Drosophila auditory system, which is largely devoted to processing courtship song. Mechanosensory neurons (JONs) in the antenna are sensitive not only to sound-induced antennal vibrations, but also to wind or gravity, which affect the antenna’s mean position. Song pattern recognition, therefore, requires adaptation to antennal position (stimulus mean) in addition to sound intensity (stimulus variance). We discover fast variance adaptation in Drosophila JONs, which corrects for background noise over the behaviorally relevant intensity range. We determine where mean and variance adaptation arises and how they interact. A computational model explains our results using a sequence of subtractive and divisive adaptation modules, interleaved by rectification. These results lay the foundation for identifying the molecular and biophysical implementation of adaptation to the statistics of natural sensory stimuli.To faithfully encode complex stimuli, sensory neurons should correct, via adaptation, for stimulus properties that corrupt pattern recognition. Here we investigate sound intensity adaptation in the  Drosophila antennae. Here the authors report two forms of adaptation in JONs that correct for antennal position (mean) as well as background sound intensity (variance) to maintain sensitivity to natural sensory stimuli.Complex auditory stimuli such as courtship song are sensed by mechanosensory neurons (JONs) in  Adaptation allows our visual systems to recognize a familiar face whether in the daytime or nighttime and our auditory systems to parse a sentence whether it is whispered or shouted. In general, adaptation corrects a neural representation for irrelevant aspects of the stimulus, such as fluctuations in stimulus mean or variance4. To ensure that the stimulus is efficiently encoded, adaptation should be both sufficiently fast and implemented as early as possible in the sensory pathway.Neural adaptation, the process of adjusting a neuron’s dynamic range to match the statistics of the sensory environment6 superimposed on sound-induced receiver oscillations, the standard deviation of which corresponds to the sound intensity. To faithfully encode the fine temporal features of sound, auditory receptor neurons should correct for both fluctuations in the mean and variance of receiver movements, without mean adaptation affecting sound sensitivity. Indeed, cochlear hair cells correct subtractively for stereocilia offsets (what we term here “mean adaptation”) and divisively for changes in intensity (what we term here “variance adaptation”)8, but how these forms of adaptation interact within hair cells has not yet been characterized. Studying these forms of adaptation in the more accessible auditory system of Drosophila melanogaster presents an opportunity to investigate their implementation.Accordingly, many sensory systems perform mean and variance adaptation within sensory receptor neurons. The nature of the physical stimulus transformed by receptor neurons into electrical activity dictates how they should implement adaptation. For auditory receptor neurons, the physical stimulus corresponds to the mechanical displacement of the receiver . Receiver displacement has two, typically independent components: a fluctuating mean or baseline10. Song bouts typically comprise two modes—“sine” and “pulse.” To detect conspecifics and assess mate quality, the female’s auditory system must reliably encode the spectral and temporal properties of song, but several idiosyncrasies of both fly ears and fly courtship behavior can interfere with a faithful representation of song. The courtship song vibrates the arista (a feathery extension of the Drosophila antenna that serves as the sound receiver)—this causes rotation of the antenna and thereby opens mechanosensitive channels housed within antennal neurons, so-called Johnston’s organ (JO) neurons (JONs)12 , which falls off strongly as the cube of the distance13, and (ii) fly ears are sensitive to sound direction since the particle velocity is a directional quantity and the sound receiver is maximally vibrated only by sound coming from a direction orthogonal to the arista14. While males dynamically adjust how loudly they sing based on their distance to the female15, this adaptation on the sender side is likely insufficient to avoid saturation of the auditory receptors during courtship. The reported dynamic range of Drosophila auditory receptor neurons is between 0.1 and 1 mm s−116, but song can be as loud as 4 mm s−1 when the male is close to the female17. In addition to intensity fluctuations, the baseline position of the antenna constantly shifts because of changes in air flow and gravity12. This baseline corresponds to the mean of the distribution of antennal displacements 12 Fig. , top. Thnts Fig. , bottom Drosophila auditory system requires a neuronal mechanism for adaptation to both fluctuations in the baseline of antennal displacements and the intensity of the acoustic signal. While a subtractive mean adaptation mechanism corrects for the displacement baseline19, no studies have yet examined variance adaptation in Drosophila. Here, using neural recordings combined with computational modeling, we find that mean and variance adaptation co-occur within JONs and both do not require spiking or synaptic transmission. Despite the compact implementation of these two forms of adaptation within JONs, we find that mean adaptation does not affect sound sensitivity. This separation is not achieved by a separation of timescales since both forms of adaptation are equally fast. Rather, using a computational model, we show that the separation of mean and variance adaptation can be achieved with a simple sequence of computations: first, subtractive adaptation to correct for antennal offset, followed by rectification for encoding sound intensity, and finally divisive adaptation to correct for sound intensity. We find that the placement of rectification after mean adaptation in the model is essential for maintaining sound sensitivity for different levels of mean adaptation. While mean adaptation first occurs during mechanotransduction itself19, our data suggest that rectification and variance adaptation arise in subsequent steps. Our analysis of the organization of adaptation in Drosophila auditory receptor neurons should now facilitate the genetic dissection of these conserved operations, especially given the extensive parallels between fly and vertebrate hearing22.Thus, the Drosophila sound receiver, its arista, is moved by sound and by slower, quasi-static stimuli, such as gravity or wind—these stimuli push and pull on the antenna, activating mechanosensory neurons (Johnston’s organ neurons or JONs) in its second segment , the first relay for auditory processing in the fly brain11. While type CE JONs encode slow antennal movements by responding tonically to static displacement of the antenna23, type AB JONs rapidly and completely adapt their responses to these static displacements and produce sustained responses primarily for fast, sound-induced antennal vibrations in the frequency range of courtship song . The chosen intensity range corresponds to that which females naturally hear during courtship17. Noise exhibits a richly patterned fine structure that evokes reproducible, temporally structured responses in the JONs and has been shown to reduce desynchronization in other systems25  of fluctuating intensities. Since recording intracellularly from individual JONs with patch electrodes interferes with the natural movement of the antenna and with mechanotransduction  neurons  and exhibit both positive and negative transients immediately after steps. While adaptation appears to be weaker than that measured with the CAP likely due to the slow dynamics of the calcium indicator , but by what arithmetic operation do they do so? Since sound intensity scales the distribution of antennal displacements, variance adaptation should be divisive, not subtractive, and result in a change of the slope of the intensity tuning curve. We therefore measured JON responses to short, intermittent probe stimuli interleaved within background noise stimuli of a particular intensity Fig. , bottom.vel Fig. . Thus, aDrosophila courtship song comprises bouts consisting of rapid alternations between two modes—sine and pulse  of ~36 ms for melanogaster. In contrast, the envelope of sine song is only weakly modulated and hence resembles the steady envelope of noise stimuli , and thereby could contribute to song evaluation.We examined neural responses for song-like stimuli across a range of intensities  nor feedback to the JON from the brain or other sensory structures36, variance adaptation is likely to arise within individual JONs, at one of the following levels  the generation of spikes.Since there are no synapses between individual JONs  the37. If the variance adaptation reported here was part of the same process underlying amplification and mean adaptation, then it should reduce the gain of antennal movement. This should decrease sound-induced antennal vibrations following a rapid increase in sound intensity and create adaptation transients in antennal position. We measured arista movement using laser Doppler vibrometry and found that the amplitude of antennal vibrations faithfully followed changes in stimulus intensity , variance adaptation was still present  and subsequently measured responses to superimposed steps  and the magnitude (or variance) of antennal vibrations independently using a piezoelectric actuator. Piezoelectric actuation reproduced previous results regarding mean adaptation studied using electrostatic forces to deflect the antennanot Fig. . The ind 18 Fig. . To gain45: The loop’s input is low-pass filtered to an adaptation signal, which is either subtracted from the input (subtractive adaptation) or used to divide the input (divisive adaptation). We also included a rectifying nonlinearity in our model, since rectification is known to be necessary for variance adaptation46.We started by defining a set of elementary computations we deemed necessary for reproducing our experimental findings. Mean and variance adaptation in our model are implemented using incoherent feedforward loopsTo explore the constraints underlying the organization of the two types of adaptation we consider here, we tested all possible sequential or parallel arrangements of these three elementary computations in our model 53 already exhibit weak band-pass tuning for inter-pulse interval. Our results suggest that this tuning may in part be inherited from the JON inputs52. The incomplete adaptation to pulse trains containing melanogaster-specific inter-pulse intervals may be useful for encoding absolute pulse intensity, and this may facilitate orientation behaviors54. In addition, first-order auditory neurons in the fly brain (AMMC neurons) show adaptation across pulses in a train9, thereby further correcting for pulse intensity variation.Variance adaptation in the auditory receptor neurons, while fast enough to correct for the weakly amplitude modulated sine song, does not correct for the intensity of individual pulses within the pulse song Fig. . However23. Thus, just like in the mammalian somatosensory system, where different neurons adapt differentially to encode different aspects of touch55, distinct sensory channels in the Drosophila antenna differ in their adaptation properties to encode distinct aspects of antennal displacement.Information about antennal position in the sound-responsive type AB JONs is ambiguous since onset responses for step deflections depend on the intensity of the superimposed sound Fig. . HoweverOur data strongly suggest that adaptation is not a population-level phenomenon, but arises within individual type AB JONs. We recorded CAP signals from only type AB or B JONs Fig. , and rec21. Mean adaptation is thought to occur via a stiffening of the gating springs that transform the antennal movement into opening and closing of mechanosensitive channels in JONs55. For mean adaptation to not affect sound sensitivity, the underlying adaptation signal must not be rectified, but must be a linear representation of the symmetrical antennal vibrations . Adaptation channels can operate in a calcium-dependent manner, but we found that variance adaptation in calmodulin mutants appeared normal —disentangling the two requires a way to measure adaptation and mechanotransduction independently.Which molecules mediate variance adaptation? One possibility includes low threshold, voltage-sensitive potassium channels that open in response to subthreshold depolarizations with a delay and can thus dynamically reduce the amplitude of the generator currents . Since homozygous nompC mutants are lethal, we used transheterozygous mutants for two different nompC null alleles  as in ref. 19. Similarly, we generated transheterozygotes for the cam locus (camn339/cam5)20. We recorded CAPs from genetically identified subsets of JONs using flies mutant for iav (iav1/iav1) and we rescued the mutation by expressing wild-type Iav under the control of JO1 (JON-ABCDE), JO2 (JON-B), or JO15 (JON-AB)70. Calcium signals of JON-AB were recorded using GCaMP6f under the control of JO15-Gal4. Patch clamp recordings from the GFN were performed in flies expressing eGFP in C17-GAL4. Since the generator currents were stronger in younger flies19, we recorded at days 1–2 post eclosion. All flies were raised on a 12:12 dark–light cycle. All fly strains were ordered from the Bloomington or Kyoto stock centers, with the exception of UAS-iav, which was kindly supplied by Rachel Wilson. The original citations for the fly strains are as follows: iav126, UAS-iav71, JO1 (NP0761), JO2 (NP1046), and JO1570, c17-Gal472, UAS-eGFP73, nompC1 and nompC382, cam[n339]74, cam[5]82, and UAS-GCaMP6f28Group housed, virgin females were used for all recordings unless noted otherwise. Extracellular recordings were performed 2–5 days post eclosion. The wild-type strain used was Canton SFigures Canton S (wild type) and iav1/iav1 (iav1) and iav1/iav1;UAS-HM-iav/+;JO1-Gal4/+ (JON-ABCDE) and iav1/iav1;UAS-HM-iav/+;JO15-Gal4/+ (JON-AB), and iav1,JO2-Gal4/iav1, JO2-Gal4;UAS-HM-iav/+;+ (JON-B)Figure Canton S ,+; UASeGFP2x/C17-Gal4 Figure Canton S (wild type) and+;nompC1,cn,bw/cyo (nompC1/cyo),+;nompC3,cn,bw/cyo (nompC3/cyo) and +;nompC1,cn,bw/nompC3,cn,bw  and nompC3/nompC1  and camn339/cam5 Supplementary Fig. UAS20x-GCaMP-6f-UAS-Tom/cyo; JO15-Gal4/Tm6,tb Supplementary Fig. ++ imaging) in a sequence that contained all transitions between ¼, ½, 1, and 2 mm s−1  whose intensity was switched every 120 ms for 20 ms to a probe intensity of 1/16, 1/8, ¼, ½, 1, 2, 4, and 8 mm s−1  was produced from a sequence of normally distributed random values by band-pass filtering using a linear-phase FIR filter with a pass band between 80 and 1000 Hz. For probing adaptation, we switched the intensity of the noise every 100 ms  exp(−(x/σ)2 with carrier frequency fc of 250 Hz, phase ϕ of 0π, standard deviation σ of 4.6 ms and an inter-pulse interval of 36 ms. These parameters were chosen to mimic the shape of song pulses produced by Drosophila melanogaster males during natural courtship33. For Fig. Artificial pulse song Fig.  was geneThe sound delivery system consisted of (i) analog output of a DAQ card , (ii) a two-channel amplifier (Crown D-75A), (iii) a headphone speaker , and (iv) a coupling tube .9. Briefly, the amplitude of pure tones of all frequencies used (100–1000 Hz) was set using a frequency-specific attenuation value measured with a calibrated pressure gradient microphone . To ensure that the temporal pattern of the noise stimuli was reproduced faithfully, we corrected the presented noise patterns by the inverse of the system’s transfer function, measured using a pressure microphone . During experiments, the sound tube was positioned behind the fly at a distance of 2 mm and an angle or 90° to the right (extracellular recordings and laser Doppler vibrometry) or left  arista.The stimulus presentation setup was calibrated as in ref. Piezoelectric actuation was performed using a single channel piezo driver  and actuator (A186). The piezo actuation was calibrated using laser Doppler vibrometry (see below) by measuring displacement and velocity of the piezo tip for all driving voltages used in the experiments . Step stimuli for piezoelectric actuation were low-pass filtered using a 1 ms Gaussian window to stay within the operating limits of the piezo driver.76.Extracellular recordings were performed using glass electrodes  pulled with a micropipette puller . The fly’s wings and legs were removed under cold anesthesia and the abdomen was subsequently fixed using low-temperature melting wax. The head was fixed by extending and waxing the proboscis’ tip. The preparation was further stabilized by applying wax or small drops of UV-curable glue to the neck and the proboscis. The recording electrode was placed in the joint between the second and third antennal segment and the reference electrode was placed in the eye. Both electrodes were filled with external salineThe recorded signal was amplified and band-pass filtered between 5 and 5000 Hz to reduce high-frequency noise and slow baseline fluctuations induced by spontaneous movement of the antenna . We ensured that the band-pass filter did not distort the recorded signal, e.g., introduce artefactual response transients. We subsequently digitized at 10 kHz with the same DAQ card used for stimulus presentation .19. After obtaining access to the membrane voltage (input resistance between 70 and 400 MΩ), we recorded in voltage-clamp mode the spike-induced current, or—after bath application of TTX  to 6 μM final concentration—the generator current.Patch clamp recordings from the giant fiber neuron were performed as described in refs.28 under the control of JO15-Gal4, which expresses in the majority of JON-AB69. Flies were anesthetized on ice and gently waxed into the hole of a perfusion chamber with the proboscis facing upward, such that both antennae were free to move on the underside of the chamber. The antennal nerves and the AMMC were exposed by removing the proboscis and surrounding tissue. Sound stimuli were presented using sound delivery tubes as described above.Calcium signals were recorded from flies expressing GCaMP6f69 . The calcium indicator was excited at 940 nm using a TiSa Laser (Coherent). GCaMP emission was detected using a resonant scanner at a frame rate of ~60 Hz. ROIs were drawn manually around the projections of type A and type B JONs in AMMCArista movement was measured using laser Doppler vibrometry (LDV) . Flies were fixed and the arista was stimulated with sound using the same protocol as for recording CAP responses. The fly was mounted on a micromanipulator and visualized using a CCD imaging system . The fly was moved using the micromanipulator such that the laser was pointed at the distal part of the arista77 since it allows estimating the amplitude without imposing a timescale through the duration of the smoothing time window.Pre-processing: CAP/LDV instantaneous amplitude was estimated as the magnitude of the Hilbert transform. This method is preferable over “root mean square” like algorithmsr(t) = r0 + rmax exp(−t/τ) to the falling/rising phases of positive/negative transients /Δx >x, where <.>x denotes the average over intensities. 1−son/sss is a measure of adaptation strength that is independent of response magnitude: It is 0 for no adaptation (son = sss), approaches 1 for complete adaptation (sss≪son), and can exceed either of these values in the presence of noise.Adaptation timescale and strength: The adaptation timescale was estimated by fitting an exponential function ts Figs. c and 3f.ts Figs.  was estiSigmoidal fits of the tuning curves: Onset and steady responses for the intensity-step stimuli Fig.  and for α and offset or shift β of the tuning curves from these fits: r(x) = r0 + rmax/(1 + exp(−α(x−β)), where x is the sound intensity on a logarithmic scale and r(x) is the CAP amplitude. Fits were excellent throughout (r2 > 0.9).Sigmoidal fits to the resulting curves were obtained using the least squares method. We extracted the slope Ifisher) is a local measure of stimulus (intensity) discriminability that can be estimated from the tuning curves. Ifisher increases with the magnitude of the local slope of tuning curve (Δr/Δx)2—steeper slopes produce larger response changes for a given stimulus change and hence facilitate stimulus discrimination. Ifisher decreases with the local variance of tuning curve σ2(x) since variability adds uncertainty to stimulus-induced response differences29. Tuning curve slope as a function of intensity was estimated by taking the derivative of sigmoidal fits to the experimental tuning curves (see above). Since in our data, the response variance is proportional to mean (r2 = 0.98), Fisher information is proportional to Ifisher(x)∝(Δr(x)/Δx)2/r(x)71.Fisher information: Fisher information  for Fig. Statistical methods: In all figures, error bars depict mean ± s.d. over animals. For the statistical tests in Fig. 72.To explore different explanations for the adaptive dynamics in the CAP, we employed regular and adaptive leaky integrate and fire (LIF) model neuronsVm) in a LIF is given by: τVd/dtVm(t) = Iin(t)−Ileak(t)−Iadapt(t), with input current Iin = σstimIstim(t) + σnoiseInoise(t) and additive noise Inoise. The leak current Ileak with membrane resistance Rm follows Ileak(t) = Vm(t)−Vrest. The dynamics of the adaptation current Iadapt evolve according to τadapt d/dtIadapt(t) = Iadapt(t). A spike is elicited when Vm reaches the threshold Vthres after which it is reset to Vres. Iadapt increments by ΔA upon each spike and is 0 for a regular LIF. For all simulations τV = 4 ms, Vthres = 1 mV, and Vrest = −65 mV, except for Supplementary Fig. tv was chosen randomly from the interval 2 and 8 ms. The time constant of the adaptation current τadapt is 25 ms for Supplementary Figs. A is 0 mV. For adaptive LIF models, ΔA is 1 mV for Supplementary Figs. The subthreshold dynamics of the membrane voltage (−1) and then scaled to an input current Istim to obtain firing rates that were comparable across conditions (regular vs. adaptive LIF models) and physiologically plausible. Noise stimuli were low-pass filtered white noise (cutoff 1000 Hz) with step changes in intensity (standard deviation of the white noise) as describe in the figure legends. For Supplementary Fig. −1 to induce constant depolarization and to promote spike time jitter to accumulate over time. For Supplementary Fig. −1) and the high-intensity population (orange) only received input during the high-intensity period (2 mm s−1). LIF models were numerically simulated using Euler’s method with a step size dt = 1/20 ms.Stimuli were defined in terms of sound intensity dt. The integration time constant τ controls how fast the adaptation signal is updated and hence determines the effective speed of adaptation. Second, the adaptation signal then either subtracts or divides the input: xout = xin−xada or xout = xin/(1/σdiv + xada). 1/σdiv is added to the adaptation signal in the divisive case to avoid division by zero in the absence of stimulation and to control completeness of adaptation.Subtractive and divisive adaptation were modeled as an incoherent feedforward loopxada ≈ 0 for stimulus symmetrical fluctuations much faster than τ. Hence, there is no adaptation for rapidly fluctuating and symmetrical stimulus components, i.e., sound-induced vibrations, and adaptation acts as a high-pass filter. Note that this model can in principle also account for power law dynamics in adaptation74, by replacing the exponential integration kernel with a power law kernel75. This does not affect the qualitative model behavior we were interested in here. Rectification in the model was implemented as either full-wave rectification y = |x| or half-wave rectification y = Θ(x), where Θ(x) = 0 for x < 0 and Θ(x) = x for x ≥ 0 , divisive adaptation (D), rectification (R). To that end, we generated computational networks using the following rules: For serial networks, the root node could have either R or Ø  followed by an adaptation node containing S or D or Ø, followed by R or Ø, followed by another adaptation node containing S or D or Ø, followed by R or Ø. More compactly, R/Ø → S/D/Ø → R/Ø → S/D/Ø → R/Ø (“/” stands for “or”). These rules yielded 22 unique serial networks. Parallel networks contained two branches (rules for each branch: → R/Ø → S/D/Ø → R/Ø →) and an input and an output node (rules for each: → R/Ø →). This resulted in 100 parallel networks. The parameters for testing all 122 computational network motifs were: τsub and τdiv on a grid (10 ≤ τ ≤ 100, 20 × 20 values, linearly spaced, σdiv = 10−4; Supplementary Fig. σdiv (10−8 ≤ σdiv ≤ 100, 20 values, logarithmic spacing, τsub = 30 ms, τdiv = 50 ms; Supplementary Fig. x-scale relative to the adaptation step (Supplementary Fig. σdiv, simply because this parameter controls adaptation strength and when adaptation is weak, tuning curves do not change much.We tested for robustness of the qualitative model behavior to changes in model parameters by running the selected network motif (Fig. All data and source code are available from the authors on request.Supplementary InformationPeer Review File


Clostridium difficile infections (CDI). In contrast to adults the microbiome of infants develops from sterility at birth toward an adult-like profile in the first years of life. The effect of PPIs on this developing microbiome has never been studied. The aim of the present study was to determine the effect of oral PPIs on the fecal microbiome in infants with gastroesophageal reflux disease (GERD). In this prospective longitudinal study 12 infants with proven GERD received oral PPIs for a mean period of 18 weeks (range 8–44). Stool samples were collected before (“before PPI”) and 4 weeks after initiation of PPI therapy (“on PPI”). A third sample was obtained 4 weeks after PPI discontinuation (“after PPI”). The fecal microbiome was determined by NGS based 16S rDNA sequencing. This trial was registered with clinicaltrials.gov (NCT02359604). In a comparison of “before PPI” and “on PPI” neither α- nor β-diversity changed significantly. On the genus level, however, the relative abundances showed a decrease of Lactobacillus and Stenotrophomonas and an increase of Haemophilus. After PPI therapy there was a significant increase of α- and β-diversity. Additionally, the relative abundances of the phyla Firmicutes, Bacteroidetes, and Proteobacteria were significantly changed and correlated to patients' age and the introduction of solid foods. PPI treatment has only minor effects on the fecal microbiome. After discontinuation of PPI treatment the fecal microbiome correlated to patients' age and nutrition.Proton pump inhibitors (PPIs) are the standard therapy for gastroesophageal reflux disease. In adults, PPI treatment is associated with  Gastroesophageal reflux (GER) is a common finding in infants caused by temporary relaxations of the immature lower esophageal sphincter (LES)  , Bacteroidetes (10–30%), Proteobacteria (up to 10%) and Actinobacteria (up to 10%), . After ethical approval  and informed consent of parents or legal guardians patients younger than 1 year with proven GERD were enrolled in this study between November 2014 and August 2016. Patients with relevant additional diagnoses were excluded. This trial was registered with clinicaltrials.gov (NCT02359604).before PPI” sample). According to our protocol all patients received 1 mg/kg body weight oral esomeprazole daily. After 4 weeks of PPI treatment a second stool sample was collected and stored as described (“on PPI” sample). The duration of PPI therapy depended on the patients' clinical symptoms. A third sample was collected 4 weeks after discontinuation of PPI therapy (“after PPI” sample). None of the patients received antibiotics or other acid suppressants during the course of the study. Dietary habits were recorded.A first stool sample was taken before initiation of PPI therapy, stored in a PSP® Spin Stool DNA Kit  and frozen at −21°C until further processing   and transferred to MagnaLyser green bead tubes  for mechanical lysis performed two times at 6,500 rpm for 30 s in a MagnaLyser instrument . After bead beating 25 μl lysozyme (100 mg/ml) were added to the samples for enzymatic lysis and incubated at 37°C for 30 min followed by incubation with 43 μl Proteinase K (20 mg/ml) at 65°C for 1 h. Heat inactivation of enzymes was performed at 95°C for 10 min. Samples were centrifuged at 13,000 rpm for 5 min and 100 μl of the lysed samples were transferred to the Magna Pure instrument and DNA was purified according to manufacturer's instructions. PCR and library preparation with hypervariable regions V1-2 were performed as described before .Sequence reads were submitted to the NCBI Sequence Read Archive  using the vegan package. Significance of differences in alpha diversity was calculated by non-parametric two-sample t-test using Monte Carlo permutations to calculate the p-value. Lefse analysis was performed for all categories as described previously  using the pick_open_reference_otus.py script and uclust algorithm based on the greengenes database (gg_otus-13_8-release) and a 97% identity threshold. OTUs were visualized as OTU tables, bar charts and PCOA plots. Alpha diversity measurements (observed species and chao1) and beta-diversity measurements (unweighted unifrac) were derived using the respective QIIME tools. Group significance for all categories was determined with the Adonis test, while individual species difference was quantified by Kruskall-Wallis tests and pairwise comparisons by Mann-Whitney-U-test. The Adonis test computes R2 (effect size) and pseudo-P values of categories by first identifying the relevant centroids of the data and then calculating the squared deviations from these points. After that, significance tests are performed using n = 12 “before PPI”, n = 12 “on PPI,” n = 12 “after PPI”) of 12 patients  were included. The mean gestational age was 38 weeks . Patients had a mean birth weight of 2,794 g  and a mean birth length of 48.8 cm .36 stool samples . All patients suffered from GERD. The data of their 24 h-pH-MII is shown in Table p = 0.729, Figure before PPI” and “on PPI” .In the within-individual comparison (“before PPI” vs. “on PPI”), oral PPI treatment did not influence α-diversity  were significantly altered  (p > 0.10).Taxa summary plots at the phylum and class level at the different time points tested are depicted in Figure us Table . Althougd Figure . There wp = 0.003 for the comparison “before PPI” to “after PPI” and p = 0.003 for the comparison “on PPI” and “after PPI”). Furthermore, the β-diversity significantly changed throughout the experiment (p = 0.003 comparing “before PPI” and “after PPI” and p = 0.001 comparing “on PPI” and “after PPI”). For the relative abundances the majority of changes was also seen in comparison to the third sample and occurred in the Firmicutes phylum (Table p = 0.062) and nutrition (p = 0.001).The α-diversity significantly increased over the time of the experiment  and decreases of Lachnospiraceae, Ruminococcaceae, and Erysipelotrichaceae  under PPI therapy has been postulated. In detail, increases of Enterococcae and Streptococcae combined with decreases of Clostridiales were reported in association with an increased risk of CDI  tract. The fecal microbiome is easily accessible and the obtained results can be compared to adult studies (Freedberg et al., on PPI” we could demonstrate that our institutional protocol and PPI dosage was effective in a previous examination (Castellani et al., Since we were not able to demonstrate any relevant changes under PPI therapy one might question whether a PPI dosage of 1 mg/kg/day esomeprazole was sufficient. Theoretically, our findings could be caused by inadequate acid suppression. Although we did not repeat impedance monitoring “In conclusion, oral PPI therapy did not have relevant impact on the development of the infant fecal microbiome at a sensitive time of life in our series. Microbial changes associated with an increased risk of CDI infection described in adults did not reach statistical significance in this study. The majority of alterations occurred through the course of time and is correlated to patients' age and nutrition representing the normal development of the microbiome. Future studies are required to investigate possible microbial changes of the upper GI-tract under PPI treatment.CC and GS performed the statistics and wrote the manuscript; KK performed the microbiome analysis and performed the biostatistics; AH recruited the patients and collected the samples; CF and MK analyzed the data and assisted to draft the manuscript; HT coordinated the project and critically reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


During the synthesis of α-Fe2O3, particle sizes, crystal phases and morphologies were significantly influenced by pH, concentrations of precursor and template. The unique shape of worm-like hematite was obtained only when a starting solution was prepared by a weakly basic pH condition and a very specific composition of constituents. The synthesized nanocrystal at this condition had a narrow pore size distribution and high surface area compared to the bulk α-Fe2O3 or the one synthesized from lower pH conditions. The hydrocracking performance was tested over the synthesized iron oxide catalysts with different morphologies. The worm-like shape of iron oxide showed a superior performance, including overall yield of liquid fuel product and coke formation, over the hydrocracking of heavy petroleum oil.This paper reports the synthetic route of 3-D network shape α-Fe Iron oxide nanostructures are very important classes of materials used in a variety of fields including catalytic123The synthetic methods of the mesoporous metal oxides based on the template used are largely classified into two major routes, i.e. hard- and soft-template methods39101112132151617Nevertheless, there have been continuous efforts to synthesize iron oxide nanostructure possessing large surface area by deriving various novel morphologies such as hollow tube152O3 (hematite) nanocrystals having 3-dimentional network with a narrow pore size distribution can be synthesized by controlling acid-base conditions and non-ionic soft template, Pluronic P123. Iron oxide can take diverse crystal phases12To our best knowledge, the synthesis of worm-like mesoporous iron oxide using a non-ionic soft template in an aqueous solution has not been reported yet. Herein, we report the α-FeThe hydrocracking is a major upgrading process to convert heavy hydrocarbon feedstock into light fuel oils by adding hydrogenation catalysts. As worldwide crudes become dirtier and heavier, the slurry-phase hydrocracking has attracted a growing attention from oil refinery industries and related societies because it has many advantages, including feedstock flexibility, high yield of valuable distillate, low coke formation, and consequent process stability, over the other hydrocracking technologies2830The mesoporous structure of hematite is expected to have improved hydrogenation ability, especially when their pore sizes are uniformly controlled to have no diffusion limitation of reactant molecules. In this study, the synthesized mesoporous iron oxides were applied to demonstrate catalytic activities in slurry-phase hydrocracking of petroleum residue in which selective conversion of targeting heavy molecules including asphaltenes  solution resulted in the crystal structure of goethite, as XRD patterns for those cases are presented in 2O3 crystalline structures (JCPDS 33-0664). However, the (110) peak has a higher intensity than (104) peak for iron oxide synthesized at pH between 7 and 9 when compared to the one at pH conditions between 5 and 6. This result indicates that the direction of α-Fe2O3 crystalline structure did not conform to the direction of the crystal growth, i.e. [001], in a bulk synthesis of α-Fe2O33 in the presence of self-assembled F88 or F127 among other Pluronics by HClThe morphology and size for calcined iron oxide nanoparticles prepared within different pH were further investigated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Corresponding images are shown in Those are originated from hematite and ferrihydrite phases in an as-synthesis sample which can be further validated by corresponding XRD patterns in 311(x−1)Sx; JCPDS 29–0723)39The synthesized mesoporous iron oxide particles and their modified forms were applied to slurry-phase hydrocracking reactions to demonstrate the feasibility of materials as a dispersed catalyst. In the catalytic activity test, a heavy petroleum vacuum residue (VR) was used as a feedstock for the hydrocracking. The detailed properties for the feedstock obtained from chemical analysis are listed in 44302O3 catalysts at the same conditions and their synthesized iron oxide supporters were prepared by starting solutions of pH5 and pH9 respectively. In both cases, the faithful improvement for coke suppression and liquid products yield can be found compared to the cases of original iron oxide particles. In particular, 2%Mo/α-Fe2O3 (prepared by pH9 solution) catalysts shows an excellent selectivity to valuable liquid products , reduction of less valuable gas products, relatively high residual conversion, and small amount of coke formation. Although we are not quite successful to analyze Mo dispersion over the iron oxides in the analysis of TEM-EDS mapping (mainly because of insufficiently strong detection of molybdenum over the iron oxide specimen), we believe that the better dispersion of limiting amount of Mo over the supporter having a higher surface area may also contribute to improve the hydrocracking performance because there is a higher chance to form single layered MoS2 by in-situ sulfidation while only a rim side of basal sites in MoS2 multilayers possesses an equivalent hydrogen activity. In summary, the results implies that the iron oxide materials having well-developed mesopores and no limitation of asphaltene mobility to internal pores can be a good candidate for the heterogeneous dispersed catalyst or its supporter.Molybdenum is known to have a higher activity than iron oxide for the hydrogenationApart from the parallel comparison between different iron oxide supporters, it is noteworthy to see how the mesoporosities of iron oxide structure affect hydrocracking performance. It is very interesting that unmodified mesoporous iron oxide catalyst  brings i2O3 phase could be achieved without significant structural deformation when exposed to abrupt oxidation at a high temperature in the presence of air. When started with the solution of [Fe precursor]/[P123] = 1:4 adjusted to pH between 7 and 9, mesoporous α-Fe2O3 nanoparticles, showing a narrow pore size distribution and 20 nm average pore size, were obtained. The proposed synthetic route is relatively facile and simple without any further uses of expensive reagents or the aid of hard-templates. In the hydrocracking tests over the synthesized iron oxide or Mo loaded iron oxide catalysts, the catalytic supporters with well-developed mesopores were more beneficial in terms of valuable liquid fuel product yield and coke suppression. Although we put only one example of hydrocracking in this contribution, we expect that the suggested fabrication methods for mesoporous hematite will be very useful for other applications, especially for catalysts requiring facilitated mass transport of massive chemical species into the pore, owing to the relatively large open pores in their structures and the potential to scale-up in an efficient and economical way.In summary, the mesoporous worm-like iron oxide crystal structures were successfully synthesized by pH control of initial solution containing a very common iron nitrate precursor and non-ionic template, P123. Their transformation to homogeneous α-Fe20PO70EO20, Mn = 5,800) and bulk hematite(Iron(III) oxide, powder, <5 μm) were obtained from Aldrich. All other materials were purchased from Samchun chemical co, Korea unless otherwise specified. All chemicals were used as received and without any further purification. 8 g of Pluronic P123 were added to 60 g of water and stirred for an hour to obtain a completely dissolved solution. The solution was mixed with 240 g of 2 M HCl aqueous solution with continuous stirring for 10 h by magnetic stirrer. Then, 2 g of Fe(NO3)3·9H2O powder was added to the resulting solution. After 5 min stirring, 10 M sodium hydroxide solution was added to the mixture till the solution reaches at pH 5. During the addition of basic solution, the solution temperature was controlled to maintain below 40 °C by adjusting the titration speed. The same procedures were repeated to prepare samples having different pH 6 to 12. After pH adjustment, each sample was transferred to the oven preheated to 100 °C and kept there for 60 h. After this aging step, a large amount of P123 can be recovered because they were entangled massively and floated on the solution. The segregated iron oxide particles were purified by repeating membrane filtration followed by removal of upper solution and replenishing DI water for three times. The final wet solid powders were dried in vacuum oven kept at 60 °C (as-synthesized iron oxide). For a complete removal of soft template residue and transformation to hematite, the calcination was carried out in a box furnace maintained at 500 °C for 5 h under air flow of 100 ml/min.The mesoporous hematite structures were prepared in the following procedures. Triblock copolymer Pluronic P123  and TEM (TECNAI G2 T-20S). From a nitrogen adsorption-desorption isotherm  for each sample, the Brunauer-Emmett-Teller (BET) surface areas, the pore diameters by applying Barrett-Joyner-Halenda (BJH) model, and the total pore volumes were determined.Crystal structures of synthesized iron oxide were characterized by XRD . The morphology of calcined α-Fe2O3 nanocrystals were prepared for dispersed catalysts. The modifications of α-Fe2O3 nanocrystals were performed based on the incipient wetness impregnation methods. For instance, 0.076 g ammonium heptamolybdate tetrahydrate  was dissolved in pre-calculated amount of D.I. water and then the solution was slowly titrated to 2.062 g of synthesized iron oxide powders with an exposure to sonication (60 Hz) and continuous mixing for 1 h at the temperature of 40 °C. Then the resulting products were dried in an air oven at 120 °C for 12 h and ground with a pestle and a mortar. The dried powder samples were placed to an air furnace, heated to 450 °C by a heating rate of 100 °C/min under the constant air flow of 100 ml/min and held for 1 h more.To explore potential applications of the materials to slurry phase hydrocracking, synthesized and modified α-Fe2 flow, the reactor was heated up to the reaction temperature 430 °C with a nominal heating rate of 15  °C/min and the hydrocracking reactions were performed for 2 h. After the reaction, the reactor was suddenly chilled with cooling water till it reaches an ambient temperature. Gas sample was collected with 20 L Tedlar bag, and liquid and precipitate samples were transferred into the glass vials for further analysis followed by weighing with a high-precision digital balance.The hydrocracking experiments were carried out using an autoclave reactor . 40 g of vacuum residue (from Hyundai Oilbank Co.) and 0.5 g of catalysts  were added to the reactor and the reactor temperature was raised to 80 °C. The hydrogen gas (99.999%) purging steps were repeated more than 3 times and the final hydrogen fill-up was made to keep 80 bars in gauge at an equilibrium temperature of 80 °C. After halting H2O3 KCl  capillary column with a flame ionization detector) calibrated with Agilent refinery gas analyzer mixture. The yield of coke was evaluated by a solvent extraction of hydrocarbon oils from a precipitate product. 0.5 g of precipitate product was mixed with excessive amount (about 100 ml) of toluene and wet solid particles were collected by filtration (with Macherey-Nagel 0.45 μm PTFE membrane filter). After complete drying of solvent, the final solid dry powder was weighed for the coke quantification. The yields for liquid fuel product were quantified by GC-Simdis  analysis for liquid and precipitate samples (only toluene soluble part).Elemental compositions of VR and hydrocracking samples (liquid and precipitate) were analyzed with an elemental analysis instrument ) and an inductively coupled plasma atomic emission spectrometry . The compositions of macromolecules, which are saturates, aromatics, resins and asphaltenes (SARA), were analyzed by a SARA analyzer (Latroscan MK6s). The compositions of gas samples were quantified with gas chromatography .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Endothelial cells (ECs) are major modulators of hemostasis by expressing and releasing pro- and anticoagulant mediators into the circulation. Previous studies showed that cultured ECs release procoagulant mediators into cell culture supernatants as evidenced by the reduction of viscoelastic clotting time. This effect was reversed with an anti-tissue factor antibody. Here, we aimed to investigate whether tissue factor (TF) was released by endothelial-derived extracellular vesicles (EVs) and which portion of the released vesicles displays the most prominent procoagulant properties. After stimulation of ECs with tumor-necrosis factor-α (TNF-α) the supernatants of EC cultures were subjected to differential centrifugation steps to collect larger and smaller EVs which were then characterised by nanoparticle tracking analysis (NTA) and flow cytometry. Mixed with fresh human blood and analysed by thromboelastometry EVs exerted a significant procoagulant stimulus, which could be partly reversed by addition of an anti-TF antibody. Moreover, TF activity was confirmed in the centrifuged fractions. In summary, our results provide evidence of the procoagulant potential of smaller and larger endothelial-derived EV fractions detected by thromboelastometry. The observed effect is most likely due to the release of TF-bearing EVs of different dimensions, which are released upon TNF-α stimulation of endothelial cell cultures. In the classical coagulation cascade model of secondary haemostasis, the initiation of coagulation is triggered via contact activation with negatively charged surfaces (intrinsic pathway) or tissue factor (extrinsic pathway). However it has been proposed by Hoffmann and Monroe that coagulation occurs not as a “cascade”, but in three overlapping stages starting with “initiation”, which occurs on tissue factor (TF) bearing cells, then “amplification”, in which platelets and cofactors are activated to set the stage for large scale thrombin generation, and “propagation”, in which large amounts of thrombin are generated on the platelet surface. The authors also state that although endothelial cells express active antithrombotic features preventing coagulation from being initiated in the intact endothelium, inflammatory cytokines can also induce endothelial cells to increase expression of TF and surface adhesion molecules as an adaptive defence mechanism facilitating hemostasis at sites of injury2. In addition, it has been recently demonstrated that extracellular vesicles (EVs) play a relevant role in coagulation3. Moreover, in work already published ~50 years ago Wolf found that platelet derived EVs (termed “platelet dust”) contribute to blood coagulation4.The endothelium modulates blood coagulation by producing coagulant and anticoagulant factors to maintain the sensitive balance between blood fluidity and its stagnancy5. Primarily based on their size and the site of cellular origin, three different types of EVs are distinguished: (I) Shedding microvesicles which directly bleb from the plasma membrane (<1000 nm in diameter), (II) exosomes derived from the endosomal compartment (approx. 30–150 nm in diameter) and (III) apoptotic bodies which are released in the late stage of apoptosis (>1000 nm in diameter)6. However, current isolation methods including differential centrifugation are not able to fully separate different types of EVs from each other7. Therefore, the collective term “extracellular vesicles” is used. Interestingly, EVs were shown to play an important role in cell to cell communication in physiological9 as well as pathological conditions such as cardiovascular disease7 and the formation of atherosclerotic plaques10.EVs are small vesicles, consisting of a lipid bilayer membrane which contain proteins, and coding as well as non-coding RNA and DNA2+-dependent factor VII activation. Besides transmembrane bound TF, a non-cell-associated isoform has been described, which is involved in thrombus formation and thus might contribute to pro-thrombotic states that are found in a number of pathological procoagulant conditions11.TF is a 47 kD sized transmembrane protein. It’s primary function is to activate the coagulation process via Ca12. Moreover, it has been reported that tumor necrosis factor-α (TNF-α) increases blood levels of EC-derived microparticles in mice13. In addition, shear stress has been reported to regulate the release of endothelial microparticles15. Besides their clinical application to detect coagulopathy during the management of bleeding patients, viscoelastic tests such as thromboelastometry (ROTEM) have recently come into focus to investigate procoagulant patterns17. In our previous study we demonstrated that ECs release procoagulant mediators into cell culture supernatants as evidenced by the reduction of whole blood clotting time in thromboelastometry. This effect can be partially reversed with an anti-TF antibody18. However, the influence of different fractions of endothelial-derived EVs on viscoelastic clotting parameters has not been investigated thus far. In the current study, we aimed to test different subsets of endothelium-derived EVs with regard to their coagulatory activity and whether this effect is mediated by TF. In order to evaluate this hypothesis, we characterised different fractions obtained by differential centrifugation with nanoparticle tracking analysis (NTA) and flow cytometry. Finally, we functionally analyzed the fractions by ROTEM and a TF activity assay.Previous studies have shown that endothelial cells (ECs) release EVs exposing TF on their surface18. Since we attributed this effect to endothelial EVs, we aimed to isolate and characterise the different fractions of EVs which caused the observed biological effects. Subsequently, we measured TF expression on cells and on particles released thereof, as outlined in Fig. +/TF+ particles in P14 fractions while only few particles could be detected in P100  in the otherwise non-activated ROTEM assay when adding TNF-α-stimulated endothelial cell culture supernatants to whole blood from healthy volunteers100 Fig. . Quantif100 Fig. . Furtherles Fig. . To analTo confirm the biological effect of isolated particles from TNF-α-stimulated EC culture supernatants we used the ROTEM (NATEM) assay. In order to prove that the cell culture medium itself does not influence the study results, we first compared unconditioned EGM-2 and PBS and did not find any significant differences in the ROTEM parameters CT (clotting time), CFT (clot formation time) and MCF  and 100,000× g (P100) centrifuged supernatant of stimulated endothelial cells exerted a clear procoagulant pattern in fresh whole blood from healthy volunteers. This effect could be partly reversed by an anti-TF antibody, suggestive of a prominent role for TF as a procoagulant stimulus of EVs. The involvement of extracellular vesicle-bound TF could be confirmed by a chromogenic TF activity test. A major aim of the current study was to evaluate which vesicle subtype in TNF-α-stimulated endothelial cell culture supernatants primarily contributed to the observed changes in thromboelastometry. Three elementary subpopulations of EVs have been defined based on their diameter and biogenesis: exosomes (30–150 nm), shedding microvesicles (<1000 nm) and apoptotic bodies (>1000 nm). In contrast to free circulating macromolecules such as hormones, larger EVs can be enriched by centrifugation at 14,000× g (P14) and smaller EVs at 100,000× g (P100). However, several centrifugation protocols exist as particle sedimentation is dependent on the respective particle density21. Although TF influences the speed of clot formation and clot strength (such as CFT and MCF) to some extent, it is the clot initiation (expressed by the CT) that is most markedly altered by the addition of TF in otherwise non-activated viscoelastic assays24. This is in accordance with the results of the current study, where relatively small amounts of TF significantly affected CT but did not consistently affect CFT and MCF. In whole blood assays, CFT and MCF are primarily dependent on platelet contribution and fibrin polymerisation26. However, it has been shown that higher levels of TF activity such as in P14 in our study may also influence clot formation/strength to some extent.Each vesicle-rich pellet was added to fresh whole blood to investigate the isolated influence of EVs on the dynamics of the blood clotting process. In contrast to the control samples using medium only, the supernatant samples of TNF-α-stimulated HUVEC caused a strong CT reduction when added to fresh whole blood. Using differential centrifugation we could provide evidence that for the most part EVs are involved in the CT reduction as the S100 containing soluble factors did not mediate CT reduction. The native, non-activated viscoelastic assay (such as NATEM used in this study) has been proposed to be the most sensitive viscoelastic test for detecting endogenous TFTo investigate the role of TF in that context, an anti-TF antibody was successfully used to inhibt its activity.To confirm that EC-derived and EV-bound TF is most likely the reason for the acceleration of coagulation in thromboelastometry, a chromogenic TF activity assay was performed. This assay is specific to membrane-bound TF, since negatively charged surfaces, such as EVs, are necessary to activate TF. This assay confirmed that EV-bound TF from TNF-α-stimulated endothelial cells could not be detected in the mainly vesicle-free S100 but a high concentration of active TF could be detected in P14. Similar concentrations of active TF could be determined in the P100 fraction as compared to the original supernatant S0.5. The control sample with the medium EGM-2, which is known to contain extracellular vesicles itself, did not show TF activity. These results also confirmed that higher TF activities are in accordance with shorter clotting times in thromboelastometry.27. Furthermore, a study by de Jong OG et al. suggests that TNF-α-exosome-driven changes are mainly on the proteome/transcriptome level when vesicles are taken up by host cells27. Due to the time frame of our ROTEM experiments (<60 minutes) we excluded any potential influence of exosomes in our setting. Indeed, it has been shown that only the microparticle fraction isolated from the blood of healthy individuals leads to a clotting time reduction while exosomes isolated from the same sample showed no effect on coagulation28. Here, we characterised the particle fraction having the strongest biological effect in our functional assay and demonstrate that this effect is at least in part dependent on TF. Furthermore, using flow cytometry we were able to co-detect Annexin V on TF-positive particles, which is both a marker for EVs and an important indicator for procoagulant activity. We additionally provide data on Annexin V-positive but TF-negative particles and vice versa indicating that many particles are secreted which are positive for only one of these markers. However, a significant increase upon TNF-α stimulation could be detected in P14 samples regardless of whether particles are single- or double-positive suggesting that particle-bound TF was detected using our flow cytometry protocol.The remaining procoagulant activity of P100 raises the issue that small particles obtained from TNF-α-stimulated HUVEC may contribute to TF-dependent coagulation in our system. However, we and others did not observe a difference in particle count and size of P100 pellets after TNF-α-stimulation of endothelial cells5. Furthermore, the method of ultracentrifugation  does not fully separate but rather enriches EVs of different sizes, thus making exact quantifications impossible. We are also aware that protein aggregates are spun down together with vesicular components when exposing supernatants to this magnitude of forces. Additionally, with the use of anti-TF antibody the largely vesicle-free S100 fraction also showed a CT-reversion. This could be attributed to the inhibition of non-cell-associated TF, which is also present in the supernatant of endothelial cells and most likely representing a secreted alternatively spliced form of TF29. In addition, besides the use of HUVEC as the “golden standard”, microvascular endothelial cells have been shown to be a physiologically relevant endothelial subtype when it comes to cytokine-triggered TF expression30. Moreover, HUVEC are derived from venous origin, and different results might be obtained when using ECs from arterial vascular beds. Finally, although we understand that the transition is fluent, we defined the fractions as either large (P14) or small (P100) and did not further fractionate the initial supernatant samples for the sake of clarity.Nevertheless, we see some limitations in our study. Using anti-TF antibody we did not observe a full but rather a partial reversion of the reduction of CT. This could be due to stochiometrical reasons, in which higher amounts of inhibiting antibody could have led to a complete reversion. On the other hand, other cytokines/mediators than TF or EVs themselves (due to their negatively charged surface) might underlie this effect as well. Nevertheless, CT-reversion in P14 and P100 indicate a potential role of TF-bearing EVs, the existence of which has been published previouslyIn summary, this study provides evidence for the existence and the pro-coagulant activity of TF- bearing EVs, collected from TNF-α-stimulated endothelial cell culture supernatants. EVs of different sizes were separated by ultracentrifugation and their activity was shown by thromboelastometry and a chromogenic TF activity assay. As TF-bearing EVs from endothelial cells may play an important role in cardiovascular disease further research is warranted in order to identify the mechanisms involved and to elucidate the role of procoagulant EVs as potential drug targets in various clinical conditions.31 or purchased from Lonza . HUVECs were grown in EGM-2  containing 5% fetal calf serum (FCS) at 37 °C and 5% CO2. The cells were tested for their pro-inflammatory responsiveness by stimulating them with 10 ng/ml TNF-α  for 4 h. Subsequently, cells were tested for their ELAM-1 expression by flow cytometry (data not shown). All cell types used in this study showed strong inducibility of this surface marker. For the isolation of EVs, conditioned medium was collected from confluent HUVEC after stimulation with 10 ng/ml TNF-α for 24 h.The experiments were conducted in accordance with the local guidelines and were approved by local ethics committees of the state of Upper Austria and of the AUVA with written informed consent by the donors (ethics committee vote #200). Cells were either isolated according to established protocols5 cells were centrifuged at 500× g and 15 minutes  for cell removal resulting in the so called supernatant S0.5. The fraction S0.5 contained both larger and smaller EVs. This S0.5 was then centrifuged at 14,000× g for 15 minutes  to collect the pellet P14, which was assumed to contain mainly larger EVs, enriched in shedding microvesicles but also apoptotic bodies and cell debris. The remaining supernatant was subjected to an additional centrifugation step of 100,000× g for 60 minutes  to obtain the so called pellet P100 enriched in exosomes. The residual supernatant S100 was intended to be mostly vesicle-free. Both pellets P14 and P100 were gently resuspended in 50 µl phosphate buffered saline (PBS). Unconditioned medium alone  was used as a negative control in all experiments and equally processed for EV isolation as described above.For EV collection from the cell culture supernatants, serial centrifugation steps were conducted Fig. . EVs wer32. Briefly, samples were diluted 1:250 in sterile-filtered 1x PBS prior to staining for flow cytometry, injected into the device and videos were recorded for 60 seconds at two positions of the measurement cell. Camera sensitivity was determined automatically for each sample. Videos were analysed using ZetaView 8.02.31 software.The size and concentration of isolated particles was determined with the Zetaview PMX 110 V3.0 particle analyser  as described previouslyk-factor 830, 4 °C). 1 ml supernatant was discarded and the bound antibody detected by incubation with 20 µg/ml anti-mouse Alexa Fluor® 488 for 30 minutes on ice. The particle suspension was again washed and centrifuged before 1 ml supernatant was discarded and 2 µl Annexin V  in a total of 300 µl 1x Annexin V Binding Buffer were added to the remaining 100 µl. This staining protocol does not result in loss of Annexin V-positive particles in P14 and P100 fractions . Cells were kept in suspension on ice during staining with 5 µg/ml anti-TF antibody . After 45 minutes of incubation, bound antibody was detected by incubation with 10 µg/ml anti-mouse Alexa Fluor® 488  for 45 minutes on ice. To stain isolated EVs, the particle pellets P14 and P100 were incubated with 10 µg/ml anti-TF antibody for 30 minutes on ice, washed with 1 ml PBS/BSA (1%) and centrifuged at 16,100× g for 20 minutes  were incubated with 1 µg anti-TF  for 30 min at 37 °C.33. ROTEM uses a rotating pin that is vertically immersed into a prewarmed cup containing the blood sample. Coagulation of the citrated blood sample is initiated by recalcification and is detectable by the reduced rotation range of the pin due to the formation of a fibrin clot between the pin and the cup wall. The generated signal is converted into a curve that indicates the time of clotting initiation, the speed of clot formation, the clot strength as well as the clot quality/lysis34. In clinical diagnostic situations, ROTEM assays are usually activated via contact activation  or with tissue factor . However, to be sensitive to minimal amounts of TF, the initially nonactivated assay NATEM was used in the current study22. Briefly, 20 µl of CaCl2 (200 mmol/l) were added to 300 µl of citrated blood  followed by addition of 50 µl of the study sample  before the rotating pin was immersed into the mixture of blood and study sample. On average, 61906 and 1246 Annexin V- and TF-double positive particles obtained from P14 and P100 fractions of TNF-α-stimulated HUVEC, respectively, were added to the assay matrix in ROTEM. Particles were always isolated from a confluent well of a 6-well plate containing 2 ml of growth medium. The parameters clotting time (CT), clot formation time (CFT) and maximum clot firmness (MCF) were evaluated. ROTEM experiments were repeated between four and six times with at least two different HUVEC donors.ROTEM  is a viscoelastic method for the analysis of the clotting dynamics during whole blood coagulation35. Briefly, 50 µl of all centrifugation samples  were incubated with either mouse anti-human TF antibody  or mouse IgG-control  for 15 min at room temperature. Subsequently, samples were incubated with 50 µl of HBSA containing 10 mM CaCl2, 10 nM Factor VIIa  and 150 nM Factor X  for 2 hours at room temperature. The reaction was stopped with 25 µl HBSA containing 25 mM EDTA and samples were incubated with 25 µl chromogenic substrate  for 15 min at 37 °C, and absorbance was measured at a wavelength of 405 nm. Samples were measured in duplicates and EGM-2, which was not incubated with cells, as well as PBS served as negative controls. TF activity was calculated by reference to a standard curve generated using relipidated rh TF . The TF-dependent FXa generation was determined by subtracting the amount of FXa generated in the presence of anti-TF antibody from the amount of FXa generated in the presence of the isotype control antibody.TF activity of EVs dervied from HUVEC stimulated with and without TNF-α was assessed as described elsewhereData were analysed with GraphPad Prism 5 . Based on distribution of data analysed with a Kolmogorov-Smirnov-test a Student t-test or a Mann-Whitney test were used to compare mean differences between corresponding groups. Fold changes were analysed by a Wilcoxon matched-pair signed rank test. P-values < 0.05 were considered to be significant.Supp Info


In this paper, we establish new Poisson type inequalities with respect to a cone. As applications, the integral representations of harmonic functions are also obtained. P and radius R in n-dimensional Euclidean space, P and Let a such that We shall also write R are denoted by The set dσ  tχ instead of We remark that the function ∂Γ. Then ,,\documensee Letgbe a measurable function onsuch thatThen the harmonic functionsatisfiesasinWe first obtain the solutions of the Dirichlet problem with continuous data on the boundary of a cone.Letandgbe a continuous function onsatisfying r  an an\documThus δ be a positive integer. Then from  mentioned above, there exists For We put If If btain by  and HöldBy , we consWe first haveb such that Next, we shall estimate ere see , 17, 18), 18\docuIf Put Since By , we havetor (see ) we obtaSoCombining -22), we, we22), mentclass2pt{minimh in We apply Lemma ult see , 19):\d:\d\docuIf Since btain by \documenlds (see )24\docuCombining , 22) an an22) anh also satisfies (Notice that the condition  is stronatisfies  by Theorentclass1pt{minimaSinceentclass1pt{minimaHence, by applying Lemma e obtain . Then Thentclass1pt{minimaIn this paper, we discussed the improved Poisson type inequalities with respect to a cone only using gradient information. They inherited the advantages of the Poisson type conjugate gradient methods for solving the unconstrained minimization problems, but they had broader application scope. Moreover, the integral representations of harmonic functions are also obtained.


Parenteral nutrition associated cholestasis (PNAC) is a frequently observed pathology in extremely low birth weight (ELBW) infants. Its pathogenesis is determined by the composition and duration of parenteral nutrition (PN) as well as the tolerance of enteral feeds (EF). “Aggressive” nutrition is increasingly used in ELBW infants to improve postnatal growth. Little is known about the effect of “aggressive” nutrition on the incidence of PNAC. We analyzed the influence of implementing an “aggressive” nutritional regimen on the incidence of PNAC and growth in a cohort of ELBW infants.n = 77) or “aggressive”  nutritional regimen that differed in the composition of PN after birth as well as the composition and timing of advancement of EFs. We analyzed the incidence of PNAC (conjugated bilirubin > 1.5 mg/dl (25 µmol/l)) corrected for confounders of cholestasis , growth until discharge (as the most important secondary outcome) and neonatal morbidities.ELBW infants were nourished using a “conservative” . Body weight (+411g), head circumference (+1 cm) and length (+1 cm) at discharge were significantly higher. Extra-uterine growth failure (defined as a Z-score difference from birth to discharge lower than −1) was significantly reduced for body weight (85% vs. 35%), head circumference (77% vs. 45%) and length (85% vs. 65%) (P < 0.05). The body mass index (BMI) at discharge was significantly higher (11.1 vs. 12.4) using “aggressive” nutrition and growth became more proportionate with significantly less infants being discharged below the 10th BMI percentile (44% vs. 9%), while the percentage of infants discharged over the 90th BMI percentile (3% vs. 5%) did not significantly increase.The incidence of PNAC was significantly lower during the period of “aggressive” vs. “conservative “nutrition (27% vs. 46%, “Aggressive” nutrition of ELBW infants was associated with a significant decrease of PNAC and marked improvement of postnatal growth. Parenteral nutrition associated cholestasis (PNAC) is the most common form of liver damage in neonates while receiving parenteral nutrition (PN) . Its preThe nutritional guidelines for ELBW infants at our unit are shown in With “conservative” nutrition, PN was started after birth with glucose only, while protein and fat were slowly introduced during the first week of life. Enteral feedings were started using an extensively hydrolyzed formula (eHF) at half strength  when the infant was clinically stable. As soon as half EF were tolerated, eHF was advanced to full strength  and switched to preterm formula  as soon as infants reached 1 kg of body weight. Advancements of EF were not defined using the “conservative” approach.With “aggressive” nutrition, full PN was started with protein and fat on the first day of life. Enteral feeds were started using preterm formula  on the first day of life. Clinicians were allowed to perform advancements of EF by a maximum of 20 ml/kg/d, which was considered to be safe in terms of triggering NEC .In both periods, mothers were encouraged to express breast milk, which was used instead of formula as soon as available. Fortification of mother’s milk was performed using a bovine multicomponent fortifier that was added as soon as 140 ml/kg/d (“conservative”) or 100 ml/kg/d (“aggressive”) of EF were reached. In both periods, the attending physicians individually prescribed EFs and PN. Physicians were trained to follow the new feeding guidelines by members of our nutrition team.The components used for preparation of PN did not change within the study period. In particular the content of taurine in the amino acid solution  and the nature of lipid emulsion  did not change. In both periods, urodeoxycholic acid was used for treatment—but not prevention —of PNAC.The study was retrospective and observational. The primary aim was to evaluate the impact of an “aggressive” versus a “conservative” nutritional regimen on the incidence of PNAC in ELBW infants. The secondary aim was to compare postnatal growth. ELBW infants born within a two-year period of “conservative” (01/2005–12/2006) or “aggressive” (07/2007–06/2009) nutrition were screened for eligibility. Patients were excluded due to: (i) cholestasis at birth, (ii) diseases associated with cholestasis  that were diagnosed while hospitalized and (iii) early death or transfer to another hospital . Patients who died (but not those who were transferred) after 28 days of life were excluded from growth analysis, due to the risk of distortion of weight measurements by perimortal edema.®, Phillips Medical Systems, Eindhoven, The Netherlands, EU), the hospital’s PN prescription software  was used in preterm infants with more than 35% oxygen on continuous positive airway pressure in the first 2 hours after birth. Starting from 2009, ELBW infants <27+6 weeks gestational age (GA) received surfactant prophylactically while spontaneously breathing as previously described , EF  and total energy and fluids in infants of the “conservative” and “aggressive” period were expressed as the median during the first week of life . Total dNeonatal morbidities including the primary outcome are presented in A multivariate analysis on the effect of “aggressive” nutrition on the incidence of PNAC corrected for confounders (see statistics) is presented in 2) with respective percentiles, the percentage of infants <10th and >90th BMI percentile as well as Z-scores at birth  are presented together with Z-scores in standard deviations (SD) and the body mass index  surgery, culture proven sepsis, birth weight, Z-score of birth weight, time on PN and male sex) and the adjusted odds ratio is reported .n = 153), “aggressive” 07/2007–06/2009 (n = 143)). Of 296 infants screened, 162 infants  were eligible. Reasons for exclusion were death <28 days of life , transfer <28 days of life  and congenital cytomegalovirus infection (“conservative”: n = 1).A total of 296 ELBW infants were born within the study period  compared to “conservative” (45.5%) nutrition . FurtherP = 0.004), while infants of the “aggressive” group significantly gained BMI percentiles , length (+1 cm) and head circumference (+1 cm). Postnatal growth faltering was significantly decreased  and the ELBW infants without PNAC  receivedImplementation of “aggressive” nutrition for ELBW infants was associated with a reduced incidence of PNAC and improved weight, head circumference, length and BMI at discharge.P = 0.246, After implementing new nutrition guidelines at our unit, nutrition of ELBW infants was performed more “aggressively.” This included to introduce parenteral amino acids and lipids earlier and at higher amounts, as well as to start enteral nutrition immediately after birth with feeding advancements up to 20 ml/kg/day. To evaluate whether the new guidelines were put into practice, we analyzed the nutritional intake in the first week of life. Here, we found that ELBW infants received significantly more parenteral amino acids and lipids, but less glucose . The lowWe were concerned about a negative impact of “aggressive” nutrition on PNAC—due to the early increase of amino acids  and lipiTo exclude that other factors besides a more “aggressive” nutritional regimen–especially the lower sepsis rate-reduced PNAC, we performed a multivariate analysis  includinAs PNAC remained significantly reduced after correction for non-nutritional confounders it seems most plausible, that nutritional factors—modified by “aggressive” nutrition—were involved. In view of the lack of literature on “aggressive” or “conservative” nutrition of ELBW infants and PNAC incidence, the putative mechanisms deserve some more attention. “Aggressive” nutrition contained a bundle of changes to our feeding regimen  that arePostnatal growth restriction was common at our unit as elsewhere . By implOur study showed that a combined approach of “aggressive” parenteral and enteral nutrition significantly reduced PNAC while improving postnatal growth in a proportionate manner.10.7717/peerj.2483/supp-1Data S1Click here for additional data file.


Due to its concern about the future of Brazilian scientific journals after new criteriawere adopted by the QUALIS system of CAPES , the Brazilian Medical Association (AssociaçãoMédica Brasileira - AMB) has held several meetings at its headquarters inSão Paulo to discuss this matter. Editors of the main Brazilian medical journals,directors of the Brazilian Association of Scientific Editors (AssociaçãoBrasileira de Editores Científicos - ABEC), and coordinators of the areasMedicine II and Medicine III of CAPES exchanged information and came out with proposalsaimed at improving the evaluation process of Brazilian scientific journals by the newQUALIS system of CAPES. The classification of the scientific production according to theQUALIS system will be one of the main items of the three-year evaluation of graduateprograms. Since most scientific articles published in Brazilian journals are producedwithin graduate programs supported by CAPES, it was very important to fine tune thespeech and make sure that all the parties involved speak the same language. The editorsof scientific journals are afraid that the new criteria adopted by CAPES may create asubgroup of journals exclusively based on the ISI Impact Factor. The previous criterionrecommended an impact factor of 1 as a cutoff point. Recently, some Brazilian journalshave achieved this goal after putting a great load of effort into it. However, inaddition to considering only the impact factor, the new criteria established much highercutoff points. If this measure is adopted, Brazilian journals will be despised bygraduate academic advisors and students - who are the main producers of science inBrazil - thus creating a vicious cycle within which Brazilian journals will hardlysurvive.Professor João Pereira Leite spoke on behalf of CAPES. In addition to being thecoordinator of the area Medicine II, he is also the current representative of the healtharea in the Technical Scientific Council, which is the main department of CAPES. Duringone of the meetings, professor Leite provided a detailed explanation about the criteriaadopted for three-year evaluation and their impact on Brazilian graduate programs. Healso explained that, in face of the evident improvement of the quality of graduateprograms, it was necessary to increase the cutoff point or the separation point in orderto better differentiate these programs and classify them in terms of their qualitylevel. Based on data from the graduate programs - collected using the data collectionsystem of CAPES - it was found that many programs had more than 50% (some of them evenhad 80%) of their scientific production published in journals classified at higherlevels of the classification scale. On its turn, CAPES decided to create a larger numberof levels with the purpose of reclassifying the Brazilian journals. A decreasing scalebased on the impact factor has been suggested: A1, A2, B1, B2, B3, B4, B5, and C. Inaddition, CAPES also created an equivalence factor according to which the number ofarticles published in journals belonging to the lower levels of the scale would beequivalent to a smaller number of articles published in journals belonging to the higherlevels of the classification scale. Therefore, for example, for a certain area, 2articles B1 would be equivalent to 1.2 article A1; 1 article B1 + 1 article A2 would beequivalent to 1.4 article A1; 3 articles B2 would be equivalent to 1.2 article A1.According to professor Leite: "Such equivalence would bring benefits for journals withdifferent qualification levels." Professor Leite also informed that the newclassification system was designed based on the median of the journals’ impact factorprovided by the Journal Citation Reports (JCR) and calculated every year by the ISI Webof Knowledge. A list of journals including each area of CAPES was made to calculate themedian. The median for each area was based on this list and on the respective impactfactors; then, a new classification system ranging from A1 to C was created.The editors reminded professor Leite that the three-year evaluation process of CAPESwould cause a relative disagreement for the reclassification of the journals, sinceseveral Brazilian journals will have their impact factor increased or published for thefirst time during 2010, mainly those that have just been indexed in the ISI. Inaddition, these journals would have to wait for three years to change theirclassification in the new QUALIS! Another aspect that was questioned by the editors isrelated to the choice of the impact factor published by the JCR as the ONLY anduniversal index to assess the quality of the journals. There is a high standarddeviation in the impact factors of different journals. Certainly, that is the reason whyCAPES used the median of these indexes to analyze the scientific production of graduateprograms. Actually, according to this criterion, some medical specialties, such as thoserelated to surgery, have their best journals with a lower impact factor, which mightresult in a bias that could be extremely unfavorable for them.Both the editors and CAPES agree that valuing the Brazilian journals is important for theBrazilian scientific growth and development. With the purpose of keeping and stimulatingthis virtuous cycle, it is necessary to promote and foster the citation of articlespublished by Brazilian authors, intensify the efforts of editors, reviewers and authorsto increase the quality of the articles and, on the other hand, make sure that thegovernmental agencies, especially CAPES and CNPq, provide support for the management ofthe financial resources and qualitative classification of the journals.The results of these discussions were presented in several meetings attended by editors,coordinators of graduate programs and researchers, and new suggestions were made. Theideas described below will be used as the conclusion of this editorial and, at the sametime, we hope that they serve as an important tool to convince the agencies to changethe criteria of journal classification in the QUALIS system of CAPES. Our suggestionsare as follows:The qualitative analysis of the Brazilian journals should be reassessed andit should not include only the Impact Factor published by the JCR;The specific characteristics of each area of interest or each specialtyshould be taken into consideration and respected;The Brazilian publishing industry, in contrast with what happens in othercountries where it is financed by private investors, is financiallysupported by public and private universities and scientificassociations;Brazilian journals need to receive more support and stimuli, which may beprovided as: financial remuneration for editors, financial support forjournals, greater visibility for national journals abroad, more objectiveand encompassing criteria for the qualitative classification, and supportbased on the performance of each journal;Support for the internationalization of scientific journals by fostering theprofessionalization of the editorial process and promotion of the journalsin other countries;Continuous update of the journal classification system within the new QUALISwith no need to wait for the three-year period of assessment;Participation of scientific associations  in thedecision-making process regarding the QUALIS system of CAPES;Strong stimulation of citations directly in the source of scientificproduction, that is, graduate programs (for instance, recommending thatgraduate programs classified as 6 or 7, in addition to being required tohave a percentage of articles published in journals with high impact factor,should also have a percentage of articles published in Brazilian journals.This measure includes both ends of the scientific production, since youngand future researchers begin their careers publishing in national journalsunder the supervision of experienced researchers.th National Meeting of ScientificEditors – 2009Forum ofthe Areas Guidelines of the 12, which will be timely sent to all the Braziliansponsoring agencies, which should be done periodically because this is a continuousprocess.In conclusion, to show its agreement with all these measures and its concern with theconsequences of the new QUALIS of CAPES and other evaluation procedures of journals,ABEC devoted three days to the forum of the areas during its last National Meeting ofScientific Editors, which was held in November 2009. During this meeting, members of thestaff of CAPES and editors of all the areas of scientific knowledge held longdiscussions on this topic and came up with the suggestion of the The following editors approved this editorial:Adagmar Andriolo [Jornal Brasileiro de Patologia e MedicinaLaboratorial] Aécio Flávio Meireles Souza[Revista GED] Alberto Queiroz Farias [Revista Arquivosde Gastroenterologia] Alfredo José Afonso Barbosa[Jornal Brasileiro de Patologia e Medicina Laboratorial] AntonioSpina França Netto [Revista Arquivos deNeuro-Psiquiatria] Arnaldo José Hernandez [RevistaBrasileira de Medicina do Esporte] Aroldo F. Camargos[Revista Femina] Benedito Barraviera [Journal ofVenomous Animals and Toxins including Tropical Diseases] Bogdana VictoriaKadunc [Surgical & Cosmetic Dermatology da Soc. Brasileira deDermatologia] Bruno Caramelli [Revista daAssociação Médica Brasileira] Carlos Eduardo AguileraCampos [Revista Brasileira de Medicina de Família eComunidade] Carlos Brites [Brazilian Journal of InfectiousDiseases] Dejair Caitano do Nascimento [HansenologiaInternationalis] Domingo M. Braile [Revista Brasileira deCirurgia Cardiovascular] Dov Charles Goldenberg [RevistaBrasileira de Cirurgia Plástica] Edmund Chada Baracat[Revista da Associação Médica Brasileira] EdnaT. Kimura [Arquivos Brasileiros de Endocrinologia eMetabologia] Edson Marchiori [Revista RadiologiaBrasileira] Eduardo de Paula Vieira [Revista Brasileira deColoproctologia] Eros Antônio de Almeida [Revista daSociedade Brasileira de Clínica Médica] Geraldo PereiraJotz [Revista Brasileira de Cirurgia Cabeça ePescoço] Gilberto Camanho [Revista Brasileira deOrtopedia] Gilberto Friedman [Revista Brasileira de TerapiaIntensiva] Giovanni Guido Cerri [RadiologiaBrasileira] Ivomar Gomes Duarte [Revista deAdministração em Saúde] Izelda Maria CarvalhoCosta [Anais Brasileiros de Dermatologia] JoãoFerreira de Mello Júnior [Brazilian Journal ofOtorhinolaryngology] Joel Faintuch [Revista Brasileira deNutrição Clínica] José Antônio BaddiniMartinez [Jornal Brasileiro de Pneumologia] JoséAntonio Livramento [Revista Arquivos de Neuropsiquiatria]José Eduardo Ferreira Manso [Revista do ColégioBrasileiro de Cirurgiões] José Luiz Gomes do Amaral[Revista da Associação Médica Brasileira]Linamara Rizzo Battistella [Revista ActaFisiátrica] Luís dos Ramos Machado [RevistaArquivos de Neuropsiquiatria] Luiz Felipe P. Moreira[Arquivos Brasileiros de Cardiologia] Luiz Henrique Gebrim[Revista Brasileira de Mastologia] Marcelo Madeira[Revista Brasileira de Mastologia] Marcelo Riberto[Revista Acta Fisiátrica] Marcus Bastos [JornalBrasileiro de Nefrologia] Mário Cícero Falcão[Revista Brasileira de Nutrição Clínica] MarioJ. da Conceição [Revista da Sociedade Brasileira deAnestesiologia] Mauricio Rocha e Silva [RevistaClinics] Milton Artur Ruiz [Revista Brasileira de Hematologiae Hemoterapia] Milton K. Shibata [Arquivos Brasileiros deNeurocirurgia] Mittermayer Barreto Santiago [RevistaBrasileira de Reumatologia] Nelson Adami Andreollo [ArquivosBrasileiros de Cirurgia Digestiva] Osvaldo Malafaia [ArquivosBrasileiros de Cirurgia Digestiva] Regina Helena Garcia Martins[Brazilian Journal of Otorhinolaryngology] Renato SoibelmannProcianoy [Jornal de Pediatria] Ricardo Baroudi[Revista Brasileira de Cirurgia Plástica] RicardoFuller [Revista Brasileira de Reumatologia] RicardoGuilherme Viebig [Arquivos de Gastroenterologia] RicardoNitrini [Dementia & Neuropsychologia] Rita CristinaMainieri R. de Moura [Revista da Associação Brasileirade Medicina de Tráfego] Rogério Dedivitis[Revista Brasileira de Cirurgia Cabeça e Pescoço]Ronaldo Damião [Urologia Contemporânea]Sergio Lianza [Revista Medicina deReabilitação] Sigmar de Mello Rode [BrazilianOral Research] Winston Bonetti Yoshida [Jornal VascularBrasileiro] Zuher Handar [Revista Brasileira de Medicina doTrabalho].


C57BL/6 and ApoE−/− mouse models are widely used to study gene expression changes in liver disease and metabolism, currently there are no validated stably expressed endogenous genes in these models, neither is it known how gene expression varies within and across liver lobes. Here we show regional variations in the expression of Ywhaz, Gak, Gapdh, Hmbs and Act-β endogenous genes across a liver lobe; Using homogeneous samples from the four liver lobes of 6 C57BL/6 mice we tested the stability of 12 endogenous genes and show that Act-β and Eif2-α are the most stably expressed endogenous genes in all four lobes and demonstrate lobular differences in the expression of Abca1 cholesterol efflux gene. These results suggest that sampling from a specified homogeneous powdered liver lobe is paramount in enhancing data reliability and reproducibility. The stability of the 12 endogenous genes was further tested using homogeneous samples of left liver lobes from 20 ApoE−/− mice on standard or high polyphenol diets. Act-β and Ywhaz are suitable endogenous genes for gene expression normalisation in this mouse model.The liver plays a critical role in food and drug metabolism and detoxification and accordingly influences systemic body homeostasis in health and disease. While the  C57BL/6 mouse model is a widely known inbred mouse strain that has been used in numerous studies to investigate disease phenotypes and outcomes in response to treatments such as in alcoholic liver diseases and dietary treatments respectively4. Due to its ability to evoke spontaneous hypercholesteraemia and arterial lesions resembling those of human atherosclerosis as well as its propensity to be induced to a full spectrum non-alcoholic steatohepatitis (NASH), an ApoE knock-out (ApoE−/−) mouse model is widely used for studying the underlying mechanisms involved in cardiometabolic disease development7. Food bioactive compounds such as polyphenols have been shown to be protective against cardiometabolic diseases through anti-lipidaemia and anti-inflammatory mechanisms8. Although the liver has several lobes with reported vascular anatomical and physiological variations which may contribute to molecular differences, numerous studies have utilised livers from both C57BL/6 and ApoE−/− models without necessarily reporting how samples were collected11. Understanding whether such molecular variations exist between and within lobes with respect to gene expression is paramount for data reliability as well as for improving data reproducibility and consequently reducing the number of animals being used in such studies in accordance with the “3 Rs”  in animal ethics12. Therefore, we determined whether there are differences in gene expression in different biopsies taken from the same liver lobe or between different liver lobes. Polyphenols are known to induce gene expression changes in the liver. However, most studies investigating the underlying mechanisms involved in the protective effects of polyphenols have used single, endogenous genes such as 18S ribosomal RNA (18S), glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and beta-actin (Act-β) for gene expression normalisation without the validation of their expression stability14. It is widely accepted that single genes may not be suitable for gene expression normalisation as certain endogenous genes including Gapdh and Act-β may be regulated under certain pathological conditions or treatment and may change depending on experimental conditions and cell type15. 18S ribosomal RNA, has also been shown to be a poor representation of mRNA expression15. As such, the use of these genes without the validation of their stability may severely compromise assay sensitivity and accuracy leading to inaccurate data interpretation. In the current study, we sought to validate stably expressed endogenous genes in the liver lobes of C57BL/6 fed a standard diet and ApoE−/− mice fed a low polyphenol diet or tomatoes containing different types but similar amounts of polyphenols  lower in the right lateral lobe than in the left lobe and it tended to be higher in the right lobe compared to the right medial lobe and the caudate lobe, although this did not reach significance.  and this also indicated that together with Hmbs, β2m, and Rpl4, the expression of Tbp was significantly different between the five groups. However, the expression of Gak was no longer different between the groups  that Act-β and Ywhaz are suitable endogenous genes for gene expression normalisation when using ApoE−/− mice livers from mice fed on a standard low polyphenol diet or those on high polyphenol diets. These findings have important implications in that using liver sample biopsies may lead to erroneous results and this may also occur where samples from different lobes are being compared in different treatments. We therefore suggest that a homogeneous sample from a specified liver lobe should be used in all studies where comparisons between treatments or disease phenotypes are required. In order to facilitate data comparisons between laboratories we recommend that where possible the left liver lobe should be used for gene expression studies as this provides a larger sample that can allow gene expression correlations with other molecular and biochemical markers. Alternatively, a homogeneous sample from the whole liver should be used and this should be specified in the literature.The main findings of this study are (1) that there are intra-lobular and inter-lobular differences in the expression of genes in the mouse liver, (2) Housekeeping genes C57BL/6 were bred inhouse and 8-week-old male homozygous B6.129P2-Apoetm1Unc/J (named ApoE−/− hereafter) mice were purchased from Jackson Laboratories . Mice were housed in a temperature-controlled environment (22 °C) with a 12 h light/dark cycle. They were kept under relative humidity of 55% and supplied with free access to water and food with the location of each cage within the experimental room being regularly moved. C57BL/6 were fed a standard breeding diet AIN-93G until aged 8 weeks when they were sacrificed . ApoE−/− mice were fed a standard breeding diet AIN-93G  for 8 weeks before being randomly divided into five groups and fed ad libitum for seventeen weeks either supplemented with a low polyphenol tomato powder, or high polyphenol tomato powders where the tomatoes were enriched in either flavonols, a mixture of flavonols and anthocyanins, resveratrol or isoflavones  at 4 °C overnight and kept at -20 °C until the day of RNA extraction.All experimental procedures and protocols used in this study were reviewed and approved by the Animal Welfare and Ethical Review Body (AWERB) at the University of East Anglia and were conducted within the provisions of the Animals (Scientific Procedures) Act 1986 (ASPA) and the LASA Guiding Principles for Preparing for and Undertaking Aseptic Surgery (2010). Care of the animals for the duration of the study was in accordance with the UK Home Office guidelines. Briefly male C57BL/6 mice  was added to 25 mg of all samples and homogenised using the Precellys 24 lysis & homogeniser at 6000 rpm for 4 cycles for 30 s  and total RNA was extracted using miRNeasy Mini Kits according to the manufacturer’s instructions . Total RNA concentration was assessed by Nanodrop and 1 µg of total RNA was used for cDNA synthesis using the Precision NanoScript Reverse Transcription kit  in a final volume of 20 µl according to the manufacturer’s instructions.For lobular differences, 4 liver lobes were collected from six ice Fig. , for witice Fig.  and for https://lifescience.roche.com/en_gb/brands/universal-probe-library.html) or the PubMed primer designing tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Table https://blast.ncbi.nlm.nih.gov/Blast.cgi) or sent for sequencing   in a final reaction volume of 10 µl, and comprised of 1 X ImmoMix PCR MasterMix , SYBR Green (0.06ul of 100x stock), ROX reference dye (175 nM) magnesium (0.5 mM), BSA (50 µg/ml) and 10 nM forward and reverse primers. The following PCR cycling conditions were used; initial denaturation at 95 °C for another 10 min, followed by amplification and data acquisition at 95 °C for 15 sec and annealing/extension at 60 °C for 1 min for 40 cycles and a melt curve. For each gene, the melt curves Fig. . Standar18. Several studies have shown that the results obtained from these packages are fairly similar29. Owing to the former two packages being freely available to be used as excel add-ins, in this study Bestkeeper and Normfinder were used for endogenous gene stability analysis. Bestkeeper computes a value termed Bestkeeper index based on the geometric mean of the cycle threshold (Ct) values of each candidate gene and carries out a pair-wise correlation and regression analysis to generate P values, with the most stably expressed gene having a P value closer to 1. Normfinder determines gene stability based on inter-and intra-group variations with the most stably expressed gene having the lowest stability value.We considered three programmes; Bestkeeper, Normfinder and GeNorm that apply statistical algorithms to determine the most stably expressed endogenous genes in different cell types, disease phenotypes and treatmentsRpl27 in the isoflavone and Gapdh in the resveratrol groups were normally distributed according to the Shapiro-Wilk test and as such a One-Way ANOVA with Bonferroni correction was used. Rpl27 AND Gapdh data were log transformed and they remained not normally distributed and as such group differences were determined by the Kruskal-Wallis test.After PCR analysis by VIIA7, data were exported to MS Excel. For analysis by Bestkeeper, raw Ct values were used to calculate the geometric mean of Ct values for all the genes to generate the Bestkeeper index, which was then correlated to the Ct values of each gene to derive the coefficient of correlation value [r]. For Normfinder a standard curve was used to calculate transcript concentrations and these were log transformed by the software and the data were used to compute stability values based on intra-group and inter-group variations. Statistics were carried out using IBM SPSS software . For comparisons between lobes a non-parametric Wilcoxon related sample test was used. For comparisons between treatment groups, all data except The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Figure 1Supplementary Figure 2