We present a tactile telepresence system for real-time transmission of information about object stiffness to the human fingertips. Experimental tests were performed across two laboratories . In the Italian laboratory, a mechatronic sensing platform indented different rubber samples. Information about rubber stiffness was converted into on-off events using a neuronal spiking model and sent to a vibrotactile glove in the Irish laboratory. Participants discriminated the variation of the stiffness of stimuli according to a two-alternative forced choice protocol. Stiffness discrimination was based on the variation of the temporal pattern of spikes generated during the indentation of the rubber samples. The results suggest that vibrotactile stimulation can effectively simulate surface stiffness when using neuronal spiking models to trigger vibrations in the haptic interface. Specifically, fractional variations of stiffness down to 0.67 were significantly discriminated with the developed neuromorphic haptic interface. This is a performance comparable, though slightly worse, to the threshold obtained in a benchmark experiment evaluating the same set of stimuli naturally with the own hand. Our paper presents a bioinspired method for delivering sensory feedback about object properties to human skin based on contingency–mimetic neuronal models, and can be useful for the design of high performance haptic devices. The use of haptic technologies to deliver tactile information in real-time has grown in recent years, because of the effectiveness of the human tactile sense as a communication channel for different kinds of object information. We use the sense of touch to interact with the surrounding world, especially through the hands, which represent the most somatosensitive part of our body ,2. IndivThe possibility to deliver tactile information directly onto the users’ skin, and especially on the hands, via tactile feedback can enhance the performance of tasks executed in different scenarios. This could contribute, amongst others, to rehabilitation procedures, as well as to the development of tactile aids for visual and audition sensory disabled persons since the seminal works of Bach-y-Rita ,6,7,8,9,In the last decades, a variety of research has focused on the development of devices for the provision of tactile information in telepresence, mainly with sensory augmentation or substitution purposes ,15,16,17Tactile information presented on the skin is collected by all the mechanoreceptors distributed in the stimulated areas and the dedicated receptors can then provide information about the characteristics of an object . In geneThe delivery of tactile feedback in telepresence operations ,28,29,30In the present study, we tested the reliability of a tactile telepresence system in delivering feedback information about the stiffness, i.e., the ratio between a compression force and the resulting deformation of the tissue, of selected polymeric samples to the hand of an operator placed remotely with respect to the inspecting system. The study was designed with possible applications in the field of industrial and surgical robotics. Our approach is based on the conversion of information about the stiffness of rubber surfaces in vibrotactile stimuli by means of neuronal spiking models. This encoding strategy enables the real-time conversion of force information in neuronal spikes, which are delivered directly on the skin of the fingertips of the hand of a remote human subject. In our novel approach, the spikes, usually delivered via neuronal electric stimulation directly on nerves to restore tactile sensation in amputees  or to inThe telepresence device is constituted of a mechatronic platform for the automatized indentation of rubber samples and of a haptic display (a vibrotactile glove) for the remote transmission of vibrotactile information . Using aThe experimental setup was composed of two main subsystems, which allowed for the execution of the experiments in telepresence. The first subsystem was a mechatronic platform for the indentation of samples of different rubber surfaces. It consisted of a cartesian manipulator with three translational degrees of freedom, a load cell for the measurement of the normal force during the indentation of the samples , a probe mounted on the load cell and a Graphical User Interface (GUI) made in LabView  in order to control the movement of the sliders and acquire data from the load cell A. This fThe second sub-setup was located in a remote environment with respect to the indentation platform, i.e., a laboratory within the Trinity College Institute of Neuroscience in Dublin, Ireland. The apparatus comprised a textile glove equipped with two vibrotactile piezoelectric elements which were placed on the index and thumb fingertips respectively, a control electronics and a GUI made in LabView, which allowed the transmission of data from the mechatronic platform to the haptic glove B. The coFz) generated during the rubber compression stage. In particular, it executed indentation protocols with controlled testing velocities and positions, and selectable force targets. The vertical element of the slider mechanism was a precision positioner, and could ensure the vertical application of loads up to 10 kg along the z-axis. The couple of sliders along the x- and y-axes had the same performance as the vertical positioner. The probe was fixed to the load cell: it was a cylindrical tip made of aluminum with a diameter of 6 mm.The mechatronic platform for the automatic indentation of the rubber samples A allowedDuring the indentation of the rubber samples, the vertical slider was driven with a constant velocity of 0.5 mm/s, which was always the same for each investigated specimen. The choice of a constant indentation velocity allowed us to characterize the stiffness of the samples without taking into account variations due to the viscoelastic behavior of the materials. In each experimental trial, the rubber pair, the force and the duration used to indent each rubber sample were randomized, in order to guarantee a very challenging task for the participants during the experimental protocol see .The tactile feedback system allowed for the vibrotactile stimulation on the remote user’s hand, giving information regarding the stiffness of the indented rubber samples B. It was3). The position of the center of each sample was represented by a couple of coordinates (x-y). These coordinates were randomized with a GUI made in LabView, which allowed for the movement of the horizontal sliders along the x-y axes. Once the target x-y position was reached, the translation mechanism allowed indenting the rubber via the probe along the z-axis. The rubber compression lasted until the measured Fz on the load cell reached a random threshold level, that was varied in the 4 to 8 N range during the experimental protocol. Once the threshold was reached, the loading phase was interrupted, and the support returned to the reference position .The mechatronic platform moved the rubber samples in order to indent those selected for the current experimental session. The six rubber samples were placed on a Delrin support (170 × 130 × 17 mm) provided with six housings The obtained spikes were then delivered to the glove by means of a piezoelectric driver , working in analog mode, so that the on–off activity of the transducers was regulated by the neuromorphic model, proportional to the magnitude of the normal force measured by subsystem I. The actuation parameters for the piezoelectric driver in analog mode were a 40.7 dB gain, a peak-to-peak voltage amplitude of 200 V and a Boost voltage of 105 V. We evaluated the efficacy of the telepresence system in delivering reliable information regarding rubber stiffness using a two-alternative forced-choice (2-AFC) psychophysical protocol. Specifically, a control test was performed investigating an active touch condition naturally with the own hand, while the actual experimental protocol was focused on the investigation of the communication of stiffness information with neuromorphic tactile telepresence. The two protocols are detailed in the following.An active touch experiment was performed in order to evaluate the benchmark performance of a cohort of participants who actively judged, via their own hand, the stiffness of the same rubber samples used in the telepresence experiment. This experimental protocol regarding active touch involved 10 participants, . Participants self-reported having no pathologies regarding the tactile sense, nor was their tactile sensitivity compromised by previous activities. The tactile task involved the index fingertip of the dominant hand (one left-handed and nine right-handed). During the active touch experiment, participants were invited to take a seat and to place their dominant arm on a table. To avoid seeing the stimuli during the experiment, participants were also invited to insert their arm in a box where a cushion was placed for positioning the arm in a comfortable manner, with their palm facing down. During the task, the experimenter inserted in the box the holder containing the selected rubber stimuli, and guided the index fingertip of the participant’s dominant hand onto the two selected rubber samples, sequentially.The tactile telepresence experiment involved ten participants . Participants self-reported having no pathologies regarding the tactile sense, nor was their tactile sensitivity compromised by previous activities. The tactile stimulation was performed on the participants reported dominant hand (one left-handed and nine right-handed).The experiment was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee for non-clinical experimentation of Sant’Anna School of Advanced Studies Pisa and by the Research Ethics Committee of the School of Psychology, Trinity College Dublin. All participants gave written informed consent.During the telepresence experiment, each participant was invited to take a seat in a remote laboratory (Ireland) with respect to the one where the indentation platform was placed , and was invited to wear the vibrotactile glove on the dominant hand. Each participant was asked to wear a headset which provided white noise in order to mask the sound from the activation of the piezoelectric transducers.The active touch experimental protocol was carried out for benchmarking purposes to evaluate the physiological acuity in discriminating stiffness variations. It consisted of a 2-AFC tactile discrimination task A. In eacThe actual experimental protocol for evaluating the proposed spike-based cutaneous tactile feedback strategy consisted of a 2-AFC tactile discrimination task B. The paBefore an experimental session, each participant underwent a training session, in order to familiarize them with the stimulation apparatus and procedure. Each training session consisted of the presentation of six randomized trials, also provided in reverse order, for a total of 12 trials . The triThe whole experiment consisted in the presentation of 72 trials in blocks of 12, with a 5 min break between the third and fourth block in order to ensure concentration and avoid distress. In comparison with the active touch benchmarking protocol, the speed constraints of the mechatronic platform in the indentation of the stimuli, as well as in the transitions between the stimuli in a pair, enlarged the time required for the presentation of 12 trials, which took about 15 min. The duration of the total experiment was about 1 h and 30 min.binofit test) of the rates of identification of stimuli with increasing stiffness . A logistic fit of the resulting psychometric curves was computed for the presented stiffness variations using the Matlab nlinfit function, using a custom fitting cumulative distribution function. The significance of participants’ responses for each normalized stiffness variation  was computed using the Matlab binofit test.Data analysis was performed using the Statistics Toolbox in Matlab . For each stiffness variation, the success rate was evaluated across the population of participants, together with the 95% confidence interval , except for the first stimulus of the explored range of stiffness variation, for which the stiffness variation was small . In bothThe experimental data were well-fitted by the symmetric logistic cumulative distribution function (CDF) (5), suggested by Ulrich and Miller , over thx, where The fitting function denotes the probability of a correct response at difference nditions  correspoThe goal of the present work was to evaluate the capability of a haptic system, a vibrotactile glove, in delivering reliable information about rubber stiffness in a telepresence configuration using a neuronal spiking model to trigger the delivery of stimuli. We investigated this topic using customized piezoelectric transducers embedded in a textile glove for the simultaneous stimulation of index and thumb fingertips. In order to do so, we compared the results from the vibrotactile stimulation of the hand with the benchmark experimental data relative to the active exploration of the rubber stimuli performed with the index fingertip.We analyzed the reliability of a telepresence system in delivering, directly on the skin of a remote hand, haptic information about the stiffness of selected polymeric samples. The stimuli were indented by an automated system and their stiffness encoded according to a neuronal spiking model heuristically adapted from our previous implementations that emulate the firing activity of human mechanoreceptors ,41,43. TWe acknowledge the several differences between the two experimental conditions: the time taken by the task (20 min for the active task vs. 90 for the telepresence task), the active vs. passive approach, the auditory masking (absent in the active task), and the control of the force and indentation velocity for the telepresence vs. no control in the active task. Despite this, with our work we aimed at demonstrating the proof of principle of a telepresence task, where tactile feedback can be representative of object characteristics . Therefore, we preferred to design our experiment so that the proposed method was assessed in more challenging conditions than the natural benchmark. This study will be complemented with future research where participants will actively control a robotic arm in immersive telepresence, mimicking an active industrial task while receiving tactile information about the robot contact forces and displacement, whereas with typical virtual-reality-based human–machine interfaces the human operator can hardly recognize states when the robot has established contact with the objects in its environment.In order to deepen the explanation of the physical determinants of the responses given by the participants, we calculated the average inter-spike interval (ISI) for each rubber stimulus in the initial phase of the stimulation . In these situations, the information coming from one sensory channel can be conveyed to the tactile sense in a perceptible manner ,42,52,53Tactile information was proven useful also in industrial environments for human–robot co-working activities. The development of alerting haptic devices, in environments where the interaction with automated machinery can be dangerous for operators, may also improve safety and the adoption of collaborative robotics in common applications.Our tactile device can be classified within state of the art technologies where haptic feedback is used to provide sensory augmentation, with a particular focus on the remote perception of stiffness and potential applications in several fields, including robotic surgery. Our preliminary experimental protocol followed a simplified characterization approach for the rubber samples. Since the stiffness of the specimens was investigated via a constant indentation velocity of the probe, we did not consider variations in dynamic deformation behavior due to the viscoelastic characteristics of the rubber samples, which is an important aspect for the tactile characterization of a biological tissue. Towards the applicability in robotic surgery scenarios, this study will be complemented with future experiments involving different indentation velocities to investigate the effects of viscoelasticity in the perception of stiffness. According to the proposed spike-based encoding paradigm, tactile displays installed on robotic manipulators are expected to allow surgeons to feel in real time the stiffness of human tissues, while force information is collected by force sensors installed on the robotic end effectors.The tactile device herein presented is grounded on a novel neuromorphic discrimination mechanism for the recognition of the stiffness of rubber samples. Accordingly, a sequence of vibrotactile pulses are delivered on the skin surface, resembling the neural spikes produced by the mechanoreceptive endings underneath. Hence, the proposed strategy for encoding haptic data is non-homologous to physiological perception via the own fingers. The assumptions made in proposing such a strategy for the development of sensory substitution and augmentation devices were very strong. As a matter of fact, tactile devices are based on cognitive discrimination mechanisms  that are


Acinetobacter species such as Acinetobacter baumanii are of increasing concern in association with ventilator associated pneumonia (VAP). In the ICU, Acinetobacter infections are known to be subject to seasonal variation but the extent of geographic variation is unclear. The objective here is to define the extent and possible reasons for geographic variation for Acinetobacter associated VAP whether or not these isolates are reported as Acinetobacter baumanii.Acinetobacter incidence within the published literature was undertaken using random effects methods. This model incorporated group level factors such as proportion of trauma admissions, year of publication and reporting practices for Acinetobacter infection.A meta-regression model of VAP associated Acinetobacter species associated VAP incidence among seven world-wide regions. The highest incidence is amongst reports from the Middle East  versus that from North American ICU’s . There is a similar geographic related disparity in incidence among studies reporting specifically as Acinetobacter baumanii. The incidence in ICU’s with a majority of admission being for trauma is >2.5 times that of other ICU’s.The search identified 117 studies from seven worldwide regions over 29 years. There is significant variation in Acinetobacter associated VAP among reports from various geographic regions worldwide. This variation is not explainable by variations in rates of VAP overall, admissions for trauma, publication year or Acinetobacter reporting practices as group level variables.There is greater than fivefold variation in The online version of this article (doi:10.1186/s12879-016-1921-4) contains supplementary material, which is available to authorized users. Acinetobacter species are opportunistic gram negative bacteria which are of emerging concern in intensive care units worldwide [Acinetobacter species accounted for 7.9 % of bronchoscopically documented ventilator associated pneumonia in a series drawn predominantly from centres in Europe and The United States of America [Acinetobacter species varies in incidence worldwide for reasons that remain to be fully defined.orldwide –102. Aci America . In contAcinetobacter baumannii has the greatest clinical importance amongst Acinetobacter species as it is typically associated with outbreaks in the hospital setting and it has major antimicrobial resistance issues. There is evidence for [Acinetobacter baumannii infections in the ICU.ence for  and agaience for  an increAcinetobacter species blood stream infections versus other bacterial isolates such as Pseudomonas aeruginosa and appears to correspond with the higher outdoor air temperature [A. baumanii at the University of Maryland Medical Center over a seven year period [Any survey of the worldwide epidemiology of Acinetobacter associated with VAP would be challenging for several reasons. First is that Acinetobacter infections in ICU are subject to seasonal influences –108. Forperature . For exaA. baumanii infection has the potential for the simultaneous occurrence of endogenous outbreak and non-outbreak strains in an ICU together with, in tropical areas, natural disasters and military deployments, occasional community acquired infections [The second challenge is its potentially complex epidemiology. fections , 109–111Acinetobacter baumannii is reported as the most frequently isolated species (>90 percent of Acinetobacter species isolates) [However, the greatest difficulty is in the vagaries and variations in the reporting practices of Acinetobacter species. The differentiation among Acinetobacter species may be challenging for a busy clinical microbiology laboratory as the microbiological identification on the basis of phenotypic characteristics is difficult , 102. Assolates) , this prRegional variation in the worldwide incidence has previously been described . HoweverAcinetobacter. Because this analysis was based on a literature survey, institutional review board approval was not required.The literature search and analytic approach used here is as described previously . In brieAcinetobacter is the number of patients with VAP having an Acinetobacter species isolated from respiratory sampling. Where necessary, this number was derived as the number of patients with VAP multiplied by the proportion of VAP isolates that were Acinetobacter species. In addition, the following were also extracted where available; the number of ICU patients surveyed, the overall incidence of VAP per 1000 mechanically ventilated day (MVD), whether the mode of diagnosis of VAP required bronchoscopic sampling and whether the ICU was a trauma ICU (defined as more than 50 % of admissions for trauma). Also, whether the mode of reporting of Acinetobacter infection was as Acinetobacter baumanii versus other modes such as Acinetobacter species was used as an indicator variable.The VAP associated The assignment of countries to near neighbour groupings was solely determined in relation to geographic proximity without regard to political, economic or other considerations. For the purpose of generating a world map of Acinetobacter VAP incidence by country, summary rates by country were estimated were at least two study reports were available for that country.Acinetobacter was undertaken. The weight in this model is the inverse of the study variance. Because heterogeneity both within and between regions is to be expected, a random effects method was used for these estimates. The predictor variables in the regression model were the region from where the study originated, the mode of diagnosis of VAP, mode of reporting of Acinetobacter infection, trauma ICU and year of publication.A meta-regression model of VAP associated The search identified 117 studies contained in 100 publications ; Acinetobacter calcoaceticus (four studies); and Acinetobacter anitratus (two studies). There was no instance of any study reporting more than one Acinetobacter species type.The Acinetobacter associated with VAP was reported most commonly as Acinetobacter without speciation . Acinetobacter VAP infections were less commonly reported as follows; p = 0.36; Table Acinetobacter across the region categories  with the Acinetobacter species associated VAP incidence being highest amongst reports from ICU’s in the Middle East  versus reports from Northern Europe  and North American ICU’s   data and more recently within The Surveillance Network-USA database [Acinetobacter species infections in these surveys were ~50 % more common in summer than winter months. The variation seen here in this worldwide survey exceeds that explainable by seasonal variation. A possible mechanism to account for this seasonal and possibly geographic variation, and by contrast to species that do not exhibit the same variation, is that Acinetobacter and particularly A. baumanii have an exceptional ability to survive desiccation. It remains to speculate how this property of Acinetobacter could account for the variation found here. Of interest in this regard however, amongst a panel of Acinetobacter isolates, this ability to survive desiccation was notable for A. baumanii that had caused an outbreak of hospital acquired respiratory tract infections [Seasonal variation is another challenge to attempts at surveillance –108. Thedatabase . Acinetofections .The advantage of a literature survey is that published data is readily available and the meta-regression methods for analysing these types of data are established. In contrast to multi-country incidence studies, which tend to be a snap shot over typically less than six months, most of the studies here extended over more than twelve months. Here a random effects methods is used. By using this method, the precision associated with each individual study estimate is incorporated in the derivation of both the overall summary estimate and in the derivation of the meta-regression model. Moreover, in contrast to a fixed effects model, a random effect meta-regression model will generate more conservative summary estimates (i.e. wider 95 % confidence limits) as the method incorporates both within and between study variability. In this way, comparisons to address questions of study specific  and contAcinetobacter species associated VAP. For example, the usage of empiric antibiotic therapy in each study is an important unknown as use or non-use may account for vulnerability to Acinetobacter species associated VAP at the level of the individual patient [There are several limitations to this literature based study. This is an analysis at the group level and is unable to take account of patient specific risk factors for  patient , 120.The grouping of countries into near neighbour groupings is somewhat arbitrary. Country and even regional groupings could be confounded by other variables such as infection control practices, prevalence of antibiotic use and standards of care for patients receiving mechanical ventilation that have not been able to be considered in the analysis here. Another limitation and difficult to exclude bias is the possible influence of publication bias.The main limitation of a literature based survey is the lack of standardization across jurisdictions. It could be anticipated that there might be a range of clinical definitions used in the diagnosis of VAP at the level of the individual patient. That the mode of VAP diagnosis was not a significant factor in the regression model Table  implies Acinetobacter, being the number of patients with VAP having an Acinetobacter species isolated from respiratory sampling, was not available. For these reports this number was derived as the number of patients with VAP multiplied by the proportion of VAP isolates that were Acinetobacter species. This is likely a reasonable approximation for a relatively rare outcome as found here. This approximation allows for VAP patients with multiple isolates.An additional limitation is that for some reports, the VAP associated Acinetobacter baumanii from studies outside of Northern European and North American centers. It is possible that second line Acinetobacter species had been identified and listed within the category of ‘other’ gram negative infections. However, it remains a plausible explanation that the common practice in the literature reported here of the listing of a single Acinetobacter species generally reflects the mode of local reporting practices. In this regard and of pertinence to this survey of Acinetobacter baumanii, the reports of Acinetobacter species cannot be easily dismissed.A more difficult issue is that of laboratory documentation and reporting of Acinetobacter species type across jurisdictions. The striking observation among this survey was that all studies reported only one classification type of Acinetobacter. This was apparent in even the surveys with the most number of isolates , 27, 63.Acinetobacter baumanii showed similar patterns of regional variation despite similar rates of overall VAP infection. Moreover, this regional variation in rates of Acinetobacter VAP were not explainable in a meta-regression model by trauma ICU, year of publication and Acinetobacter reporting practice as group level variables whereas a comparable meta-regression model of VAP showed no major regional variation in overall VAP rates (Additional file Of particular note, the rates of Acinetobacter VAP reported from studies reporting as Acinetobacter species versus studies reporting as Acinetobacter calcoaceticus-A. baumanii complex was suggested at one point [The methods in use in clinical microbiology laboratories likely varied not only geographically but also likely temporally over the three decades encompassed in this survey –125. Becne point . These pne point –125. MorAcinetobacter in cooler seasons [Acinetobacter species are gram negative bacteria which have important additional international public health issue for two further reasons. The transportation of patients around the world create the potential for infection control failures [Acinetobacter species are commonly multi-drug resistant although rates of resistance vary from region to region [The lower prevalence of  seasons –108 is cfailures . Moreoveo region .Acinetobacter associated VAP among published reports from various geographic regions worldwide. This variation is not explainable by variations in rates of VAP overall, admissions for trauma, publication year or Acinetobacter reporting practices as group level variables.There is a greater than fivefold variation in


In the original publication of this article , the accThe correct details of the NCBI accession numbers can be found below:Availability of data and materialsThe genomic sequencing data and assembled and annotated genomes are deposited at NCBI under bioproject accession numbers PRJNA304627 (K. pastoris), PRJNA304977 (K. phaffii wildtype), and PRJNA304976 (K. phaffii GS115). RNA-seq data are deposited at NCBI under the bioproject accession numbers PRJNA311606.In addition to this, please find the direct links to the data below:Transcriptome study:http://www.ncbi.nlm.nih.gov/bioproject/PRJNA311606http://www.ncbi.nlm.nih.gov/sra?linkname=bioproject_sra_all&from_uid=311606Genome assemblies:http://www.ncbi.nlm.nih.gov/bioproject/PRJNA304627http://www.ncbi.nlm.nih.gov/bioproject/PRJNA304977http://www.ncbi.nlm.nih.gov/bioproject/PRJNA304976


Pseudo-nitzschia blooms often occur in coastal and open ocean environments, sometimes leading to the production of the neurotoxin domoic acid that can cause severe negative impacts to higher trophic levels. Increasing evidence suggests a close relationship between phytoplankton bloom and bacterial assemblages, however, the microbial composition and succession during a bloom process is unknown. Here, we investigate the bacterial assemblages before, during and after toxic and non-toxic Pseudo-nitzschia blooms to determine the patterns of bacterial succession in a natural bloom setting. Opportunistic sampling of bacterial community profiles were determined weekly at Santa Cruz Municipal Wharf by 454 pyrosequencing and analyzed together with domoic acid levels, phytoplankton community and biomass, nutrients and temperature. We asked if the bacterial communities are similar between bloom and non-bloom events and if domoic acid or the presence of toxic algal species acts as a driving force that can significantly structure phytoplankton-associated bacterial communities. We found that bacterial diversity generally increases when Pseudo-nitzschia numbers decline. Furthermore, bacterial diversity is higher when the low-DA producing P. fraudulenta dominates the algal bloom while bacterial diversity is lower when high-DA producing P. australis dominates the algal bloom, suggesting that the presence of algal toxin can structure bacterial community. We also found bloom-related succession patterns among associated bacterial groups; Gamma-proteobacteria, were dominant during low toxic P. fraudulenta blooms comprising mostly of Vibrio spp., which increased in relative abundance (6–65%) as the bloom progresses. On the other hand, Firmicutes bacteria comprising mostly of Planococcus spp. (12–86%) dominate during high toxic P. australis blooms, with the bacterial assemblage showing the same bloom-related successional patterns in three independent bloom events. Other environmental variables such as nitrate and phosphate and temperature appear to influence some low abundant bacterial groups as well. Our results suggest that phytoplankton-associated bacterial communities are strongly affected not just by phytoplankton bloom in general, but also by the type of algal species that dominates in the natural bloom. Phytoplankton plays an important role in global carbon cycling by consuming carbon dioxide from the atmosphere for photosynthesis and sequestering the fixed carbon as cells sink down into the deep ocean. Phytoplankton bloom, happening either in the coast, upwelling regions or open ocean, not only provides organic materials to the higher trophic food web but also provides photosynthate-released dissolved organic matter  and orgaMany bacteria associate with phytoplankton  and are Pseudo-nitzschia (Pn) and studied the phytoplankton-associated bacterial community from four independent natural bloom events. We asked what bacterial groups dominate in bloom events formed by different Pseudo-nitzschia species. We determined how bacterial composition changes before, during and after the decline of Pseudo-nitzschia bloom, as it was replaced or co-dominated by other phytoplankton species. Aside from phytoplankton biomass (as chlorophyll a), we also looked at environmental variables such as nutrients , and temperature to determine if these physical factors also play a role in bacterial succession. Our results indicated that bacterial composition and structure are strongly influenced by the Pseudo-nitzschia species in bloom and domoic acid can play a role in limiting bacterial diversity.In this study, we focus on the domoic acid producing a and fecal indicator bacteria are measured. Water condition in Monterey Bay is characterized by seasonal upwelling that brings low temperature, high salinity nutrient-rich water from approximately February to August, followed by an oceanic period characterized by upwelling relaxation in August to mid-November. In winter (mid November–mid February), the Davidson Current surfaces along the coast and brings in relatively warm, high salinity, nutrient-rich water  in Monterey Bay, California, one of the ocean observing sites in Central and Northern California (CeNCOOS). The site was established to monitor the presence, abundance and population dynamics of red tide forming species, where weekly monitoring of nutrients, environmental parameters, phytoplankton abundance, chlorophyll ch water . Upwellech water . Differe in 1991 .Pseudo-nitzschia cells per liter and are as follows: No bloom – 0 to 10000; Low Bloom – 10, 001 to 30,000; Medium bloom – 30,001 – 100, 000; High Bloom – 100001 to 300000; Highest Bloom- >300, 000. Closure of shellfish harvesting commonly occurs when DA-producing Pseudo-nitzschia bloom is seen at 100, 000 cells L1  using a 20 μm mesh with a cod end volume of 300 mL. A 2-L Niskin bottle was used to collect water samples at the same integrated depth and used for nutrients and chlorophyll analysis. Temperature was measured immediately after water collection. For this study, opportunistic sampling was carried out at different phases of the bloom, from the beginning and end of the bloom in November to December 2010  and peak and demise in March (Spring), July and August 2011 (Summer). Bloom phases were categorized based on total number of cells L1 .Pseudo-nitzschia cells by microscopy and counting of toxic Pseudo-nitzschia species using DNA fluorescent in-situ hybridization (FISH)  . Domoic n (FISH) . Nutrieng for 10 min. Algal pellets were finally resuspended in 500 μL of sterile TE buffer and stored at -80°C. For sequencing, raw samples were sent to Research and Testing Laboratory  for 454 pyrosequencing using the universal primers Yellow939F and Yellow1492R. Sample preparation for sequencing and sequencing methods are described in Phytoplankton tow samples were first pre-filtered with 300-μm mesh net (bleached and UV-exposed) to remove debris and zooplankton. Three of 30-mL tow samples were filtered with sterile 5 μm Durapore filters  and washed with sterile seawater to filter out unassociated bacteria. Algae were collected by resuspending filters in 10 ml of PCR water. Samples were then pelleted via centrifugation at 3000 × Pseudo-nitzschia were manually isolated from bloom samples with a microcapillary tip from a sterile glass Pasteur pipette. Prior to cell isolation, an aliquot of the pre-filtered tow sample was filtered with 100 μm nylon mesh (bleached and UV-sterilized) and washed two times with autoclaved-sterilized 0.2 μm filtered seawater (FSW) to remove unassociated bacteria. Algal suspension was diluted with FSW. Isolated cells were washed 10 times with FSW, inoculated in F/10-Se medium and incubated under growth conditions of 12:12 photoperiod, 15°C and 80 μE m-2 in an algal incubator. Successful isolates were transferred in F/2-SE medium for maintenance. The Pseudo-nitzschia cultures were identified by morphology using light microscopy and genotyped by sequencing the 18S rRNA gene.Single cells of Pseudo-nitzschia genomic DNA was extracted with PowerSoil DNA Isolation Kit . P. pungens and P. fraudelenta were amplified with primer pairs 1360F (5′-GCGTTGAT/ATACGTCCCTGCC- 3′) and ITS055R (5′-CTCCTTGGTCCGTGTTTCAAGACGGG-3′), while P. australis and P. multiseries was amplified with primer pairs 18S-F (5′-CTGCGGAAGGATCATTACCACA-3′) and ITS055R using EconoTaq DNA Polymerase  under the following PCR conditions: 94°C for 2 min, 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min and 72°C for 10 min extension. The products were sent to a service facility  for direct sequencing after labeling with the Big Dye Terminator with AmpliTaq FS Sequencing Kit  using the PCR primers as sequencing primers.E-value 10_20 (megablast only); minimum coverage 97%; minimum pairwise identity 90–97%]. Singletons and chloroplast sequences were filtered from the OTU table prior to bacterial diversity analyses. A phylogenetic tree of the OTUs was generated and viewed with Fast Tree 2.1.3 (Short sequence reads (length <150 bp), low quality sequences , SAMN05410842 (Tow_12.01.10), SAMN0541084 (Tow_12.08.10), SAMN05410844 (Tow_12.15.10), SAMN05410845 (Tow_12.22.10), SAMN05410846 (Tow_03.16.11), SAMN05410847 (Tow_03.23.11), SAMN05410848 (Tow_07.06.11), SAMN05410849 (Tow_07.13.11), SAMN05410850 (Tow_08.24.11), SAMN05410851 (Tow_08.31.11).t-test were carried out using the software JMP 12.0.Correlation tests using multivariate non-parametric Spearman rank’s correlation coefficient and Student’s Pseudo-nitzschia bloom. In mid-November 2010, phytoplankton diversity was high with the community being co-dominated by Pseudo-nitzschia, Prorocentrum and Ceratium . The assemblage changed into mostly Pseudo-nitzschia as the massive bloom progressed for 4 weeks. At the end of the bloom at week 5, there was an equal presence between Pseudo-nitzschia and dinoflagellates. Single algal cell isolations followed by genotyping indicated that P. fraudulenta dominated the Pseudo-nitzschia community and FISH probing and counting indicated that P. australis were also present in small number .Opportunistic sampling was done in Santa Cruz Municipal Wharf during the occurrence of P. australis occurred in mid-March, but this bloom did not last as the population of the dinoflagellate Ceratium co-dominated the following weeks. In July 2011, a small bloom of P. australis was seen together with Chaetoceros and Eucampia, but noticeably decreased the following weeks and was replaced by higher diversity of phytoplankton with the co-dominance of Guinardia, Chaetoceros and Eucampia. In August 2011, a massive P. australis bloom occurred that lasted for 3 weeks. The algal community shifted and co-dominated both by the dinoflagellates Protoperidinium and P. australis during the fourth week, but phytoplankton biomass has decreased. P. australis were the dominant Pseudo-nitzschia species in 2011 , with a small number of P. multiseries (3450 cells/ L) seen on March 23, 2011. Chlorophyll levels for the data gathered during opportunistic sampling showed that overall phytoplankton biomass range from 2.8 to 11.7 mg m-3, with the highest chlorophyll level observed in 2010 during the bloom dominated by P. fraudulenta, where positive correlation existed . Temperature range during late Fall 2010 sampling was between 11–14°C, while it was 12°C during Spring 2011 and 14–15°C in the Summer 2011. There was no correlation seen between temperature and chlorophyll level, or between temperature and total Pseudo-nitzschia cells, Pseudo-nitzschia species or DA. Additionally, a negative correlation of temperature with nitrate was seen .In 2011, an early spring bloom of Pseudo-nitzschia cell number and the presence of toxic Pseudo-nitzschia species . Interestingly, none of the nutrients  were correlated with cell abundance of Pseudo-nitzschia in a natural bloom and the five nutrients did not show any correlation with P. fraudulenta, P. australis, P. multiseries cell numbers (data not shown). Notably, DA level was positively correlated only with P. australis cell numbers  but not with total Pseudo-nitzschia number or with P. fraudulenta cell number, even though this low-toxin producing species was present in very high numbers in 2010 bloom . Likewise, temperature did not show any correlation with total Pseudo-nitzschia cell numbers, P. fraudulenta or P. australis cell numbers or with any of the aforementioned parameters, except for nitrate, to which it showed negative correlation .We looked at the correlation between nutrients and total Pseudo-nitzschia bloom events captured at different bloom phases . These sequences were assigned into 4732 OTUs (>97% ID), but reduced to 2621 OTUs after removing singletons and chloroplast sequences. The remaining OTUs were analyzed for bacterial diversity in every sample. Statistical analysis indicated that there was no significant correlation between phytoplankton biomass  and bacterial diversity  suggesting that total phytoplankton biomass did not influence bacterial diversity (Table 1).A total of 83,284 clean 16S rDNA sequences was obtained from 11 samples belonging to four Pseudo-nitzschia bloom. Although bacterial diversity was lower during Pseudo-nitzschia bloom , comparison of four bacterial indices  between Pseudo-nitzschia bloom and no-bloom suggested that bacterial diversity was not significantly different between the two events . However, the correlation analysis at the Pseudo-nitzschia species level indicated that the presence of high DA-producing P. australis negatively affected bacterial diversity (Table 1). Bacterial diversity was generally lower when the Pseudo-nitzschia bloom was dominated by P. australis versus P. fraudulenta . Because low DA-producing P. fraudulenta were only present in 2010, correlation analysis for P. fraudulenta number versus bacterial diversity was carried out on this small data set. We found that as P. fraudulenta increased in number, bacterial diversity also declined (Table 1). Also, closer inspection indicated that bacterial diversity was lower as DA accumulated, due to increasing number of P. fraudulenta and the combined presence of P. australis . Bacterial diversity also negatively correlated with temperature while it positively correlated with phosphate and nitrate concentrations (Table 1).We determined if bacterial diversity was specifically affected by the presence of Pseudo-nitzschia bloom and looked at other environmental variables that may facilitate their dominance. At least ten bacterial phyla (or class) were frequently found associating with phytoplankton during non-bloom events . However, during Pseudo-nitzschia bloom events, only a few bacterial phyla dominated the bacterial assemblages  and these bacterial assemblages changed depending on the dominant Pseudo-nitzschia species that comprised the bloom. In 2010, Gamma-proteobacteria (65–85%) were prevalent when a low-toxic species, P. fraudulenta, dominated the Pseudo-nitzschia populations  and decreased in relative abundance during P. australis bloom (0–38%). These bacterial groups were also correlated with nitrate concentrations . The Firmicutes phyla (20–95%) dominated in 2011 during high toxin blooms and were highly correlated with P. australis cell number . Bacteroidetes (0–32%) and Epsilonbacteria (0–19%) groups were mostly present in the background but were not significantly correlated with the environmental conditions studied; Bacteroidetes, however, had higher relative abundance during non-Pn bloom events (14–32%) than during Pn bloom . Similarly, Alpha-proteobacteria were also always present but their abundance was negatively correlated with the total number of Pseudo-nitzschia cells especially in the presence of high number of P. fraudulenta . On the contrary, Fusobacteria (1–2%) were only present in 2010 bloom and positively correlated with P. fraudulenta abundance . Bacterial groups that were present but in low relative abundance such as the Chloroflexi (0–3%), Acidobacteria (0–12%), and Betaproteobacteria (0–4%) groups were negatively correlated with temperature . Actinobacteria (0–6%), another low abundant bacterial group, was only present when there was low number of Pseudo-nitzschia cells in the water (in 2011) . The presence of the very low abundant group Verrucomicrobia (1–4%) and Planctomycetes (0–1%) were not correlated with any of the aforementioned environmental parameters.We asked which bacterial groups dominate before, during or at the decline phase of Vibrio spp. was the Gamma-proteobacteria that dominated the Pseudo-nitzschia bloom in 2010 . Vibrio increased in relative abundance (6–65%) as the bloom of Pseudo-nitzschia progressed into its peak, and positively correlated with the increase of P. fraudulenta cells, the abundant Pseudo-nitzschia species in 2010 bloom . Vibrio was also positively influenced by phytoplankton biomass (chl)  but it did not dominate in P. australis bloom in 2011. Planococcus spp. (12–93%), bacteria from the Firmicutes group, was positively correlated with the bloom of P. australis in 2011 , where it dominated in three independent P. australis blooms. Temperature might have also significantly influenced its dominance .At the genus level, Shewanella (1–27%), the third most abundant bacteria in the samples, is an Alpha-proteobacteria that was present in 2010 bloom and showed positive correlations with P. fraudulenta , together with nitrate and phosphate . Erythrobacter (0–8%), another Alpha-proteobacteria, was present only in 2011 blooms and was negatively correlated with nitrate and ammonium . The abundant Gamma-proteobacteria bacteria, Psychromonas (1–21%), was mostly seen in 2010 and its presence was not significantly influenced by any of the environmental variable studied other than P. fraudulenta cell number . Psychrobacter (0–9%), another Gamma-proteobacteria, was ubiquitous in many samples but was not specifically influenced by any of the environmental variables. The Lutimonas bacteria and other Flavobacteriaceae (0–10%) also did not show any correlations with any of the environmental variables. Rubricoccus (0–18%), another Bacteroidetes, showed positive correlation with temperature . The Epsilonbacteria, Arcobacter (0.4–19%), was abundant in 2010 blooms and was positively correlated with P. fraudulenta and nitrate .Pseudo-nitzschia blooms in the same biogeographical location sampled before, during and after bloom and looked at how harmful algal bloom species and phytotoxin, domoic acid, influence phytoplankton-associated bacterial communities. Our study showed that the composition of the bacterial communities is driven by the phytoplankton species that comprised the bloom, while some less-abundant bacterial genera are also driven by phytoplankton biomass, temperature and nutrients . Two species of Pseudo-nitzschia were found to dominate in the four bloom events, with the low-DA producing P. fraudulenta dominating in late Fall 2010 bloom , and the high-DA producing P. australis dominating in three blooms in the spring, mid-summer and late summer of 2011. The dominant bacterial group changed with the dominant algal species that comprised the algal bloom. Moreover, with each bloom phase shift (increasing or decreasing of Pseudo-nitzschia cell numbers), these dominant bacterial groups also followed the same trend with the algal shift, that is high P. fraudulenta or P. australis cell number also increased the abundance of the respective dominant bacterial group. In this study, our approach did not allow us to study the composition of the bacterial community associated to a specific algal species but rather from a community of phytoplankton species that was dominated by Pseudo-nitzschia during bloom events. The Gamma-proteobacteria Vibrio spp., for instance, increased in relative abundance as P. fraudulenta cells increased in number, while the Firmicutes bacteria, Planoccocus spp. decreased in abundance as P. australis decreased in abundance. Vibrio spp. are known to associate with phytoplankton and zooplankton  or dinoflagellates , the bacteria Planococcus spp. was also replaced or co-dominated by the abundance of other bacterial groups from Bacteroidetes, Gamma- and Alpha-proteobacteria. This suggests that specific bacteria and phytoplankton groups can influence each other and their abundance does not only depend on phytoplankton biomass abundance during bloom but that the relationship can also be dictated by the diversity of algal species in the phytoplankton community. a, Table 1), hence a closer look at the phytoplankton species composition during and after phytoplankton blooms is necessary if we want to understand the factors that facilitate the dominance of phytoplankton-associated bacterial groups and their role in the degradation of phytoplankton-derived organic matter.We also observed that when Pseudo-nitzschia cell number, nutrients  and temperature, but nitrate and temperature did show negative correlation. Hence, a longer time series is suggested to give sufficient evidence to confirm the direct influence of these physical factors on bacterial community composition and succession.Temperature, nitrate and phosphate seem to play a role in facilitating the abundance of some bacterial groups, but our data set is not enough to resolve if these abiotic factors can drive bacterial succession. Indeed, these physical factors affect the metabolism of phytoplankton and influences algal growth, which ultimately can influence phytoplankton-associated bacterial composition and diversity. In our study, we found no correlation between phytoplankton biomass, P. australis depressed bacterial diversity when it dominated the bloom in 2011. P. fraudulenta, a low-DA toxin producing Pseudo-nitzschia, also depressed bacterial diversity as DA increased when P. fraudulenta increased in cell number, but the bacteria diversity is not as low as that observed in P. australis bloom . Interestingly, the presence of P. australis , with P. fraudulenta in the December 15, 2010 bloom sample increased the DA level and drove the bacterial diversity further down . These field-based results agreed with our past laboratory study, where we found a higher bacterial diversity harbored by cultured P. fraudulenta strains and a lower bacterial diversity in cultured P. australis strains (Pseudo-nitzschia-associated bacterial communities. Domoic acid may act as a selective agent that can structure bacterial communities either by serving as a nutrient source for associated bacteria that can effectively assimilate DA or the toxin may act as deterrent to other associative bacteria. Previously, domoic acid has been hypothesized to be anti-bacterial (Our current study showed that the neurotoxin domoic acid tend to limit bacterial diversity or bacterial OTU richness. For instance, the abundance of high-DA producing  strains . This leacterial  but concRoseobacters and Gamma-proteobacteria to determine their genetic capability of degrading phytoplankton-derived material (Sulfitobacter and P. multiseries (Phaeobacter and Emiliania huxleyi (Pseudo-nitzschia and its microbiome (Current efforts are now focused on understanding which bacterial groups can degrade various types of phytoplankton-derived organic matter. The pervading principle is that bacterial diversity and composition is largely driven by the availability of organic material from phytoplankton biomass, and the bacteria that can degrade this copious amount of algal-derived organic material will get selected and proliferate in the marine environment. material . We can material . Associatiseries  or to co huxleyi , or hostcrobiome . To undeMSM and SJ conceived the project. MSM and RK executed the project, MSM and SM did the data analysis. MSM, SJ, and RK wrote the paper.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.


R2 = 0.83) and 59.6 ± 0.7% , respectively. Thereby, detectable total amount of starch decreased from 77.7 ± 0.8% linearly to 67.6 ± 1.7%, and sigmoidal, to 69.4 ± 0.4%, respectively. Increase in pressure caused a linear decrease in gelatinization enthalpy of 33.2 ± 5.6%, and linear increase in hydration properties by 11.0 ± 0.6%. The study revealed structural and technological relevant alterations of starch-based food matrices with low moisture content by HPT, which must be taken into consideration during processing and preservation of food.In recent years, high-pressure treatment (HPT) has become an established process concerning the preservation of food. However, studies dealing with the structural, and consequently functional modification of non-hydrated starchy matrices (moisture content ≤ 15%) by HPT are missing. To close this knowledge gap, pressure  and pressurization time depending  alterations of wheat flour were investigated. Pressure rise from 0 to 600 MPa or pressurization time rise from 0 to 20 min resulted in a decline of amylopectin content from 68.3 ± 2.0% to 59.7 ± 1.5% (linearly,  In recent years, consumer expectations on safety, sensory, and nutritive characteristics of processed food have increased, resulting in the evolution of novel processing technologies as high-pressure treatment (HPT). Thereby in the beverage industry, HPT diminishes the microbial load of products without significantly affecting quality parameters ,2. HowevAlthough in some food matrices, HPT should not cause alteration of covalent bonds and applied pressure should have minimal effects on processed food , researcTo predict the extent of high pressure-induced starch modification, the knowledge of influencing factors is indispensable. Kudta and Tomasik observed a pressure and time dependent structural modification of starch . TherebyTo date, investigation on the impact of HPT on starch were performed mainly in starch–water slurries . HoweverHigh pressure was applied to commercial wheat flour  provided by Rosenmühle GmBH . The moisture content of the flour was determined according to the approved method of the AACC 44-40 (2000). Moisture content was constant at 14.64 ± 0.27% before and after treatment. The protein content (10.55 ± 0.03% flours DM (dry matter)) was evaluated using Kjeldahl method AACC 46-12.01 with a conversion factor of 5.7. Ash content was 0.67 ± 0.02 g/100 g analyzed in accordance to AACC 08-12.HPT experiments were performed on a self-constructed high-pressure device at the Chair of Technical Microbiology at Technical University of Munich. Wheat flour was filled in elastic cryotubes, slightly compressed and hermetically sealed. The surrounding housing was filled with polyethylene–glycol–water to achieve a satisfying pressure transfer to the sample tubes. In two experimental test series , the impDetermination of glucose, maltose, maltotriose, as well as fructose and saccharose, was performed using HPAEC-ED . Flours were dissolved in a methanol–water (1:1) solution (1 g/flour/8 mL solvent) to inhibit enzymatic reactions. Afterwards, samples were filtered through 0.45 μm syringe filters and stored at −18 ± 1 °C before the analysis. Relative amylose content was determined with an amylose/amylopectin assay kit . Total starch content was analyzed according to the AACC Methode 76-13.01 using Total Starch Enzyme Kit from Megazyme International Ireland Ltd. . To purify starch and remove interfering dextrin of the sample, flour samples were purified in concentrated ethanol solution (80%). Impact of HPT on crystalline structures of starch was studied using dynamic scanning calorimetry (DSC)  equipped with a cooling system (Intracooler 2P). Flour samples were mixed with distilled water (flour/water = 1:3), filled in aluminum pans (20–35 mg) and hermetically sealed. After 2 min equilibration time at −40 °C, samples were heated from −40 °C to +95 °C, and held again for 2 min. The gelatinization enthalpy  describes the amount of energy which is necessary to melt crystalline parts of starch during heating. Particle size distribution prior and after HPT was analyzed by static light scattering using Mastersizer 3000 equipped with AeroS unit for dry measurements . “Mie Theory” was applied to calculate the particle size distribution using a refractive index of 1.59, the “general purpose mode”, and an obscuration ranging between 2 and 8% during measurements. All measurements were performed in triplicates.Starch modification degree (formerly known as starch damage) was determined according to the approved method (AACC method 76-31.01) using the Starch Damage Assay Kit . The starch modification degree (SMD) specifies the amount of starch which can be hydrolyzed at 40 °C by fungal α-amylase to low molecular weight dextrin, and is calculated using Formula (1):The solvent (water) retention capacity (WRC) of flours states the amount of water which can be bound by the sample after centrifugation at low rotational speed (AACC 56-11.02). The approved method had to be modified due to the low sample amount. The ratio flour/solvent remained constant, however, flour weight was reduced to 1.00 ± 0.05 g, and distilled water to 5.00 ± 0.05 g. ® Pro . The effects of HPT on starch structures and functionality were determined using Tukey’s pairwise comparisons with a confidence level of 95.0% with ANOVA.The statistical analysis was performed using JMPR2 = 0.834), and decrease of total starch from 77.7 ± 0.8% to 67.6 ± 1.7%  occurred. The simultaneous increase of percentage amylose content and decrease of total starch can be reduced to the destruction of the starch polymer amylopectin. It is well known from literature, that the degradation of the highly-branched polymer amylopectin (~108 Da in comparison to amylose ~106 Da) is favored when physical forces are applied to pure starch or flours [Starch mainly consists of the highly branched polymer amylopectin and the linear glucan amylose, which determine the functional properties of starch. The polymer constitution of starch in flour samples gives information of the origin of starch and provides fundamental insights into the extent of stress acting on starch during physical treatments. As shown in r flours ,22, explr flours . HoweverAlthough the determination of total starch content using a Megazyme assay kit presents a fast and reproductive method, it is questionable if an underestimation of the dextrin content after severe physical treatment of starchy matrices occurs. In the first step of this assay, interfering substances are removed by washing the sample in ethanol. The cleavage of starch leads to the formation of low molecular weight fragments showing a higher solubility than starch. Those dextrins are removed prior to the enzymatic digestion, and thus, not detected. A slight (significant) decrease of maltose content was further investigated, when pressure increased from 0 to 600 MPa (0.8 ± 0.1 to 0.6 ± 0.1 mg/g flour). To date, it remains unclear if the reduction of maltose content is due to a mechanically induced splitting of the disaccharide. An enzymatic hydrolyzation of starch was excluded, since enzymatic degradation of maltose would cause an increase in glucose content, which could not be determined so far (data not shown), and furthermore, temperature did not rise during the pressure treatment above (30 ± 1 °C). Although decrease in maltose content is negligible for practical application, it provides insights into the mechanism of high pressure on the molecular modification of starch. Surprisingly, decrease in maltose content could not be determined with an increase in pressurization time from 0 to 20 min at a pressure level of 450 MPa. Furthermore, the analysis of the impact of pressurization time on starch structures elucidates the time-dependent destruction of starch. Due to the natural logarithmic destructive behavior of starch with increase in pressurization time, during the first 5 min of the treatment, the total starch content decreased about 5.1%; within the following 15 min (min 5–20), starch content only decreased about 5.8%. Due to the huge impact of HPT, even in low moisture matrices on starch, pressure treatments of food systems should not be neglected. Since a favored destruction of amylopectin occurred during HPT, measurements of starch crystallinity are necessary to predict the modified behavior of HPT starches during non-isothermal processes.R2 = 0.851)  and X-ray method . These r= 0.851) . Thus, tR2 = 0.97). A dependency of SMD from the applied pressure was also revealed by Ahmed et al. on matrices containing 20–50% wheat flour [The starch modification degree (SMD) increased with pressure level (up to 600 MPa) by 37%  [Wheat flours, modified by grinding procedures, showed in previous studies a good correlation of SMD and the particle size distribution, so that authors assumed that rise in SMD was caused by the surface enlargement due to the particle size reduction . Howevert series . Low prey (>50%) , an incry (>50%) ,29. Howey (>50%) .Since particle size distribution (PSD) values are in contrast with the development of SMD under pressure treatments in non-hydrated matrices, where a continuous increase in SMD over the whole pressure range was measured, PSD is not, in general, an adequate analytical method for the prediction of the accessibility of particles for enzymatic digestion. Consequently, surface alterations of wheat flour particles occur, which is not related to the particle size reduction. Changes of starch–gluten agglomerates are known to occur by mechanical treatments, for example grinding, even in low moisture matrices, due to the removal of starch-covering proteins by friction forces. However, since during HPT, 3-dimensional and low friction forces are applied on starch–protein agglomerates, no removal of starch-covering proteins was expected, increasing the accessibility of starch. Therefore, possible explanations for the rise in SMD are microcracks and the deformation of starch granules, which enlarge the surface of starch, and consequently, enzymatic accessibility, without altering noticeably the particle size distribution.To summarize, high pressure-treated flours show comparable molecular , crystalline  and surface-related (enzymatic accessibility) alterations, which cannot be correlated to the modification of particle size. Thus, using static light scattering, HPT-caused alterations of wheat flours cannot be determined. For the application of pressure-treated starchy matrices in food industry, beside structural alterations, the functional properties of HPT flours are of special interest, too.R2 = 0.942) from 65.68 ± 0.43% to 73.77 ± 0.92%. This is in agreement with findings from authors dealing with alterations of flour hydration introduced by high-pressure treatments in starch suspensions. However, less effects of high-pressure on starch or flour are expected, as in comparison to HPT of starch suspension, since the hydration highly depends on the availability of free water [The water retention capacity (WRC) of flours describes the amount of water which can be bound by flours after centrifugation. For an increase in WRC, a high accessibility of flours to water is prerequisite. Existing studies in high-moisture matrices have already shown a dependency of pressure and the swelling index of wheat starch , and a hee water . A pressee water . Hence, High-pressure treatment of non-hydrated starch–protein particles causes structural modifications of starch, and consequently, functionality of the matrix. Thus, evaluation of high-pressure treatments of starchy food should not exclusively focus on the improvement of the microbiological stability of food products, but also consider functional alterations of the matrix by HPT. These findings are of special interest for the practical, industrial application. The reduction of microbiological stability, in combination with a better enzymatic accessibility of starch, facilitates long-term fermentation processes under controlled conditions. Since high-pressure treatments of starchy matrices, even under low moisture conditions, evoke modifications of starch, grinding processes with  compressive forces—as found in roller grinding processes—should be examined and adjusted precisely, to prevent undesired alterations. This knowledge can lead to a reconsideration of high-pressure treatments of food systems, and to a targeted selection of monitoring parameters during HPT.


Although the authors provided the journal with the underlying blots for all the figures, the journal and authors decided to retract the manuscript. The replacement blot for Figure 2(b) and the underlying blots for all figures are available in the Supplementary Materials. The authors stated that the representative loading control bands in Figures 1(c) and 2(b) were improperly assembled, leading to repetitions of bands, and that these errors do not affect the results, once several papers have demonstrated that exercise does not change both, ACC [Mediators of Inflammation has retracted the article titled “Treadmill Training Increases SIRT-1 and PGC-1 Manner” –9. Due toth, ACC , 11 and oth, ACC –14 prote


Vascular cognitive impairment/vascular dementia have been the subject of a largenumber of studies, due to their high prevalence and broad preventive andcompensatory therapeutic potential. The knowledge of the cerebral anatomycorrelated to the vascular territories of irrigation enables understanding ofclinical manifestations, as well as classification into the several types ofsyndromic presentations. The central cholinergic system exercises importantneuromodulatory functions on cerebral circuits related to cognitive andbehavioral integration, as well as on vasomotor control related to cerebralblood flow adjustments. The acquisition of data on the anatomy of thecholinergic pathways, including the localization of the nuclei of the basalprosencephalon and the routes of their projections, established an importantmilestone. The knowledge of the vascular distribution and of the trajectories ofthe cholinergic pathways allows identification of the strategic points where avascular lesion can cause interruption. The ensuing denervation leads tocholinergic hypofunction in the involved territories. This information provesimportant to better evaluate the sites of vascular lesions, emphasizing theirstrategic localizations in relation to the cholinergic pathways, and offeringmore robust foundations for treatment aiming at enhancing cholinergicactivity. Dementia & Neuropsychologia, a newscientific periodical.In the last two decades there has been a considerable increase in the Brazilianscientific production in many areas of knowledge, including the two closely relatedfields that constitute the core of It is important to present the other main reasons for launching this new journal inBrazil:There is no journal specifically dedicated to these areas in the country,especially for research on dementia;Versions to Brazilian-Portuguese or adaptations of internationally utilizedtests, scales or questionnaires and studies focusing on peculiarities of thelanguage problems of Portuguese-speaking patients may prove very difficultto publish in an international journal or in the national general journalsof neurology, psychology or psychiatry;When papers are not published in high level international journals, but inless well known international indexed journals, the paper often ends upvirtually unknown by the national community;Maybe these and other reasons have contributed to the low number of Brazilianstudies that progress from poster form to full papers;Last but not least, selected reviewers may improve the quality of themanuscripts, providing suggestions on how to adapt the scientific writing,statistical analyses and proper citation of the literature.Dementia & Neuropsychologia was approved as theofficial journal of the Cognitive Neurology and Ageing Department of the BrazilianAcademy of Neurology and is receiving support from the most important groups related tothe dementia and neuropsychological fields in Brazil, with an editorial board includingmembers from most regions of Brazil, Latin America and abroad, it probably will receivemanuscripts within the first year. In the near future, indexation may be achieved if westrictly follow a few rules. The journal has received support from Professor AntonioSpina-França Neto, editor-in-chief of Arquivos deNeuro-Psiquiatria, the most prestigious neurological journal in Brazil, andalso from Abel Laerte Parker and other expert librarians of the Pan-American HealthOrganization and from the SCIELO (Scientific Library On-Line), a very successfulBrazilian initiative designed for the on-line diffusion of Brazilian and Latin Americanscientific papers. If their guidance is followed,Dementia&Neuropsychologia will be on-line even before the end ofits first year. Then, the first published papers will be retrospectively included in theon-line library and will be freely available to both national and internationalcommunities.When this landmark is accomplished, the journal will be attractive for thespeed of on-line publication. The next challenge will be international indexation.Of course, it will be difficult for a not yet indexed journal to attract goodmanuscripts. As Papers will be published in English, but versions of tests, scales or special papers withfeatures peculiar to Brazilian or Latin American spheres may be published in Portugueseor Spanish.Dementia & Neuropsychologia is published quarterly and follows theUniform requirements for manuscripts submitted to biomedicaljournals. The first issues are including reviews, original reports, shortcommunications and case reports. In future issues, other sections will be included.In this first issue it is necessary to also thank the Brazilian scientific community, theinternational members of the editorial board, and the advertisers for making possible togive this first step in a long, but hopefully very significant journey.Ricardo NitriniEditor-in-Chief


Per/Arnt/Sim), GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) and PHY (phytochrome-specific) domains but with the C-terminal PAS repeat and transmitter-like module deleted] auto-assembled in vitro with 13C- and 15N-labeled phycocyanobilin (PCB) chromophore. Thereafter, quantum mechanics/molecular mechanics (QM/MM) enabled us to refine 3D structural models constrained by the NMR data. We provide definitive atomic assignments for all carbon and nitrogen atoms of the chromophore, showing the Pfr chromophore geometry to be periplanar ZZEssa with the D-ring in a β-facial disposition incompatible with many earlier notions regarding photoconversion yet supporting circular dichroism (CD) data. The Y268 side chain is shifted radically relative to published Pfr crystal structures in order to accommodate the β-facial ring D. Our findings support a photoconversion sequence beginning with Pr photoactivation via an anticlockwise D-ring Za→Ea photoflip followed by significant shifts at the coupling of ring A to the protein, a B-ring propionate partner swap from R317 to R287, changes in the C-ring propionate hydrogen-bonding network, breakage of the D272–R552 salt bridge accompanied by sheet-to-helix refolding of the tongue region stabilized by Y326–D272–S554 hydrogen bonding, and binding of the NTE to the hydrophobic side of ring A. We discuss phyA photoconversion, including the possible roles of mesoscopic phase transitions and protonation dynamics in the chromophore pocket. We also discuss possible associations between structural changes and translocation and signaling processes within the cell.We present structural information for oat phyA3 in the far-red-light-absorbing (Pfr) signaling state, to our knowledge the first three-dimensional (3D) information for a plant phytochrome as Pfr. Solid-state magic-angle spinning (MAS) NMR was used to detect interatomic contacts in the complete photosensory module [residues 1–595, including the NTE , PAS ( Although both represent singlet electronic ground states (S0), Pfr slowly reverts to Pr in darkness or can be photoconverted back to Pr by FR light. The situation is complicated significantly, however, by the existence of two functionally dominant phytochromes in plants, namely phyA and phyB, each with very different physiological properties and ecological roles, yet likely undergoing similar photochemical processes. Although plant phytochromes have been studied intensively for many years on account of their importance in natural ecosystems and agriculture, many fundamental aspects are still scarcely understood. As expected, the discovery of Cph1 phytochrome from the cyanobacterium Synechocystis 6803  which functions as a light-repressed HK  phyA, the material on which most of the pioneering phytochrome work was focused. Indeed, it was the first phytochrome to be purified intact  experiments allowed the detection of direct and indirect 13C–13C correlations of the PCB chromophore within its binding pocket  then allowed 1H intramolecular contacts between the chromophore carbons and the retained NH (HN21-N24) and OH protons to be established, the spectrum with a cut-off distance of ∼2.8 Å  revealing four clear pyrrolic 1H resonances. The NH proton resonating at 9.0 ppm was assigned to HN24 via its contacts with C19 (169.5 ppm), C16 (150.3 ppm), and C18 (140.7 ppm) at ring D. These carbons showed no contacts to other NH protons even with a ∼3.5 Å cut-off distance . The integrated intensity ratio of the HN21 contacts to C1 and C4 is ∼45:55, implying that the two A-ring carbons are roughly equidistant from this proton  were seen: -0.3, +0.6, -0.4 and +0.8 ppm as Pfr minus Pr for HN21 to HN24, respectively. The lack of correlations involving OH protons of the propionic side chains of rings B and C in the two MELODI–HETCOR spectra implies that both are deprotonated, as in earlier studies  allowed us to assign the nitrogens themselves straightforwardly, with N21–N24 located at 159.0, 140.7, 157.3, and 136.9 ppm, respectively .From the sonances , in whicsonances  were intδC and δN changes (Δδ) of the chromophore associated with photoconversion . Whereas in the A–B ring region most carbon atoms show only small changes upon photoconversion, larger ΔδC values are seen for the A-ring linkage to the protein at C3, C31, and C32. State-related δC changes in the conjugated π-system of the chromophore are also apparent .The A-ring region, the C-ring propionate and several protein residues nearby showing at least two conformations associated with different hydrogen bonding networks  is asymmetrical and significantly broader than the other three, deconvolution revealing components at 140.7 and 142.1 ppm with an intensity ratio ∼0.7:0.3 , perhaps implying heterogeneity at this point. Moreover, the 15N line-shapes become more Gaussian and less Lorentzian at -40°C relative to those obtained from the spectra recorded at temperatures just below the freezing point of the sample solution , indicating an increased distribution of chemical shifts at lower temperatures. On the other hand, Pfr heterogeneity was apparent in neither the DARR spectra  nor interfacial 1H contacts of the Pfr chromophore (see below). Indeed, the line-widths of the 13C and 15N signals of the chromophore are distinctly narrower in Pfr than in Pr . For instance, the side chain of conserved H323  showed contacts to the pyrrolic carbons of rings B and C as well as their propionates in Pr, the corresponding 1H shifts nicely distinguishing cationic and neutral τ tautomers of the imidazole in the two Pr isoforms , consequently with ring D on the α-face (D-αf) relative to rings B and C  allowed most of its 1H contacts to be assigned straightforwardly  and none within ∼6.0 Å of its immediate protein environment, indicating that the pocket is much more rigid in Pfr. The NTE too is likely to be less mobile in Pfr, as a single 1H contact with C2 of ring A was seen in Pr, yet 15 SIDY contacts (mainly with the ethylidene and methyl substituents of ring A and C5) were apparent in Pfr . R548, another tongue residue seen by SIDY only in Pfr, showed two contacts to ring A at C2 and C21  and Y326 (Hη). The B-ring propionate likely forms a salt bridge with R317 in Pr, but the ΔδH values of ∼1.5 ppm associated with photoconversion and the contacts between the B-ring propionate and R287 seen only in Pfr indicate a light-driven partner swap associated with a shift of the two inner rings, also implied by changes in 1H contacts to I273 and P274  are radically different in Pr and Pfr  for this tyrosine in Pr and a g-,t rotamer  in Pfr , associated with a reduction of ∼1.5 Å in the -dependent C𝜀2–Nα internuclear distance. Similarly, large changes were observed for Y268: in Pr its phenolic ring is close to C171, C181 and C182 of the ring D, whereas in Pfr only a single 1H contact to C18 was apparent . Further prominent δH changes associated with photoconversion were detected for the imidazole rings of H372 and the neighboring H323  in both Pr and Pfr .All the chromophore carbon and nitrogen atoms in both states are now assigned definitively (troscopy . In that1H contacts (1HN21-N24) of the A-ring carbons are unaffected by photoconversion  in plant phytochromes. Our Pr model for oat phyA3 predicts 57°, whereas 46° was predicted for Arabidopsis phyA  give clear evidence that a protonated cationic bilin ring system is present in both Pr and Pfr, paralleling conclusions from earlier work on prokaryotic phytochromes  reflect changes in the pocket, in particular for the highly conserved D272 and H323 as well as the pyrrole water, all of which are within hydrogen-bonding distance . Indeed, the δN values calculated quantum-mechanically for the chromophore nitrogens in the Pfr state agree well with those measured experimentally  and the pyrrole water  fragment . The Pfr peaks are also narrower than those in Pr , consistent with reduced mobility. The state-dependent changes in dynamics of the NTE based on HDX-MS data for Arabidopsis phyB  NTE seems to be influenced critically by subtle interactions with the tip of the tongue-like hairpin extending from the PHY domain. For example, the PAS–GAF and PHY domains in wild-type (2VEA) and Y263F mutant (3ZQ5) crystal structures of the Cph1 Pr sensory module  are almosis phyB  showed t551-555 tongue motif is also seen to contact ring A in the SIDY spectrum, commensurate with a salt bridge to D272 . In the corresponding Pfr spectra, however, only R548, M549 and S554 were detected . The S554 hydroxyl group probably hydrogen bonds to D272 and via a strategically-placed water to rings A–C in Pfr , replacing the D272–R552 salt bridge which connects the chromophore to the tongue in Pr. These findings accord with the likely changes associated with photoconversion derived from Pr and Pfr structures of Cph1 and bacteriophytochromes. A particular R residue in the tongue specific to the ‘bathy’-subgroup of bacteriophytochromes has been suggested to be an important factor in promoting Pfr formation in darkness , as has been proposed for prokaryotic phytochromes . Although tongue refolding and associated shifts of the PHY domains in the dimer offer an attractive model for downstream intramolecular signaling at least in prokaryotic phytochromes, we note that the Deinococcus BphP (DrBphP) Pfr crystal structure was an inappropriate model for the small-angle X-ray scattering data  transcription factors (see below). surface . Althougfragment  is ambigδH changes upon photoconversion were also seen for H372 and the neighboring H323. In the case of H372 this is due to the hydrogen-bonding partner-swap of its imidazole ring (N𝜀2) with the chromophore during the photoflip. Specifically, H372 hydrogen-bonds to the D-ring carbonyl group in Pr, but to the C-ring propionate carboxylate group in Pfr . C123 interacts in Pfr with H372 at H𝜀2, and accordingly, the model predicts a C123-O…H372-N𝜀2 distance of 3.2 Å, consistent with a hydrogen bond. The formation of such a bond in Pfr is facilitated by the positional shift of the inner rings and the ring rotameric flip of H372 .Prominent δH changes associated with the imidazole ring of H323, particularly the prominent ΔδH of -1.2 ppm seen at H𝜀2 . As in other phytochromes, this histidine is strategically placed above the A–C ring nitrogens, an intricate web of hydrogen bonds connecting them to the histidine via the pyrrole water and thence to the C-ring propionate carboxylate group. Conversely, in Pfr, H323 is bridged via another water molecule to the C-ring propionate and further to H372 . In this way, the bilin chromophore protonates itself rather than the wall of the binding pocket providing an appropriately acidic side chain. Moreover, in H260Q of Cph1 pH values above 7.5 lead to chromophore deprotonation in both states  phenotype, implying that they mimic Pfr in darkness . Applying QM/MM calculations to the two different starting points consistent with the NMR data reported here for Pfr, QM/MM calculations yielded similar optimized structures in which the Y268 ring is roughly perpendicular to the chromophore plain, as in Pr . Given that the known Pr structures of bacteriophytochromes are quite similar to those of Cph1 and Arabidopsis phyB in this region, in Pfr the radically different positioning of Y268 in phyA relative to that in bacteriophytochromes is remarkable – especially as the photochromicity seems to be similar too. However, the CD data implying that the chirality of the chromophore inverts upon photoconversion in Cph1 and plant phytochromes but not bacteriophytochromes is unambiguous  and chromophore C15 and C16, indicating proximity to rings C and D , QM/MM robustly exclude hydrogen bonding. Nevertheless, this tyrosine is perfectly conserved, so what important role does it play if not in hydrogen bonding to N24 in Pfr? In accordance with our data and QM/MM calculations, we propose that it is involved in hydrogen bonding to S554 of the tongue , thereby stabilizing its Pfr-specific helix, the formation of the hydrogen bond being supported by a +1.7 ppm ΔδH of the Y326-Oη group . The observed 13C and 15N line-narrowing in Pfr results from greater structural homogeneity and reduced mobility of the surrounding protein relative to Pr in which the chromophore is loosely embedded in a soft pocket. Resonance Raman spectra of Xanthomonas BphP also imply increased dynamics in the Pr chromophore , sharp absorption peak at ∼660 nm corresponding to a cationic bilin chromophore with periplanar ZZZssa geometry. The proton probably originates from the C-ring propionic carboxyl itself and is transferred via H323 and the pyrrole water above the A–B–C-ring nitrogen triad. It seems that Pr comprises at least two sub-states, Pr-I and Pr-II . Instead, it seems to be stabilized by van der Waals interactions with Y268.The photoflip necessarily breaks the Pr-stabilizing hydrogen bond between the ZZEssa ground-state photoproduct, Lumi-R, is followed by spectroscopic Meta-R intermediates, the last of which prior to Pfr formation shows weak absorbance probably as a result of chromophore deprotonation accompanied by proton loss and re-uptake to/from the medium  is hypsochromically shifted relative to ZZZssa (as in Pr) under denaturing conditions , constraining the 15Ea photoproduct and perhaps providing an explanation for the enigmatic red shift of the Pfr state.The initial e medium , the caunditions . We acknD relative to the protein pocket  in association with the movement, thereby perhaps inducing a small shift at the PAS–GAF domain interface and thereby Pfr-dependent binding to the PIF transcription factors : accordingly, in Arabidopsis phyB, the S554-homologous mutant S584A shows extremely rapid Pfr→Pr dark reversion  and implied by the highly intense 814-cm-1 hydrogen-out-of-plane mode observed in Raman spectroscopy; Fundamental questions arise regarding the actual mechanism of photoconversion. The energy of a red photon is about 100-fold larger than necessary to break a hydrogen bond, but why should the ring somerize . Superfidarkness ; thirdlydarkness , 2013 wodarkness . The ‘coFigures 5, 6). Phosphorylation of the N-terminal S/T residues in phyA is associated with reduced sensitivity to light , and the refolding of the tongue region . Like Cph1 but in contrast to bacteriophytochromes, ring D is β-facial in Pfr. The far more numerous atomic contacts between the ring A and the NTE region in Pfr imply that the latter interacts more extensively with the protein in Pfr than Pr . The different mobilities of the Pr and Pfr states might represent processes fundamental to photoconversion and intramolecular signal transduction in phytochromes. Thus, although complete 3D protein structures cannot yet be derived from MAS NMR, the interfacial 1Hresidue–13CPCB contacts revealed  of Avena sativa (oat) phytochrome A3 expressed in yeast and assembled with u-13C,15N-PCB chromophore] used in our earlier MAS NMR study  phyA3 PCB chromophore in the Pfr state was constructed in four steps: A preliminary model based on the oat phyA3 sequence and published crystal structures was produced using MODELLER and Chimera programs, then improved by crude QM/MM geometry optimization of the chromophore-binding pocket using a small QM region. That model was used for preliminary assignments and refined on the basis of NMR-determined chromophore interfacial 1H contacts in an iterative fashion. Finally, the refined model was subjected to QM/MM geometry optimization of the chromophore-binding pocket using a 198-atom QM region  of holophytochrome (4:1 Pfr/Pr ratio) was loaded into a 4 mm zirconia MAS rotor and frozen in liquid N2. In the magnet, the sample was kept at -40°C. The MAS rate of 2D 13C–13C DARR, 1H–15N HETCOR , and 1D 13C /MAS  experiments was 13000 Hz. Typical 1H π/2 and 13C π pulses were 3.1 and 5.2 μs, respectively. The 1H power followed a tangent amplitude ramp (100–80%) during 1H–X (X = 13C or 15N) CP. The 1H–13C MELODI–HETCOR  operating at Larmor frequencies of 750.13 MHz for Arabidopsis phyA with the native PΦB chromophore (1H–13C heteronuclear correlation spectra using the PCB model corresponding to the u--PCB chromophore investigated experimentally (Supplementary Figures In our earlier paper  the Pr somophore . Here weCS, WG, JM, and JH conceived the experiments. CS, MM, WG, and JM designed the experiments. CS, MM, CL, and JK performed the experiments. CS, MM, WG, JM, and JH analyzed the data. CS and JH wrote the manuscript with extensive input from MM, WG, and JM. All the authors read and corrected 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.


International Brazilian Journal of Urology with quality international submissions to the video section from across the World. It highlights the commitment by the urology community in continually striving to improve the outcomes of surgical patients. We should never accept that good is good enough particularly with the plethora of surgical tools at our disposal. In this regard, it gives me a distinct pleasure to introduce the three best videos for 2017. These selections were made based on their novelty and potential to favorably impact the surgical care of urological patients. Making these selections is always challenging, being that all accepted videos are already of an incredibly high quality; hence, I would like to congratulate all authors for accepted videos over the past year and look forward to receiving your future submissions in the months to years to come. The selection of best videos of the year are as follows:This year has been another memorable year for the The third prize goes to the video entitled “Laparoscopic partial nephrectomy for multiple (four) tumors” by Lessandro Curcio et al. from the Hospital Federal Ipanema in Brazil, published in the May-June issue (43(3): 567)  of our JThe second prize is awarded to the video entitled “Feasibility of robot-assisted segmental ureterectomy and ureteroureterostomy in a patient with high medical comorbidity” by Raheem Ali Abdel et al. from centers of excellence in Egypt and South Korea (published in the Jul-Aug issue (43(4): 779-80) . This suThe first and top prize is awarded to Peter Caputo et al. from the Cleveland Clinic in Ohio in their video entitled “Robotic assisted laparoscopic augmentation ileocystoplasty” which was published in the Sept-Oct issue of our Journal (43(5): 994) . This viInternational Brazilian Journal of Urology, ultimately insuring its growing inter-national success and regard among the superior peer reviewed urological journals. On behalf of our entire editorial team, we hope each of you and your family’s had happy holidays and the very best wishes for the upcoming year.Finally, I would like to thank each one of you for your continual support of the video section of the