AITC (100 μM) increased (231%) area of spontaneous bladder contractions (SBCs) an effect reduced by a TRPA1 antagonist  and reversed to inhibition by indomethacin  a cyclooxygenase inhibitor. The post-INDO inhibitory effect of AITC was mimicked (39% depression) by calcitonin gene-related peptide  and blocked by a CGRP antagonist . CAPS (1 μM) suppressed SBCs by 30% in 81% of strips, an effect blocked by a TRPV1 antagonist  or BIBN. SBCs were suppressed by OA-NO2  or by OAG  an effect blocked by BIBN. OA-NO2 effects were not altered by HC3 or diarylpiperazine. OA-NO2 also induced excitation in 23% of bladder strips. These observations raise the possibility that guinea pig bladder is innervated by at least two types of afferent nerves: [1] Type A express TRPA1 receptors that induce the release of prostaglandins and excite the detrusor, [2] Type B express TRPV1, TRPA1 and TRPC receptors and release CGRP that inhibits the detrusor.Activation of TRP channels expressed in urinary bladder afferent nerves and urothelium releases neurotransmitters that influence bladder function. Experiments were undertaken to examine the mechanisms underlying effects of TRPA1 , TRPV1 , and TRPC  agonists on guinea pig bladder activity. Effects of these agonists were compared with effects of nitro-oleic acid (OA-NO The urinary bladder is innervated by small myelinated (Aδ) and unmyelinated (C-fiber) afferent nerves that respond to mechanical and chemical stimuli and then transmit information to the central nervous system about bladder filling and the chemical environment in the bladder  channels and synthesizes and releases neuropeptides such as neurokinins or calcitonin gene-related peptide (CGRP) . The effects of four agonists  were examined on spontaneous activity of bladder strips with intact mucosa from adult guinea pigs.The present experiments were undertaken to determine the mechanisms underlying effects of OA-NOn = 11) and female (n = 47) Hartley guinea pigs  were prepared as described previously  the bladders were removed and placed in room temperature Krebs solution . Each bladder with the mucosa intact was cut into eight longitudinal strips, extending from the bladder neck to the dome and were mounted in a vertical double-jacketed organ bath with oxygenated Krebs solution (15-ml volume) and kept at 37°C via a circulating water pump. Tissue was stretched by applying 10 mN (1 g) baseline tension and allowed to equilibrate for 1–2 h prior to drug testing. Antagonists were applied in the bath 15–60 min prior to or following agonist application.All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Bladder strips from adult 200 to 400g male -9- and 10-nitro-octadec-9-enoic acid] synthesized as described previously , 1-oleoyl-2-acetyl-sn-glycerol , human α calcitonin gene-related peptide , olcegepant , substance P , prostaglandin E2 (500 nM) and indomethacin  were obtained from Sigma Aldrich  or Tocris . Vehicles: distilled water for CGRP and SP; DMSO  for all other substances, did not significantly change the activity of bladder strips. Chemicals were used in concentrations shown to be effective in preliminary experiments or in concentrations used in previously published studies  which reflects changes in amplitude and frequency of SBCs and as a change in baseline tension. Data are reported as percent change from basal activity. Eighty percent of the experiments were conducted on tissue from female animals; and the data from both sexes were combined for data analysis because strips from male and female guinea pig did not exhibit obvious differences in response to agonists and antagonists. Statistical significance was tested on raw data using a paired two tailed t-test, *P < 0.05 and **P < 0.001, a one-way ANOVA with Dunnett's multiple comparison post-hoc test (+P < 0.05) or a two-way ANOVA and Bonferroni multiple comparisons post-hoc test  when appropriate. Excel and Graph Pad Prism 7 software were used for analysis. Data were considered statistically significant when P < 0.05.All data are presented as mean ± S.E.M. To study the onset and duration of agonist-evoked responses, majority of the bladder strip activity was measured in 5 min intervals immediately before  and at varying intervals after application of the agonists, during the time period when they exerted their maximal or prolonged effects: [a] 0–30 s for CAPS early transient response , [b] 0-5 min for OA-NOn = 16 strips from nine female and two male guinea pigs; P <0.001; When stretched with 1 g tension, guinea pig bladder strips displayed spontaneous bladder contractions (SBCs) of variable amplitude and frequency  that perP < 0.002) followed by a more prominent inhibition in 81% of 26 strips from 1 male and 16 female guinea pigs .CAPS at a concentration (1 μM) that enhances rat SBCs . Inhibition was present in 77% of strips (P < 0.001) measured 0–5 min after application, while excitation occurred in 23% of strips (P < 0.01). Neither response was accompanied by a significant change in the baseline tension . Oleic acid (30 μM), a non-electrophilic fatty acid control, did not alter AUC or baseline tension .In contrast to its excitatory effect in rat bladder strips , a TRPC3,6,7 channel agonist .To examine the role of efferent neurons or an intramural neural network, in the responses of TRP channel agonists the agonists were administered in the presence of TTX. TTX did not significantly alter: [1] control SBCs . On the other hand, the second application of AITC elicited a response , that was not significantly different (P > 0.05) from that elicited by the first application (data not shown).In a separate group of experiments, agonists were applied multiple times to the same bladder strip after washout and long recovery periods (60–100 min). Neither OA-NO2, even at a time point when the SBCs had recovered from the initial inhibitory effect of CAPS . On the other hand, OA-NO2 did not alter the inhibitory response to the subsequent application of CAPS (n = 17 strips from 13 guinea pigs). Administration of AITC during the inhibition induced by the combined application of OA-NO2 and CAPS rapidly reversed the inhibition .In another group of experiments where multiple agents were administered sequentially to the same bladder strip  applicatn = 12 strips from five guinea pigs) and the facilitatory response of AITC was significantly reduced (n = 16 strips from four guinea pigs). Application of DP or HC3 alone or in combination did not change the SBCs , a TRPV1 antagonist and HC3-03001 , a selective TRPA1 receptor antagonist, at concentrations used in previous studies  or TRPA1  antagonists , an agent known to block various subtypes of TRPC channels , whereas, 50 μM La3+ blocked OAG suppression of SBC AUC  alone slightly decreased AUC  but did not affect baseline tension . Higher concentrations of La3+ could not be tested because they further suppressed SBCs.The inhibitory effect of OA-NOn = 9 strips from nine guinea pigs, P < 0.001) and this inhibition was reversed , CAPS  and OAG . Thus, the block of inhibition by BIBN did not unmask prolonged facilitatory effects of CAPS or OANO2 that occurred in the minority of untreated preparations  but only slightly increased baseline tension . To determine the role of SP release in AITC induced excitation, bladder strips were pretreated with a combination of neurokinin receptor 1, 2, and 3 antagonists  which blocks the excitatory effect of substance P in the guinea pig ileum  decreased AUC by 33 ± 9% (P < 0.05), without changing the baseline tension and significantly reduced the SP induced enhancement of SBCs  indicating that SP released from bladder afferents is not the primary mechanism for AITC induced excitatory responses.Substance P (SP) released from bladder afferents contributes to the excitatory effects of CAPS, OA-NO2 (500 nM) significantly increased the SBC AUC by 173 ± 76% as well as the baseline tension by 66 ± 40% . BIBN (25 μM) blocked the AITC inhibition unmasked by INDO or converted the inhibition to excitation  suppress the contractile activity in the majority of bladder strips by releasing CGRP [3]. The inhibitory effect of CAPS is preceded by a transient facilitation that is blocked by TTX and therefore likely mediated by activation of neurons in the bladder wall.This study revealed that activation of TRP channels in guinea pig urinary bladder can either enhance or suppress spontaneous contractile activity by releasing prostaglandins or CGRP, respectively. The main findings are: [1] a TRPA1 agonist (AITC) enhances SBCs by releasing prostaglandins. This effect is blocked by indomethacin (INDO) a COX inhibitor. AITC also inhibits SBCs by releasing CGRP an effect blocked by BIBN, a CGRP receptor antagonist. [2] A TRPV1 agonist (CAPS), and TRPC agonists (OA-NO2 induced inhibition of bladder activity (The mechanisms underlying the effects of the TRP agonists on bladder activity are commonly ascribed to activation of transmitter release from intramural afferent nerves because: [1] TRP channels are expressed in bladder afferent neurons Maggi, , 2018,  CGRactivity , 1989, activity . Howeveractivity , which cactivity , it is p2 responsive guinea pig DRG neurons also respond to CAPS indicating co-expression of TRPV1 and TRPC channels in these neurons (Zhang et al., 2 (Assuming that afferent nerves are the principal target for the agonists , it is lt al., 2  further 2 or CAPS (CGRP could act at multiple sites to modulate the contractile activity of the bladder strips. CGRP has excitatory or facilitatory effects on peripheral (Cornelissen et al.,  or CAPS , 3E. Thu or CAPS .AITC has facilitatory and inhibitory effects on bladder activity . The facThe distribution of TRPA1 channels in two populations of bladder afferents  is consi2+ in the sensory nerve terminals or non-neural cells.In the majority of strips CAPS produced a small initial transient enhancement followed by the CGRP mediated inhibition. A similar bimodal effect of CAPS in guinea pig bladder preparations was reported in earlier studies (Maggi et al., TRPV1 and TRPA1 antagonists did not change spontaneous activity , 3E; howThe main limitation of our study is the use of a single technique, i.e., the measurement of spontaneous contractions of bladder strips and the use of only pharmacological approaches to examine the functions of TRP channels in the bladder. More detailed information about the distribution of TRP channels in the bladder will be obtained in future experiments using immunohistochemical techniques. Mucosa free bladder strips will also be used to limit the possible sites of drug action.2, to selectively target the type B afferent population and induce CGRP mediated inhibition combined with its known anti-inflammatory actions might generate a synergistic interaction that could result in an effective treatment for some types of overactive bladder dysfunction.In summary, activation of TRP channels in guinea pig bladder strips enhances or suppresses SBCs. It is likely that one mechanism underlying these effects is release of signaling molecules from bladder afferent nerves which express multiple TRP channels. Activation of TRPV1, TRPA1 and TRPC channels expressed by one type of afferent nerve releases CGRP that elicits inhibition; while activation of TRPA1 channels in another type of afferent nerve and/or in non-neuronal cells releases prostaglandins that elicit excitation . CGRP anThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Ethics Committee of Pittsburg University.WG, BF, and XZ designed experiments. SD, KK, and JL carried out experiments and analyzed data. JB, SW, and XZ wrote the manuscript. WG critically edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Altered functioning of the inhibition system and the resulting higher impulsivity are known to play a major role in overeating. Considering the great impact of disinhibited eating behavior on obesity onset and maintenance, this systematic review of the literature aims at identifying to what extent the brain inhibitory networks are impaired in individuals with obesity. It also aims at examining whether the presence of binge eating disorder leads to similar although steeper neural deterioration. We identified 12 studies that specifically assessed impulsivity during neuroimaging. We found a significant alteration of neural circuits primarily involving the frontal and limbic regions. Functional activity results show BMI-dependent hypoactivity of frontal regions during cognitive inhibition and either increased or decreased patterns of activity in several other brain regions, according to their respective role in inhibition processes. The presence of binge eating disorder results in further aggravation of those neural alterations. Connectivity results mainly report strengthened connectivity patterns across frontal, parietal, and limbic networks. Neuroimaging studies suggest significant impairment of various neural circuits involved in inhibition processes in individuals with obesity. The elaboration of accurate therapeutic neurocognitive interventions, however, requires further investigations, for a deeper identification and understanding of obesity-related alterations of the inhibition brain system. Over the last decade, health care professionals have been exposed to the emergence of a new form of addiction—food addiction . Eating Parallel to reward system hyperactivity, but less extensively investigated, a hypoactivity of the inhibition system was observed in individuals with obesity. Specifically, (f)MRI studies reported decreased gray matter volume  and funcSuch imbalance between the two systems will behaviorally translate into an impulsive way to act , charactInterestingly, the presence of binge eating disorder (BED) leads to even greater impulsive traits , 36. ThiRecent brain stimulation studies provided promising results on the control of food craving and food intake . SpecifiConsidering the importance of a precise identification of the different brain areas involved in the functioning of inhibition processes for the elaboration of relevant and accurate neurocognitive therapies, we propose in this review to gather the results provided by neuroimaging studies. The main aims are to [1] provide a detailed report of the brain regions showing obesity-related impaired function during response inhibition and [2] examine whether BED leads to similar although steeper neural deterioration.To identify studies aiming at investigating the neural basis of the inhibitory system in obese individuals, we used Google Scholar and PubMed databases using the following keywords: impulsive, impulsivity, inhibition, inhibitory system, and executive functions, each combined with the following terms: food, feeding behavior, obesity, and obese. Additional papers were also found from the reference lists of the selected papers. To comply with inclusion, studies had to [1] use a behavioral task specifically designed for the assessment of impulsivity, which [2] participants completed during functional magnetic resonance imaging (MRI) or magnetoencephalography (MEG), and [3] entail a lean control group (except in the specific case of comparison between obese with and without binge eating disorder). Moreover, participants from the experimental group had to [4] be obese (BMI ≥ 30) and not “only” overweight (25 < BMI < 29). Nonetheless, due to the lack of studies investigating impulsivity in individuals with BED, we decided to also include studies where participants were normal-weight binge eaters. Brain activation data were extracted from the MNI coordinates reported in the results sections of the original articles.The initial search identified 3,009 studies. After removal of duplicates and exclusions through title and abstract screening, 30 studies were assessed for eligibility. For design, population, and control reasons, 19 studies were excluded. After we further searched for articles on lean individuals with BED, one more article was selected. Twelve studies were hence included in this systematic review of the literature .n = 8) or in obese with BED in comparison with obese without BED (n = 2) and one in lean individuals with BED in comparison with lean without BED. Differences were mainly observed in the frontal  and stop signal tasks (action cancellation) engage overlapping but also different brain activations during successful inhibition. More precisely, while the medial prefrontal cortex and the insula were found to be engaged during both tasks, the go/no-go task triggers further activation in the fronto-parietal network and the stop signal task in the cingulo-opercular network . The StrFrontal regions, including the prefrontal (PFC) and premotor cortices, have been consistently found to be involved in the inhibitory system –62. ThisCompelling data from the studies that investigated the neural correlates of behavioral inhibition in obese in comparison with lean individuals clearly emphasize a hypoactivity of the PFC in the totality of both its medial and dorsal parts, suggesting obesity to be linked to a global impairment of this brain region, known to be a central hub for inhibitory processes.Located in the inferior part of the frontal lobe, the orbitofrontal cortex (OFC), known to be involved in encoding reward value and decision-making , plays ainter alia decision-making (The anterior part of the PFC (BA10) plays a central role in cognitive processes. BA10 is parcellated into an inferior and a superior part, respectively, named ventromedial and dorsomedial PFC. While each of these subregions endorses distinct cognitive roles, such as emotional regulation, salience attribution , and foon-making –70 and un-making  for the n-making , 73 and n-making , 75 weren-making , 77, thin-making , 48.The lateral part of the PFC entails the dorsolateral and ventrolateral PFC. Postulated to be involved in cognitive inhibition processes , 79, theDecreased patterns of activity were also reported in parts of both the left  and rigOn the other hand, Levy and Wagner's meta-analysis  revealedSituated in the superior part of the frontal cortex, Brodmann area (BA) 6 is located on the posterior part of the premotor cortex and corresponds to the supplementary motor area, also described as secondary motor cortex. Several behavioral tasks, such as the go/no-go and stop-signal task, emphasize its importance for response inhibition , 91, 92.The limbic system is composed of a set of interconnected subcortical but also cortical structures. Those numerous connections form complex circuits , known tIn the present review, we report the major contributions of the limbic system to eating behavior control, but impaired among the obese population, either showing hypo- or hyperactivity in different limbic regions according to their functional role in inhibitory control .As part of the gustatory cortex , the insConsistent with the results suggesting increased insular activations during inhibition to be characteristic of inhibitory control difficulties  and to cThe posterior part of the cingulate cortex is involved in processing emotionally relevant stimuli and memory-related functions , 105. InBesides forming an integral part of the limbic system, the anterior cingulate cortex is often considered as belonging to the frontal cortex  and assovia bottom-up and top-down control  with the putamen, cingulate cortex, supplementary motor area, precuneus, and inferior parietal cortex  Figure , suggestConversely, a U-shaped pattern of functional connectivity was reported between the amygdala, involved in motivational salience encoding , and regPart of the obese population is confronted with an aggravated form of excessive eating behavior, consisting in consuming a large amount of food within a short period of time, paired with a sensation of loss of control. This pathological eating behavior, named BED, is referenced as a stand-alone illness in the DSM-5 . As bingBinge eating episodes can also occur in normal-weight individuals . They arThis review article entails studies that used general as well as food-specific paradigms to investigate inhibition processes, despite coming from different methodological paradigms . Thus, this precludes obtaining information on the general or specific aspect of inhibition impairment in individuals with obesity and BED. Moreover, only 12 studies could be included, hence not allowing detailed and firm conclusions to be drawn. That is why we aimed at additionally adding EEG studies. However, all studies we found, although they showed impaired inhibition response, did not report on precise brain regions. Thus, we did not include EEG studies in this review.In this review, we gathered further evidence of shared neural impairment underlying drug and food addiction, mainly targeting the frontal and limbic regions. Specifically, obesity is linked to alterations of the dopaminergic system during response inhibition, translating into hypoactivity of the frontal regions and either hypo- or hyperactivity of the limbic regions according to their role in behavior control. We further conclude that the presence of BED may lead to similar although greater impairment of the inhibition system. We however suggest that a deeper and more grounded understanding of the inhibition system impairment related to obesity should constitute the subject matter of future studies. Investigating whether alterations of the dopaminergic function and its related inhibition processes in obese individuals are rather general or specific to eating behavior, and also whether it varies within different classes of obesity, could provide valuable insights for the comprehension of inhibition impairment in the obese population.In summary, inhibition processes are found to rely on a complex neural network involving the prefrontal but also the frontal and subcortical regions. Whether stimulation of the identified key areas, such as the striatum, orbitofrontal, medial prefrontal, cingulate, or insular cortex, may lead to substantial improved eating control deserves future non-invasive  as well as invasive brain stimulation studies (deep brain stimulation).The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.ES wrote the manuscript. BP revised the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ABSTRACT IMPACT: This study will answer key questions that spine surgeons have regarding techniques used in cement augmentation of vertebral compression fractures and will ultimately advance patient care for such injuries. OBJECTIVES/GOALS: The objective of this study is to determine if a difference exists in load-bearing characteristics and load-to-fracture between injecting cement anteriorly prior to screw placement versus cement augmentation via fenestrated pedicle screws. We also expect differences in load-to-failure characteristics between different cement volumes. METHODS/STUDY POPULATION: This study will be performed in a bioengineering laboratory that has access to a Materials Testing System (MTS). Eight cadaveric specimens will be selected from our stock after pre-screening via CT for inclusion and exclusion criteria. The levels T8-L1 will be dissected from the vertebral column along with any soft tissue structures. The vertebral bodies will be potted in an epoxy mold. From each spine, there are 2 groups of three. One vertebral body from each spine will serve as an internal control, one will be augmented with cement via a cannula and then instrumented with a non-fenestrated screw and the third will be instrumented will a fenestrated screw and then augmented with cement. After appropriate curing time, repeat CT imaging will be completed. The specimens will then be loaded to failure and the results analyzed. RESULTS/ANTICIPATED RESULTS: We hypothesize that we will see a better anterior spread with the cannula/non-fenestrated screw method as compared to the fenestrated screw. The reason being is that we would expect the fenestrated screw to experience more cement extruding from the fenestration rather than being directed anteriorly. We believe a better anterior spread of the cement will lead to a greater load-bearing capacity for the vertebral body. We also believe that a difference will exist in load-to-failure testing with the two volumes being tested, though we cannot predict to what a degree this difference will be impactful as there have been few studies prior looking at this. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study is significant because it will aid in determining the optimal technique to implement in the setting of vertebral compression fractures. This will lead to improved patient care as well as a greater understanding of the instrumentation used in such procedures. The results will lay the groundwork for future research on this procedure.


Cerebrovascular reactivity (CVR) is defined as the ratio of the cerebral blood flow (CBF) response to an increase in a vasoactive stimulus. We used changes in blood oxygenation level-dependent (BOLD) MRI as surrogates for changes of CBF, and standardized quantitative changes in arterial partial pressure of carbon dioxide as the stimulus. Despite uniform stimulus and test conditions, differences in voxel-wise BOLD changes between testing sites may remain, attributable to physiologic and machine variability. We generated a reference atlas of normal CVR metrics (voxel-wise mean and SD) for each of two sites. We hypothesized that there would be no significant differences in CVR between the two atlases enabling each atlas to be used at any site. A total of 69 healthy subjects were tested to create site-specific atlases, with 20 of those individuals tested at both sites. 38 subjects were scanned at Site 1  and 51 subjects were tested at Site 2 . MRI platforms were: Site 1, 3T Magnetom Skyra Siemens scanner with 20-channel head and neck coil; and Site 2, 3T HDx Signa GE scanner with 8-channel head coil. To construct the atlases, test results of individual subjects were co-registered into a standard space and voxel-wise mean and SD CVR metrics were calculated. Map comparisons of z scores found no significant differences between white matter or gray matter in the 20 subjects scanned at both sites when analyzed with either atlas. We conclude that individual CVR testing, and atlas generation are compatible across sites provided that standardized respiratory stimuli and BOLD MRI scan parameters are used. This enables the use of a single atlas to score the normality of CVR metrics across multiple sites. Presently, there are a number of techniques that have been developed for the assessment and mapping of CVR. Of these, blood oxygenation level-dependent (BOLD) magnetic resonance imaging (MRI) signal intensity as a surrogate measure of cerebral blood flow (CBF) is in common use. However, the vasodilatory stimulus in most instances is uncontrolled, impeding test standardization, which is required for research and clinical use. Stimulus standardization requires precise, repeatable changes in PaCO2 independent of subject, or patient, efforts. A standardized “brain stress test” would provide a uniform platform from which to assess and manage a wide variety of disorders affecting brain blood flow regulation, such as in steno-occlusive disease . Whereas absolute values for CVR may vary from center to center with the stimulus protocol and scan parameters, as long as the protocol for the CVR test and the atlas are the same, the z scores should be comparable across platforms. However, it would be very burdensome, expensive, and inefficient if an atlas had to be generated for every scanner. We hypothesized that if a repeatable and quantitative ventilatory protocol could be applied in conjunction with matching of critical MRI scan parameters, the variability in time-point to time-point and scanner-to-scanner variances could be minimized. A single healthy control atlas derived from one MRI system could then be used to assess single subjects across platforms. It would also enable the comparison of individuals and cohorts across institutions for use in multi-center trials.The first step for comparing data across scanners required the generation of an atlas consisting of merged CVR data from healthy subjects from which was calculated a voxel-wise mean and standard deviation of CVR. Single subject’s CVR metrics can be compared to those of the atlas, analogous to the way blood clotting tests are compared to the normal range for The aim of this study was to evaluate whether the CVR atlases generated on different MRI platforms with the same, reproducible stimulus and the same scan parameters would have sufficient uniformity to be interchangeable across platforms for normalizing individual CVR data.This study conformed to the standards set by the latest revision of the Declaration of Helsinki and were approved by the Research Ethics Board of the University Health Network and Health Canada. All subjects provided written and informed consent to participate in this study. We recruited 69 voluntary, healthy participants  between the ages of 18–80 years old by advertisement and word of mouth. Each subject was in good health with no history of neurological or cardiovascular disease, non-smokers taking no medication. The demographics for the cohort are represented in CVR studies were performed on two separate MRI platforms. At Site 1, a 3T Magnetom SKYRAfit Siemens scanner with a 20-channel head and neck coil  was used, while at Site 2, a 3T HDx Signa GE scanner with an 8-channel head coil  was used.2 protocols. A total of 69 healthy subjects were scanned to create site-specific atlases . From the 69 participants, 20 of those individuals completed CVRs at both sites within a 2-month time frame.All CVR studies reported in this study at both Site 1 and Site 2 consisted of matching MRI parameters and identical experimental CO2 stimulus consisted of clamping end-tidal partial pressure of CO2 (PETCO2) at the subjects resting value for 2 min, followed by a step increase by 10 mmHg for 2 min and returned to baseline for another 2 min. PETCO2 was then reduced by 10 mmHg for 1 minute, followed by a steady rise in PETCO2 to 15 mmHg above baseline over ∼5 min, and returned to baseline for 2 min. The stimulus pattern shown in TM, Thornhill Research, Toronto, Canada) that runs a prospective gas targeting algorithm  that is also precisely controlled, independently of CO2, by the RespirActTM system.Subjects breathed through a fitted facemask connected to a sequential gas delivery breathing circuit . The CO2lgorithm , which cto PaCO2 . The ent1. 3D T1-weighted Inversion-Recovery Prepared Fast Spoiled Gradient Echo (IR-FSPGR) with TI = 450 ms, TR = 7.88 ms, TE = 3 ms, flip angle = 12°, voxel = 0.859 × 0.859 × 1 mm, matrix = 256 × 256, 146 slices, field of view = 24 × 24 cm, no interslice gap, acquisition time = 7.4 min. 2. T2∗-weighted gradient recalled echo sequence with echo-planar readout (EPI-GRE) with TR = 2400 ms, TE = 30 ms, flip angle = 70°, 41 slices, voxel = 3.5 mm3, matrix = 64 × 64, number of volumes = 335, field of view = 24 × 24 cm, acquisition time = 13.4 min.The protocol consisted of: ETCO2 data were analyzed using AFNI software 1 . CVR was expressed as the percent change in BOLD signal per change in PETCO2 (%/mmHg). Step and ramp CVR maps were color-coded based on a spectrum of colors denoting the magnitude of response in either the positive or negative direction (The acquired BOLD MRI scan and Pryland)1 . BOLD imlsewhere . CVR forlsewhere , and theirection .2. A voxel-by-voxel mean CVR, and associated standard deviation (SD), was calculated for the step and ramp potions of the stimulus, to form site-specific atlases  standard space using an analytical processing software software . Four sesoftware . The co-t-tests (α = 0.05) were used to determine if significant differences existed between the 20 person atlases of each CVR parameter (step and ramp), which contained only subjects that were scanned at both sites. Permutation testing was then applied to the height of the maxima of the resulting statistic image, using the “randomize” permutation-based inference tool . To evaluate measurement reproducibility and investigate possible bias in the CVR data, Bland-Altman analysis was used to calculate the two sites limits of agreement between the 20 person atlases for GM and WM of each CVR parameter. A Two-Way repeated measures analysis of variance (rmANOVA) with the Holm-Sidak method of multiple comparison correction was performed on the 20 person atlases to determine if any significant differences existed between the groups (α = 0.05). For the full site atlases, a Two-Way ANOVA was performed with the Holm-Sidak method of multiple comparison correction (α = 0.05).Voxel-wise paired nce tool  in FSL vIndividualized z scores were calculated on a voxel-wise basis by co-registering the subject’s CVR map to the same space as the reference atlases followed by scoring the subject’s CVR values by assigning a z-score according to the mean and SD of the corresponding voxel of the atlas . A healtt-test for each of the CVR parameter maps between Site 1 and Site 2 was preformed, and significant differences (p < 0.05) are displayed in CVR step and ramp atlas maps were created for the 20 individuals that were scanned at both sites and compared to one another to discern site-related differences. –5 ± 0.068%/mmHg in GM and –0.009 ± 0.035%/mmHg in WM. Mean site difference for CVR ramp was 0.037 ± 0.099%/mmHg in GM and 0.017 ± 0.050%/mmHg in WM.The results of the Bland-Altman analysis for between site reproducibility for the 20 individuals that were scanned at both sites are illustrated in P-values presented in P-values presented in Mean step and ramp CVR values of the 20 subjects scanned at Site 1 and Site 2 for GM, WM and each hemispheric vascular territory, were calculated. GM and WM values are presented in CVR step and ramp atlas maps were created for all subjects scanned at either site, with 38 subjects in total scanned at Site 1 and 51 subjects scanned at Site 2. p-values for GM and WM are presented in P-values presented in P-values presented in A Two-Way ANOVA was performed for each of the CVR parameter maps comparing all subjects scanned at each site for GM, WM, and each hemispheric vascular territory. The overall means, SD, and associated t-tests applied to the atlases showed minimal differences . The ramp CVR atlases which use only a few points of the full ramp stimulus (resting PETCO2 + 10 mmHg) and are consequently more variable than the step CVR measurements, also showed several ROIs that were significantly different between sites, with the majority again in the ACA territories. However, interaction between site and ROI was also found and so these findings are not interpretable with respect to site difference.With respect to further atlas comparison using two-way rmANOVA, although such analysis of the step CVR atlases found significant differences for most of the GM and WM ACA territories (areas prone to susceptibility artifacts), these differences were not interpretable with respect to site difference since there was a statistically significant interaction between factors Site and ROI  between machines, the z-map images will be otherwise the same. The variability between subjects is expected to be greater than the experimental noise within a subject even with a lower SNR coil. The effect of using different scan parameters such as voxel size on the individual scans and comparing them to the atlases was not examined.If these two technical issues are standardized, the remaining sources of variability between scans must arise from differences in physiological factors affecting the MR signals. These include the physiological expression of the variety of day-to-day experiences such as amount of sleep the previous night, foods eaten, tobacco use, emotional state, and ingested medications. However, the effects of these experiences may be dampened or cancel each other out. In any event, their effect size will be small compared to those of the technical conditions. Indeed test-retest differences were measured and quantified in a single scanner over weeks to months as mean ± SD for each voxel by When z scoring of scans was first contemplated, it was considered that each institution would need to use their own standardized stimulus pattern for patients and repeat that pattern on a control group to generate an atlas for each scanner. Our findings suggest that for a standardized stimulus and MRI sequence, the variation in CVR is sufficiently small that a single atlas would be suitable across platforms. In addition, specialized atlases such as those controlling for sex, age, or medication, can be distributed across scanner locations. These considerations imply that all CVR data generated by a standard stimulus and scanner settings would be comparable, enabling a standard CVR test.For all intents and purposes, the CVR atlas functions as the “control” cohort for all CVR studies, thereby requiring smaller test cohorts. The reduced test variability would also enable the detection of smaller effect sizes with smaller cohorts. Data from various centers would be directly comparable, as is the case with other standardized tests such as the clotting test prothrombin time. The atlas can be made to have greater specificity for a given condition by narrowing the variability of the cohort to control for specific factors. For example, for examining CVR in patients with multiple sclerosis, the atlas can be restricted to subjects of younger age, and if examining females, then restricted by eliminating data from males.15O–)H2O positron emission tomography (PET)  is assumed for matching BOLD to PETCO2, this relationship is very complex and not necessarily well approximated by assuming a GLM  . With reng a GLM .Atlases were also constructed and compared between sites using all subjects tested so that they included CVR data from the 20 subjects studied in both locations. While this comparison is like that of the matched 20 atlases, nevertheless, we feel that these atlases were worth examining because they contained many subjects unique to a particular test site, and even when used to examine a cohort, the data from the overlapped subjects were gathered at the alternate location.Small differences were apparent in both the atlas maps and the z score maps. The two scanner specific atlases derived from the same group of 20 individuals tested on both 3T scanners from different vendors were virtually identical but exhibited small differences . HoweverComparing CVR z score maps  of a sinVariability within the atlases may be attributed to differences in age and sex, although we have not found differences in CVR across age ranges . SubjectThis study has demonstrated the feasibility of using a single atlas across platforms. A single atlas would be useful for normalizing clinical studies of individual patients as well as pooling compatible data in multicenter trials.joe.fisher@utoronto.caAnonymized data will be shared by request from any qualified investigator for purposes such as replicating procedures and results presented in the article provided that data transfer is in agreement with the research institution on the general data protection regulation. Requests to access the datasets should be directed to JAF, The studies involving human participants were reviewed and approved by the University Health Network. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.OS and KS were involved with data collection. OS, ES, JD, and JAF were involved in the data analysis and drafted the manuscript. All authors participated in the feedback and writing process following the initial drafting of the manuscript, contributed to the design and conceptualization of the study.TM  used in this study and have equity in the company. OS and JD received salary support from TRI. TRI provided no other support for the study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.JAF and DM contributed to the development of the automated end-tidal targeting device, RespirAct


Our research was designed to decide whether the application of C2 pars screws is an alternative choice for patients with OPLL involving the C2 segment.A total of 40 patients who underwent cervical laminectomy with fusion (LF) from C2 to C6 for OPLL were reviewed. Among them, C2 pedicle screws were placed in 23 patients, who were the pedicle group, and C2 pars screws were placed in 17 patients, who were the pars group. The screw placement and vertebral artery (VA) anatomy presented by standard CT. General clinical characteristics and health-related outcomes were evaluated and compared preoperatively and during the follow-up period.p = 0.044; blood loss: 383.53 ± 116.19 vs 457.83 ± 145.45, p = 0.039). Operation time and blood loss were both independently related to the pars group . The idealization and acceptability of C2 screws in the pars group exceeded those in the pedicle group (100% vs 91.3%). However, no statistically obvious variation in the included complications or health-related outcomes between the pedicle and pars groups was observed.The Pars group tended to have a shorter operation duration and less blood loss than the pedicle group (operation time: 115.29 ± 28.75 vs 133.48 ± 26.22, In the treatment of patients with OPLL involving the C2 segment, the application of C2 pars screws is an alternative choice, which is not only safer but also reduces the amount of blood loss, shortens the operation time and obtains a more ideal screw placement. As a multifactorial disease caused by ectopic bone hyperplasia and posterior longitudinal ligament calcification , OssificA total of 40 patients who received cervical laminectomy with fusion (LF) for OPLL between January 2010 and January 2017 were reviewed retrospectively. Among them, C2 pedicle screws were placed in 23 patients, and C2 pars screws were placed in 17 patients. All the surgeries were finished successfully  leakage provided clinical data. Clinical and neurological observations of each patient’s postoperative process were performed to ensure that there was no evidence of nerve death or stroke due to VA injury before discharge. Radiological analysis includes evaluating cervical CT scans for the quantification of the patient’s bone and vascular anatomy, the classification of the accuracy of C2 screw placement and the judgement of screw looseness and failure.The classification of screw positioning was made based on the modification below of the All India Institute of Medical Sciences result-based classification described for grading pedicle screws : Type I:C2 pedicle screw: The reference frame was connected to the Mayfield clamp or spinous process after exposure. The intraoperative CT images were got by O-arm navigation . Then, the transfer of images to the operational guidance system  of the stealth station was made, and the registration and verification of instruments were made for accuracy. To optimize the screw length and avoid neurovascular structure, the entry point of the pedicle screw was determined by CT image guidance. Pedicle screw placement entered the medial and upper quadrants of the pars surface. When the image guidance system is used, the navigation drill and power drill advance along the required trajectory. Then, the ball-tip probe was applied for the exploration of whether there was any breach. The screws were placed on the same track after tapping.C2 pars screw: After the C2 lamina and lateral mass were exposed, C2 pars were dissected directly under the periosteum. Then, a pilot hole was drilled with a high-speed drill. The entry point of the pars screw is usually inferior and lateral to that of the pedicle screw, about 4 mm lateral and cranial to the inferomedial edge of the C2–3 facet joint. Under direct anatomical visualization, the screw was inserted freehand, and the trajectory pointed to the junction of the pars and superior facet joint  (score 0–17) and recovery rate (RR) (postoperative score-preoperative mark)/(17-preoperative mark) × 100%.). An RR higher than 75% was regarded excellent, 50 to 75% good, 25 to 49% fair, and less than 25% poor. Axial pain around the posterior neck or suprascapular areas was assessed by visual Analog Scale (VAS) scoring system (range 1–10) and Neck Disability Index (NDI) (range 0–50).p < 0.05 level.Data are presented as the number of subjects in each group or mean ± standard deviation and were explored with the SPSS program . Each independent variable was compared between the two groups using the independent-sample t-test or Mann-Whitney U test for continuous variables and the χ2 test, continuity correction test or Fisher’s exact test for categorical variables. Each continuous variable was compared preoperatively and at follow-up using the Wilcoxon signed rank test. Relevant factors of pars screw placement for patients with OPLL were analyzed applying multivariate logistic regression analysis, and the outcomes are shown as odds ratios (ORs) and 95% confidence intervals (CIs). Significance was indicated at the Forty patients with multilevel cervical OPLL received surgeries with cervical LF and selected for the current study. Twenty-three patients were treated with C2 pedicle screws, and 17 patients were treated with C2 pars screws. The patients’ features are summarized in Table p = 0.055). The pedicle group was related to a longer operation duration and more blood loss than the pars group . Of all the C2 screws with postoperative CT imaging, none were regarded unacceptable (Type III). In the pedicle group, 42 pedicle screws were considered ideal (Type I), and 4 pedicle screws were considered acceptable (Type II). However, all pars screws were considered ideal (Type I) in the pars group. Following surgery, the idealization and acceptability of C2 screws in the pars group exceeded those in the pedicle group (91.3% vs 100%). Complications included VA injury , spinal cord injury , neurologic injury , CSF leakage  and screw looseness/failure   postoperatively was 5.87 in the pedicle group and 5.18 in the pars group   and posterior procedures  have been established . HoweverThere have been many reports on the placement technology of C2 pars screws and C2 pedicle screws, with slightly different starting points and placement trajectories –23. CompIn the past 10 years, we have noticed some general trends in our hospital for C2 posterior fixation in patients with OPLL involving the C2 segment. The use of C2 pedicle screws seems to decrease with the appearance of C2 pars screws, indicating that the latter has become the preferred fixation method for patients with challenging VA anatomy. Especially in elderly patients, due to the abnormal and fragile vertebral bone caused by osteoporosis, the use of pedicle screws will have a high risk of VA injury, while short pars screws are usually used when the VA is abnormal or pedicle screw fixation is used when the risk is too high, which indicates that it has become the screw of choice for patients with challenging VA anatomy. Our study found that with the same segmental fixation, the postoperative health-related outcomes of the two groups did not show significant differences, which may be related to the fact that both groups of patients received adequate decompression. However, the operation time and blood loss related to C2 pars screws were significantly less than those of C2 pedicle screws. This may be because compared with a C2 pedicle screw, a C2 pars screw has less muscle dissection on C2 lateral mass and back of lamina and retains the C2 attachment muscle group to a large extent, which reduces partial blood loss. At the same time, the tail direction of C2 pars screws is more medial than C2 pedicle screws, so it is more conducive to the connection of the screw rod system, which saves time in bending and inserting the connecting rod. In addition, O-arm navigation is required for C2 pedicle screw placement, which undoubtedly increases the operation time and repeated adjustment of the screw placement direction also indirectly increases unnecessary blood loss. However, as long as the C2 epidural venous plexus is not damaged, there will be no more blood loss during C2 pedicle screw placement. We think this is an invasion of the muscle, but if it is separated under the periosteum, the muscle will hardly bleed. So the difference in our research may be related to exposure. In addition, for highly skilled surgeons, the implantation of C2 pedicle screws is performed without O-arm navigation and normal fluoroscopy imaging. Therefore, the advantages of pars screw technology in operation time and blood loss may be weakened. According to previous reports, the rate of VA damage and imaging screw dislocation rate of C2 pars screws were both significantly lower than those of C2 pedicle screws. None of the C2 pedicle screws or pars screws had VA, spinal cord, neurologic injury or screw looseness/failure in our study. One patient in both groups showed CSF leakage. The incidence of screw misalignment is also much lower than that reported in previous studies. The high extent of accuracy was attributed to the excellent surgical technique. The ideal and acceptable degree of screw placement in the pedicle group was 91.3%, while the ideal and acceptable degree of screw placement in the pars group was as high as 100%. Although it has not been proven, we believe it is due to the emergence of C2 pars screw technology. Further, the high rate of success also demonstrates that when we have thorough knowledge of pivotal anatomy, all C2 pars screws and C2 pedicle screws can be placed accurately and risk of VA damage or serious clinical dislocation can be avoided. However, biomechanical studies have proven that C2 pedicle screws have greater rigidity and higher load failures than C2 pars screws , 29. TheHowever, there are still several weaknesses to be further emphasized and discussed regarding the current research. The first limitation is related to the small-sized retrospective sample. All patients were chosen based on strict inclusion and exclusion criteria. Second, the average follow-up time was too short. A prospective research with a longer follow-up period is necessary to further observe the postoperative clinical results of patients with OPLL. Third, we can only judge that the biomechanical strength of C2 pars screws is sufficient from the previous literature on atlantoaxial internal fixation. Although our study showed that there was no screw looseness or failure in either group within 2 years after the operation, it has not been proven by finite element analysis that patients with OPLL can obtain enough holding force, similar to C2 pedicle screws, after inserting C2 pars screws. A longer follow-up period  to observe the screw loosening is very necessary, which is also the focus of future research. Nevertheless, despite these restrictions, our research is valuable in demonstrating the advantages of C2 pars screw placement in patients with OPLL involving the C2 segment.In the treatment of patients with OPLL involving the C2 segment, the application of C2 pars screws is an alternative choice, which is not only safer but also reduces the amount of blood loss, shortens the operation time and obtains a more ideal screw placement.


Antifreeze proteins(AFPs) can bind to ice nuclei thereby inhibitingtheir growth and their hydration shell is believed to play a fundamentalrole. Here, we use molecular dynamics simulations to characterizethe hydration shell of four moderately-active and four hyperactiveAFPs. The local water density around the ice-binding-surface (IBS)is found to be lower than that around the non-ice-binding surface(NIBS) and this difference correlates with the higher hydrophobicityof the former. While the water-density increase (with respect to bulk)around the IBS is similar between moderately-active and hyperactiveAFPs, it differs around the NIBS, being higher for the hyperactiveAFPs. We hypothesize that while the lower water density at the IBScan pave the way to protein binding to ice nuclei, irrespective ofthe antifreeze activity, the higher density at the NIBS of the hyperactiveAFPs contribute to their enhanced ability in inhibiting ice growtharound the bound AFPs. Despite their variability in terms of primary, secondary, andtertiary structures, AFPs share a common mechanism of action whichconsists of binding to small ice nuclei, preventing their furthergrowth at the adsorbed positions.3 Because the ice-crystal growth is thermodynamicallyless favorable on a curved ice surface than on a flat one, the icegrowth stops until the temperature drops. This leads to a local differencebetween the freezing and melting temperature, ΔT, known as thermal hysteresis (TH). On the basis of the TH values,the AFPs can be classified into moderately active (ΔT < 1 K), mostly alanine-rich α-helical AFPs, and hyperactiveAFPs (ΔT > 1 K), mostly threonine-rich β-helicalproteins found in insects. At low concentrations (<0.5 g/L), hyperactiveAFPs greatly outperform more traditional antifreeze agents, makingthem of potential interest for use in medicine, agriculture, foodprocessing, and surface protection.4Bindingof AFPs to specific ice planes is ruled by both hydrogenbonds and hydrophobic interactions with a complementary protein surface,termed the ice-binding surface (IBS). The mechanism proposed for theantifreeze activity is that, by binding to a growing ice nucleus,AFPs cause a microcurvature of the ice surface between the adsorbedAFPs.9 pointing to a critical role for hydrogen bonds. At the same time,a relevant hydrophobic content in the IBS is required, providing anentropically driven thermodynamic contribution to ice binding. Althougha crucial role played by the AFP’s hydration layer in the molecularmechanism of the antifreeze activity is recognized,11 its structural and dynamical characterization is still incomplete.On the basis of molecular dynamics (MD) simulations, it has been proposedthat hydration water at the IBS could form an ice-like layer alreadyin solution, thereby easing ice recognition.19 However, other MD simulations23 and an NMR study24 didnot find any indication of ice-like structure in liquid water aroundAFPs. Concerning the dynamics, there is both computational25 and experimental evidence11 that waternear the IBS displays exceptionally slower hydrogen bond reorientationdynamics compared with other protein surfaces.A general consensus has been recently reached on the nature ofthe interactions that determine the binding to the ice surface: aspatial match between the polar residues of the IBS and the oxygen–oxygendistance in the ice lattice is needed,28 showed thatthe solvent at the IBSs features a lower density increment with respectto the bulk density and a higher degree of order with respect to thesolvent at the NIBSs. In particular, we recently showed for two AFPsthat although the overall hydration shell density does not differfrom the one of non-AFPs,23 the higher contentof hydrophobic (at IBS) and hydrophilic (at NIBS) residues in AFPsdetermines a slightly inhomogeneous density inside the hydration shellof AFPs (lower at IBS and higher at NIBS).22 While it is widely accepted that the lower density at the IBS favorsbinding to ice, the higher water density at the NIBS might discourageice growth around the bound AFPs, as was suggested many years agobut on the basis of dynamical analysis,15 thus contributing to the curvature of the ice surface at the basisof TH. The coupled features of ice-binding and non-ice-binding surfacesin AFPs and the resulting density unbalance could thus contributeto the antifreeze activity by providing protection against ice growth.At variance withthe IBS, detailed studies of the role of the non-ice-bindingsurfaces (NIBSs) of AFPs are few. Previous computational workHere, we provide further evidence of a clear correlation betweenthe hydration density at the IBS and the antifreeze activity for fourmoderately active samples. More importantly we show that, rather thandifferences in the hydration structure at the IBSs, what actuallydiscriminates between moderately active and hyperactive AFPs is anincreased hydration density at the NIBSs of the latter. This increaseddensity around the non-ice-binding parts of the hyperactive AFPs mightcontribute to enhance their capability of preventing the adsorbedprotein from being overgrown by a growing ice surface.Typhula ishikariensis isoform6 , the fish type I AFP from Pseudopleuronectes americanus , the fish type III AFP from Zoarces americanus  and the snow mold fungus AFP from Typhula ishikariensis isoform 8 . The hyperactive AFPs are the spruce budworm AFP from Choristoneura fumiferana isoform 337 , the beetle AFP from Tenebrio molitor , the longhorn beetle AFP from Rhagium inquisitor , the spruce budwormAFP from Choristoneura fumiferana isoform501 .EightAFPs (four moderately active and four hyperactive) are simulatedat a constant pressure and at room temperature see . The modSupporting Information), and the solvent density, ρ,around them is calculated as a function of the distance from the ellipsoidsurface . To compare the properties of water within thehydration shell with those of the bulk water, a previously employedstrategy23 was followed, which is based on a fictitioushydration shell filled with water molecules distributed as they wouldbe in the bulk solvent. This strategy permits removal of possibleeffects arising from the different topological features of the proteinsurfaces on the hydration shell features and hence the removal ofthe effect of the spatial arrangement of the protein atoms that protrudefrom the protein ellipsoid surface on the density profiles. More detailson the computational procedure are presented in In order to characterize theirhydration shell, the proteins aremodeled as ellipsoids  ataround 1 nm from the protein surface. Furthermore, our results confirmthat, as previously reported for several non-AFPs and a few AFPs,the ratio ρ/ρb,fict is always greater than1; that is, the solvent density around the proteins is always higherthan in the bulk.23The variations of the hydration density with respect to bulkwaterare followed by computing the ratio ρ/ρVshell, we use the distancefrom the protein ellipsoid surface of 1 nm at which the layer solvent-densityreaches the bulk plateau value. The relative density increment, η,inside the hydration shell with respect to the bulk density, ρbulk, is calculated asshell, the densitywithin the hydration shell, is estimated asVex is the protein-excluded volume ; Nshell is thenumber of water molecules inside the hydration shell, and angle bracketsindicate ensemble averages. It is noteworthy that exact and approximatedformulations lead to identical results within the estimated errorbar . From the above data, we can also estimate the partial molar volume, v, through the following expression, which was previouslyshown to be affected by small statistical errors31 (its derivation is given in the Supporting Information):In order to define the protein hydration shell thickness, and correspondingshell volume v, alongwith the quantities needed for their calculation, for the eight studiedAFPs are reported in 39 and previous calculations.41 When η is reported as a function of the partial molar volume v  at a protein solution concentrationof 0.3 g/L).The values of the relative increment in the hydration-shelldensity,η, and of the partial molar volumes, me v see 3, a cleame v see 1B. The dSupporting Information).Thelack of any significant correlation between the whole hydration-densityof the AFPs and their activity prompted us to analyze the local densityaround the IBS, which is the specific surface involved in ice binding,and compare it to that around the NIBS, i.e., the rest of the proteinsurface . We chose a 0.55 nmcutoff, which is less than the 1 nm thickness of the whole hydrationshell, to define the first hydration layer with the aim of maximizingthe local differences in water density at the different surfaces . The surface density calculation is also performedfor the “fictitious” configurations in which the firsthydration layer is filled with bulk water (ρsurffict). In this way the localsurface density of the IBS and NIBS can be compared among the differentproteins by calculating the relative increment with respect to thebulk, ηsurf, assurf is calculatedfor boththe IBS and the NIBS of each protein, and the results are reportedin surf with respectto the NIBSs, and for each protein, the IBS always displays a lowerrelative increment with respect to the corresponding NIBS (with theonly exception being TisAFP6 for which they are comparable).For each AFP,the solvent-density around a given surface, ρsurf is reported as a function of thefraction of the accessible hydrophobic surface area, Spho/S, for all the IBSs and NIBSs, itcan be seen that ηsurf is inversely proportionalto Spho/S. A lower incrementcorresponds to a higher Spho/S, and the ice-binding surfaces display the highest hydrophobic content.Moreover, a highly hydrophobic IBS is coupled to a highly hydrophilicNIBS, conferring to the AFPs a strong amphipathic character.42 Similar results were previously found on a smallerset, namely two of the eight AFPs studied here.22Further analysis shows that the surface density increment withrespect to the bulk density correlates with the chemical nature ofthe exposed surface 3. From Fsurf and Spho/S as a functionof ΔT (see Spho/S in the range of 0.54–0.70)but also similar between the two classes (being on average slightlyhigher for the moderately active ones). On the other hand, the hydrophobiccharacter of the NIBS (lower than that of the corresponding IBS) issignificantly higher for the moderately active than for the hyperactiveAFPs.In order to understand whetherthe properties of the local densitiesand the chemical character of the IBSs and NIBSs can be related tothe antifreeze activity of the AFPs, we report ηf ΔT see 4. For bosurf and Spho/S shown above,similar results are obtained also for the relative increment of solventdensity: while around the IBSs the ηsurf is similarbetween moderately active and hyperactive AFPs, with on average slightlylower values for the moderately active AFP, around the NIBS ηsurf differs, being higher for the hyperactive AFPs.Together with the strong correlationbetween η44 However, here we show that rather than this, it isthe higher hydrophilic content and consequent higher relative densityincrease of the NIBS that differentiate the hyperactive AFPs fromthe moderately active ones.These results show that what actually differentiates moderatelyactive from hyperactive AFPs are the properties of the NIBS, ratherthan those of the IBS. Indeed, for both classes of proteins the IBShas a high hydrophobic content, and a consequent lower water density,as previously reported by different groups.45 inconjunction with the OPLS-AA46 and SPC47 force fields for the proteins and water, respectively.The neutrality of all the simulated systems was ensured by adding(when needed) the suitable number of counterions. A constant temperatureof 300 K was always enforced by the velocity-rescaling algorithm.48 An isothermal–isochoric (NVT) reference simulation of 100 ns was first performed for pure SPCwater at 300 K with a density corresponding to the experimental liquidwater density at the same temperature (∼33.3 molecules pernm3). The resulting pressure of ∼560 bar was thenused to calibrate the densities of the boxes employed to simulate(in the NVT ensemble) the different proteins in SPCwater. All bond lengths were frozen by means of the LINCS algorithm,49 thus allowing the safe use of an integrationstep of 2 fs. The particle mesh Ewald method50 was used to compute long-range interactions above a cutoff radiusof 1.1 nm. After equilibration of the isolated protein and then ofthe full solute–solvent system, production simulations of 100ns were run for all the systems.The MD simulations of theeight proteins were performed with thehelp of the Gromacs 5.1.4 softwareb,fict, as a function of thedistance from the protein ellipsoidal surface was then calculatedfor these fictitious “protein + bulk-solvent” configurationswith the same procedure employed for conventional simulations. Detailsabout the calculation of the protein hydration shell densities areprovided in the Supporting Information .The MD simulation of the pureSPC water described above was usedto create the fictitious protein + bulk-water configurations. Foreach AFP, the protein coordinates extracted from 10 000 framesof the protein–solvent MD simulation were overlaid on 10 000configurations extracted from the bulk-water simulation, and the watermolecules overlapping the protein  were removed. This procedure provides 10 000“fictitious” configurations of protein–solventin which the hydration shell was filled with bulk-like water molecules.The solvent density, ρZaAFP and the hyperactive CfAFP501 proteins performedwith the TIP4P/2005 model for water in conjunction with the amber03force field for the protein.51 The resultscollected in Table S2 of the Supporting Information show that the relative density-increment in the hydration shellwith respect to the bulk density, η, obtained by the above forcefield and by the SPC/OPLS-AA model employed in the present Letterare comparable concerning both the absolute η values for eachprotein and of the difference of η between the two proteins.The possible roleof the specific force field employed in the MDsimulations in determining the calculated densities was assessed analyzingtwo previous simulations for the moderately active gmx sasa, which employs a definitionof polar (hydrophilic) and nonpolar (hydrophobic) surface areas basedon atomic partial charges . A test of the dependence of thefraction of the hydrophobic SASA on the choice of the hydrophobicitydefinition is reported in Figure S5 of the Supporting Information showing that differences arising from this choiceare small and systematic, thus not influencing appreciably the comparisonbetween different proteins.The fraction of the hydrophobicSASA was calculated with the GROMACSbuilt-in module


Lactiplantibacillus plantarum 299v. The product was tested for the survival of bacteria in various conditions, and the influence of the probiotic on the physicochemical and organoleptic properties of non-dairy ice cream was assessed. The dessert with probiotic throughout the storage period (8 weeks) kept the therapeutic minimum defined for probiotic food products. It was found that the addition of the probiotic did not deteriorate either the color or the sensory profile of the dessert. There was also no increase in the redox potential nor the acidity of the product with the addition of a probiotic.Food enriched with probiotics and prebiotics belong to the class of novel foods. Functional food, apart from its nutritional function, has an additional pro-health effect. The aim of the presented study was to create a concept of a functional dessert—avocado-based non-diary ice cream enriched with probiotic bacteria  Today, access to highly processed food is common, therefore carrying the risk of disturbances in carbohydrate and fat metabolism, and, ultimately, also leads to the development of the obesity epidemic. These are far-reaching effects of the growing wealth of the population, especially the population of highly developed countries. It is a Western-style diet based on flour and sugar, rich in monosaccharides, glucose-fructose syrup, and saturated fats . NegativLactobacillus and Bifidobacterium [Lactiplantibacillus plantarum is commonly found in vegetables and fermented products. It is also present in the mucosa of the human small intestine, and its functional properties have been confirmed by in vitro and in vivo tests [L. plantarum 299v is used as a starter culture in the technology of fermented food of plant origin [The proper functioning of the intestines, and, more specifically, the colonizing microorganisms, can be supported by probiotic therapy. A good solution is including in daily diet products that are source of probiotic lactic acid bacteria (LAB) with prebiotics that feed them. The health benefits of consuming probiotics and prebiotics are well known and include obesity and the metabolic syndrome prevention . Productacterium . Lactiplvo tests ,7,8,9. Tt origin . Althougt origin . Ice cret origin . The devt origin .L. plantarum 299v.There are milk-based ice cream and frozen dairy-free desserts made from soy or coconut vegetable proteins and traditional fruit-based sorbets ,14. AvocThe research material was an avocado-based non-dairy ice cream dessert with the addition of banana and lime juice. The basic ingredients of the dessert were fresh fruit—banana and avocado (in a 2:1 ratio), obtained from a local grocery store. The choice of plant raw materials was guided by the fact that the banana is a sweet fruit (contains on average about 22 g of digestible carbohydrates in 100 g of product ) and, thLactiplantibacillus plantarum 299v (DSM 9843) . According to the manufacturer’s declaration, one capsule of Sanprobi IBS contains at least 10 billion colony forming units (CFU). The preparation of the probiotic began 24 h before its application to the non-dairy ice cream dessert. All laboratory equipment used for this process was autoclaved at 121 °C (for 20 min). Exactly 90 g of the peeled banana were blended  and put into 3 jars which were then autoclaved (121 °C for 20 min). In this way, a matrix for the probiotic was obtained. Next, the jars were cooled to 30 °C. One capsule of Sanprobi IBS was dissolved in 1 mL of sterile phosphate buffered saline (PBS), pH 7.4, and was added to the first jar (Lb1) only. Pure lyophilizate (content of 1 capsule without PBS) was added to the second jar (Lb2). The control sample (CON) was peeled banana with addition of 1 mL of sterile PBS solution.A probiotic inoculum was added to the product in the form of a lyophilized or reconstituted forms strain of The technological scheme of the product is shown in To assess the energy and nutritional value of an non-dairy ice cream dessert, the tables of nutritional values for banana and avocado  and the The pH value of the non-dairy ice cream dessert was measured using a digital pH meter  equipped with a combined pH glass electrode  in a suspension (sample/distilled water 1:10) homogenized for 1 min using a disperser . The oxidation-reduction potential (Eh) was measured in a homogenate containing 10 g of sample and 30 mL of deionized water. Eh measurements of the homogenates were taken using digital pH-meter  set to the millivolt scale and equipped with platinum redox electrode . All measurements were performed in triplicate and expressed as a mean ± standard deviation (SD).The total viable counts (TVC) were quantified by means of the colony-count technique (PN-EN ISO 4833-1:2013-12) . The numThe color of a dessert was examined using the instrumental method—reflectance . The conditions were 13 mm port size, illuminant D65 and 10° standard observer. The X-Rite’s white and black standards were used to calibrate the spectrophotometer. To determine the color parameters of the non-dairy ice cream dessert, a portion (5 g) was placed in a clean glass beaker, and then the beaker was placed in the measuring gap. The results were expressed on the CIEL*a* b* scale, where L* means lightness, and a* means green-red chromaticity, while b* means blue-yellow. All measurements were performed fifteen times and expressed as a mean ± standard deviation (SD).E*) was calculated based on ΔL*, Δa*, and Δb* results for each sample depending on the storage time in the freezer, as follows: ΔE* = ((ΔL*)2 + (Δa*)2 + (Δb*)2)1/2. The total color difference was interpreted as follows: invisible (0 < ∆E < 1), very small (1 < ∆E < 3.5), clear (3.5 < ∆E < 5), and large (∆E > 5).The total color difference . All samples were coded with two random digit numbers, and the serving order was also randomized. Each panelist received only two non-dairy ice cream samples at −5 °C for evaluation. A 5-point hedonic scale was employed for the evaluation of appearance, texture (structure and consistency), color, smell, and taste of non-dairy ice cream samples. After receiving the evaluation results of individual panels, the average values of individual evaluations were calculated.p ≤ 0.05. Two-way analysis of variance (ANOVA) was used for statistical analysis. The effect of added antioxidant and storage time on changes in selected parameters was considered. The experiment was realized in two series. Calculations were made using Microsoft Office Excel 2007  and Statistica 10  software. The post hoc Tukey’s procedure was used to find patterns and relationships between subgroups. Differences among groups were determined as statistically significant at a level of The suggested portion of the product (90 g) can be a light dessert as it provides about 109 kcal, including about 5 g of fat, about 1 g of protein, and 16 g of carbohydrates . Comparep ≤ 0.05) oxidation-reduction potential was found in sample Lb1, but only in the study conducted before freezing the dessert. As the 8-week freezing time elapsed, differences in redox potential between all samples were not found (p > 0.05).All analyzed desserts had a similar (~5.2 units) active acidity, stable during freezing . The relp ≤ 0.001. After freezing, the number of LABs significantly decreased in all variants, compared to the pre-freeze test, but then remained stable, on average: 7.3 for the Lb1 sample, 5.8 for the Lb2 sample, and 1.4 for the control sample. The Lb1 variant of non-dairy ice cream dessert, in which probiotic was dissolved in 1 mL of sterile phosphate buffered saline (PBS), turned out to be the best. In this sample, the number of lactic acid bacteria was the highest, even during freezing.Immediately after production, the same number of LAB 7.3–8.1 log CFU/g was found in both samples with the addition of probiotic (Lb1 and Lb2) . In the p ≤ 0.05) and more yellow (p ≤ 0.05). The total color difference (∆E) of the desserts after freezing was calculated based on the individual parameter results. Color of the CON sample was the most stable (∆E = 1.7), and the probiotic samples showed clear (∆E = 3.6 or 4.0) color deviation.In the visual evaluation of the desserts, it was noted that they had a similar tint . The insAccording to the respondents’ assessments, the differences between the two non-dairy ice cream samples were imperceptible . It is tLactobacillus rhamnosus LOCK900 in fruit and tea sorbets. The product, after 12 weeks of storage, was characterized by the content of live bacterial cells at the level of >7 log CFU/cm3, similar to obtained in this experiment. According to Mohammadi et al. [The production of probiotic food with the use of fruit and vegetable raw materials as carriers, particularly fruit juices, may be associated with difficulties in the survival of bacteria. There are substances in plant raw materials that act as a carrier for microorganisms, such as fiber, vitamins, and minerals, as well as lipids . The litLactobacillus acidophilus compared to the reference sample.TVC is an important determinant of microbial quality, and results are confirmed by the results for the number of lactic acid bacteria. It was shown that, in the sample with the probiotic activated in PBS solution, the total number of bacteria remained unchanged throughout the storage period. The values of both, the sample with the pure lyophilizate of bacteria and the control sample free from probiotic LAB, decreased over time. In the studies of Senanayake et al.  also obtLactobacillus rhamnosus LOCK900. Moreover, Nousia et al. [Lactobacillus acidophilus LMGP-21381, also did not notice significant differences from the original values. Ervina et al. found that the addition of avocado to non-dairy ice cream reduces the acidity of the product, but no statistically significant differences were found compared to the control sample [Lactobacillus strains. There was checked the survival of bacteria under simulated gastrointestinal digestion. Inulin had a positive effect on the growth of bacteria survival and improved their resistance to gastrointestinal conditions during simulated digestion. From the observed results of the acidity, it can be clearly noticed that, immediately after production, the probiotic sample with PBS (Lb1) reached the lowest pH, which proves the high acidity and undeniable fermentation activity of the lactic acid bacteria. Although the value increased after the first 4 weeks of freezing, it remained at the same level after the next 4 weeks, which may suggest the inhibitory effect of low temperature. Szydłowska and Kołożyn-Krajewska  noticed a et al. , after 4l sample . This isl sample . Howeverl sample , who anaThe oxidation-reduction potential is an important indicator for monitoring and estimating oxidative changes in the product. In our study, this parameter is of particular importance due to the used raw material—avocado, which mainly contains monounsaturated fatty acids susceptible to oxidation processes . The facLactobacillus casei fermented plant-based ice cream (soybean) and the unfermented control product. On the other hand, in the studies by Norouzi et al. [Lactobacillus paracasei had a positive effect on the organoleptic characteristics of desserts. The authors’ own research positively accepted the fact that the taste and smell of probiotic desserts were highly rated. It is also promising that the acidic reaction of ice cream with probiotics was not perceptible by the respondents. It is assumed that the plant material is capable of masking undesirable odors that may appear during fermentation processes [LAB can find application in the production of fermented plant food if they meet the important aspect of maintaining the desired sensory quality . The pani et al. , fermentrocesses . The resBifidobacterium lactis BB-12 sweetened with polyols, not with sucrose, for the creation of sugar-free product. Ice cream produced in this experiment does not contain any additional amount of sugar other than naturally present in the fruit. The main source of carbohydrates in products are bananas, which have a high glycemic index (GI), but the presence of avocado fat may slow down the absorption of glucose into the blood and be a factor lowering the predicted GI of a meal [Ice cream enriched with probiotics, as a product intended for people who respect the impact of nutrition on the body, should have an attractive nutritional value. It should be noted that conventional ice cream contains mainly milk fat rich in saturated fatty acids. Avocados, which were a component of the ice cream in this study, are distinguished by a high content of monounsaturated fatty acids; moreover, they contain many antioxidant compounds that can be used in an anti-inflammatory diet . The enef a meal . Non-daiL. plantarum 299v probiotic strain is presumably able to multiply and survive in non-dairy ice cream dessert based on avocado. Dissolving the bacterial lyophilizate in PBS improved the viability of the bacteria in the product. After several weeks of freezing, the number of LABs in this variant was, on average, 7.4 log CFU/g and was over 1 logarithmic order higher than in the variant without PBS addition. It is true that the presence of exactly L. plantarum 299v in the dessert was not determined. However, the analysis of lactic acid bacteria, to which this strain belongs, was performed. As differences in the number of LABs between the dessert with the probiotic and the control sample were noticed, there is a strong supposition that the L. plantarum is in the product. Further studies are needed to confirm this hypothesis. The presence of probiotic bacteria in the non-dairy ice cream dessert and their survival in the freezing process is the most important factor and the starting point for further research work on composing a potentially functional fruit dessert enriched with probiotics.It was found that the use of the probiotic did not deteriorate the overall quality of the product, including sensory evaluation. The non-dairy ice cream dessert based on avocado with the addition of probiotic LAB was similar, both terms of physicochemical properties and organoleptic evaluation, to the control sample. Both mean pH values (5.2 ± 0.1) and mean oxidation-reduction potential values (454 ± 11 mV) were similar in all variants. The