalent g−1 cocoa, with a standard deviation of 54.5, a minimum 2.9 and maximum 204.5 mg ferulic acid equivalent g−1 cocoa. The reference methods used in the present study are well established procedures, and the parameters analysed are accepted by the industry as cocoa quality indicators. The direct measurement of total polyphenols on the cocoa beans was previously reported by Reference measurements for total phenolic compounds, antioxidant activity and fermentation index for single, shelled cocoa beans are shown in −1 cocoa (fat free) in fresh beans , with Pearson r = 0.876. Similarly, a strong statistical correlation was observed between polyphenols and fermentation index, with r = −0.570, and between FI and AA, r = −0.443. These results were expected, as polyphenols are known to be a major source of the antioxidant activity in many food products. In addition, a strong decrease of polyphenols is expected to occur during cocoa bean fermentation, from an initial content of 150–200 mg gsh beans , to 60–1ed seeds .FI is a parameter commonly assessed in research laboratories and at the industrial level, as the degree of fermentation is related to differences in composition. For example, fermentation causes dramatic changes in the phenolic profile. Estimation of the degree of fermentation is commonly carried out by assessing the colour changes inside the cocoa bean, or the use of phenolic compounds as an indication of fermentation, as well as using fermentation index as an analytical parameter . Anthocy−1 cocoa  had 33.1 mg polyphenol g−1 in the cocoa powder, and ∼250–300 mg g−1 in the other sources. The highest value was observed for cocoa nibs, with higher concentrations than for beans due to the dilution effect of the shell. Lower levels were found in the cocoa mass and powder probably due to degradation during processing. Previous literature also suggested the absence of qualitative difference among different genotypes of cocoa or fermentation processes, but dramatic quantitative differences are often observed, as a result of growing and fermentation conditions , attributed to carbohydrates  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z M0 280 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z"/></g></svg>O stretching second overtone in the carbonyl groups (–CO2H or CONH) (2) absorbs at this wavelength. The CH2 group also absorbs at 1725 nm, due to the C–H stretch first overtone  . Howeveror CONH) , and theovertone .Fig. 2AvPLS regression was used to build prediction models. The performance of the predictive models is shown in 2 = 0.498 and RMSEP = 0.272, with RPD of 1.40). Better performance was achieved for TP and AA. Area normalisation pre-processing gave the best prediction for TP, with Rc2 = 0.817 and Rp2 = 0.700; the prediction error was RMSEC = 23.4 and RMSEP = 34.1 mg Ferulic acid equivalent g−1. The best performance was achieved for AA prediction, using second derivative treatment, which gave Rc2 = 0.764 and Rp2 = 0.735. The calibration error was 55.3 and the prediction error was 60.0 mmol Trolox kg−1 cocoa.Good prediction of cocoa bean composition was achieved. However, the model for FI was less strong, although it could be acceptable for separating high and low FI samples  as well as O–H polymeric groups, which can be due to complex carbohydrates .For other agricultural commodities, similar large heterogeneity in polyphenol content was reported by 2 = 0.927 and 0.909 for polyphenol content and antioxidant activity. The RMSEP was 1.97 (range: 25–50) g gallic acid/kg coffee, and 95 (400–1250) mmol Trolox kg−1 of coffee, respectively.The results herein presented are comparable with previous reports where NIRS was used for the prediction of polyphenol content and antioxidant activity. For example, The possibility of predicting fermentation index in cocoa beans using colour measurement has been recently reported by 3. Very good performance was shown for this parameter, with R2 = 0.97, SECV = 24 ppm (range 25–441) and RPD = 3.9, however the NIR spectra were acquired from bulk ground samples of cocoa beans (Other authors have proposed different indicators of fermentation degree, in particular the amount of nitrogen compounds, and some authors also proposed a traditional NIR spectroscopy approach to estimate the concentration of NHoa beans . Therefo3.3Once calibrations had been developed for cocoa constituents, they were applied by HSI. It is possible to apply the prediction models for single cocoa beans to estimate the total phenolic content, antioxidant activity and fermentation index in a rapid way. In addition, HSI allows visualisation of the composition of a target compound or property within single beans, by applying the calibration at a single pixel level, as shown by an example in 4We report, for the first time, quantitative predictions based on HSI for whole shelled cocoa beans on a single bean basis. We used a wide range of samples to develop a comprehensive model which includes much of the expected variation in commercial cocoa beans. Our research demonstrated for the first time that estimation or prediction of fermentation index, total polyphenols and antioxidant activity is possible by NIR-HSI on a single cocoa bean level, thus allowing cocoa beans to be individually analysed without any previous grinding or extraction process. In addition, the method can be applied at a single pixel level, with potential to provide information on the distribution of composition within beans.The bean-to-bean variability was found to be particularly wide, possibly due to the natural variation within and among plants due to environmental conditions, but also to the post-harvest practice. Our approach can be of interest for the scientific community, as it can be used to further elucidate the consistency of the fermentation/drying process to obtain higher quality product. In addition, it can also be used to identify cocoa nibs with particularly high or low polyphenol content or antioxidant activity in a non-destructive manner, which could in turn lead to the production of functional foods with increased nutritional properties, and improved product consistency.


Aplidium species was studied using conventional solution-phase and solid-state techniques, based on the voltammetry of immobilized particles methodology. The interaction with O2 and electrochemically generated reactive oxygen species (ROS) was electrochemically monitored. At the same time, a molecular modeling study including density functional theory (DFT) calculations was performed in order to analyze the conformational and electronic properties of the natural thiazinoquinones, as well as those of their reduced intermediates. The obtained electrochemical and computational results were analyzed and correlated to cytotoxic activity of these compounds, highlighting some features possibly related to their mechanism of action.The electrochemical response of four natural cytotoxic thiazinoquinones isolated from the  Since the early times of organic polarography , conside2•−), and regenerate the parent molecule .,27.1) [2The methodology previously used to study several synthetic quinone analogues  was expa1–4) immersed into aqueous PBS at physiological pH. 2.In view of the low solubility in water of the studied compounds, solid state electrochemistry was performed on microparticulate films of the compounds (1) and −1.03 V (C2) vs. Ag/AgCl. These signals are coupled, in the subsequent anodic scan, with a weak shoulder at −0.80 V (A2) and a well-defined, although weak, oxidation peak at −0.45 V (A1) which precedes a broad oxidation signal at −0.04 V (A3). This peak is absent in initial anodic scan voltammograms  in aqueous solution occurs typically via two rounds of electron-transfer (E) coupled with proton uptake , i.e., ECEC mechanism. This reduction pathway produces the radical anion Q●−, the semiquinone radical QH●, and the anion QH− species . The peak A3, which is depleted when the peak A2 is well developed (1 through the NH motif. In some cases (4) appears. This process can be assigned to the oxidation of the NH unit of the parent quinone [As judged upon comparison with the electrochemistry of synthetic thiazinoquinones,  the aboveveloped b, can beme cases b,d, an i quinone .In order to test the possible interaction of the studied compounds with ROS, the voltammetry of microparticulate films of the same compounds in contact with air-saturated PBS was carried out. ox). This process has been described in literature in terms of an initial one-electron reduction of O2 to the radical anion superoxide O2•− which is rapidly protonated and reduced to H2O2 (Equations (1)–(6)) [ox may be, however, complicated by adsorption processes, so that the first step can be represented as [At the unmodified glassy carbon electrode, dissolved oxygen yields an apparently irreversible reduction wave at ca. −0.85 V (C(1)–(6)) ,40,41. Tented as ,39,40O2According to literature ,40,41, t2O2:Alternatively, it undergoes disproportionation:2• and HO• can be formed at the electrode surface, in agreement with literature on electrochemical generation [ox in 2 resulting in the generation of ROS, the signals Aox and Ahox being attributable to the oxidation of O2•− and HO2•, respectively [Through this series of processes, reactive radicals HOneration ,30,31,32neration ,43 of ROectively . These sox peak shows a weak anodic counterpart (Aox) and is accompanied by an oxidation peak at +0.75 V (Ahox). At films of the natural thiazinoquinones (ox is preceded by the first reduction process of the compound (C1), corresponding, in PBS, to the formation of HQ•, and the peak Aox is depleted. The depletion of the signal is relatively large for 2 and 3 and much less intense for 4 and 1. Interestingly, in the anodic scan, the oxidation peak at ca. +0.75 V (Ahox), appearing in the voltammograms at the unmodified glassy carbon electrode  by undergoing equilibrium reactions with superoxide radicals [At a relatively high frequency a, i.e., ow 10 Hz b, i.e., radicals ,45.4NPF6/DMSO solutions  yielding a superoxide anion radical. In the absence of oxygen, the studied compounds produce an essentially reversible one-electron reduction (CQ1/AQ1) to the corresponding semiquinone at ca. −0.75 V followed by a second one-electron reduction at ca. −1.5 V (CQ2/AQ2). Voltammograms of air-saturated DMSO solutions of such compounds consist of superimposed signals for O2 and Q reduction but, remarkably, in the anodic scan, the anodic signal AQ1 is entirely or almost entirely depleted after passing the peak AO which remains essentially unaltered, while the couple CQ1/AQ1 is also decreased.It is well known that the electrochemical reduction of dissolved oxygen in aprotic solvents occurs reversibly at potentials ca. −0.9 V  is recorded, corresponding to the overall reduction to hydroquinone, appearing as a unique voltammetric couple in the presence of water, that is, when the protonated semiquinone species can be formed  attributable to the formation of some radical hydroperoxide as a result of the presence of residual water, was recorded at the unmodified electrode  in an aqueous system. This involves a sequence of steps in which reversible one-electron reduction reactions are followed by proton uptake reactions producing different intermediate species, as illustrated in 1–4, two possible protonated semiquinone radicals can be produced, i.e., QH•i, QH•ii, respectively  to hydroquinones , molecular mechanic (MM), and PM7 calculations. Obtained conformers were grouped into conformational families and ranked by their potential energy values. Secondly, the lowest energy conformer of each family was selected to be subjected to DFT full optimization. Finally, the DFT global minimum (GM) conformer of each compound was used to generate the reduced species Qmization .rG° = Σ(ε0 + Gcorr)Prod. − Σ(ε0 + Gcorr)React. of each step of the reduction pathway reported in •i and QH•ii   as the snd QH•ii , two difQH•ii  cancer cell line [3 shows a higher propensity to acquire both the first and the second electron  with respect to 4  than for 4 (less active).Some considerations can be made by comparing the results obtained for ell line . Consideect to 4 . On the EA)Q, as well as the vertical EA and ionization potentials (IP) of the one-electron reduced forms Q•− and QH•i. The vertical EA measures the propensity of a molecule to acquire one electron without considering subsequent electronic and/or structural rearrangements, which may drive the structure to a lower energy minimum . Under the same conditions, the vertical IP measures the propensity of a molecule to lose one electron. The resulting ω, EA, and IP values are reported in •− (SOMO); and (iii) the radical QH•i (SOMO).To further investigate the redox properties of the studied bioactive quinones, we calculated their electrophilicity index (ω)  and vert3 (more active) presents higher vertical EA and electrophilicity index and lower ELUMO than those of 4 (less active). Moreover, the corresponding Q•− and QH•i radicals of 3 present higher vertical IP and lower ESOMO than those of 4 , a platinum wire auxiliary electrode, and an Ag/AgCl (3 M NaCl) reference electrode were used in a typical three-electrode arrangement. 0.10 M phosphate-buffered saline (PBS) at pH 7.4  was used for experiments in aqueous solution. Experiments in DMSO  were performed using 0.10 M Bu4NPF6  as a supporting electrolyte. Prior to electrochemical runs, all solutions were deaerated by bubbling Ar for a duration of 15 min.Electrochemical experiments were performed at 298 ± 1 K in a thermostated cell with CH I660 equipment . A BAS MF2012 glassy carbon working electrode (GCE) . The following protocol was applied: The system was heated up to 1000 K over 2000 fs (time step = 3.0 fs). The temperature of 1000 K was applied to the system for 2000 fs (time step = 3.0 fs) with the aim of surmounting torsional barriers. Successively, the temperature was linearly reduced to 300 K in 1000 fs (time step = 1.0 fs). The resulting conformations were then subjected to molecular mechanic (MM) energy minimization within an Insight 2005 Discover module   until the maximum RMS derivative was less than 0.001 kcal/Å, using Conjugate Gradient [E from the global energy minimum) and grouped into conformational families. The occurrence rates and ΔEGM range for each conformational family were calculated. The lowest energy minima of each family were then subjected to DFT calculations. All structures were fully optimized at the B3LYP/6-31 + G level [−4 a.u. The calculations were carried out using the Gaussian 09 package [Apparent psoftware . The comsoftware . The conGradient  as the mGradient  in the MGradient  and EF (Gradient  as the gp) level ,55 usingp) level . The SCR package .•−, QH•i, QH•ii, QHi−, QHii−, QH2 were generated. In particular, following the reduction pathway of quinones reported in . In particular, Gibbs free energy calculations for electron attachment were accomplished by determining ΔG of the reaction Y + e− → Y− that is, ΔrG° (298.15 K)= Σ (ε0 + Gcorr)Y− − Σ (ε0 + Gcorr)Y, where Y is the Q or QH• DFT conformer. Gibbs free energies of reaction for proton attachment were calculated by determining ΔG of the reaction Y + H+ → YH that is ΔrG° = Σ (ε0 + Gcorr)YH − Σ (ε0 + Gcorr)Y, where Y is Q•− or QH− DFT conformer [Starting from the structure of the Q  GM conformers, the redox states Qonformer .IP); and (iii) the vertical electron affinity (EA). The electrophilicity indices (ω) were calculated following the expression ω = (μ2/2η), where μ is the chemical potential given by μ = −(IP + EA)/2 and η is the chemical hardness given by η = (IP − EA) [IP = G(N = N0−1) − G(N = N0) and EA = G(N = N0) − G(N = N0+1). Here, N0 is the number of electrons in the ground state of the  system. Thus, the single point Gibbs free energies (G) of the cationic (N0−1 electron system), and anionic (N0+1 electron system) forms for each considered species were calculated for each compound.For each considered redox state, the energy of the frontier molecular orbitals  were used to calculate the following parameters: (i) the electrophilicity index (ω); (ii) the vertical ionization potential (IP − EA) , where I1–4 in aqueous environments, such as PBS, differs from that in polar non-aqueous solvents, such as DMSO, and such behavior affects their reaction with O2 and electrochemically generated ROS. Indeed, the whole of the voltammetric studies indicate that, upon one electron reduction, 1–4 form a semiquinone radical intermediate, which, depending on the considered solvent, can be reduced (when protonated) or oxidized (in the un-protonated form) by ROS and O2, respectively. In agreement with the electrochemical results, computational studies suggest that the cytotoxic activity of the natural thiazinoquinones may be related to their ability to undergo a one-electron reduction, as well as to the reactivity of the generated radical species according to their protonation state.The electrochemical response of the natural thiazinoquinones 1–4, interpreted in the light of computational studies, pointed out some redox features of natural thiazinoquinones possibly related to their cytotoxic mechanism of action.The whole of our studies demonstrates the potential of the use of electrochemical techniques, combined with DFT calculations, to gain information on bioactive compounds whose mechanism of action involves a redox reaction. In our case, the electrochemical response of thiazinoquinones


Sorghum bicolor as a case study.We provide an overview of the current knowledge on cytochrome P450-mediated metabolism organized as metabolons and factors that facilitate their stabilization. Essential parameters will be discussed including those that are commonly disregarded using the dhurrin metabolon from Sessile plants control their metabolism to prioritize their resources between growth and development, or defense. This requires fine-tuned complex dynamic regulation of the metabolic networks involved. Within the recent years, numerous studies point to the formation of dynamic metabolons playing a major role in controlling the metabolic fluxes within such networks.b5. Solvent environment, lipid composition, and non-catalytic proteins guide metabolon formation and thereby activity, which have important implications for synthetic biology approaches aiming to produce high-value specialized metabolites in heterologous hosts.We propose that P450s and their partners interact and associate dynamically with POR, which acts as a charging station possibly in concert with Cyt Saccharomyces cerevisiae [As sessile organisms, plants produce an impressive diversity of toxic chemical compounds to defend themselves from the continuum of biotic and abiotic challenges they are exposed to. The specialized metabolites produced depend on the specific plant species and type of challenges encountered, and their biosynthesis demands a high degree of functional organization to offer swift metabolic plasticity. Delicate mechanisms regulate the complex metabolic grids organizing biosynthesis and storage ••. Organrevisiae . Interesrevisiae , 40–42. Sorghum bicolor. Dhurrin is produced from the amino acid l-tyrosine and synthesized by two membrane-anchored cytochrome P450 enzymes (CYP79A1 and CYP71E1) and by a soluble UDP-glucosyltransferase (UGT85B1). Although multiple studies have focused on the formation of metabolons involving P450 enzymes, and even if metabolon formation seems essential to organize and direct flux across biosynthetic networks [In this review, we will focus mostly on recent papers and use the dhurrin metabolon as a case study. Dhurrin is a cyanogenic glucoside present in the cereal crop plant networks , it is sThe expansion of the plant-specialized - metabolites during evolution notably results from the massive recruitment of cytochromes P450 (P450s) that catalyze multiple types of catalytic reactions . EukaryoMetabolons are defined as organization of sequential biosynthetic enzymes into complexes and involve substrate channeling and protein-protein interactions. Furthermore, metabolons involved in specialized metabolism are commonly considered highly dynamic, responding to environmental conditions. Initially, substrate channeling studies pioneered the investigation of metabolons , providiThe catalytic cycle of all microsomal P450s requires two single-electron transfer reactions catalyzed by the shared NADPH-dependent cytochrome P450 oxidoreductase (POR). This makes POR an essential component of P450-mediated metabolism, even if it is typically not considered in the understanding of metabolic networks and metabolons. POR, as P450s, is an ER-anchored protein, and the physical interactions between P450s and POR are facilitated and modulated by the membrane environment , 54. It Accumulating experimental data support the importance of individual protein-protein interactions in order to accomplish substrate channeling. However, the experimental approaches used typically involve targeted screening based on prior knowledge. Very little is known about the triggering mechanisms that stimulate assembly of metabolons and the biomembrane microenvironments required to stabilize these dynamic complexes. Here, we will highlight three areas that remain poorly understood: the neighboring proteins not essential for in vitro functional studies, the lipid bilayer environment, and the local solvent medium properties.b5 (Cytb5), reticulons, synaptotagmin (SYTA), band7 protein, sterol methyltransferase (SMT), vesicle-associated protein (VAP), membrane steroid-binding protein (MSBP) and others [In animals and yeast, a small number of proteins have been found associated to P450s without being directly involved in their catalytic function. BiP proteins are co-detected with P450s and seem important for their ER retention . CYP2C2 d others •, 75. Thb5 in P450-catalyzed reactions [b5 is a small (15 kDa) hemoprotein known to enhance the activity of several P450s, thus increasing the complexity of the electron donation chain of P450-mediated metabolism, but still largely overlooked. While Cytb5 is usually considered as non-essential for P450 activity in vitro, several studies have demonstrated the complex in vivo functional role of Cytb5 in P450 catalysis [b5 can provide the electron for the second electron-transfer step. Cytb5 has been shown to stimulate, inhibit, or have no effect on various P450 reactions [b5 has a significant role in forming hetero-dimers with P450s either to provide electrons or induce conformation changes augmenting interaction between POR and selected P450s [b5 reductase and Cytb5 may even serve as sole electron donors to the human CYP1A1 [b5 proteins. However, several isoforms of Cytb5 are also found in plants and most likely display similar roles [b5 importance for P450 function in plant phenolic metabolism [b5 is not essential in glucosinolate biosynthesis but can modulate P450 activities and change the glucosinolate pattern [b5 isoforms have been found associated to the lignin metabolon [Arabidopsisthaliana Cytb5 proteins interact with a series of other proteins in addition to the P450s, such as a plasma membrane-localized sucrose transporter [b5 could play a broader important physiological role. Understanding the role of the Cytb5 isoforms may have huge implications for synthetic biology approaches to boost P450 metabolism [Hildebrandt and Estabrook first reported the involvement of Cyteactions . Cytb5 iatalysis . POR is eactions , 78. Desn CYP1A1 . Most ofar roles . Varioustabolism  and in ftabolism –84. Cytb pattern . Cytb5 i a small  kDa hemonsporter  and the nsporter . This sutabolism ••.2+ signaling and exo/endocytosis [Reticulons contribute to ER tubule shaping , 88. Vesocytosis , 91. Retocytosis . The banocytosis , 94. AccSeveral biosynthetic pathways are distributed across different organelles and even different cells or have products excreted by cells –100. MCSThe ER obviously functions as a highway for delivery of a diverse range of proteins and molecules ••. QuantEukaryotic P450s and POR are both membrane anchored and thought to partly interact via their transmembrane N-amino acid terminus ••, 71. LCells have generally been thought of as composed of two solvent environments, namely the hydrophilic aqueous phase and the hydrophobic lipid phase. Increasing evidence suggests that this is a too simplified view and that the cellular cytosol is a marbled soup of solvent microenvironments. This implies that local concentrations of solutes, salt and pH may differ and control metabolism in a hitherto unknown fashion ••. IntraER components might be associated with NADES, e.g., with the positively charged choline head groups in the ER membrane associating with sugars and acids and contributing to the formation of a NADES. P450 metabolons might be dissolved in this solvent together with substrates and intermediates. Thus, NADES could make a kind of shield around metabolons and create a microenvironment within the cytoplasm, making experimental studies to isolate the metabolon even more complex . NADES aThe plant ER is a multitasking membrane network connected to several organelles and involved in most cellular processes . ConsequThe ER membrane system can form diverse structures, e.g., sheets, cisternae, and tubules, but most commonly is tubular with a reported average ER tubule diameter of 50 nm –134. WitFor clarity of the further models, we do not display UGT85B1 and other soluble proteins or scaffold proteins that might mediate metabolon formation as discussed earlier.In order to propose a model describing the current experimental data on the dhurrin metabolon, we have defined seven constraints essential for the validity of the model. We explore five models and validate them according to the constraints, where green “√” indicates that the constraint is fulfilled, the orange “(√)” indicates that the constraint may be satisfied in very specific conditions, and the red “X” indicates that the constraint cannot be explained by the configuration proposed.S. bicolor total extract data, we calculated an abundance of 134 ± 2 UGT85B1 molecules within a total number of 100,000 proteins. Accordingly, we define constraint #1: ER membrane surfaces harboring a total number of 4960 proteins must display 4 ± 0 POR2b, 12 ± 2 CYP79A1, 22 ± 9 CYP71E1, 26 ± 5 other P450s and 4896 other ER proteins. P450s are in large excess compared to POR, whereas the UGT85B1 abundance is similar to that of CYP71E1. More than half of the total P450s are CYP79A1 and CYP71E1, as previously determined by spectroscopy [Constraint #1: Based on quantitative proteomics data ••, we catroscopy . Most imConstraint #2: Using the Förster resonance energy transfer (FRET) equation with parameters of the eGFP-mRFP1 pair ••, we haR0 representing the Förster radius and R the distance between donor (eGFP) and acceptor (mRFP1). For the eGFP-mRFP1 couple, R0 is equal to 4.7 nm [with o 4.7 nm . Therefoo 4.7 nm ••. This Constraint #3: Background FRET values measured between two unrelated ER proteins ranging from 0 to 5% •• defineConstraint #4: When a protein A interacts with a protein B and concurrently protein B interacts with protein C, proteins A and C may be envisioned to be drawn very close to each other resulting in indirect FRET. High indirect FRET values were measured between non-partners at some very specific conditions ••, and tConstraint #5: At the resolution of the confocal microscope, a uniform fluorescence emission was observed from the ER membrane system expressing the fluorescent-labeled dhurrin pathway enzymes ••, althoConstraint #6: FCS experiments highlighted high dynamics of the target proteins, high variability of diffusion speeds, and decreased speeds of each target protein when partner proteins were co-expressed ••, whichConstraint #7: Metabolomic data demonstrated efficient channeling upon metabolon formation without leakage of intermediates or by-products •• and deConsequently, this model was discarded.Model 1 Fig. a assumesConsequently, this model was discarded.Model 2 Fig. b represeConsequently, this model was discarded.Models 3a, b, and c display three different configurations of P450s organized in highways of large clusters  P450 metabolons are localized at the dynamic ER surface, (2) metabolon components are highly diluted in plant cells, (3) the ER is highly crowded, and (4) the ER is a multifunctional and dynamic organelle site of protein production, lipid biosynthesis , auxin rCellular metabolism is highly modular utilizing relatively few enzyme classes to generate an impressive diversity of metabolites. Therefore, we hypothesize that other biosynthetic pathways, especially those involved in formation of specialized metabolites, may be organized in a similar fashion as the dhurrin metabolon. Understanding the mechanisms that govern metabolon assembly has huge implications for synthetic biology approaches aiming to produce high-value plant specialized metabolites in heterologous hosts, which often suffer from low yield and release of toxic intermediates , 143••.


Aggregation of the amyloid-β (Aβ) peptide is strongly correlated with Alzheimer’s disease (AD). Recent research has improved our understanding of the kinetics of amyloid fibril assembly and revealed new details regarding different stages in plaque formation. Presently, interest is turning toward studying this process in a holistic context, focusing on cellular components which interact with the Aβ peptide at various junctures during aggregation, from monomer to cross-β amyloid fibrils. However, even in isolation, a multitude of factors including protein purity, pH, salt content, and agitation affect Aβ fibril formation and deposition, often producing complicated and conflicting results. The failure of numerous inhibitors in clinical trials for AD suggests that a detailed examination of the complex interactions that occur during plaque formation, including binding of carbohydrates, lipids, nucleic acids, and metal ions, is important for understanding the diversity of manifestations of the disease. Unraveling how a variety of key macromolecular modulators interact with the Aβ peptide and change its aggregation properties may provide opportunities for developing therapies. Since no protein acts in isolation, the interplay of these diverse molecules may differentiate disease onset, progression, and severity, and thus are worth careful consideration. Amyloid plaques, first identified over 100 years ago  by β and γ secretase enzymes to a length of 38–43 amino acid residues  , which fine-tune protein expression (Audas and Lee RNA aptamers are short (<100 bp) segments of selection-enriched nucleic acid sequences which are able to bind tightly and specifically to amyloid proteins  generation, particularly as a source of oxygen radicals. ROS damage has been linked to membrane binding by both Aβ42 oligomers and fibrils in cell culture , which has been shown to affect Aβ production, deposition, and clearance in sporadic Alzheimer’s disease and is also found in senile plaques. In APP transgenic mice, knockout of ApoE prevented amyloid deposition; instead, the animals formed only diffuse plaques , but not neutral (pH 7.4), conditions   showed that other regions of the Aβ40 peptide, particularly residues D23–G29   Fig. g, a chelPlaques are complicated assortments of aggregated protein and other co-effectors of the aggregation process Fig. . The balTo date, no therapeutic has been identified which is able to fully mitigate AD. Perhaps this is because many drugs to date Fig.  have tar


Rickettsiales has constantly grown. Since 2006, 18 novel Rickettsiales genera inhabiting protists, such as ciliates and amoebae, have been described. In this work, we characterize two novel bacterial endosymbionts from Paramecium collected near Bloomington, IN. Both endosymbiotic species inhabit the cytoplasm of the same host. The Gram-negative bacterium “Candidatus Bealeia paramacronuclearis” occurs in clumps and is frequently associated with the host macronucleus. With its electron-dense cytoplasm and a distinct halo surrounding the cell, it is easily distinguishable from the second smaller symbiont, “Candidatus Fokinia cryptica,” whose cytoplasm is electron lucid, lacks a halo, and is always surrounded by a symbiontophorous vacuole. For molecular characterization, the small-subunit rRNA genes were sequenced and used for taxonomic assignment as well as the design of species-specific oligonucleotide probes. Phylogenetic analyses revealed that “Candidatus Bealeia paramacronuclearis” clusters with the so-called “basal” Rickettsiales, and “Candidatus Fokinia cryptica” belongs to “Candidatus Midichloriaceae.” We obtained tree topologies showing a separation of Rickettsiales into at least two groups: one represented by the families Rickettsiaceae, Anaplasmataceae, and “Candidatus Midichloriaceae” (RAM clade), and the other represented by “basal Rickettsiales,” including “Candidatus Bealeia paramacronuclearis.” Therefore, and in accordance with recent publications, we propose to limit the order Rickettsiales to the RAM clade and to raise “basal Rickettsiales” to an independent order, Holosporales ord. nov., inside Alphaproteobacteria, which presently includes four family-level clades. Additionally, we define the family “Candidatus Hepatincolaceae” and redefine the family Holosporaceae.In the past 10 years, the number of endosymbionts described within the bacterial order IMPORTANCE In this paper, we provide the characterization of two novel bacterial symbionts inhabiting the same Paramecium host . Both symbionts belong to “traditional” Rickettsiales, one representing a new species of the genus “Candidatus Fokinia” (“Candidatus Midichloriaceae”), and the other representing a new genus of a “basal” Rickettsiales. According to newly characterized sequences and to a critical revision of recent literature, we propose a taxonomic reorganization of “traditional” Rickettsiales that we split into two orders: Rickettsiales sensu stricto and Holosporales ord. nov. This work represents a critical revision, including new records of a group of symbionts frequently occurring in protists and whose biodiversity is still largely underestimated. Bacteria and Archaea, are found to populate ciliated protists  and several clades formerly represented by the family Holosporaceae . In the past years, the molecular characterization of novel endosymbiotic species and the increasing genomic and metagenomic data have changed our view of this order. More complex analyses based on different genetic markers . This model is usually supported by phylogenetic analyses based on prokaryotic small-subunit (SSU) rRNA gene sequences  rRNA gene sequences, and other studies support this notion . We characterized these two novel bacterial endosymbionts by using molecular and ultrastructural methods. In accordance with the guidelines of prokaryotic nomenclature, we propose the names “Candidatus Bealeia paramacronuclearis” and “Candidatus Fokinia cryptica.” A second Paramecium isolate harboring only “Candidatus Bealeia paramacronuclearis” was sampled nearby and was used for comparative analyses. According to our phylogenetic analyses of “basal Rickettsiales,” we propose a taxonomic reorganization of the order Rickettsiales by excluding all “basal Rickettsiales” and grouping them in the novel order Holosporales ord. nov., as suggested by Ferla and colleagues  in 2011. The single-infected isolate US_Bl 15I1 was sampled from approximately 17 km away, in the 0.54-km2 Yellowwood Lake, IN  in 2011. Monoclonal mass cultures were established and maintained in 0.25% Cerophyl medium inoculated with Raoultella planticola , following the cetyltrimethylammonium bromide-based protocol for fungi.The double-infected c oxidase subunit 1 (COI) gene  gene , and theOI) gene  and ITS OI) gene . The COIOI) gene .Alphaproteobacteria-specific forward primer 16Sα_F19b and the Bacteria-specific reverse primer 16S_R1522a  in a total volume of 15 μl for 20 min at 72°C. This DNA was cloned into the pGEM-T vector system (Promega) and transformed into competent TOP10 Escherichia coli cells following the manufacturer's instructions. Three clones derived from DNA of US_Bl 11III1, showing identical restriction patterns after digestion with MboI  but different from restriction patterns of food bacteria and the endosymbiont of the single-infected host (strain US_Bl 15I1), were sequenced from both directions using M13 primers. The resulting three sequences were used to produce a consensus sequence for the second endosymbiont isolate, US_Bl 11III1.Two different approaches, cloning and direct sequencing, were used to obtain the prokaryotic SSU rRNA gene sequence of the second endosymbiont in isolate US_Bl 11III1. For cloning, PCR amplification was carried out with Phusion high-fidelity DNA polymerase . The universal primers Bac16SFor and Bac16SRev  were useIn order to confirm the obtained SSU rRNA gene sequence, direct sequencing was performed on the same PCR product employed for cloning, using specific internal primers Fokinia_F434 (5′-CTCTTTTGGTAGGGATGATAAT-3′), Fokinia_R434 (5′-ATTATCATCCCTACCAAAAGAG-3′), Fokinia_F1250 (5′-AGAAGGCTGCAACAGGGT-3′), and Fokinia_R1250 (5′-ACCCTGTTGCAGCCTTCT-3′).mT], 55.9°C) was designed. The species-specific probe FokCry_198  was designed for the second endosymbiont present only in isolate US_Bl 11III1. Both probes were obtained from Eurofins Genomics GmbH . Fluorescence in situ hybridization (FISH) was performed with each newly designed probe, in combination with EUB338 (fluorescein isothiocyanate [FITC] labeled at the 5′ end) (in silico by using the TestProbe Tool 3.0 (SILVA rRNA database project)  , accordiproject)  and the project) , allowinproject) .Candidatus Fokinia solitaria,” the previously published probes Fokinia_198, Fokinia_434, and Fokinia_1250 , 68 sequences of “basal Rickettsiales” . Seven representatives of other Alphaproteobacteria orders were chosen as the outgroup (data set 1). A second set of sequences (data set 2) was chosen to analyze the prokaryotic SSU rRNA gene sequence of the second endosymbiont of isolate US_Bl 11III1. The sequence was aligned with 23 other sequences of “Candidatus Midichloriaceae.” Nine sequences of Anaplasmataceae were chosen as an outgroup. For phylogenetic inference, alignment of both data sets was performed with the automatic aligner of the ARB software and further refined manually, to optimize stems and loops according to the predicted secondary structure of the prokaryotic SSU rRNA of E. coli. The alignments were trimmed at both ends to the shortest shared sequence, and gaps were treated as missing data. Additionally, for data set 1, hypervariable nucleotide positions  were left out.Phylogenetic analyses were performed with the ARB software package version 5.2 . The proThe optimal substitution model for each alignment was selected with jModelTest 2.1 , using tParamecium cells of both isolates were fixed as described by Szokoli and colleagues , whereas ITS regions, including 5.8S and a part of the LSU rRNA gene, showed one nucleotide difference, located in the partial 28S rRNA gene sequence. The joined sequences of the eukaryotic SSU, ITS, and partial LSU for each of the two isolates were submitted to NCBI GenBank . Eukaryotic SSU rRNA gene and ITS sequences supported both isolates belonging to the Paramecium aurelia group. Sequence identity with respect to other published ITS sequences of the P. aurelia complex ranged between 98 and 100% and dropped to 94 to 95% for Paramecium caudatum and Paramecium multimicronucleatum. Generally, COI gene sequences showed a higher resolution power than eukaryotic SSU rRNA gene and ITS regions. Thus, partial COI genes of isolates US_Bl 11III1 and US_Bl 15I1 were sequenced (760 bp). No differences between the two sequences were observed. Their sequence identities to Paramecium biaurelia sequences ranged from 99 to 100%, whereas identities with other members of the P. aurelia complex dropped to 80 to 85%. Therefore, based on COI gene sequences and in accordance with morphological features, we identified our isolates as P. biaurelia.General morphological features of the isolates, including body size and shape, number and shape of micronuclei, and location of the cytoproct, were typical for representatives of the Candidatus Bealeia paramacronuclearis.” Bacterial SSU rRNA gene sequences of the endosymbionts from P. biaurelia US_Bl 11III1 and US_Bl 15I1 were identical through their entire length . The species-specific probe Bealeia_1245 hybridized with its target organism in formamide from 0 to 25% (vol/vol) . The described bacterial endosymbionts showing a higher sequence identity  were Caedibacter caryophilus (accession number NR_044847) from the macronucleus of Paramecium caudatum (Candidatus Nucleicultrix amoebiphila” (accession number KF697195) infecting the nucleus of an amoeba  , with th allowed . Comparin amoeba .Candidatus Bealeia paramacronuclearis” was always associated with the previously mentioned uncultured bacteria sequences, forming a genus-level “Candidatus Bealeia” clade (99.8% bootstrap support for ML and posterior probability of 1.00 for BI). All tree topologies obtained supported an association of “Candidatus Bealeia” with the genera Holospora, “Candidatus Gortzia,” “Candidatus Paraholospora,” and “Candidatus Hepatobacter” and with several sequences from uncultured organisms in a rather well-supported clade (62.3% bootstrap for ML and posterior probability of 1.00 for BI), for which we propose a family-level status and the name Holosporaceae. Another family-level clade , containing endosymbiont species such as Caedibacter caryophilus and “Candidatus Nucleicultrix,” has sufficient support (79.8% for ML and 0.81 for BI) and branched as a sister group to Holosporaceae. The recently introduced family “Candidatus Paracaedibacteraceae” of Hess and colleagues  was unstably associated with “Candidatus Paracaedibacteraceae” (AY328720 branches independently from “Candidatus Paracaedibacteraceae”). A well-supported clade (99.6 for ML and 1.00 for BI) of putative fast-evolving bacterial sequences associated with different metazoan organisms, including “Candidatus Hepatincola porcellionum,” could additionally be retrieved. Phylogenetic positioning of this clade was unstable, being sister to all other previously mentioned clades  in ML   and Holosporaceae, Caedibacter-Nucleicultrix clade, “Candidatus Paracaedibacteraceae,” and “Candidatus Hepaticolaceae” .In our phylogenetic analyses  and 3, “lleagues  was conferaceae”  and 3, b”) in ML , or sistBI trees . We alsollionum” . Our treHolosporaceae, the Caedibacter-Nucleicultrix clade, “Candidatus Paracaedibacteraceae,” and “Candidatus Hepaticolaceae” on the other side, were compared with the order values retrieved by Yarza and coauthors  whose sequences slightly differ.The second bacterial SSU rRNA gene sequence, derived only from isolate US_Bl 11III1 , was found to be closely related to “litaria” , as it sCandidatus Fokinia cryptica” sequence, the two previously published probes Fokinia_434 and Fokinia_1250  were targeted only by EUB338. Similar experiments, in which genus-specific “Candidatus Fokinia” probes were used in combination with the Bealeia_1245 probe, showed that the “Candidatus Fokinia” and “Candidatus Bealeia” probes targeted different symbionts within the same host cell without any overlap . The species-specific probe FokCry_198, targeting the same region, was designed for “Candidatus Fokinia cryptica” and was validated to have a high specificity (Candidatus Fokinia solitaria” at any stringency (data not shown).Given the “nia_1250  turned o overlap . The precificity . Its optCandidatus Fokinia cryptica” is clearly inside the family “Candidatus Midichloriaceae”  and associated with high support values by both inference methods (100% for ML and 1.00 for BI) to “Candidatus Fokinia solitaria” , and an environmental sequence isolated from Green Lake, NY (accession number FJ437943).The phylogenetic position of “litaria” . Both seP. biaurelia isolate US_Bl 15I1, only one kind of endosymbiont was observed in the cytoplasm. Symbionts had the two typical membranes of Gram-negative bacteria, reached 1.8 to 2.4 by 0.4 to 0.5 μm in size, and were devoid of flagella. The endosymbionts had an electron-dense cytoplasm  were never found in clusters, were enclosed in a symbiontophorous vacuole  represent highly supported monophyletic clades  consisted solely of Holospora spp. and Holospora-like bacteria (HLB), such as “Candidatus Gortzia infectiva,” while other species should be placed inside Rickettsiales as incertae sedis. With the ongoing accumulation of data, we believe that this Holosporaceae family definition is too strict. Indeed, it would exclude all phylogenetically related species lacking the typical Holospora life cycle. Although subfamily rank is seldom used nowadays in bacterial taxonomy, we suggest establishment of the subfamily Holosporoidea, to include Holospora spp. and Holospora-like bacteria with the typical features mentioned above; Holosporoideasensu Szokoli (this study) would correspond to Holosporaceaesensu Boscaro and colleagues , and one consisting of “basal Rickettsiales.” Among the sequences included in our analysis, several display high evolutionary rates, especially those derived from environmental origin. They were included in order to get a comprehensive and representative selection of “basal Rickettsiales” and its subclades. As a probable and unfortunate consequence, the presence of such fast-evolving sequences can also produce instabilities in phylogenetic reconstructions and is likely causing relatively lower support values of some branches in the ML tree . Within each group, the average identities are above the threshold retrieved by Yarza and colleagues , limitinHolosporales ord. nov.  and the relationship of Holosporales with respect to the Rickettsiales sensu stricto herein defined and to all other Alphaproteobacteria in general. Nevertheless, we emphasize that to our best knowledge the vast majority of published analyses  are overall much scarcer than those for Rickettsiales sensu stricto, some quite well-defined differences are already evident. From an ecological point of view, the host range of described organisms appears to include, with a much higher prevalence, aquatic organisms  than typical Rickettsiales sensu stricto sequences (mostly in the range 1 to 1.5 Mb) , includlleagues ) agree arganisms  and in prganisms , 52, 53. 1.5 Mb) . Rickettffectors –59. On tdibacter , 61, HolSpirostomum minus (Heterotrichea) was found with bacteria colonizing both the cytoplasm and the macronucleus (Euplotes spp. (Spirotrichea) infected by Polynucleobacter necessarius and other bacteria have been reported as well (Euplotes aediculatus isolate In from New Delhi (India) bore three different cytoplasmic endosymbionts genomic sequences, could help in clarifying the role of each symbiont and the interactions among them and with the host.It has been suggested that if endosymbionts colonize different compartments, they probably depend on different metabolites and, therefore, do not compete directly with each other . The intytoplasm , 22. Theytoplasm , who sugCandidatus Bealeia paramacronuclearis” was often found in association with the host macronucleus. It is intriguing to observe that most bacteria described within Holosporaceae (sensu in this report) show nuclear forms or at least nucleus-associated forms. Referring to the close association of “Candidatus Bealeia paramacronuclearis” with the host's macronucleus, we propose the name “Candidatus Bealeia paramacronuclearis,” in accordance with the guidelines of the International Committee on Systematic Bacteriology  suggests that this bacterium might be horizontally transferred, as for Holospora and “Candidatus Gortzia infectiva”  have sofectiva” , 61.Candidatus Fokinia cryptica” showed several similarities to the formerly described species “Candidatus Fokinia solitaria” . Conseqoworkers , by inclCandidatus Fokinia” . The description of “Candidatus Fokinia” (Candidatus Fokinia solitaria” (type species) and “Candidatus Fokinia cryptica.”“Fokinia”  is emendFokinia” , with boCandidatus Fokinia cryptica” (cryp'ti.ca. N.L. fem. n. kryptikós hidden). Short rod-like bacterium (1.1 by 0.35 to 0.40 μm in size). Cytoplasmic endosymbiont of the ciliate Paramecium biaurelia , residing in symbiontophorous vacuoles. Basis of assignment: SSU rRNA gene sequence (accession number KU736846) and a positive match with the specific FISH oligonucleotide probe FokCry_198 (5′-CTCGCAGTAACATTGCTGC-3′). Belongs to “Candidatus Midichloriaceae” family, order Rickettsiales, class Alphaproteobacteria. Identified in Paramecium biaurelia isolate US_Bl 11III1 from a small pond in the Miller Showers Park in Bloomington, IN. Uncultured thus far.“Candidatus Bealeia” .“Paramecium biaurelia. Belongs to the family Holosporaceae within the order Holosporales, class Alphaproteobacteria. The type species of the genus is “Candidatus Bealeia paramacronuclearis.”The new genus encompasses endosymbiotic bacteria associated with the ciliate Candidatus Bealeia paramacronuclearis” .“Paramecium biaurelia . Rod-like bacterium (1.8 to 2.4 μm by 0.4 to 0.5 μm in size) with an electron-dense cytoplasm and conspicuous ribosomes and nucleoid. Devoid of flagella. Often forms clusters of up to 7 or 8 cells, generally in close proximity to the host macronucleus. Basis of assignment: SSU rRNA gene sequence (accession number: KU736844) and positive match with the species-specific FISH oligonucleotide probe Bealeia_1245 (5′-CCTATTGCTTCCTTTTGTCAC-3′). Identified in Paramecium biaurelia isolate US_Bl 11III1 from a small pond in the Miller Showers Park in Bloomington, IN, and isolate US_Bl 15I1 from Yellowwood Lake . Type strain is US_Bl 11III1. Thus far, uncultured.Cytoplasmic endosymbiont of the ciliate Holosporaceae .Holosporaceae is emended. Basis of assignment to this family is a stable clustering in phylogenetic trees with the type genus Holospora and not with the Caedibacter-Nucleicultrix clade; showing a prokaryotic SSU rRNA identity with Holospora higher than 81%. All described members of this family are obligate intracellular bacteria. Several possess two morphologically distinct forms and/or life cycles. The family currently comprises the genera Holospora, “Candidatus Gortzia,” “Candidatus Paraholospora,” “Candidatus Hepatobacter,” “Candidatus Bealeia,” and several sequences from uncultured organisms. Type genus is Holospora .“Candidatus Hepatincolaceae” is defined based on phylogenetic analysis of SSU rRNA gene sequences of the type genus and uncultured representatives from various environments; most of the sequences are apparently associated with Ecdysozoa. The family currently contains one genus, “Candidatus Hepatincola” .Holosporaceae, “Candidatus Paracaedibacteraceae,” and “Candidatus Hepatincolaceae”), and the Caedibacter-Nucleicultrix clade, including Caedibacter caryophilus, Caedibacter varicaedens, “Caedibacter macronucleorum” , “Candidatus Nucleicultrix amoebiphila” (endosymbiont of Hartmannella sp.), and several uncultured sequences. The order Holosporales is a member of the class Alphaproteobacteria. The type genus is Holospora.Defined by phylogenetic analyses based on SSU rRNA gene sequences. The order contains three families (


Although traditionally viewed as the “powerhouse” of the cell, an accruing body of evidence in the rapidly growing field of mitochondrial biology supports additional roles of mitochondria as key participants in a multitude of cellular functions. While it has been well established that mitochondria in different tissues have distinctive ultrastructural features consistent with differential bioenergetic demands, recent and emerging technical advances in flow cytometry, imaging, and “-omics”-based bioinformatics have only just begun to explore the complex and divergent properties of mitochondria within tissues and cell types. Moreover, contemporary studies evaluating the role of mitochondria in pluripotent stem cells, cellular reprogramming, and differentiation point to a potential importance of mitochondrial subpopulations and heterogeneity in the field of stem cell biology. This review assesses the current literature regarding mitochondrial subpopulations within cell and tissue types and evaluates the current understanding of how mitochondrial diversity and heterogeneity might impact cell fate specification in pluripotent stem cells. Mitochondria are ubiquitous across eukaryotic organisms and are critical for cellular bioenergetics. The classical mitochondrial ultrastructural features, consisting of an outer membrane and an inner membrane containing invaginations that comprise the matrix-rich cristae, are specifically modified in a tissue-specific manner in order to meet cellular-explicit energy demands. The differences in mitochondrial morphology between cell and tissue types as well as in localization and distribution within the cell have been well documented , 2, and ψm)), as each cell type examined contained distinct populations of mitochondria having both high or low Δψm, and no single mitochondrion contained regions of high and low Δψm. Quantitative analysis of Δψm in HeLa cells demonstrated that mitochondria localized to the perinuclear region had a greater percentage of mitochondria with low Δψm and were more closely associated with endoplasmic reticulum (ER) than their peripherally located counterparts [Although the specifics regarding how these nuclear-encoded proteins have been shown to participate in mitochondrial remodeling have been studied for almost two decades and extensively reviewed elsewhere , 20, farterparts .Morphological changes in the physical appearance of mitochondria during the process of differentiation are not well characterized, although it is well understood that mitochondrial ultrastructure varies dramatically between tissue types , Figure . In a reψm [ψm are generally regarded as metabolically quiescent (“resting” or damaged), whereas those with a high Δψm are viewed as metabolically active (“respiring”). However, there are caveats to this generalization, including that Δψm is maintained by the balance between the electron transport chain ATP synthases. Accordingly, even well-coupled (respiring) mitochondria can have a decreased Δψm. The heterogeneity in Δψm both within individual cells and between neighboring cells can be observed in vitro using fluorometric compounds, such as JC-1 . JC-1 undergoes a shift in spectral fluorescence from green  to red-orange upon conversion from monomeric (green) to aggregate (red-orange) form in actively respiring mitochondria. Utilizing the aggregate fluorescent properties of JC-1 to detect differences in mitochondrial respiration status in undifferentiated human ESCs (H9), Kumagai et al. have recently demonstrated that shifts in Δψm  within undifferentiated ESC colonies may serve as a simple visual predictive indicator of cells that are undergoing the earliest stages of differentiation [ψm associated with oxidative phosphorylation is one of the first markers to distinguish differentiating cells in a mixed culture. Accordingly, mitochondrial metabolic changes have been proposed as early markers of stem cell differentiation [ψm, Schieke et al. obtained two distinct populations of ESCs which were indistinguishable based on morphology, yet had remarkably different resting oxygen consumption rates . Analysis of differentiation potential revealed that in the presence of bone morphogenetic protein 4 (BMP-4) in the absence of leukemia inhibitory factor (LIF) to promote mesoderm specification, ΔψmL ESCs exhibited a markedly greater mesodermal differentiation capacity (10-fold) than ΔψmH ESCs, whereas ΔψmH ESCs had a greater propensity for teratoma formation than ΔψmL ESCs [ψm and oxygen consumption in undifferentiated mouse ESCs and augmented BMP-4-induced mesodermal differentiation [Although metabolic and structural remodeling during cell differentiation represents emerging fields of study in mitochondrial and stem cell biology, mitochondrial heterogeneity as it pertains to specific mitochondrial subpopulations within a single tissue or cell remains a considerably under-characterized component in cell biology. It is well understood that under physiological conditions, cells possess a heterogeneous mitochondrial population based on differences in membrane potential Δψm , 35, 36), 36ψm [3ntiation . Due to ntiation –41, the ntiation –41, althψmL ESCs . Furtherntiation . Togetheψm in human ESCs have also been demonstrated as a consequence of in vitro age . In undifferentiated H9 and PKU1 cell lines, Δψm increased significantly in late-passage cells as compared to their younger counterparts. The elevated Δψm also correlated with increases in total mitochondrial volume and generation of reactive oxygen species (ROS) [2O2-dependent Δψm depolarization occurred at a faster rate in aged iPSCs than that observed in their “younger” counterparts, demonstrating a diminished ability to counteract oxidant exposure with in vitro age. Moreover, the capacity for in vitro neurogenesis was diminished in the aged iPSCs versus the young iPSCs [Alterations in Δes (ROS) . Late-paes (ROS) . Given tng iPSCs .ψm in the maintenance of stemness, the impact of mitochondrial subcellular heterogeneity on cellular function, including differentiation, has not been evaluated. Based on a series of studies examining mitochondrial properties within individual cells [ultrastructure—mitochondria range in size (0.2 μm to 1.0 μm), shape (circular to elongated and tubular), and cristae density (no visible cristae/vacuole-like appearance to dense cristae) and can be found alone or physically networked; ultrastructure varies across tissue types, as well as within individual cells; and fission-fusion events, as well as respiratory status, also impact on morphology; (2) functional properties—mitochondria can differ in redox state, respiration, intramitochondrial Ca2+ and reactive oxygen species (ROS) levels, mitochondrial protein composition and content, and Δψm; (3) behavior—mitochondria respond differently to oxidative stress, starvation, apoptotic stimuli, and mitophagy signals and exhibit selective responses to toxins and substrates; (4) dynamics—cell type-specific intracellular localization, oscillatory movements, translocation events, filament extension and retraction, and fission and fusion events [ψm, Wikstrom et al. propose that low Δψm represents a mechanism by which mitochondria are selectively targeted for autophagy [Although emerging evidence supports a role for Δal cells , 35, Kuzal cells ): (1) uln events . Addition events , bringinn events –47, and utophagy . Althougutophagy , 49 and utophagy . It has utophagy .2+ regulation, oxygen sensing, and ROS generation, while others are cell and tissue type-specific, such as steroid hormone biosynthesis, hormone signaling and responsiveness, thermogenesis, hemesynthesis, and processing of toxins [In addition to the differences in mitochondrial morphology mentioned above, mitochondria also differ functionally and are known to be involved in cellular processes beyond metabolism. Such functions are well characterized and ubiquitous, including cell death and differentiation, intracellular Caf toxins . These cf toxins  encoded f toxins , with thf toxins , 55. Howf toxins , 56, detf toxins . Mitocho2+ uptake capability of IFM and SSM was comparatively demonstrated to differ, as a further indicator of mitochondrial heterogeneity between the two subpopulations [Given the complexity and diversity of mitochondrial proteomic profiles amongst mitochondrial subtypes, it is not surprising that mitochondria serve to perform a diverse spectrum of functions—with many more remaining to be discovered. Among the more well-characterized mitochondrial types within a single tissue are two cardiac mitochondrial subpopulations, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). Initially characterized by ultrastructural differences observed between the two populations located beneath the sarcolemma and between the myofibrils, respectively, physical isolation of each mitochondrial subtype revealed distinct biochemical functional properties . Specifiulations , althougulations . Howeverulations . Althougulations . Additioulations . As moreThe current understanding of mitochondrial function in stem cells is limited in scope as compared to the broader field of mitochondrial biology. Given the highly specialized features of mitochondria in differentiated cell types, it stands to reason that a separate field of study dedicated to mitochondrial fate specification and differentiation might coevolve with the stem cell field. In this way, stem cells serve as an excellent model to study “mitochondrial differentiation.” As each mitochondrion contains DNA encoding for only 13 proteins, yet contains a subtype-specific proteomic profile of up to 1500 nuclear-encoded proteins, the significance of nuclear communication in the regulation of mitochondrial function becomes increasingly clear. How the mitochondria and nucleus communicate on a per organelle basis  remains to be determined. As nanoscaled technologies emerge for the study of subcellular organelles, the mechanisms that govern mitochondrial heterogeneity and function will be elucidated and perhaps provide additional platforms and metrics for stem cell reprogramming and differentiation.


Stair walking is a hazardous activity and a common cause of fatal and non-fatal falls. Previous studies have assessed the role of eye movements in stair walking by asking people to repeatedly go up and down stairs in quiet and controlled conditions, while the role of peripheral vision was examined by giving participants specific fixation instructions or working memory tasks. We here extend this research to stair walking in a natural environment with other people present on the stairs and a now common secondary task: using one’s mobile phone. Results show that using the mobile phone strongly draws one’s attention away from the stairs, but that the distribution of gaze locations away from the phone is little influenced by using one’s phone. Phone use also increased the time needed to walk the stairs, but handrail use remained low. These results indicate that limited foveal vision suffices for adequate stair walking in normal environments, but that mobile phone use has a strong influence on attention, which may pose problems when unexpected obstacles are encountered. Stair walking is a common, but surprisingly hazardous activity. In the UK, more than 1000 people die every year from falls from stairs , they can walk stairs safely when given specific fixation or secondary tasks, with very few direct fixations on the stairs  took part in the study, recruited using posters, an online research participation system and by word of mouth. Participants were a mixture of undergraduate and postgraduate students, university staff and members of the general public, and were between 19 and 68 years of age. Their vision was either corrected with contact lenses, or participants had normal vision without correction, as the eye tracker used did not allow for correction with glasses. Participants recruited via the poster received Eye movements were recorded with a Tobii 2 glasses eye tracker .Participants all provided written consent before taking part in the study. They were fitted with the Tobii 2 glasses and the calibration procedure was performed (involving the fixation of a single fixation target at about an arm’s length). Two stairs were climbed without specific instruction, whereas for a third set of stairs, the experimenter informed participants that they had to try and construct a text message on their phone while climbing the stairs with information about themselves , i am (age), i am a (gender), and i am working at/i am a student”). Participants were guided from one staircase to another by the experimenter, who also made sure that participants were safe while climbing the stairs. Participants only ascended staircases to avoid falls, and lifts were used to move participants down floors. While participants climbed the stairs, the experimenter filmed the participant using the in-built camera of a mobile phone (1080p video image at 30fps), so that the handrail and mobile phone use could be analyzed offline and any other unexpected events were documented. After the stair climbing tasks, participants completed a short questionnaire about their experience walking stairs.Data were preprocessed to extract the horizontal and vertical gaze location for each sample from the raw eye movement recordings, using a custom-built Perl script, after which gaze locations were combined with the head-centered video images using a Matlab script. Using another custom-built script in the same programming language, gaze locations were manually classified on a frame by frame basis according to what area they were directed to, with the following categories: one step ahead, two steps ahead, three steps ahead, four or more steps ahead, the handrail, the phone, the wall, the floor, people, “other”, and “unclear” category .κ of 0.704, considered to be ‘substantial’  eye movements, and where fixation on an object can be affected by head and body movements.t tests for each regions of interest (ROIs). Besides a significant difference for the ROI, “phone” (t(23) = 5.19, p < 0.001), significant differences were found for “floor” (t(62.6) = 9.87, p < 0.001), “other” (t(42.8) = 6.11, p < 0.001), “rail” (t(37.7) = 5.23, p < 0.001), three steps ahead (t(61.6) = 5.80, p < 0.001), four steps ahead (t(55.0) = 11.9, p < 0.001), and “wall” (t(55.1) = 11.8, p < 0.001).Figure t tests of dwell times on ROIs across the stairs showed significantly more time spent looking at the wall (t(43.2) = 3.53, p < 0.001) without a phone and significantly more “unclear” gazing with a phone (t(44.4) = 4.97, p < 0.001), which could be fixations on the phone that fell outside the recording window.The question arises whether, when not looking at their phone, people distribute their attention similarly to walkers without a phone. Figure Figure t(5.62) = 1.05, p= 0.34). For walking with a phone, there are no obvious differences between the two groups.In our questionnaire (see later in the results section), 14 of our 40 participants reported having experienced a fall on stairs sometime in the past. To examine whether such experience influences gaze behavior on stairs, Fig. t tests showed a marginally significant effect of phone use for the stair 1 (t(13.8) = 2.65, p = 0.019, d = 1.17), and significant increases in walking times when using a phone for stairs 2 (t(22.4) = 4.84, p < 0.001, d = 1.71) and stairs 3 (t(12.1) = 3.04, p = 0.010, d= 1.48). Figure t tests revealed no significant differences in dwell times to the various regions of interest for slow and fast walkers .Figure s 2  = 8.64, p= 0.0033). Fisher exact tests revealed a marginally significant effect of phone use on the use of the handrail for stair 1 (p= 0.060), but no significant effects for stair 2 (p= 0.67) and stair 3 (p= 0.23). Figure p= 0.81). The same holds for past experience with falls, which did not lead to increased use of the handrail .Figure p= 0.082), in agreement with the large number of people in the general population presenting to healthcare providers with injuries from such falls. Around three quarters of our participants reported being engaged in other tasks when walking the stairs, which involved looking at phones, talking on phones and eating. Finally, around three quarters thought lighting conditions influenced walking on stairs.Figure p= 0.55). The same holds for talking on the phone (p= 0.087). The younger group appear to eat less often while walking stairs, but also this difference is not significant (p= 0.32). Gender differences in the three behaviors  were smaller and not significant either. Similar results were obtained when splitting the data for past falls, where people with no experience of falls tended to report less eating (p= 0.076).Figure The present study extended earlier work on visual attention during stair walking and the influence of concurrent tasks , while more controlled stair walking studies found previews of between two to four , medication , medical conditions  or reduced vision , nutritional , and lack of exercise (Masud and Morris The present work extends earlier studies on visual attention during stair locomotion to a real world situation. The results show that mobile phone use strongly attracts attention away from the stairs, but that when gazing away from the phone, people distribute their attention similarly to when no phone is used. Handrail use is low, but slightly higher than in studies in more controlled settings, which could be due to the visual complexity of real world stairs, or the presence of other people on the stairs. The findings could be used to inspire mobile phone app developers to include the detection of stair locomotion in their apps, and to warn walkers not to use their phone while walking the stairs.