The iPhone X application delivers specific sound parameters and detects blinking responses to acoustic stimulus (in millisecond resolution) to study the responses of the blinking reflex to acoustic sounds in standing and sitting positions by using multiple acoustic test sets of different frequencies and amplitudes introduced as acute sound stimuli (<0.5 s). The single acoustic battery of 15 pure-square wave sounds consisted of frequencies and amplitudes between 500, 1000, 2000, 3000, and 4000 Hz scales using 65, 90, and 105 dB . (4) Results: Results show that there was a synchronization of amplitude and velocity between both eyes to all acoustic startles. Significant differences (p = 0.01) in blinking reaction time between sitting vs. standing at the high intensity (105 dB) 500 Hz acoustic test set was discovered. Interestingly, a highly significant difference (p < 0.001) in response times between test sets 500 Hz_105 dB and 4000 Hz_105 dB was identified. (5) Conclusions: To our knowledge, this is the first mobile phone-based acoustic battery used to detect and report significant ASR responses to specific frequencies and amplitudes of sound stimulus with corresponding sitting and standing conditions. The results from this experiment indicate the potential significance of using the specific frequency, amplitude, and postural conditions (as never before identified) which can open new horizons for ASR to be used for diagnosis and monitoring in numerous clinical and remote or isolated conditions.(1) Background: Acute acoustic (sound) stimulus prompts a state of defensive motivation in which unconscious muscle responses are markedly enhanced in humans. The orbicularis oculi (OO) of the eye is an easily accessed muscle common for acoustic startle reaction/response/reflex (ASR) investigations and is the muscle of interest in this study. Although the ASR can provide insights about numerous clinical conditions, existing methodologies  limit the usability of the method in real clinical conditions. (2) Objective: With EMG-free muscle recording in mind, our primary aim was to identify and investigate potential correlations in the responses of individual and cooperative OO muscles to various acoustic stimuli using a mobile and wire-free system. Our secondary aim was to investigate potential altered responses to high and also relatively low intensity acoustics at different frequencies in both sitting and standing positions through the use of biaural sound induction and video diagnostic techniques and software. (3) Methods: This study used a mobile-phone acoustic startle response monitoring system application to collect blink amplitude and velocity data on healthy males, aged 18–28 community cohorts during ( Dysfunctional mental health affects nearly 300 million people globally with the World Health Organization defining mood and cognitive disorders as the largest contributors to human disability . The burThe current means to examine mental health disorders are not as easily identifiable as symptoms, for example, as physical asymmetry in stroke, but usually rely on the blend of patient history, mental, and physical status examination, and laboratory and/or neuroimaging methods to detect impairments ,4,5,6. Hgold standard towards psychiatric diagnosis as well as what is the laboratory or neuroimaging test’s expected utility, or the difference between benefit and cost [While combining structured interviews with patient records, and laboratory and imaging review appears to produce more accurate primary and secondary diagnoses than routine clinical methods, there is still significant controversy as to what is considered the and cost .The two major diagnostic manuals for mood disorders: The Diagnostic and Statistical Manual of Mental Disorders Fifth Edition (DSM-V) and the International Classification of Diseases provide classification systems for clinical identification which encourage self-reporting and questionnaire-literary responses ,10. HoweThe ASR is an aversive response which is enhanced during a fear state and is diminished in a pleasant emotional context . The neuWith EMG-free muscle recording in mind, our primary aim was to identify and investigate potential correlations in the responses of individual and cooperative OO muscles to various acoustic stimuli using a mobile and wire-free system. Our secondary aim was to investigate potential altered responses to high and also relatively low intensity acoustics at different frequencies in both sitting and standing positions through the use of biaural sound induction and video diagnostic techniques and software. Sitting and standing methods provide flexibility for the use of devices for people with disabilities. These aims may then clarify the use of the ASR to researchers, medical care providers, and scientists in using sounds and postures to differentiate populations, and/or subpopulate groups into distinct neurophysiologies. We hypothesize that we may find significant details of sound amplitudes and frequencies for use in future experimentation.The ASR has been investigated to a high degree using a number of tools . HoweverThe outcomes of the present study may provide a detailed profile of the startle reflex which has various clinical and therapeutic significances.All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Human Ethics Committee of the University of Otago . ASR investigations were carried out in a testing environment <65 dB under interior-overhead lighting, and the environment remained at a stable temperature (21 °C).n = 15) or standing (n = 15) positions.Thirty male subjects (Otago University volunteers) between the ages of 18 and 28  3.7) were recruited and required ~15 min of ASR collection. With each participant, the subject was briefed of the proceedings, signed the consent form, and was alternated to either sitting  medical history of neurological disease, and (b), having active stimulants and/or depressants in their system during testing time.Due to the novelty of this method and testing equipment, we had to design and cooperate individual equipment and software in order to deliver pure and repeatable sound sets (variable in frequency and amplitude) as well as collect the blinking responses of the participants. To perform these tasks, the iPhone X and iPhone X insert earphones  were used as the hardware platforms to both deliver the acoustic battery and collect the blinking amplitude (magnitude of the blink) and response time of each blinking reflex.https://www.nch.com.au/tonegen/index.html) into the 2018 iPhone X operating system (iOS11.4.1) and collect the ASR blinking data. As acoustic outputs may differ between different phones, operation platforms, speakers, and sound files, we utilized iPhone X insert earphones to limit possible cross compatibility limitations.The Sound Stimulus App was created  to integrate the acoustic test sets from In order to collect blinking data, the Sound Stimulus App identified the eyes of an individual and geometrically designated points along the eyelids to measure the movements between geometric anchors (P1–6) against others across time. This method enabled the ability to draw parameters describing the blink completeness as well as the reaction time of both eyes to each test set. The app uses a data computing Dlib library and the included default face landmarking model file . Dlib isThe idea and formula were based on the work by Soukupová and Čech (2016), who developed a real-time algorithm to detect eye blinks in a video sequence from a standard camera . We adapThis algorithm calculates the distances between vertical and horizontal eye feature points  and computes the aspect ratio of acquired distances. The aspect ratio is approximately constant while the eye is opened and rapidly falls when the eye blinks. This change in EAR during a blink is used as the blink response amplitude.The use of this novel mobile ASR monitoring system enabled the introduction of a sound stimulus battery  of 15 puTo measure the dB output of our mobile ASR monitoring system, 500, 1000, 2000, 3000, and 4000 Hz sounds were selected at 100% (Sound Stimulus App specific) volume for each test set delivery. The side buttons on the iPhone X controls the volume of the speaker  which we had to manually select for each test set to deliver either 65, 90, or 105 dB.We used a Digitech professional sound level meter (SLM) and a sound level calibrator  to monitor the loudness of the testing environment as well as authenticate the dB output from the insert earphones. To accomplish this, the SLM was configured to record the highest dB output, set to “C” weighting (for checking the low-frequency content the sound), and set to “Fast” for normal measurements (fast varying noise) sound recording prior to each use. We obtained laboratory acoustic background noise using the SLM throughout a workday and measured the highest dB readings in the laboratory with the door closed to be <65 dB with the SLM placed 1 m from the door 1 m high.For both postures, the mobile device was placed at eye level on an adjustable tripod ~30 cm away from the volunteer’s face for optimum ASR measurements .During this time, the app was set to record maximum frames per second (120 fps) for increased data collection. The participant inserted the earphones and placed over the ears further insulating (–28 dB) noise reduction cups  prior to the stimulus delivery.The variables: Hz, dB, delay, and volume scale for each test were manually inputted for each delivery. Following input settings and initiation of the application, the camera begins recording. After establishing these stimulus parameters, the volunteer was instructed to remain still, and look at eye level with the iPhone X until the stimulus delivery and recordings were concluded.The sound stimulus battery encompasses a set specific order of acoustic signals which were initially randomized to determine delay and sequence . Each oft-tests and a repeated measures one-way ANOVA for the responses for both left/right eyes and sitting/standing with Bonferroni adjusted and Tukey post-hoc test were conducted.To determine the significance of these data, each recording was transferred from the Sound Stimulus App to a laptop computer  and underwent statistical analysis and cross platform data interpretation using MATLAB , and Microsoft Office 365 computing . SPSS  was utilized for data management as well. Pairwise comparisons using two tailed The use of our mobile acoustic-startle reflex monitoring system (MARS) allowed the collection of right and left eye responses for the acoustic battery . The actp < 0.05) between these two postures post 500 Hz_105 dB stimuli (p = 0.01) between sitting and standing only with a stimulus of 1000 Hz_65 dB (Blink reaction times (RT) between both eyes showed no significant differences across the ASR sound stimulus battery. Whereas quickest blink reflexes for the standing and sitting postures occurred after 4000 Hz_105 dB and 4000 Hz_90 dB respectively . The slowest RT between postures were 500 Hz_105 dB for standing , and 4000 Hz_65 dB for sitting . A comparison between sitting and standing blink reaction time identified a significant difference ( stimuli . AdditioHz_65 dB .p = 0.018); 4000_65 dB–4000 Hz_105 dB, (p = 0.002) and a highly significant difference (p = 0.000022) between test sets 500 Hz_105 dB and 4000 Hz_105 dB with the faster response at 4000 Hz_105 dB  between frequencies when analyzing the specific acoustic intensities of 65, 90 and 105 dB. At 65 dB: 500 Hz–1000 Hz and 1000 Hz–4000 Hz. At 90 dB: 500 Hz–4000 Hz and 1000 Hz–4000 Hz. At 105 dB: 500 Hz–2000 Hz, 500 Hz–3000 Hz, 500 Hz–4000 Hz, 1000 Hz–4000 Hz . These results were unexpected but may explain a sound set specific postural connectome in the ASR network similar to that described by [In the standing and sitting analysis of the data, the 500 Hz_105 dB cued a faster blink reflex in the sitting posture yet the 1000 Hz_65 dB sound-set cued a stronger response for amplitude for standing , 4000 Hz_65 dB–4000 Hz_105 dB (p = 0.02) and 500 Hz_105 dB-4000 Hz_105 dB (p = 0.000022). These unique sound sets may shift attitudes towards testing toward the higher and lower frequency (±4000 and 500 Hz) with different decibel ranges for future investigations, contrary to previous ASR literature using 1000 and 2000 Hz frequencies for induction. The differences in reactions to these sounds may be significant in terms of cueing the unconscious regions of reactive “survival” limbic, reticular, and autonomic systems for investigating neuronal operating bands in both healthy and disordered (hypo/hyperactive) states. Additionally, these unique sounds may establish further population-specific common ranges of activity such as those identified in children with autism spectrum disorders where hyperreactivity to weak acoustic stimuli and prolonged acoustic startle latency were found in a specific age and condition (autism) cohort [Between both postures, pairwise comparisons for the different frequencies concluded significant differences between the following test sets: 500 Hz_65 dB–500 Hz_105 dB  cohort . Using v) cohort ,44,45,46) cohort ,44, or m) cohort ,48.p < 0.05) at 65 dB: 500 Hz–1000 Hz and 1000 Hz–4000 Hz. At 90 dB: 500 Hz–4000 Hz and 1000 Hz–4000 Hz. At 105 dB: 500 Hz–2000 Hz, 500 Hz–3000 Hz, 500 Hz–4000 Hz, and 1000 Hz–4000 Hz for our sound sets. These correlations are not yet understood but may represent connectome-specific tonotopy thresholds or transitions between specific frequency groups of stereocilia or ASR processes of deeper brain centers [We found additional significant differences , using unique sound sets where sitting or standing postures are altered or not always possible. The ability to remotely and conveniently monitor for ASR-spectrum deviations and establish individual normative ASR reaction time and intensity ambits may also amplify performance training/conditioning schemes or establish neurosystem measurement parameters for elite programs . The means of wire-free monitoring the state of the nervous system using long established and rugged hardware (smart phone with protective case) opens horizons to remote monitoring in extreme environments.The results from this investigation indicate the potential significance of using specific frequency, amplitude, and postural conditions in ASR studies in addition to diversifying the remote monitoring capabilities of biometric devices from smart devices (iPhone). Utilizing sound parameters with posture related responses while monitoring the acoustic startle reflex with a mobile phone may open new horizons in ASR monitoring across a multitude of populations in order to identify biometric parameters of healthy responses.This was a pilot study, hence the sample size (N) was relatively small; nevertheless, our investigations were able to show usability and effectiveness in determining differences in responses to acoustic battery and posture. Future studies will utilize a larger N. Although the acoustic batteries used to induce the ASR were more diverse than those used in previous literature, time and resources limited the use of additional frequency and amplitude investigational acoustic sets, notably the use of more numerous higher or lower frequency sounds (>500 Hz and <3000 Hz). Age, sex, sleep schedule, and emotional state have been shown to affect ASR to certain degrees: lower response magnitude in aging, pre-pulse inhibition anomalies in sleep deprivation, and hyper/hypoaroused systems in emotion. However, blink response latency to diverse acoustic stimuli in these states is largely undescribed. While limitations of this study were considerations within these behavioral, mood, sleep, sex, and stress hormone variables at the time of testing, our focus and resources remained on the ASR delivery and capture system (MARS) with utility considerations of sitting and standing. As such, to reduce the variability within this pilot study, we utilized a specific age and sex cohort.To the best our knowledge, we are the first group to produce a mobile phone based device for ASR monitoring; hence, other direct comparisons to such systems were difficult to find in the literature. In this context, limited comparisons with EMG based ASR studies ,34,35,36These ASR latencies have the potential to be used as a diagnostic or monitoring adjunct which uses left and right eye responses to determine the presence or severity of brain injury victims . AlternaWe found significant details in the responses of cooperative OO muscles to various acoustic stimuli and identified altered responses to high and low intensity acoustics at different frequencies in both sitting and standing postures. Results suggested substantial links between individual differences in frequency, amplitude, sitting, and standing. However, further research is needed to disentangle the specific nature of these associations to one another as well as identify arousal and comfort relationships to specific frequency and amplitude acoustic test sets. To our knowledge, this is the first mobile phone-based monitoring system used to detect and report significant ASR responses to a variety of frequencies and amplitudes in sitting and standing postures. Therefore, it should be kept in mind that further replications are needed to contrast the present techniques and results or address any other potential topics. By visually recording through the use of a smart phone app, we demonstrated that it can be possible to detect and monitor the ASR in healthy population through the use of a mobile device. This opens new horizons for the ASR to be used for diagnosis and monitoring in numerous clinical conditions . The findings in the present study suggest that MARS is a simple and mobile methodology used to study the links between acute acoustic variables and their subsequent effects on the human blink response.


Posidonia oceanica that accumulate in large amounts along the coastal beaches in the form of tightly packed and dry materials of various dimensions. Both egagropili fractions have been shown to be able to improve the physicochemical properties of biodegradable films prepared from protein concentrates derived from hemp oilseed cakes. These materials, manufactured with a biodegradable industrial by-product and grafted with equally biodegradable waste-derived additives, exhibited an acceptable resistance with a still high flexibility, as well as they showed an effective barrier activity against water vapor and gases (O2 and CO2). Furthermore, both NC and LF decreased film moisture content, swelling ability and solubility, thus indicating that both additives were able to improve water resistance of the hydrocolloid films. The exploitation of egagropili, actually considered only an undesirable waste to be disposed, as a renewable source of reinforcing agents to blend with different kinds of polymers is suggested.Nanocrystalline cellulose (NC) and a lignin-containing fraction (LF) were obtained from egagropili, the so called sea balls produced from rhizome and stem fragments of  Plastics pollution has become a global threat, mostly to marine ecosystems . TherefoPosidonia oceanica (PO) is one of the most abundant Mediterranean endemic species, covering 60% of the seabed from 0 to 40 m depth [2 of coastal sandy areas [Biocomposites containing plant- or wood-based fibers have been exploited thus far in an increasing range of items to reinforce plastics, having several advantages over synthetic additives. Plant cell walls consist essentially of rigid cellulosic microfibrils embedded in a soft matrix of hemicellulose and lignin, and cellulose is the major component in lignocellulosic biomass which is localized in the cell wall at around 35–50% . Althougdy areas ,8,9,10. dy areas ,12 and sdy areas  used as dy areas , the munTherefore, the search for turning egagropili into a resource that provides economic benefits and positive feedback from an environmental point of view is strongly advisable because egagropili do not significantly differ from other lignocellulosic materials . Their sIn this paper both cellulose and lignin containing fractions obtained from egagropili fibers were investigated to assess their properties for a possible use as reinforcement additives of protein-based biodegradable/edible films, potentially able to replace oil-based plastics in packaging systems.Hemp oilseed cakes, a generous gift of prof. Daniele Naviglio, were purchased from Consorzio Goji Italia . Egagropili sea balls were collected in the sardinian Poetto beach  and stored at 4 °C until used. Sodium hydroxide, hydrochloric acid, sodium chlorite, acetic acid glacial, potassium hydroxide, sulfuric acid and glycerol (GLY) were purchased from Sigma Chemical Co . All other chemicals were of analytical grade.v/v) during 24 h and oven dried overnight at 105 °C. Afterwards, delignification process has been carried out by dispersing at 70 °C for 2 h the dewaxed egagropili powder in 600 mL of 1.7% sodium chlorite solution brought at pH 3.5 by acetic acid. This process was repeated 3 times consecutively until the color changed from brown to white/yellowish. The resulted bleached fibers, known as holocellulose, were filtered by using filter paper and washed with distilled water until the filtrate became neutral. The first filtrate obtained in the delignification step was referred as the lignin containing fraction (LF). A quantitative analysis, carried out by calculating the dry weight of LF, indicated that 152 mg of LF were obtained from 1 g of grinded egagropili powder. To remove hemicellulose and residual pectin, the obtained holocellulose was treated with 5% potassium hydroxide for 24 h at room temperature followed by an exposure for 2 h at 90 °C. Finally, the obtained cellulose fraction was filtered and washed several times until pH neutralization, and finally dried at 55 °C for 18 h. Egagropili balls were reshaped to rhizome fibers by hands, then washed and rinsed vigorously in distilled water in order to remove sand, salts and other soil contaminants, and finally dried in an oven at 80 °C for 24 h. The dried egagropili fibers were grinded in a rotary mill  at a speed of 1000 rpm for 3 min to a 60-mesh sieve size. Cellulose extraction was carried out as reported by Ilyas et al.  with somw/w) and by stirring the reaction mixture at 45 °C for 45 min in order to totally hydrolyze the amorphous regions of cellulose. Hydrolysis reaction was stopped by diluting ten-times the suspension by cold distilled water, followed by repeated washing of the pellet obtained by centrifugation at 10,000 rpm for 10 min, until the neutral pH was reached. The resulting precipitate containing NC was finally freeze-dried and, then, the obtained powder was dispersed in distilled water and subjected to ultrasonication for 10 min at 400 W to stabilize NC dispersion by eliminating its excessive aggregation. The quantitative gravimetric determination indicated that 225 mg of NC were obtained from 1 g of grinded egagropili powder. The entire extraction process, summarized in Nanocrystalline cellulose (NC) was obtained by sulfuric acid hydrolysis of the egagropili cellulose fraction according to the procedure described by Sanyang et al. . Acid hyv/w) were prepared by dissolving the protein concentrate in distilled water and by adding 1 N NaOH, under constant stirring at room temperature, until the pH of the solution was brought at pH 9 or 12. Film forming solutions (FFSs) (25 mL) were prepared at the two different pH values by using 400 mg of HPs in the presence of 50% GLY, used as plasticizer, and different amounts of either NC (at pH 9) or the extensively dialyzed LF (at pH 12) extracted from egagropili. All kinds of FFSs were cast onto 8 cm diameter polycarbonate Petri dishes and allowed to dry in an environmental chamber at 25 °C and 45% relative humidity (RH) for 24 h. The dried films were peeled, intact, from the casting surface and analyzed after their conditioning at 50% RH and 25 °C by placing them in a desiccator over a saturated solution of Mg(NO3)2·6H2O for 24 h.Hemp proteins (HPs) were extracted from the industrial hemp seed oilcake, a byproduct obtained after the extraction of the hemp seed oil. Defatted hemp seed flour was treated under alkaline conditions (pH 11), and the supernatant obtained after centrifugation was precipitated at acidic pH (5.4). The obtained pellet (HP concentrate) was finally dried and used to obtain hydrocolloid films. The protein content of the derived final HP concentrate was determined by the Kjeldahl’s method , using a®, Worcestershire, UK) in the wavelength of 633 nm using a helium-neon laser of 4 mW output power. Zeta potential was calculated by the instrument software programmer trough the electrophoretic mobility at a voltage of 200 mV using the Henry equation. Zeta potential and particle size measurements were performed at each pH in triplicate and all the results were reported as mean ± standard deviation.The effect of both NC and LF extracted from egagropili on zeta potential and particle size of the HP containing FFSs at pH 9 or 12, respectively, was studied. To this aim each FFS (1.0 mL), containing 50% GLY and 16 mg HPs, was tested in the absence or presence of different amounts of either NC or LF. Zeta potential and particle size values were measured with a Zetasizer Nano-ZSP , elongation at break (EB) and Young’s modulus (YM) were measured by an Instron universal testing instrument . Each prepared film was cut into strips with a width of 10 mm and a length of 60 mm and at least three samples of each film were then tested, by using 1 kN load and 5 mm/min speed, as previously described . Film th2 samples according to the method described by Zahedi et al. [Wi and Wd represent the film weights before and after drying, respectively.Film moisture content was analyzed on 3 × 3 cmi et al. . Films wWi) was immersed in 30 mL of distilled water at 25 °C for 1 h and, after drying of its surface by an absorbent paper, each film was finally weighed again (Ws). The swelling ratio was calculated using the following equation:Film swelling ratio was determined by a gravimetric method . Each fiWi), immersed in 30 mL distilled water and shaken for 24 h at 25 °C. The undissolved film residues were finally dried in the oven at 105 °C to calculate the film dry weights (Wf). Film solubility was determined as the percentage of total weight by using the following equation [In order to determine film water solubility, the dried film was weighed (equation :SolubiliAll the experiments were repeated at least three times.2. All the experiments were repeated at least three times.Film water vapor (WV) and gas permeability was investigated, after film conditioning for 24 h at 50% RH, as previously described ,26 by usMicrostructures of both grinded egagropili fibers and nanocrystalline cellulose were observed by using a Philips-FEI SEM . The dried samples were coated with thin layers of gold and platinum using a sputter coater at a current of 20 mA for 90 s and then the images were taken at an accelerating voltage of 3 kV, (4.4–5.2) mm working. Further surface and cross-section images were also obtained by analyzing hemp protein films derived from FFSs prepared in the absence or presence of either 6% NC or LF. For cross-section imaging, the samples were previously frozen using liquid nitrogen and then cryofractured.p < 0.05) were used to determine the significant difference among the samples.SPSS19  software was used for all statistical analyses. One-way analysis of variance (ANOVA) and Duncan’s multiple range tests  were prepared at pH 9 and 1 mL of each FFS was analyzed to determine their zeta potential value and the particle size. Furthermore, Preliminary experiments carried out by using NC or LF as additives in HP-based FFSs gave rise to manipulable films, whereas the presence of egagropili-derived fibers or holocellulose, hemicellulose and cellulose fractions in FFSs produced films difficult to handle, being fragile or sticky, as well as not homogeneous in their matrix. However, being biodegradable, non-toxic and possessing emulsifying properties, the extracted hemicellulose might find applications in different area of interest, such as papermaking and cosmetics industries.Therefore, the HP-based composite films under investigation were divided into two groups based on their grafting with only two egagropili-derived additives: NC and LF. It is worthy to note that, in the presence of NC, the optimal pH value of the FFS to give rise to HP films with the best performance was pH 9, whereas the optimal pH was 12 when the reinforcement agent tested was LF.The HP-based films prepared in the presence of either NC or LF were peeled intact from the casting surface and characterized for their mechanical properties. Conversely, no significant effect on the film thickness was observed in the films prepared in the presence of different amounts of LF . MoreoveThese findings are in agreement with those previously reported by Zadeh et al. , who invAs water sensitivity is one of the main drawbacks of hydrocolloid bioplastics, the effect of loading NC and LF on film resistance to moisture was investigated by measuring film moisture content, water solubility as well as swelling ratio. As matter of fact, the addition of fillers generally aims not only to enhance film mechanical properties but also to confer them stability for various applications that often require film exposure to a high moisture environment. Thus, In addition, Finally, reduced WV and gas permeability was detected in the films by increasing the concentration of both NC and LF into the FFSs , indicatSEM analysis was carried out to examine the surface and cross-section microstructure of hemp protein-based bioplastics prepared in the absence or presence of 6% of either NC or LF. Control film, prepared in the absence of egagropili-derived fractions, was found to contain holes that interrupted the continuity of the matrix network, as evidenced in panels 1B and 1C of Nanocrystalline cellulose and a lignin enriched fraction were isolated in good yield from egagropili, better known as sea balls, and used as reinforcement additives of a protein-based films. To this aim, new bioplastics composed of proteins extracted from hemp oilseed cakes were prepared. Film microstructure analysis indicated a good interaction of the two additives tested, as both were properly dispersed in the protein film matrix. The improved mechanical and barrier properties, as well as the weakened water trapping ability, exhibited by the grafted biocomposites suggest a potential use of egagropili, presently considered only an undesirable waste to be disposed, as a valuable renewable source for obtaining novel biodegradable materials alternative to the conventional plastics. The possible synergistic effects of nanocrystalline cellulose and lignin enriched fraction, as well as an improvement of their yield in the extraction process, remain intriguing speculations which would deserve to be further investigated.


Bos taurus cattle are one of the earliest domestic cattle in Africa, followed by the arrival of humped Bos indicus cattle. The diverse indigenous cattle breeds of Africa are derived from these migrations, with most appearing to be hybrids between Bos taurus and Bos indicus. The present study examines the patterns of admixture, diversity, and relationships among African cattle breeds.Humpless Bos taurus, 4 Bos indicus, and 5 African crossbred cattle populations. Genetic diversity and population structure were assessed using principal component analyses (PCA), admixture analyses, and Wright’s F statistic. The linkage disequilibrium and effective population size (Ne) were estimated for the pure cattle populations.Data for ~ 40 k SNPs was obtained from previous projects for 4089 animals representing 35 African indigenous, 6 European Bos indicus from European Bos taurus, and African Bos taurus from other breeds. PCA and admixture analyses showed that, except for recently admixed cattle, all indigenous breeds are either pure African Bos taurus or admixtures of African Bos taurus and Bos indicus. The African zebu breeds had highest proportions of Bos indicus ancestry ranging from 70 to 90% or 60 to 75%, depending on the admixture model. Other indigenous breeds that were not 100% African Bos taurus, ranged from 42 to 70% or 23 to 61% Bos indicus ancestry. The African Bos taurus populations showed substantial genetic diversity, and other indigenous breeds show evidence of having more than one African taurine ancestor. Ne estimates based on r2 and r2adj showed a decline in Ne from a large population at 2000 generations ago, which is surprising for the indigenous breeds given the expected increase in cattle populations over that period and the lack of structured breeding programs.The first two principal components differentiated Bos taurus or admixtures of African Bos taurus and Bos indicus. This provides a rich resource of potentially valuable genetic variation, particularly for adaptation traits, and to support conservation programs. It also provides challenges for the development of genomic assays and tools for use in African populations.African indigenous cattle breeds have a large genetic diversity and are either pure African The online version contains supplementary material available at 10.1186/s12864-020-07270-x. Bos taurus) are thought to be the earliest domestic cattle in Africa [Bos taurus and modern populations, Verdugo et al. [Bos taurus cattle may stem from roots in the southern Fertile Crescent. In their review of the evidence, Stock and Gifford-Gonzalez [Bos taurus cattle likely spread across the Sinai and into the Nile Delta 7000 to 8000 BP, then across North Africa, and subsequently into the Nile and the then-grassy Sahara, possibly with additional inputs through the Horn of Africa. Humpless, presumably Bos taurus cattle first appear to be present south of the Sahara about 4500 to 4000 BP [Bos indicus cattle in Egypt from 3500 BP [Bos indicus cattle in sub-Saharan Africa is in East Africa, where all samples, that could be analyzed from two sites dated around 2000 to 2500 BP, were of Bos indicus or Sanga (a hybrid of Bos indicus and Bos taurus) type [Bos indicus genes were already predominant in the pastoral systems in this region. Payne and Hodges [Bos taurus cattle, however, remained predominant in Ethiopia and East Africa until recently despite many waves of Bos indicus introductions to the region from about 2500 BP onwards.Based on skeletal evidence, Sahara rock art, and Egyptian dynastic representations, the humpless taurine cattle  type . This sud Hodges  concludeBos taurus, Bos indicus (zebu), Sanga (Bos taurus × Bos indicus hybrid), and Zenga (Sanga×zebu hybrid). According to Lenstra and Bradley [Bos taurus breeds are those that have short ears and no hump, while zebu breeds are those that have long floppy ears and a prominent hump. Subsequent results based on molecular marker data [Bos indicus. Thus, the term “zebu”, as applied to African cattle breeds, means that the breed has a hump, but it does not imply that the breed is pure Bos indicus, despite much of the literature using zebu and Bos indicus as synonymous when applied to African cattle.Currently, Africa is home to more than 180 cattle breeds or distinct cattle populations , and sev Bradley , Africanker data  and resuBos taurus and Bos indicus, were genetically distinct before domestication [Bos indicus and Bos taurus divide. These authors also showed, using mtDNA sequence data, that there was male-driven Bos indicus introgression into the Near East Bos taurus populations. Studies of microsatellite DNA and Y-chromosomal markers showed extensive introgression of male Bos indicus genes into existing African cattle populations [Bos taurus mtDNA, indicating male-driven introgression of Bos indicus genes into the previously Bos taurus African cattle populations. Based on genome-wide autosomal SNP markers, Weerasinghe et al. [Bos indicus and African Bos taurus.Studies of mitochondrial DNA (mtDNA) variation indicated that the two major groups of cattle, tication –16. A PCtication  using geulations –19, all e et al.  showed tF statistic (FST and FIS), and linkage disequilibrium (LD) analyses to obtain a clear picture of the patterns of admixture and genetic diversity of African indigenous and crossbred populations and to compare their diversity to exotic breeds.The present study provides one of the most extensive analyses of the genetic diversity of African cattle breeds based on genome-wide SNP data to date. We undertook admixture and principal component analyses, Wright’s Bos indicus, European Bos taurus, and African Bos taurus breeds from each other as the apexes of a triangle in the plot area  obtained from an analysis of all indigenous and crossbred cattle populations from East and West Africa, and including African and European taurine reference breeds as well as indicine reference breeds, explained a total of 96.1% of the variation in the genomic relationship matrix (GRM). The first two components accounted for 88.7 and 5.7% of the total genetic variation, respectively, and differentiated the A separate PCA was performed to evaluate in more detail the genetic structure among the eight African taurine reference populations Figure S. The firBos indicus admixture) and spread between the two axes that connect the indicine to the first African taurine group (axis 1) versus the second African taurine group (axis 2). Most of the hybrid animals between Gobra and Maure (Gobra x Maure) sit in this second cluster, aligning with axis 1. The Madagascar-zebu is distinct from all the zebu breeds being the only zebu breed that sits on axis 2.The majority of the East African indigenous breeds that are classified as zebu breeds , clustered together on or slightly to the right and at the indicine end of the axis between indicine and the first African taurine group , and Bororo and Fulani (zebu breeds) form the third cluster located more towards the African taurine breeds, with Ankole and Djakore close to axis 1 and the other breeds on or slightly to the left of axis 2  was used together with a pooled indicine sample and five European taurine reference breeds. Model 2 included an additional four African taurine reference breeds to differentiate the African Bos taurus background. Consistent with the PCA, all African indigenous breeds, other than the pure African taurine breeds, were estimated to be an admixture of indicine and African taurine ancestries. Some breeds also showed small admixture with European taurine. Absolute estimates of ancestral proportions differed substantially between Admixture Model 1 versus Model 2, with Model 2 giving lower estimates of indicine ancestry. However, the ranking of breeds for indicine ancestry proportion was very similar between Model 1 and Model 2, and the following results summarised here are for Model 1. Overall, the indicine proportion was lower in West and South African breeds compared to East African breeds. However, the West African breeds, especially from Senegal, showed a wide range of Bos indicus ancestry. For example, the indicine component in Gobra ranged from 48.5 to 79.8% (average 65.3%), from 64.8 to 70.3% (average 67.8%) in Maure, and from 56.0 to 77.3% (average 66.2%) in Gobra x Maure crosses  and indicine (87%) ancestry  and West  African crossbred animals. Admixture Model 3 with  Senegal .Figure With Admixture Model 3, the crossbred animals from Kenya showed an average exotic dairy proportion of 69%, mainly from Friesian 23%), followed by Ayrshire (23%), and Guernsey (16%). The Ugandan crossbreds showed an average exotic dairy proportion of 62% with the main contribution from Friesian (38%) and Holstein (14%). The Ethiopian and Tanzanian crossbred animals showed an average of 72% exotic dairy proportion; Ethiopian crossbreds had 36% Friesian and 30% Holstein, and Tanzanian crossbreds had 31% Friesian, 19% Ayrshire, and 12% Holstein Fig. , Table 23%, folloFIS value, was close to zero (between − 0.006 to 0.009) for most breeds across all breed groups .Inbreeding, as represented by the s Table S. The higFST values, showed a strong divergence within different groups of breeds  and adjusted squared correlation (r2adj) between phased alleles of pairwise SNP loci over increasing genome distances is illustrated in Fig. r2 and a lower rate of r2 decay, and Gobra showed lower r2 and a higher rate of r2 decay than the other populations across all distances . Later principale components could be able to tease out smaller genetic differences, such as dairy vs beef breeds, however, coressponding beef reference breeds were not included. Additionally, some beef breeds are closer related to dairy breeds than other beef breeds or vice versa [The PCA and  studies , 26–32.  studies . As showce versa –36 whichBos indicus populations in our sample. Based on the range of samples included in our study, it is unlikely that any indigenous breeds of Africa are pure Bos indicus. As outlined in the background section, it has been assumed in the literature that the admixed indigenous breeds of Africa arose from Bos indicus cattle entering Africa and breeding with existing African Bos taurus populations [Bos taurus populations in the Near East became admixed with Bos indicus likely due to human migrations around 4200 years BP. The populations they sampled in the Levant, which were the closest samples to the putative first route of Bos indicus into Africa through Egypt, showed a Bos taurus genotype that was closest to modern African Bos taurus. Therefore, it is possible that the first humped cattle entering Africa around 4000 years BP [Bos taurus and Bos indicus rather than pure Bos indicus.Among the African cattle populations, there are no pure ulations , 9. Verdulations  concludeyears BP  may alreBos taurus before the rinderpest virus (RPV) epidemic of 1887–1897, which was reported to have more severely affected the taurine cattle populations of the East and South than the zebu populations. However, inferences about the types of cattle present in Africa until recent times are largely based on sparse depictions of humped (inferred as Bos indicus types) versus non-humped (inferred as Bos taurus type) cattle.Payne and Hodges  state thBos taurus populations, all African indigenous cattle populations are admixtures of Bos indicus and African Bos taurus, with West African and Southern African populations showing lower Bos indicus admixture than East African populations consistent with recent studies by Pitt et al. [FST values. This differentiation can be attributed at least in part to their admixture with European taurine breeds. Some indigenous breeds such as the Gobra show a surprisingly wide distribution of African taurine and indicine ancestry  that was imported from Brazil into the region in which the Gobra samples used here were collected [Our results show that, other than the pure African t et al.  and Verdt et al. . The Souollected .Bos taurus populations and our reference European Bos taurus populations exhibit much greater diversity than the set of Bos indicus populations used in our analyses. Studies that included much larger samples of European Bos taurus cattle similarly showed greater diversity among European Bos taurus versus Bos indicus cattle . The distribution of indigenous breeds in the PC plots  suggest a different African Bos taurus ancestry for West African versus East African indigenous.Populations with relatively high heterozygosity levels were Ankole-Watusi and N’Dama3, which show substantial admixture between populations with a large genetic distance  clustered closely with the Fulani (sampled in Benin) and had very similar breed proportions, indicating that they likely form a single population. According to Grema et al. , Bororo us breed , but ourBos taurus ancestry and being an admixture between African and European Bos taurus. Gautier and Naves [Bos taurus. The rather dispersed cluster of the Oulmes Zaer samples in the PC plots suggests the possibility that the breed might have been deliberately created by the crossing of African and European Bos taurus cattle in relatively recent times.Results for the Mpwapwa showed that this synthetic dual-purpose breed, first created about 60 years ago, aligns with its reported genetic history of 35% Sahiwal, 20% Red Sindhi, 35% East African zebu breeds, and 10% Ayrshire . The Oulnd Naves , who anaCrossbreeding is widely used to achieve a suitable balance of productivity and adaptation in African smallholder dairy farming. According to the literature, the predominant exotic dairy breeds used in Kenya have been Holstein-Friesian, Ayrshire, Guernsey, and Jersey , and in r2adj > 0.2) for very short distances (about 10 Kb) with an exponential decline with increasing distance. The European reference dairy breeds showed a higher level of LD across all interval sizes compared to Bos indicus reference breeds and most African indigenous breeds  than the European Bos taurus breeds , and crosses between the indigenous breeds and other exotic dairy breeds, such as Montbeliarde and Holstein-Friesian.This study analyzed data on 4089 animals representing a diverse set of African indigenous and crossbred plus exotic cattle populations. In addition to the crossbreds, exotic reference breeds, one African synthetic breed, and one unclassified African indigenous breed were used, and 669 samples representing 33 breeds or populations within the three main African indigenous cattle breed groups of African Bos indicus and European Bos taurus breeds were included to represent the major known anchor points of global cattle diversity against which African cattle diversity can be assessed. The European Bos taurus breeds were chosen because these breeds are known to have contributed to the crossbred dairy populations included in our study, allowing clearer interpretation of results than inclusion of other breeds or a larger sample of breeds. The reference breeds included five African Bos taurus , four Bos indicus , and six European Bos taurus dairy breeds . The reference data were obtained from the Bovine HapMap Consortium [nsortium , Decker nsortium , the ScoThe Senegal animals were genotyped using the Illumina BovineSNP50v2 BeadChip array  comprising 54,609 SNPs. Quality control was carried out using the GenABEL package  in R CorSamples from the DGEA project were genotyped with the Illumina BovineHD Beadchip  and sourced from Strucken et al. . The DGEBos taurus reference populations to be used in the Admixture analysis. The GRMs were constructed according to the first method of VanRaden [M. Missing genotypes were replaced with the average allele frequencies across all animals for each given SNP. The GRM was then calculated as:Principal component analyses were performed using a GRM to define the covariance between animals. Two separate PCA were conducted: 1) where the GRM was based on all cattle populations; 2) the GRM was built using only African taurine breeds. The second GRM was used to evaluate the genetic structure and diversity among the African taurine breeds to select the African VanRaden . GenotypZ) was constructed by subtracting the P matrix from the genotype matrix M, where P = 2 ∗ (pi − 0.5), and pi is the allele frequency at locus i.The centred genotype matrix , and the use of supervised analyses allows clear dissection of these contributions. A pooled sample of Bos indicus reference (indicine) was created with eight animals per breed because of the four indicine reference breeds clustered very closely together in the PCA  were: N’Dama (African taurine), Indicine (pooled Bos indicus samples), Ayrshire, Friesian, Guernsey, Holstein, and Jersey. In Model 2 (K = 11), N’Dama1, Lagune, Baoule, and Somba were added as African taurine reference breeds. In Model 3 (K = 12), Montbeliarde was added as another European dairy breed, because Montbeliarde has been used for crossbreeding to indigenous cattle in Senegal.To investigate the genetic admixture of the indigenous and crossbred cattle populations, a maximum likelihood model implemented in the software ADMIXTURE 1.23  was applredicted . Also, predicted , 66 and FST values were calculated according to Weir and Cockerham [FST is defined as the genetic variance between populations expressed as a proportion of the total genetic variance. The degree of inbreeding was inferred from the FIS coefficient calculated according to Nei [FIS is defined as one minus the observed proportion of heterozygotes divided by the expected proportion of heterozygotes. To explore genetic differentiation among breeds, FST values were visualized in a heatmap, and the complete-linkage method was used for hierarchical clustering as provided in the R package “pheatmap” [Pairwise ockerham , where Fg to Nei , where theatmap” .r2 between phased alleles of pairwise SNP loci. The genotypes were phased using Eagle v2.4 [r2 was estimated separately for each breed between all pairs of SNPs with a distance of up to 50 Mb using markers with minor allele frequency ≥ 5% according to the Hill and Roberson formula [f(AB) is the observed frequency of haplotype AB [f(A), f(a), f(B) and f(b) are observed frequencies of alleles A, a, B, and b, respectively. For the purpose of graphical display, the distance of pair-wise LD was binned into 10 Kb intervals up to 1 Mb.The extent and magnitude of LD within different breeds were determined using gle v2.4 , and thegle v2.4 . The r2  formula :\documeotype AB , while fr2 values combined with marker distances were used to estimate the approximate Ne at a given time point in the past, assuming a model without mutation, and using the formula of Sved [Ne is the effective population size, and c the marker distance in Morgans assuming 1 M = 100Mbp. The time (number of generations ago) at which Ne was estimated as 1/2c [r2 across the autosomes. This model does not account for sampling bias of r2 due to small population sizes. We extended the model to account for sample size according to Weir and Hill [Ne, Ne was estimated only for populations that have a sample size ≥20. The inclusion of related animals in the sample can cause an upwards bias of Ne (particularly recent Ne). Estimates of Ne presented in this paper are from samples that exclude animals that have a GRM relationship > 0.2 with one or more animals.The  of Sved :\docume as 1/2c . For estand Hill :\documeAdditional file 1: Figure S1. PC1 vs. PC2 when using the whole dataset. (a) Expanded plot Bos indicus breeds section. (b) Expanded plot African Bos taurus breeds section.Additional file 2: Figure S2. PCA using African Bos taurus populations (a) Plot of PC1 vs PC2. (b) PC1 vs PC3.Additional file 3: Figure S3. Decline of linkage disequilibrium (r2) with physical distance (kbp). (a) Bos taurus dairy breeds. (b) Bos indicus reference breeds. (c) East and South African indigenous breeds. (d) West African indigenous breeds.Additional file 4: Figure S4. Effective population size over past generations using r2  for (a) Bos taurus dairy breeds, (b) Bos indicus breeds, (c) East and Southern African indigenous breeds, and (d) West African indigenous breeds.Additional file 5: Figure S5. Effective population size over past generations using r2adj  for (a) Bos taurus dairy breeds, (b) Bos indicus breeds, (c) East and Southern African indigenous breeds, and (d) West African indigenous breeds.Additional file 6: Table S1. Estimates (±SD) of FIS  within the breeds and pairwise FST  values between the breeds.


Pleomorphic lung carcinoma is a rare and aggressive neoplasm accounting for <1% of all lung tumors. It is more common in men and consists of spindle and/or giant cells with an epithelial component. In patients with known histories of malignancies at other sites, diagnosis of a new lung lesion may prove challenging with respect to classification as either primary or metastatic disease, especially in cases with overlapping immunohistochemical staining patterns. This was a case of a 67-year-old female with a newly discovered 1.5 cm nodule in her left lower lung lobe. Her past medical history was significant for an intracystic papillary carcinoma of the right breast diagnosed 8 years prior. Histopathologic examination of the new lung lesion revealed highly pleomorphic cells composed predominantly of neoplastic giant cells and atypical mitotic figures, with geographic areas of necrosis. However, no areas reminiscent of intracystic papillary carcinoma or other forms of breast carcinoma were seen. Immunohistochemistry showed that the tumor cells were immunoreactive for GATA3, TTF1, and napsin A and nonimmunoreactive for p40. Therefore, although this index lung tumor did show positivity with GATA3 staining, it was morphologically different from her original intracystic papillary carcinoma of the breast. In addition, intracystic papillary carcinomas are known to rarely metastasize to other organs, and GATA3 staining has been rarely reported in lung carcinomas. In summary, this case typifies the overlapping immunohistochemical staining patterns that may be seen in different tumors and the role of histopathologic morphology in arriving at the correct diagnosis. Pleomorphic lung carcinoma (PLC) is rare and accounts for 0.1 to 0.4% of all lung cancers . PLC is Intracystic papillary carcinoma (IPC) of the breast is a rare malignant tumor, constituting 1-2% of all breast carcinomas in women . IPC oftGATA-binding protein 3 (GATA3) is a transcription factor of the GATA family and thus far has been used in surgical pathology as a marker for breast and urothelial carcinomas . HoweverA 67-year-old female with past medical history significant for right-sided IPC of the breast, status post-lumpectomy, and radiotherapy, who was being managed with anastrozole presented with a new lung lesion discovered on a CT scan 8 years after her original breast cancer diagnosis. She was undergoing routine management for general malaise and decreased appetite. She had a significant past 40-year smoking history. Following an unsuccessful bronchial biopsy, a diagnostic thoracotomy with lobectomy was performed. On gross examination, a 1.5 × 1.2 × 1.1 cm well-defined tan-white solid mass was identified in the left lower lobe. A frozen section diagnosis of poorly differentiated carcinoma with extensive necrosis was made. Histopathologic examination of the permanent sections revealed highly pleomorphic cells composed predominantly of neoplastic giant cells and atypical mitotic figures, with geographic areas of necrosis, with a minor component of adenocarcinoma . HoweverPleomorphic lung carcinoma (PLC) is defined as poorly differentiated non-small-cell lung carcinoma (NSCLC), containing spindle cells and/or giant cells, or a carcinoma consisting of spindle and giant cells alone, with the pleomorphic component comprising at least 10% of the neoplasm , 3. The PLC may pose diagnostic problems, and immunohistochemistry is largely used when pathologists deal with these tumors in routine practice . By immuThe diagnostic surprise encountered in this case was when the patient's tumor cells were found to be strongly immunoreactive for GATA3. GATA3 has been thus far explored in surgical pathology as a marker for breast and urothelial carcinomas  and is fUltimately, based on the histopathologic examination and immunohistochemical results, a diagnosis of a primary PLC was made. Furthermore, a diagnosis of IPC was considered less likely because IPCs are known to rarely metastasize to other organs. In addition, GATA3 staining has been rarely reported in lung adenocarcinomas , 18. In GATA3 expression has been routinely used in surgical pathology to discriminate between tumors of breast and urothelial origins from other tumors. However, this case typifies the rare instance in which GATA3 may be positive in primary NSCLC. Therefore, additional studies are needed to further explore the diagnostic utility of GATA3 expression in primary lung tumors.


The relative mRNA expression levels of CCL5, OPRL1, CXCL13, and SST were measured by quantitative real-time PCR in the in vitro analysis of the osmotic pressure experiments. Finally, correlation analysis and a neural network module were employed to explore the linkage between significant gene biomarkers and pain. A total of 371 DEGs were identified, including 128 downregulated genes and 243 upregulated genes. Furthermore, the four genes  were identified as significant gene biomarkers of low back pain (P < 0.001) based on univariate linear regression, and CCL5  and OPRL1  were significantly related to low back pain through multivariate logistic regression. The expression of CCL5 and OPRL1 might be correlated with low back pain in patients with IVD degeneration disease caused by changes in the osmotic pressure of NP cells.The incidence of intervertebral disc (IVD) degeneration disease, caused by changes in the osmotic pressure of nucleus pulposus (NP) cells, increases with age. In general, low back pain is associated with IVD degeneration. However, the mechanism and molecular target of low back pain have not been elucidated, and there are no data suggesting specific biomarkers of low back pain. Therefore, the research aims to identify and verify the significant gene biomarkers of low back pain. The differentially expressed genes (DEGs) were screened in the Gene Expression Omnibus (GEO) database, and the identification and analysis of significant gene biomarkers were also performed with various bioinformatics programs. A total of 120 patients with low back pain were recruited. Before surgery, the degree of pain was measured by the numeric rating scale (NRS), which enables comparison of the pain scores from individuals. After surgery, IVD tissues were obtained, and NP cells were isolated. The NP cells were cultured in two various osmotic media, including iso-osmotic media (293 mOsm/kg H NP survives in a special ecological niche containing no distribution of blood vessels and nerves in which the osmotic pressure is significantly higher than that of plasma, enabling proteoglycans to be present in high concentrations3. Increasing with age, the osmotic pressure changes, and the interverbal disc degenerates5, which may be related to low back pain7. Previous studies have shown that the osmotic environment can influence the gene expression of NP cells7; thus, we hypothesized that there are pain-related genes among these affected genes, which might provide new therapeutic strategies and drug targets for low back pain.IVD is fibro-cartilage tissue located between vertebrae and is composed of the nucleus pulposus, annulus fibrosus and endplate8. The scale requires patients to describe pain intensity using the numbers 0–10 or 0–100, while a higher number means more pain 9. Therefore, the NRS was used to evaluate the degree of low back pain in this study.In addition, the NRS is ubiquitous in the clinic as a means of evaluating low back painThrough bioinformatic analysis, we screened enrichment pathways, such as the regulation of extracellular matrix organization and the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathway, as well as key genes, such as CXCL13, SST, CCL5 and OPRL1, between NP cells cultured in hyperosmotic media and NP cells cultured in iso-osmotic media. In addition, through quantitative real-time PCR and statistical analysis, we found that CCL5 and OPRL1 might be potential biomarkers of low back pain.After analysis of the datasets GSE1648) with the limma package, the difference between NP cells cultured in hyperosmotic media and NP cells cultured in iso-osmotic media could be presented in a volcano plot. After setting the threshold value, a total of 371 DEGs were reserved, including 128 downregulated DEGs and 243 upregulated DEGs  Fig. . Six MCONTS Fig. .Figure 6The VENN diagram showed that there were four significant common genes among “Metascape_MCODE”, “Cytoscape_MCODE”, and “Cytoscape_cytoHubba”, including CCL5, OPRL1, SST, and CXCL13 Fig. . SummariThe relative expression levels of CCL5 Fig. , OPRL1 , and SSTThe miRNAs that regulate the four significant genes were screened out with TargetScan Table .Table 4SThe NRS was negatively associated with osmotic pressure  Fig. . The NRSUnivariate linear regression showed that the NRS was significantly correlated with CCL5 , OPRL1 , SST , and CXCL13 . Furthermore, the NRS remained related to CCL5 , OPRL1 , and CXCL13  in the multivariate linear regression model . Additionally, the OR of high OPRL1 was 19.875   Fig. , and theAfter training, the neural network prediction model reached the best effect, in which the mean squared error was 0.0076566 at epoch 2000 Fig. , and theThrough the cubic spline interpolation algorithm, we found the high-risk warning indicator of NRS: CCL5 <7 or> 8.5, and 3.5 <OPRL1 <8 Fig. . Further10. The aetiology and mechanism of low back pain are highly complicated. Previous studies showed that disc degeneration is closely related to low back pain11. The intervertebral disc is in a special hypertonic environment2, and its degeneration includes changes in the number of intervertebral disc cells and gene expression, as well as changes in extracellular matrix synthesis and catabolism containing changes in osmotic pressure5. The aim of this study was to explore the effects of intervertebral disc osmotic pressure on intervertebral disc cells from gene expression levels to identify potential therapeutic targets and disease-associated genes.Low back pain is a common symptom experienced by people of all ages. The median prevalence rate in high-income countries is as high as 30.3%, which considerably increases the economic burden14. A total of 371 DEGs, including 243 upregulated and 128 downregulated DEGs, were screened between NP cells cultured in hyperosmotic media and NP cells cultured in iso-osmotic media by analysing the biological data of the gene expression profile GSE1648. Through gene enrichment analysis of DEGs, the influence of osmotic pressure on gene expression in NP cells was comprehensively summarized: regulation of extracellular matrix organization and the JAK/STAT signalling pathway.Biological information analysis has been widely applied to explore gene changes in the process of disease development, and this method may help scientists to identify new therapeutic targets15. Changes in osmotic pressure could affect the expression of some molecules in the extracellular matrix, and a large number of previous experiments demonstrated this phenomenon19. Ishihara used the NP of bovine tailbone for culture and found that the rate of proteoglycan synthesis was significantly increased when the osmotic pressure was 430 mOsm compared to 280 mOsm17. Takeno used bovine NP cells for in vitro culture and found that glycosaminoglycan products were in the highest concentration at 370 mOsm and in the lowest concentration at 270 mOsm19. Neidlinger-wilke obtained similar results by using bovine NP. When osmotic pressure increased from 300 mOsm to 500 mOsm, the expression of proteoglycan and matrix metallopeptidase-2 was upregulated, while the expression of matrix metallopeptidase-3 was downregulated18. However, Haschtmann found that different osmotic pressures had no significant effect on proteoglycan, but collagen I expression was upregulated when osmotic pressure was increased (485 mOsm compared to 335 mOsm)16. In addition to animal experiments, Wuertz found that collagen II was upregulated while collagen I was downregulated when the osmotic pressure was increased (300 mOsm compared to 500 mOsm) by using human NP cells7. The above evidence indicated that proteoglycan synthesis increased under hypertonic conditions. Some studies pointed out that the decrease in proteoglycan could damage the function of interverbal discs, aggravate disc degeneration, and promote angiogenesis20. Therefore, we believed that a decrease in osmotic pressure affected the synthesis of extracellular matrix and thus produced a negative impact on the disc. Artificial regulation of osmotic pressure under pathological conditions could possibly improve the function of the disc indirectly, thereby achieving pain relief and treatment. Although the molecular mechanism of osmotic pressure affecting the extracellular matrix has not been determined, some scholars have proposed that Tonicity-responsive enhancer binding protein (TonEBP) might play an important role in this process21. The survival of intervertebral disc cells in hypertonic media was closely related to the activation of TonEBP. TonEBP not only activates a series of gene products to make NP cells adapt to changes in the microenvironment but also plays an active role in protecting nucleus pulpotomy from apoptosis and regulating extracellular matrix components22. TonEBP is involved in inducing the expression of beta 1,3-glucuronosyl transferase 1, which is a key enzyme for glycosaminoglycan synthesis23.The extracellular matrix is mainly composed of water (60–99% by weight), collagen and proteoglycan24. When ligands bind to receptors, JAKs are activated, and the activated JAKs self-phosphorylate and phosphorylate the receptors. JAK-mediated phosphorylation activated STAT and thus led to the formation of dimers that directly bind to DNA and regulate gene expression25. Gabr showed that the expression of interleukin-17 increased in lumbar disc herniation and degeneration26, while Hu found that interleukin-17 upregulated the expression of VEGF in rat nucleus myeloid cells through the JAK/STAT pathway27. Chen proposed that interleukin-21 played a role in the pathological process of disc degeneration and could aggravate disc degeneration by stimulating TNF-α through the JAK/STAT pathway28. The study of Osuka showed that the JAK/STAT pathway was activated in lumbar disc herniation and played a role in inflammation, while the activation of the JAK/STAT pathway might be related to interleukin-629. In conclusion, the expression of the JAK/STAT pathway might adversely affect the intervertebral disc, but few relevant studies could be retrieved at present; this topic might be one of our future research directions.The JAK/STAT signalling pathway, as an important pathway of intracellular signal transduction, is a downstream mediator of a variety of cytokines, hormones and growth factorsIn addition, we found that there was a positive association between NRS and the relative expression of CCL5  in low back pain, as well as OPRL1 , and CCL5 <7 or> 8.5 and 3.5 <OPRL1 <8 were high-risk warning indicators of NRS. These findings might assist us in evaluating patients’ pain more objectively and provide timely medical assistance, but its clinical applicability remains to be discussed.30 activation of normal T cells expressed and secreted), functions as a chemoattractant for blood mononuclear cells, memory T cells and eosinophils, enables basophils to release histamine and activate eosinophils and not only plays an important role in the inflammatory response31 but also has a non-negligible influence on different pathological pain processes, which has been described in the literature. Oh found that subcutaneous injection of CCL5 in mice produced allodynia32. Pevida found that intraplantar injection of CCL5 caused thermal hyperalgesia in mice, which might be induced by activation of CCR1 or CCR533. Kepler noted that CCL5 was significantly higher in human-derived painful NP cells than in painless discs34. Liou demonstrated that the lack of CCL5 reduced the recruitment of inflammatory cells to painful and inflamed sites and reduced pain in a mouse model of chronic neuropathic pain35. In addition, the analgesic activity of morphine could be attenuated by chemokines, especially CCL5 and CXCL1236. The above evidence suggested that the expression of CCL5 was associated with pain production or hyperalgesia. Phillips reported that CCL5 was upregulated in degenerate intervertebral disc samples compared to nondegenerated samples, but it was greater in infiltrated samples compared to degenerated samples. In addition, the team also presented the expression patterns of other cytokines and chemokines between different samples, indicating that it was necessary to investigate the relationship between the cytokine and chemokine and intervertebral disc degeneration37. CCL5 and other cytokines and chemokines are produced and secreted by intervertebral disc cells, and its release is increased in response to degenerative conditions38. CCL5 could promote matrix degradation, changes in cell phenotype, and infiltration and activation of inflammatory cells, further amplifying the inflammatory cascade, which is attributed to degeneration39. In the present study, the expression of CCL5 in isotonic samples was higher than that in hypertonic samples, indicating that changes in osmotic pressure affected the expression of pain-related factors in intervertebral disc cells, which were involved in the low back pain that occurred under pathological conditions and might become a new target for treating related diseases.CCL5, a protein-coding gene also known as RANTES  in mice48. However, Andero pointed out that the OPRL1 agonist SR-8993 could prevent the accumulation of fear memory in the amygdala in a mouse model of PTSD and thus might prevent the occurrence of PTSD49. Ko found that subcutaneous injection of the OPRL1 agonist Ro64–6198 in monkeys produced antinociceptive effects against acute noxious stimuli and capsaicin-induced allodynia50. Rutten found that the OPRL1 agonist Ro65–6570 had an analgesic effect in diabetic mice51. In addition, changes in the expression of OPRL1 have also been reported in the literature. OPRL1 gene expression was increased after sciatic nerve ligation in mice52, and OPRL1 was upregulated on the dorsal root ganglion (DRG) in mice with neuropathic and inflammatory pain53. Anand found that OPRL1-positive nerve fibre content in the urothelium of the bladder was significantly increased in patients with hyperactivity and bladder pain syndrome, and capsaicin responses of rat dorsal root ganglion (DRG) neurons in the presence of N/OFQ were dose-dependently inhibited, indicating that activating OPRL1 represents a potential clinical pain management strategy54. However, studies by Seo indicated that curcumin and Boswellia serrata acted as nociceptin receptor antagonists to reduce pain by downregulating OPRL1 expression55. From the above results, the function of OPRL1 in modulating pain was complex and controversial, showing both analgesic and pain-promoting effects in different experimental settings, but it was undeniable that it had potential in pain treatment; however, before this step, we need to conduct more research to clarify the role of OPRL1.The protein encoded by OPRL1 is a member of the 7 transmembrane G protein-coupled receptor family, which acts as a receptor for endogenous nociceptin/orphanin FQ and is involved in a variety of biological functions and the regulation of neurobehavioral behaviours, including depression, anxiety, learning, memory, motor activity and drug dependence and addiction56 and plays a role in the pathogenesis of autoimmune diseases, inflammatory diseases and tumours57. It was reported that proteoglycan biglycan induced CXCL13 expression and aggravated lupus nephritis in mice through TLR2/4 on macrophages and dendritic cells58. However, no similar finding was found in the intervertebral disc. However, the role of CXCL13 in pain was contrary to our expectation. CXCL13 and CXCR5 produce orofacial pain through ERK-mediated proinflammatory cytokines59. After ligation of the spinal cord nerve in mice, it was found that CXCL13 was upregulated in the spinal cord, and intrathecal injection of CXCL13 could cause hyperalgesia and activation of astrocytes60. This finding showed that the role of CXCL13 in intervertebral discs merits further study.CXCL13 was mainly expressed in secondary lymphoid tissues, such as lymph nodes, spleen and gut-associated lymphoid tissues. CXCL13 selectively attracts and guides B lymphocytes by acting on CXCR5 in lymphoid follicles61. Susan found that somatostatin and its receptors helped to inhibit cutaneous nociceptors62. Carlto63 and Heppelmann64 found that somatostatin not only worked as a peripheral analgesic agent but also served as a tonic control on peripheral nociceptors66. Huang found that pain sensitivity increased when eliminating somatostatin expression from primary afferent neurons, which indicated that somatostatin released from primary afferents was involved in inhibiting pain behaviour67. Thomas found that J-2156, a somatostatin receptor type 4 agonist, could relieve mechanical low back pain in a rat model68. However, surprisingly, the present study showed that somatostatin was lower in hypersamples, indicating that further research is warranted in this area, not only regarding the role of SST in nociception.SST is an important regulator of the endocrine system with diverse effects and is widely distributed in the brain and periphery. Somatostatin inhibited the release of numerous secondary hormones by binding to high-affinity G-protein-coupled somatostatin receptors. Chapman reported that somatostatin has a marked antinociceptive function at the spinal level and a weaker inhibitory action at the peripheral nociceptor terminal but only in nociceptive states associated with peripheral inflammationDespite rigorous bioinformatic analysis performed in this study, there were still some shortcomings in this work. Specifically, size of the dataset was small.Bioinformatic analysis could screen and identify significant gene biomarkers of low back pain caused by changes in the osmotic pressure of NP cells. The four genes  were identified as significant gene biomarkers of low back pain. In particular, the expression of CCL5 and OPRL1 was most correlated with low back pain. Furthermore, this research could provide a reference for future in-depth research to identify gene biomarkers of low back pain.https://www.ncbi.nlm.nih.gov/geo/) was produced by the [HG-U133A] Affymetrix Human Genome U133A Array (Platform GPL96). The gene profile consisted of 4 isotonic samples and 4 hypertonic samples. The samples were intervertebral disc NP cells obtained from patients undergoing discectomy prior to surgery for lumbar interbody fusion or lumbar disc herniation. The iso-osmotic media consisted of a defined cell culture medium . The hyperosmotic media consisted of the same cell culture media supplemented with sucrose to a final osmolarity of 450 mOsm/kg H2O. The above data were obtained from L AWRENCE M’s cell culture69, which could be downloaded from the website (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE1648).The gene profile GSE1648 downloaded from the GEO database . As a fully functional package, the limma R package included the original data input and preprocessing capabilities of complementary DNA (cDNA) chips extracted from NP cells, as well as a linear model for analysing differentially expressed genes. We screened DEGs between isotonic and hypertonic NP cells by utilizing the limma package with an adjusted P-value < 0.05 and a log (fold change) >1 or log (fold change) <−1 as the cut-off criteria. The volcano plot was drawn by R language.The limma  R packagehttps://david.ncifcrf.gov/home.jsp) (version 6.8)71 was one online analysis tool suite with the functional annotation for Gene Ontology72 and Kyoto Encyclopedia of Genes and Genomes (KEGG)73. To perform the Gene Ontology and KEGG analysis of DEGs, the DAVID online tool was implemented. First, we clicked on the “Functional Annotation” on the website (https://david.ncifcrf.gov/home.jsp). Second, we entered the gene list, selected identifiers as official gene symbols, selected list types as gene lists, and submitted lists. Third, we selected to limit annotations and background by Homo species. Finally, the enrichment results of Gene Ontology and KEGG are presented. P-values < 0.05 indicated significance.The DAVID (http://metascape.org/gp/index.html#/main/step1)74. For given DEGs identified between the isotonic NP cells and hypertonic NP cells by the above analysis (see the “Identification of DEGs” section), function and pathway enrichment analysis was carried out with the following ontology sources: Gene Ontology and KEGG Pathway. Terms with a P < 0.01, a minimum count of 3, and an enrichment factor >1.5 were collected and grouped into clusters.Furthermore, pathway and process enrichment analyses were performed by Metascape 74 was used to construct the PPI network and screen the significant module.First, Metascape (http://string.embl.de/) was also applied to construct the PPI network, and Cytoscape was used to present the network73. Cytoscape (version 3.6.1) was a free visualization software76. The Molecular Complex Detection tool (MCODE) (version 1.5.1)77 could screen and identify the most significant module in the PPI network. When the degrees were set (degrees >10), the hub genes were excavated.Moreover, the Search Tool for the Retrieval of Interacting Genes . Summaries for the function of 4 significant genes were obtained via GeneCards (https://www.genecards.org/). The R language was used to perform the clustering analysis of significant genes based on the gene expression level. Pearson’s correlation test was performed to complete the correlation analysis among the hub genes.A Venn diagram was delineated to identify significant common genes among “Metascape_MCODE”, “Cytoscape_MCODE”, and “Cytoscape_cytoHubba” by FunRich software (http://ctdbase.org/) is a web-based tool that provides information about interactions between chemicals and gene products and their relationships to diseases78. The relationships between the significant genes and “pain and intervertebral disc degeneration” were analysed via the tool.The comparative toxicogenomics database (2. Cell culture was performed under normoxia. Cell culture medium was exchanged for either hyperosmotic (450 mOsm/kg H2O) or iso-osmotic media (293 mOsm/kg H2O) after 24 h69. The osmolarity was altered by using both sucrose and NaCl17.Human IVD tissue was obtained from patients (60±5 years old) undergoing discectomy prior to surgery for lumbar disc herniation (n = 50) or lumbar interbody fusion (n = 70). Before the surgery, NRS was obtained from all the patients. The research conformed to the Declaration of Helsinki and was authorized by the Human Ethics and Research Ethics Committees of Beijing Ditan Hospital. Informed consent was obtained from all patients. All tissue was harvested from the central NP region. Sequential pronase–collagenase digestion was used to isolate NP cells. The osmotic pressures between inside and outside of the NP cells were measured by an osmometer . The NP cells were cultured in RPMI-1640  containing 1% penicillin-streptomycin and 10% foetal bovine serum  at 37 °C with 5% CO79. The expression level of mRNA was demonstrated as the fold change relative to the control group80. The primer sequences used in the qPCR are shown in Table The relative mRNA expression levels of CCL5, OPRL1, CXCL13, and SST were measured by quantitative real-time PCR. Total RNA extraction, cDNA synthesis, and qPCR were performed. Total mRNA was extracted from NP cells with TRIzol reagent  according to the manufacturer’s protocol. One microgram of total RNA was used to generate first strand cDNA using random primers and SuperScript II reverse transcriptase (Invitrogen). Real-time PCR was performed in triplicate using the SYBR PrimeScript RT-PCR Kit . The expression of GAPDH was measured as an internal control. Thermocycler conditions included an initial hold at 50 °C for 2 minutes and then 95 °C for 10 minutes followed by a two-step PCR programme of 95 °C for 15 seconds and 60 °C for 60 seconds repeated for 40 cycles on an Mx4000 system , on which data were collected and quantitatively analysedwww.targetscan.org) is a web-based database that can predict biological targets of miRNAs. In our study, the miRNA-gene pairs of the significant genes were screened out with TargetScan.TargetScan  of each variable for NRS. A receiver operating characteristic curve analysis was performed to determine the ability of the expression of CCL5 and OPRL1 to predict the NRS.81.All statistical analyses were conducted using SPSS software, version 23.0 . A p-value < 0.05 was considered to be significant82.The training group was divided into training data and calibration data randomly according to the proportion of 7:3. There were 77 individuals in the training data and 33 individuals in the calibration data. Furthermore, a total of 10 individuals were used as the validation data. MATLAB  was used to accomplish the normalization processing of variable values, network initialization, network training, and network simulation. The number of input neurons in the input layer was the same as the number of input variables, and the number was two. The hidden layer was designed as 1 layer, and the output layer was also designed for 1 layer. One output variable was NRS. Then, the forecast model was established with a hidden unit number of 6. When training to 2000 steps after repeated training, the falling gradient is 0, and the training speed is uniform. At the same time, the training error was 0.0076566, and the R (relativity) value reached 0.98987. Cubic spline interpolation was performed to predict the high-risk warning range of NRS by using the expression of CCL5 and OPRL1The data of this research were downloaded from the GEO database public website. The research conformed to the Declaration of Helsinki and was authorized by the Human Ethics and Research Ethics Committees of Beijing Ditan Hospital. All institutional and national guidelines for the care and use of participants were followed.


Sex and heart rate recovery, HRR), which achieved a suitable discrimination performance between groups; sensitivity = 80% and specificity = 75% (AUC = 83%). However, after controlling the model for possible confounding factors, only one feature (Brugada syndrome (BS) is a genetic pathological condition associated with a high risk for sudden cardiac death (SCD). Ventricular depolarization disorders have been suggested as a potential electrophysiological mechanism associated with high SCD risk on patients with BS. This paper aims to characterize the dynamic changes of ventricular depolarization observed during physical exercise in symptomatic and asymptomatic BS patients. To this end, cardiac ventricular depolarization features were automatically extracted from 12-lead ECG recordings acquired during standardized exercise stress test in 110 BS patients, of whom 25 were symptomatic. Conventional parameters were evaluated, including QRS duration, R and S wave amplitudes ( Brugblockers , 2. Accoblockers , the impA number of depolarization disorders have been observed on BS patients, including a high prevalence of QRS late potentials (LP), fragmented QRS complexes , prolongRecent works from our group have shown that symptomatic BS patients exhibit greater fluctuations in sinus node response to ANS in 24-h Holter recordings  and an iIn this study, we propose a complementary analysis with respect to our previous works, in which advanced signal-processing methods are applied to extract quantitative markers of ventricular depolarization during exercise stress tests in patients with BS. The dynamic changes of these electrophysiological markers, which are associated with ventricular electrical conduction velocity, were then analyzed to assess differences between symptomatic and asymptomatic BS patients. Clinical characteristics of the population as well as the HRR index were finally integrated into a combined multivariate model to improve discriminative performance among these two groups of patients.110 consecutive patients  diagnosed with BS were enrolled in a prospective, multicentric study, led by the Cardiology department of the Rennes University Hospital (CHU de Rennes) between 2008 and 2013, in France. It was conducted in accordance with applicable good clinical practice requirements in Europe, French law, ICH E6 recommendations, and the ethical principles of the Helsinki Declaration (1996 and 2000). Participants were enrolled in 6 French hospitals located in Rennes, Saint Pierre de la Réunion, Nantes, Bordeaux, Brest and La Rochelle. The study protocol was approved by the respective local ethics committees: Comité d’Éthique du CHU de Rennes (ID RCB 2007-A00887-46), Comité d’Éthique du CHU Saint-Pierre, Comité d’Éthique du CHU de Nantes, Comité d’Éthique du CHU de Bordeaux, Comité d’Éthique du CHU de Brest and Comité d’Éthique du Centre Hospitalier de La Rochelle. All patients provided their written informed consent before participation.nd, 3rd or 4th intercostal space, or induced during a sodium channel-blocking administration, according to the current guidelines [Patients mean age was 44.6±13.7 years. BS was diagnosed when a coved ST-segment elevation ≥0.2 mV) was spontaneously observed in ≥1 right precordial leads located in the 2 mV was sStress Test Protocol: A standard 12-lead ECG acquired at a sampling rate of 1000 Hz was recorded for each patient using a standardized exercise stress test performed on a cycle ergometer , defined by the following protocol:HRmax = 220–age).Exercise period (EX): Comprised of an initial workload of 2 minutes pedaling at 50 W (30 W for women) and followed by successive increments of 30 W (20 W for women) every 2 minutes, until the patient reached at least 80% of the theoretical maximum HR (Recovery period (RE): It included two successive three-minute periods, one of active recovery with the patient pedaling at a workload of 50 W, followed by a passive recovery at rest.All ECG signals were preprocessed before the automatic extraction of the analyzed indices. This step included automatic QRS complex detection  and subsQRSd). QRSd was calculated by using a multilead-based approach that determines the earliest QRS onset, and latest QRS end among the available ECG leads [Conventional ECG markers associated with ventricular depolarization were assessed from the ECG signals: the amplitude of the R and S waves ; 2) the time of maximum effort (EXmax); 3) and the end of the complete recovery period, including both the active and passive recovery periods (REend). The mean values were calculated in 15-s windows duration for each phase. Throughout the manuscript, these values will be referred as i and j represent the involved phases. Two transition periods were thus analyzed: i) between the EXbeg and EXmax phases, indicating the change occurred during the effort  the ptomatic  and asymptomatic  groups.HRR), an autonomic-related measure resulting from a combination of the vagal activation and sympathetic withdrawal during the recovery period after effort, was also assessed in this study [HRR is defined as the heart rate decay occurred during the first minute of recovery evaluated immediately after maximal effort, as illustrated in The heart rate recovery index  integrating only selected ventricular depolarization markers and a second model (Model2) integrating ventricular depolarization markers and clinical features.The depolarization markers showing statistically significant differences between groups during the univariate analysis were used as input to a multivariate logistic regression analysis (MLRA). Two MLRA models were created: a first model (Model1 and Model2) meeting the above criterion was selected by splitting the entire population in 10 folds, and performing the cross-validation process that generates 10 testing errors, obtained from different, equally split population subsets not seen during learning.The LASSO L1-regularization technique was applied in both cases to find the most predictive features, while protecting against overfitting . This meModelXR, with X = {1, 2}. Moreover, potential confounding factors such as SNC5A mutation, ICD data and BMI values, were used for correcting the models, which were defined as The features selected according to the optimal λ were then used to obtain the final model applied to the whole dataset. In this last step, since standard statistical inference cannot be applied to LASSO coefficients, logistic regression was conducted without regularization, allowing for the detection of the most significant characteristics and further reducing the initial subset of predictive characteristics, yielding to even more reduced models denoted as beg). Moreover, the asymptomatic group showed significant variations from the maximal effort to the end of recovery, while little or negligible changes were observed during incremental exercise, particularly in lead V2. A complete table of the observed values of each marker is presented in the Dynamic changes quantified between the exercise and the recovery periods for all markers are presented in Model1 was obtained with the predictive parameters subset selected with the LASSO approach. By using the most relevant variables reported in Model2 was obtained following the same strategy as for Model1. In this case, the clinical characteristics reported in To assess the added value of depolarization dynamic features, Model1, were retained for this combined model. The most relevant features were Sex and HRR, as reflected by their p-values. Concerning HRR, negative coefficients indicate that for each one-unit increase in these features, the risk of being symptomatic decrease by a 0.01 and a 0.06-fold, respectively. This means that one-unit increase in HRR, increases the odds of not being symptomatic over 100-fold and 16.7-fold, respectively (1/0.01 = 100 and 1/0.06 = 16.7). Finally, concerning the Sex variable, the OR = 5.67 means that men have 5.67-fold higher risk of being symptomatic patients than women. By using only these three significant features, further reduced models were considered bellow, following the flowchart presented in As it can be seen from N = 79). Only significant parameters in Model2R_79. For this smaller subgroup, all markers remained significant, with Sex having an OR of 13.33 (p = 0.036), while for HRR it was <0.01 (p = 0.003) and 0.01 (p = 0.010) for p-values associated with HRR and Sex, whose OR increased from 13.3 to 20.6 (>50%). This suggests that association between male gender and symptoms is enhanced by the presence of the SCN5A mutation.Because the genetic screening was not performed in all patients, the multivariate analysis was separately applied to the subgroup of patients for which SNC5A mutation information was available , including thus patients for which no genetic screening was performed. Unlike the unadjusted model, Model2R_110, only p = 0.002) and its protective role was reinforced for the adjusted model . Conversely, BMI and ICD data confound the role of Sex  as a potential risk factor as well as the protective role of HRR  in terms of symptoms association, which were no longer meaningful after adjustment. Statistics about these two models are summarized in Since mutation data had no significant impact on two features of the reduced model, and the association of Model2—Model1—Model1 may still be suitable for discriminating between BS patient groups, using half the number of parameters with respect to Model2. Performance metrics remained similar when non significant features in Model2, in order to create the even more reduced model (Model2R_110). However, this reduced model with only 3 features, tested on the population subset that underwent genetic screening (N = 79), presented similar overall performances before (Model2R_79) and after .oved see , with AUStratifying the level of risk in asymptomatic Brugada patients is still a major clinical challenge. Better discriminative markers are needed to improve prognosis and to optimize the therapy for a given patient. Apart from classical high-risk markers reported in previous BS-related studies, such as spontaneous type-I ECG pattern and VF history, the analysis of ventricular depolarization in this population may provide an interesting source of information, especially if combined with ANS-related parameters. In fact, it has been previously shown that the application of multivariate methods that combine clinical and electrophysiological analysis provide more robust estimations for risk stratification in BS , 20.Regarding depolarization analysis, many previous studies have reported noninvasive risk markers for arrhythmic events in patients with BS, related to the QRS complex. For instance, a longer QRS duration in symptomatic patients have been observed in lead V2 . In factIn our study, we have investigated the potential usefulness of more robust and refined markers of ventricular depolarization, mainly related to conduction velocity and associated with the R and S waves shape. Unlike some previous studies , 28, we orted in  about detentials . The roltentials , 29. Indtentials . This fatentials . In thatet al. [ICa [The analysis of the dynamics of ventricular depolarization markers along the main phases of a stress test is another contribution of this work. In general, the major differences found between the two patient groups were associated with significantly larger modifications of depolarization markers through time (depolarization dynamics), observed in asymptomatic patients with respect to the symptomatic group. This large dynamicity in the asymptomatic group was even more pronounced during recovery, especially for markers o et al. . In thatsing ICa . The aboModel2) was able to discriminate among groups reaching a suitable performance outcome . Nevertheless, when only depolarization features were used, the performance of the model (Model1) was slightly smaller, despite the fact that only four features were included . This highlighted the significant contribution of dynamic depolarization properties in terms of discrimination between symptomatic and asymptomatic patients. Further multivariate analyses performed on the smaller subgroup screened for SCN5A mutations, and using only significant features from Model2, showed similar results even after correcting by mutation data. Indeed, this characteristic did not have a strong impact on the overall performance of the model since all the involved features remained significant after adjustment. Only the sex’s association with the presence of symptoms was, in fact, positively impacted . However, its association with the symptoms was dampened after controlling the model for potential confounding factors.In addition to the relevant parameters found when exploiting only the ventricular depolarization markers, the clinical characteristics of the population were also included in a multivariate logistic regression analysis. While none of the clinical parameters were significantly different between groups see , their cIt is well-known that early recovery of the heart rate, occurring immediately after cessation of exercise, is due to parasympathetic reactivation. Such reactivation is somehow captured by the HRR marker, that turned to be a protective factor in our combined model before adjustment. To some extent, this finding may be linked to the results reported in , where aFinally, as suggested by Postema et al. , repolarSymptomatic and asymptomatic patients with Brugada syndrome studied in this work have shown significantly different ventricular depolarization dynamics during exercise and mostly during recovery. Such differences are mainly observed through the proposed novel depolarization indices, associated with the second half and/or terminal part of the QRS complex. These findings may be useful to improve risk stratification for malignant arrhythmic events, specially in individual asymptomatic patients. Since the obtained results came from a retrospective data, further work is warranted to evaluate the proposed indices in a prospective study, with a larger patient population, and to assess the added value of a combined analysis of cardiac depolarization and repolarization parameters, together with different clinical factors and genetic status.S1 Table(PDF)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.