Moral orientation refers to moral values that have a consistent guiding orientation toward an individual's moral cognition and behavior. Gilligan  proposed Moral orientation refers to moral values that have a consistent guiding orientation toward an individual's moral cognition and behavior and consists of two types: reward good cops and punish bad ones. Kyte  suggestePiaget and Kohlberg are recognized as representatives of the “justice” moral orientation. Kohlberg's  proposedMoral orientation refers to the manner in which an individual handles dilemma decisions. This topic is not often discussed in the context of cognitive scientific development and practice. In addition, when the stimuli related to an individual's self-concept differ , the individual's judgment about the orientation of justice and care also differ. Studies have demonstrated that individual factors (such as attitudes and motivations) and cultural values are two integral sources of influence  cultural values can predict the self-reference effect on moral justice or moral care; (2) participants in the traditional culture group exhibit more differential effects on different self-referential stimuli  at the implicit justice level, whereas those in the modern culture group exhibit less differential effects at the implicit justice level; and (3) participants in the traditional culture group exhibit more differential effects on different self-referential stimuli  at the implicit care level, whereas those in the modern culture group exhibit less differential effects at the implicit care level.We collected data using two experiments with similar procedures. Therefore, the procedures and results of both experiments are reported together. We used moral justice as the trait category label for an SA-IAT in Experiment 1, whereas moral care was used as the trait category label for Experiment 2. These trait categories were represented using single-attributes . We informed the participants that the experiments focused on personality traits. The experiments lasted for approximately 25 min, and we promised and delivered feedback to the participants about their results after they completed the tasks. The cultural value icons and order of combined tasks within IATs were counterbalanced among participants. We designed each experiment using a 3  × 3  mixed factorial design, with repeated measures on the first factor. The dependent variables were the response time of the implicit moral justice and moral care experiments.A total of 126  and 114  healthy college students participated in Experiment 1 and 2, respectively. The participants were right-handed, had normal or corrected-to-normal vision, and had no history of neurological or psychiatric disorders. After the experiments concluded, we paid the participants ¥50. The Ethics Committee of Hunan Normal University approved this study.Based on the cultural icon paradigm, cultural icons were used as a stimulus  = 25.34, p < 0.001], and the ratings of modern cultural icons (8.16) was significantly higher than that those for similarly sized sheets of blank paper . Therefore, we used 72 pictures  as the cultural cue stimuli for these Chinese participants. After reviewing recent studies  was significantly higher than the sheets of similarly sized sheets of blank paper . Moreover, the modernity group indicated smaller IAT effects than the control group  and the traditionality group . However, the correlation between the control and traditionality groups was not significant (p > 0.05). In addition, the interaction between self-relevance and cultural values were significant . A simple effects analysis revealed that regardless of the priming conditions, the target stimulus elicited more strict justice judgments toward strangers than friends and themselves. Specifically, the target stimulus under traditional culture conditions elicited stronger justice awareness  than the control group under neutral conditions  and modern culture conditions . The post hoc multiple comparisons indicated that the highly self-relevant (self) stimulus elicited more strict justice judgments than other names, whereas moderately self-relevant names (friend) elicited more strict justice judgments than non-self-relevant names (stranger) .To examine the influence of self-relevance on implicit justice judgment, we used the effect size measure “d” as the dependent variable, whereas the correlation between self-relevance and cultural values was determined using a 3 × 3 ANOVA with repeated measures on the first factor . The resF = 234.26, p < 0.001, F = 4.86, p < 0.05, post hoc comparisons indicated that participants' implicit care judgments varied among the self-referential stimuli, as presented in the following differential order: self > friend > stranger, thus suggesting that the participants showed less concern attitudes toward strangers than those for friends and themselves. . Besides, the traditionality group indicated larger IAT effects than the modernity group , the control group indicated larger IAT effects than the modernity group . However, the correlation between the traditionality and control groups was not significant (p > 0.05). In addition, the interaction between self-relevance and cultural values were significant,  than the control group under neutral condition  and modern culture conditions . The post hoc multiple comparisons indicated that the highly self-relevant stimulus (self) elicited more care attitudes than other names, while as moderately self-relevant names (friend) elicited more concern judgments than non-self-relevant names (stranger) .To examine the influence of self-relevance on the implicit care judgment, the effect size measure “d” was used as the dependent variable, the correlation between self-relevance and cultural values was determined using a 3 × 3 ANOVA with repeated measures on the first factor see . The resWe employed the SA-IAT and the cultural icon prime paradigm to examine whether and how self-relevance and cultural values influence implicit moral orientation (justice and care) processing. The findings showed a clear self-relevance effect: the highly self-relevant stimulus (self-names) elicited a stronger justice attitude than the names of friends and strangers in the modern cultural setting (context), whereas they elicited a weaker care attitude than the names of friends and strangers in the traditional cultural context. Moreover, the findings demonstrated variations in the justice and care orientation of the participants of the control and modernity groups, thus indicating that different cultural value orientations influence the moral justice or care attitudes of the target objects of different self-relevance at the implicit level.p > 0.05). This result is consistent with that of explicit justice orientation (Hu and Liu, The results of Experiment 1 indicate that participants' implicit justice judgments varied among the self-referential stimuli, as presented in the following differential order: The correlation between self-relevance and cultural values was significant, thus demonstrating that both cultural values and self-relevance influenced the implicit justice attitude. Compared with the control group, the priming of traditional cultural values aggravated the differential order of justice attitude, and the differential sequence was performed from near to far and from close to sparse, which caused differences in the justice attitude toward different self-relevant stimuli to further expand (Otten and Epstude, However, the results indicate a significant difference in the justice attitude toward self-relevant stimuli between the modern culture and control groups. The differences in the attitude tendency gradually decreased, indicating that the modernity context moderated the differential order of justice attitude. Moreover, regardless of the self-relevance level, the priming of modern cultural values can weaken the performance of the differential order and reduce the degree of “privateness.”The results of Experiment 2 indicated that participants' implicit care judgments varied among the self-referential stimuli, as presented in the following differential order: Qinqin,” “Zunzun,” and “Renlun,” which have a deep-rooted influence on people's care orientation (Yan et al., Consistent with the aforementioned findings, we also found that the priming of traditional cultural values accelerated the differential order in care orientation, whereas this “privateness and differential order” phenomenon was weakened in the modern culture context. The reason may be that traditional cultural values activate Confucian beliefs, such as “Studies have also noted that participants responded with increased positivity toward those they had personal relationships with; whereas they responded with increased negativity toward those they were unfamiliar with (Fan et al., Taken together, in addition to the weakening effect of partial on justice and care orientation processing such as task-switching, perceptual matching reported in the previous studies, the present study further showed that culture values can weaken social cognitive processing, such as that associated with self-relevance. This self-related weakening effect occurred at both implicit justice and care orientation in the modern culture context, but not in the traditional culture context, whereas traditional culture aggravates the self-related effect in justice orientation. These findings suggest that culture values can influence self-perception in an implicit manner. Future studies should focus on specific characteristics of modern culture and self-relevant stimuli to investigate the weakening effect of moral orientation on self-processing, particularly using high-spatial-resolution fMRI to uncover neural substrates that mediate this weakening effect, and how these neural activities are related to the justice and care attitudes.JB and LY: conceived and designed the experiments. JB, LL, QL, and JS: performed the experiments. JB, LL, XZ, and JD: analyzed the data. JB and LY: contributed reagents, materials, and analysis tools. JB and LL: wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In most of the MRI-based studies, an important requirement to analyze the acquisitions is an accurate parcellation of the considered anatomical structures. Manual segmentation is time-consuming and typically poorly reproducible, while state-of-the-art automated segmentation algorithms rely on available atlases. In this work we introduce the first digital neonatal rabbit brain atlas consisting of 12 multi-modal acquisitions, parcellated into 89 areas according to a hierarchical taxonomy. Delineations were performed iteratively, alternating between segmentation propagation, label fusion and manual refinements, with the aim of controlling the quality while minimizing the bias introduced by the chosen sequence. Reliability and accuracy were assessed with cross-validation and intra- and inter-operator test-retests. Multi-atlas, versioned controlled segmentations repository and supplementary materials download links are available from the software repository documentation at https://github.com/gift-surg/SPOT-A-NeonatalRabbit.The rabbit model has become increasingly popular in neurodevelopmental studies as it is best suited to bridge the gap in translational research between small and large animals. In the context of preclinical studies, high-resolution magnetic resonance imaging (MRI) is often the best modality to investigate structural and functional variability of the brain, both  •In magnetic resonance image analysis accurate automatic segmentations tools are critically important.•Multi-atlas based methods are valuable tools to obtain an automatic MRI segmentation.•The rabbit model has become increasingly popular in neuro developmental studies.•Currently there is no multi-atlas of the newborn rabbit available in literature.•In this manuscript we provide the first high resolution MRI multi atlas for the newborn rabbit brain. Animal models are a crucial component for the advancement of neuroscience and although rodent neurocognitive models are well established, their translation value is limited, especially in view of their prenatal myelination, lissencephalic brain structure and low proportion of white matter . As an aDigital atlases are a widely used tool for most studies related to volume, shape and microstructure quantification of specific brain regions . To the The lack of a digital atlas prevents from applying the state-of-the-art automatic methods. This is particularly critical when considering the growing range of studies involving neonatal rabbits, especially in the investigation of pre-term birth related perinatal brain injury , cerebraThe preferred strategy to obtain a segmentation is through manual delineation based on multiple MR modalities . This chWhen a comprehensive MR atlas or a probabilistic atlas  is available, it can be transposed over the image awaiting to be parcellated, with a segmentation propagation method . In a siAccuracy can be further improved by a multi-atlas based approach , hereby In this paper we present the first micro-MR multi-modal multi-atlas for the neonatal rabbit brain, delineating 89 areas for 12 subjects. Alongside we provide an open-source segmentation propagation and label fusion algorithm. Combined together they serve as a tool for the automatic segmentation of the newborn rabbit brain.1.Animalium of the Biomedical Sciences group at the KU Leuven, Belgium. Animals were treated according to current guidelines for animal well-being, and all experiments were approved by the Ethics committee for Animal Experimentation of the Faculty of Medicine (P062/2016). Does were housed in separate cages before delivery, with free access to water and chow and a light-dark cycle of 12 h. The does underwent a cesarean section on either 28 (pre-term) or 31 (term) days of gestation. Thereafter, newborn rabbits were nursed in an incubator with twice daily gavage feeding until harvesting on post-conceptional age of 32 days. This established an equivalent time-point at harvesting for both term  and pre-term .Time-mated pregnant does  were obtained from the 2.®, Dimeglumine Gadopentetate 0.5 mmol/mL, Bayer HealthCare Pharmaceuticals, Germany).Neonatal rabbits were actively perfused with a mixture of formalin and gadolinium to increase MRI signal to noise ratio while preserving the tissue . Kits we3.in situ was then immersed in this solution for another 48 h followed by a rehydration phase in a 1:200 solution of Magnevist ®/PBS for 48–72 h. The head was trimmed and placed in a sample holder, surrounded by proton-free perfluoropolyether solution  that minimizes susceptibility artefacts at the interface.The head with the brain The proposed atlas was produced as part of a wider study, evaluating encephalopathy of prematurity in a pre-term newborn rabbit model. Initially, 17 subjects, both born either term (gestation 31 days) or pre-term (gestation 28 days), were prepared according to the following protocol see :1.Time-m▷Structural T1 weighted image acquisition: Three-dimensional MR images were acquired using a gradient-echo sequence (3D Flash) with Time of Echo (TE)/Time of Repetition (TR) 5.5/50 ms; flip angle 70°; bandwidth 50 kHz; acquisition matrix of ▷Diffusion-weighted image acquisition: After the T1 acquisition, diffusion-weighted images (DWI) were acquired using a 3D segmented spin-echo version of the echo-planar imaging (SE-EPI) sequence with 8 segments; TR/TE: 280/24 ms; FOV: Micro-MRI scans  version 5.0.9 , mean diffusivity map (MD) and RGB encoded direction of the main eigenvalue map (V1) were resampled in the T1 weighted image space to overlay structural information. Due to sample as well as frequency drifting occurred during the acquisition, anatomies resulting from different sequences were not perfectly spatially aligned. Therefore the B0 was rigidly aligned with the T1  and the same transformation was applied to the remaining DW modalities.The available adult atlas  could noITK-SNAP v2.2.0  (Similarity and Truth Estimation for Propagated Segmentations (STEPS)   and Simi (STEPS) , with a Majority voting is the simplest label fusion method. When applied, the final label selected for each voxel is the one that have appeared more often when propagating the segmentation of each subject in the multi-atlas. STAPLE estimates the value of the final label adding at the spatial distribution of the segmented structures a spatial homogeneity constraint within a probabilistic model optimized with an Expectation-Maximisation algorithm. STEPS extends STAPLE, considering a ranking strategy based on the local normalized cross correlation (LNCC) measure of similarity applied to the propagated anatomies on the new subject anatomy.With the proposed iterative method, schematically represented in The time of the manual adjustment has decreased significantly, from a time range between 20 and 25 h for the first subject to a time range between two to 6 h, depending on the outcome of the propagation.The same procedure developed to initialize the segmentations for the manual adjustment can be extended to approximate the segmentation on a new subject, constituting an automatic segmentation algorithm. The first step consists in the diffeomorphic registration of the region of interest (brain and skull) mask with the subsequent propagation of the labels from each element of the multi-atlas to the new subject.Among the several label fusion methods employed to produce the resulting parcellation from the stack of the aligned elements of the multi-atlas, the multi-modal Majority Voting provided the best result, both scored with visual assessment and leave-one-out cross validation. Accordingly, this is the proposed choice for the automatic segmentation method.NiftyReg . It is open to further improvements and to evolve according to future needs of structural and functional investigations. In analogy with what is currently employed in software development, possible further upgrades will be tracked in the hosting repository with a version control system.As called for by 1.Inter-operator variability assessment and comparison with the automatic method via hippocampi segmentation.2.Visual scoring of unlabeled segmentations of the same subject.3.Intra-operator consistency assessment for the manual adjustment of the automatic initialization via test-retest.4.Fully automated segmentation assessment via cross validation.When the same protocol is applied, the manual segmentation variability between and within operators can be significant, even when segmenting a single region. Taking into account this known limitation, to assess the validity and robustness of an automatic method respect to the manual counterpart we performed five experiments:The comparison of their outcomes provides an assessment of the coherence between the manual and the automatic approach. Before detailing them, we briefly recall the definitions of a range of measures to compare two segmentations . These wAs discussed in Dice's score: it measures the number of overlapping voxels of two regions, over the mean of the voxels. It is defined asCovariance distance: the segmented regions are considered as clouds of points, whose covariance matrices are compared. It provides an idea of how well the relevant features of the distributions are aligned when orthogonally translated onto the same centre, and it is defined as:j in the 3D space, Tr is the matrix trace, or the sum of the diagonal elements, α is a multiplicative factor corresponding to the maximal possible dissimilarity  and the two manual adjustments (adj1 and adj2) in three different boxplot.In this experiment, the rater did not consider necessarily to adjust every region: the macro regions 1, 2, 4, 6, 8, 11 and 12 required no manual intervention after the automatic segmentation. In a leave-one-out cross validation , each suTo simplify the representation, the delineated regions were grouped into a set of 16 macro-regions, proposed in The boxes show the interquartile interval, whiskers the minimum and the maximum. The central bar shows the median. Macro regions that were better aligned, according to all the selected metrics, were regions 1, 2, 4, 6, 8, 11 and 12.in vivo acquisitions were planned. Four neonatal rabbits underwent high resolution MRI with the following protocol:▷Structural T1 weighted image acquisition: MRI was performed on living animals under isoflurane anaesthesia using a Bruker Biospec 9.4 T small animal MR scanner  equipped with actively shielded gradients (600 mT/m) utilising a rat brain surface receiver decoupled to a volume quadrature transmit coil . Field homogeneity was corrected using fieldmap correction . Anatomical reference was obtained through a 3D T2 weighted RARE sequences  were acquired using a spin-echo version of the echo-planar imaging (SE-EPI) sequence with 4 segments; TE/TR: 27/5000 ms; 2 averages; FOV: To provide an assessment of the quality of the proposed automatic method and to validate the feasibility on a different dataset further ex vivo example. The propagation was therefore based only on the T1 modalities. Moreover, the non-rigid bending energy parameter was increased from 0.5 of the ex vivo setting to 0.8 for the in vivo. The low resolution of the DWI made also difficult to quantify the quality of the segmentation alongside the borders that are visible only in the DWI-based modalities .As the resolution of the DWI resulted too low compared to the T1, it was not possible to obtain satisfactory results with the multi-modal approach described for the Results are shown in coronal section in The proposed high-resolution MRI-based multi-atlas provides a tool to create an automatic parcellation for neurological research using MRI imaging of newborn rabbits. It may be beneficial for the translational research focusing on perinatal brain injury and on MRI-based pre-clinical trials.in skull acquisition, to avoid an overall shape deformation found in ex skull protocols due to extraction artefacts and the long time of the brain in the scanner.Acquisition quality is a major determining factor in creating a reference atlas. Herein we opted for an in vivo acquisition of future studies. Preliminary results in this direction, shown in in vivo multi-atlas.Another benefit could entail the better segmentation propagation when translating the multi atlas on an Susceptibility artefacts have had an impact on the design of the propagation algorithm as well. These artefacts, caused by local magnetic field inhomogeneities resulted in unwanted misalignment in the non-rigid registration processing phase. Furthermore the presence of artefacts especially affecting in the DWI-based modalities was problematic.Susceptibility artefacts were most likely a consequence of the presence of air bubbles in the gadoteridol-paraformaldehyde composition, which can accumulate between the brain and skull during the perfusion. An improvement in image quality was observed during the study, when applying degasification process to the gadoteridol-paraformaldehyde composition prior to the perfusion. Further studies should therefore consider the inclusion of this step in the sample preparation.The customary Dice score alone is not enough to provide a numerical representation of the differences between two shapes. The four selected metrics, other than quantifying the overlap, provide the overall shape direction distribution, the upper bound and the average discrepancy between borders.In the inter-rater variability test-retest, the expert rater rater1,  appears The strategy of iterating three times between automatic method and manual adjustments led to a continuous improvement, as proved in the anonymized segmentation visual scoring .The outcome of the leave one out cross validation for the 12 subjects shown in other Fibretracts. This region includes most of the elongated anatomies (such as Optic tracts and anterior commissure), whose misalignment is better captured by the covariance distance. If measured with the Hausdoroff distance, the same region performed better than the ventricular system, that is the most difficult to delineate manually, in particular in the region between the skull and the retrosplenium left and right.The macro region with the worst alignment was The normalized symmetric contour distance can be seen as robust to outliers Hausdoroff distance. If for example two regions are perfectly aligned apart from one column of voxels protruding in radial direction from one of the regions, their Hausdoroff distance equals the length of the protruding line of voxels. The normalized symmetric contour distance in the same situation is almost zero, not being influenced by this single roughness. Comparing the results of these two measurements in the leave-one-out experiment, the Hypotalamus results to be the region less affected by outliers respect to the other regions.In general, the high scores in the leave-one-out highlight a stability in the regions propagation. Moreover the higher Dice score and the consistency in the intra-rater manual adjustment variability shows that the outcome of the automatic algorithm is qualitatively high, requiring small amount of manual adjustment.The key question relates to the representativeness and the consequent transferability of the proposed multi-atlas on a different dataset. The uneven distribution between term and pre-term and among male and female subjects  may biasin vivo experiments showed that even if the proposed algorithm is applied over an image acquired with an different protocol, most of the regions were subjectively considered well aligned.The On the algorithmic side, for the fine-tuning parameters we mostly relied on visual assessment. As the manual segmentation quality went through a process of gradual improvement throughout the study, a ground truth had not been available to apply a more sophisticated numerical method. Grid-based or random search methods may, if applied in future studies, contribute to improve the propagation results over a wider range of acquisitions.In this paper we have proposed the first new-born rabbit brain multi-atlas, acquired at 9.4 T and segmented with a semi-automatic method using the T1 weighted images and a range of DWI modalities.Due to the differences between rat and rabbit brain anatomy, we proposed a stereotaxic orientation based on readily identifiable landmarks and compatible with longitudinal studies. This is also aimed at facilitating any future comparison or study involving histology and MRI in the newborn rabbit and longitudinal studies.https://github.com/gift-surg/SPOT-A-NeonatalRabbit). Each further improvement and each different taxonomical subdivision can be uploaded, while keeping track of the previous multi-atlas version with a unique identifier, to guarantee reproducibility.Subsequent studies, which may potentially benefit from the work presented here, might require their own specific refined segmentation, parameter tuning and taxonomy. For this reason, in analogy with collaborative software development, the segmentations are proposed in a versioned controlled open repository (


Such events result in significant mortality, physical and/or mental incapacityand costs for the individual and society.2 For patientsat very high risk for premature events, such as those with familialhypercholesterolemia (FH), an elevated LDL-C level is an extremely prevalent riskfactor.3The causality of plasma LDL-C levels and reduced LDL-C uptake mediated by the LDL-Creceptor in the pathophysiology of CVD has been very consistentlyestablished.4A relevant clinical question is the difficulty of achieving the LDL-C levelsrecommended by the guidelines for patients at very high cardiovascular (CV) risk.Even using high-potency statins, a substantial proportion of those patients will notachieve the LDL-C target, partially because of the pharmacogenetic effects thatdetermine wide inter-individual variability in the response to statins. Thisemphasizes the need for an additional reduction in LDL-C levels with new therapeuticoptions aimed at those atherogenic particles.The proprotein convertase subtilisin/kexin type 9 (PCSK9), a member of the serineprotease family, plays a central role in the regulation of the liver LDL-C receptoractivity. Individuals with mutations in the PCSK9 gene and function loss, andconsequent lower LDL-C levels, have a substantially reduced risk of developingcoronary artery disease. Inversely, heterozygous individuals for the PCSK9 mutation,with function gain, have a phenotype consistent with FH.6Those findings have stimulated the investigation of the use of PCSK9 inhibitors(PCSK9-I) as an innovative therapeutic alternative to improve the control ofelevated LDL-C levels.8Several clinical studies with different monoclonal antibodies against circulatingPCSK9, both in isolation and combined with statins, have confirmed significantreductions in LDL-C levels, reaching up to 60%.9The FOURIER trial, published in 2017, showed a significant reduction in relevantclinical events, such as acute myocardial infarction (AMI) and atherothromboticischemic stroke, in patients with established CVD and plainly treated with moderate-and high-potency statins, associated or not with ezetimibe. The median LDL-C levelwas 92 mg/dL, and patients receiving evolocumab reached a median LDL-C level of 30mg/dL. Safety data showed no significant adverse effect, except for injection-sitereactions, in the median follow-up of 2.2 years.10The recently published EBBINGHAUS trial, assessing patients who received eitherPCSK9-I or placebo in addition to statin therapy, has shown no significantdifference in cognitive function, even in individuals with very-low LDL-Clevels.11Another study using the monoclonal antibody bococizumab has shown a significantreduction in CV events, which is aligned with the result obtained with evolocumab inthe FOURIER trial. However, the SPIRE trial was interrupted because of thedevelopment of high rates of antidrug antibodies and consequent reduction in thetherapeutic response.12The trial has shown a significant reduction in non-fatal AMI, unstable angina andischemic stroke in patients randomized to receive the PCSK9-I alirocumab. Thesubgroup of individuals with LDL-C levels greater than or equal to 100 mg/dL and receiving alirocumab had the highest benefit,with a 29% reduction in total mortality as compared to placebo.The ODYSSEY trial, recently presented as a late-breaking clinical trial at theAmerican College of Cardiology Scientific Session, has assessed patients who hadacute coronary syndrome within 1 to 12 months before randomization. All individualswere on moderate- and high-potency statins, associated or not with ezetimibe. Themean follow-up was 2.8 years.Although the advent of precision medicine and the innovative treatments have guidedto an individualized approach in prevention and patient’s management, the financialrestrictions to the progressive increase in health system costs worldwide oftenrequires balancing the therapeutic benefit and the cost of a certainintervention.13The recently updated Brazilian Guideline on Dyslipidemias and AtherosclerosisPrevention recommends the use of PCSK9-I  only topatients at high CV risk, receiving optimized treatment with statins at the highesttolerated dose, either associated or not with ezetimibe, and who have not met therecommended LDL-C or non-HDL-C targets.The Brazilian guideline, however, does not indicate which individuals will benefitmost from the use of that new class of drugs.14Some studies have demonstrated that the quantification of the absolute benefit of anadditional therapy is an important factor to support the clinical decision of usingor not the new treatment. In addition, financial aspects should be taken intoaccount, but so far cost-effectiveness analyses of the PCSK9-I in Brazil have notbeen made.15Considering that the costs of PCSK9-I are greater than those of the other drugs forthe treatment of CVD, it is important to identify among high-risk individuals thosewhose treatment is associated with greater clinical relevance, which can beestimated by the number needed to treat (NNT) to prevent the first outcome within acertain time.In addition, calculating the NNT can help identify groups of patients who willbenefit most from the addition of a non-statin therapy, by combining absolute riskand LDL-C thresholds.In this position statement of the Atherosclerosis Department of the Rio Grande do SulSociety of Cardiology, the selection of patients to use PCSK9-I is more conservativethan that of most current guidelines on the use of those drugs. Thus, it is worthemphasizing that the use of antibodies against PCSK9 for individuals who do not meetthe criteria presented in this document is not contraindicated, because thetherapeutic decision involves clinical judgement and consensus between physiciansand patients.Therefore, this position statement was aimed at identifying the individuals who willbenefit most from the use of that new class of drugs to treathypercholesterolemia.This first position statement will not address indications of that new class of drugsfor statin-intolerant individuals or those on high-risk primary prevention, such asFH.Considering that evolocumab and alirocumab consistently reduce LDL-C levels by 50% atleast, two factors should be observed to quantify the benefit of the treatment: theindividuals’ clinical characteristics and the LDL-C levels obtained after maximumtreatment with statin/ezetimibe.16 The greatest benefit derived from the use ofPCSK9-I is obtained in individuals at CV risk higher than 20% in 10 years. Thus,patients with previous coronary events or procedures, stroke or aortic aneurysmare classified as “high risk” (20-29% in 10 years).The clinical characteristics of the patients at CV risk should be identifiedbased on the absolute risk of CV events in 10 years.Patients at “very high risk” for CV events (over 30% in 10 years) are those withrecurrent acute coronary syndrome, repeated arterial revascularization orrepeated strokes within the first year from the initial event. Advanced age, andthe association of diabetes or peripheral occlusive arterial disease areaggravating factors.In addition to the patients’ clinical characteristics, the LDL-C cutoff pointsfrom which the treatment with PCSK9-I provides the greatest benefits should beindicated.The FOURIER trial has shown that even individuals in the lowest quartile of LDL-Clevels had a significant reduction in CV events when receiving evolocumab.17However, when assessing that variable, it is worth noting that the reduction inthe absolute risk for the same relative reduction in LDL-C level will be smallerwhen the baseline LDL-C level is lower . In othe18The relative reduction in CV events resulting from the use of statins, ezetimibeand monoclonal antibodies against PCSK9 has shown consistency with therelationship reported in the Cholesterol Treatment Trialists meta-analysis, inwhich every 39-mg/dL reduction in LDL-C was associated with a 21% reduction inmajor CV events.16 have estimated the NNTsin 5 years to prevent a CV event.16 (By associating the two variables presented in an excellent analysis and according tothe CV risk and LDL-C levels of patients receiving treatment with statins, Robinsonet al.event.16  19 for new interventions, it is worthnoting that PCSK9-I are high-cost drugs. On the other hand, NNTs under 20 are rarelyobtained for interventions to treat or prevent CVD resulting from the currentstudies.20Although there is consensus that NNTs up to 50 are acceptableAssuming that PCSK9-I consistently reduce LDL-C levels by at least 50%, we considerthat NNTs under 30 are totally acceptable and identify a subgroup of individuals whowill greatly benefit from receiving that new class of drugs.Therefore, patients at high CV risk, plainly treated with high-potency statinassociated with ezetimibe, and whose LDL-C levels are higher than 130 mg/dL, willsignificantly reduce their risk of CV events by adding PCSK9-I to theirtreatment.Similarly, individuals at very high CV risk, treated with statin and ezetimibe, andwhose LDL-C levels are higher than 100 mg/dl, have a very good chance ofsignificantly reducing outcomes and the residual CV risk by adding that new class ofdrugs to their treatment.This position statement of the Atherosclerosis Department of the Rio Grande do SulSociety of Cardiology identifies patients who can derive the greatest secondaryclinical benefit from PCSK9 inhibition. Those patients have higher CV riskassociated with the highest probability of achieving a significant LDL-C reduction.In addition, this document takes into account the financial limitations of thehealthcare system and the current economic scenario.It is worth emphasizing that the use of antibodies against PCSK9 for individuals whodo not meet the criteria presented in this document is not contraindicated, becausethe therapeutic decision involves clinical judgement and consensus betweenphysicians and patients.


Scientific Reports 10.1038/s41598-018-36158-w, published online 14 December 2018Correction to: The Acknowledgements section in this Article is incomplete.“The authors wish to thank Mr. Paolo Giannotti for the technical assistance, the panelists and the volunteers who participated to the repellence tests.”should read:“The authors wish to thank Mr. Paolo Giannotti for the technical assistance, the panelists and the volunteers who participated to the repellence tests. This research was supported by Italian Ministry of Education, University and Research (MIUR) .”


In the harsh application environment, improving the mechanical properties of liquid crystal materials is a fundamental and important problem in the design of anti-counterfeit materials. In this paper, by a stepwise polymerization of first, photo-polymerization and subsequently thermal-polymerization, a coexistent polymer dispersed network was first constructed in cholesteric liquid crystal materials containing a photo-polymerizable system of urethane acrylate and a thermo-polymerizable system of isocyanate. Results revealed that the coexistent polymer dispersed network exhibited largely enhanced mechanical performance, and the networks obtained by different methods had different contributions to the enhancement of the peel strength and toughness of the composite films. Then an angular photochromic anti-fake label based on a coexistent polymer dispersed network with enhanced mechanical and apparent angular discoloration characteristics, suitable for practical applications, was successfully achieved. Anti-fake technologies such as holograms , watermanPsinθ, where n = (no + ne)/2 is the average of the ordinary (no) and extraordinary (ne) refractive indices of the ChLC, P is the cholesteric pitch corresponding to the length of a 2π molecular rotation, and θ is the angle between the surface and viewing direction. Accordingly, for a specific ChLC material, the reflection colors (supposing that the reflection wavelength is in the visible region) is directly proportional to the viewing angle, which is very suitable for anti-fake purposes.Liquid crystal (LC), which exhibits excellent optical, controllable, characteristic, and self-assembled soft matter properties, can be used as anti-fake material and has been widely reported previously ,5,6. AttHowever, the mechanical properties of pure LC materials are not satisfactory for application. Reports have shown that there are many different methods to reinforce the mechanical properties of a material such as nanoparticle filling ,11,12,13In the present paper, a series of polymer dispersed cholesteric liquid crystalline film was prepared. Different from the work based on a single polymer dispersed network by photo-polymerization or thermal-polymerization previously reported, a coexistent polymer dispersed network by a stepwise polymerization of first photo-polymerization and subsequent thermal-polymerization was first realized; the impact of different polymer dispersed networks and ChLC materials on the mechanical and optical properties of the ChLC films was systematically investigated. Then an angular photochromic anti-fake label based on a coexistent polymer dispersed network with enhanced mechanical and apparent angular discoloration characteristics suitable for practical applications was successfully prepared.Cr-N <−40.0 °C, TN-I = 91.8 °C) was purchased from Shijiazhuang Yongsheng Huatsing Liquid Crystal Co., Ltd. . S811  and urethane acrylate  were commercially available and used without any further purification. Poly-isophorone di-isocyanate , tetra-ethylene glycol , and other chemical reagents were used as received. ChSCLCP was obtained via conventional free radical polymerization of different liquid crystalline monomers—for details of the synthetic route refer to our previous work [The nematic LC SLC1717  was carried out on a Carl Zeiss Axio Vision SE64 polarized optical microscope  with a Linkam LTS420 hot stage. Spectral characterization was done by an unpolarized UV/Vis/IR spectrophotometer  in transmission mode at normal incidence. The peeling strength experiment was practiced on a universal tensile test machine  and the rate of extension was 0.5 mm s−1. The samples were sandwiched between two PET films to perform the peeling strength experiment from the horizontal direction of the film. The horizontal cross-section area was 1 cm2.A PerkinElmer DSC8000  with a mechanical refrigerator was used to obtain the phase transition of the polymers under dry nitrogen at a heating and cooling rate of 20 °C minThe prepared samples were mixed thoroughly in the specified proportions according to Previous reports have shown that the center reflection wavelength of chiral compounds strongly relied on the content of the chiral component . AccordiIn our previous studies, we revealed that the introduction of a polymer dispersed network into LC could considerably improve the peeling strength of the material . HoweverAs shown in As illustrated in To further investigate the light transmittance properties of the samples, UV/Vis/IR spectra were utilized and the results are shown in However, although the overall optical properties of the samples with a polymer dispersed network by photo-polymerization were better than that with a polymer dispersed network by thermal-polymerization, the mechanical performance of the samples showed an interesting phenomenon. As shown in From the above results, we expect that there may be some interaction between them if we introduce the two kinds of polymer dispersed networks simultaneously in one system, and an equilibrium point may exist. So a coexistent polymer dispersed network by a step polymerization of first photo-polymerization and subsequent thermal-polymerization was attempted and the effect of different ratios between the photo-polymerizable and thermal-polymerizable monomers on the mechanical properties was investigated. According to the results obtained above, when the weight ratios of photo-polymerizable and thermal-polymerizable monomers were similar, the coexistent polymer dispersed network exhibited the optimal mechanical performance, which was favorable for the anti-fake application. Thus, two kinds of angular photochromic films based on ChLC of SLC1717/S811 and 3HG2080 with a coexistent polymer dispersed network  were prepared. In summary, employing the selective light reflection characteristic of ChLC, by introduction of a coexistent polymer dispersed network via a stepwise polymerization of first photo-polymerization and subsequently thermal-polymerization, an angular photochromic anti-fake film with enhanced mechanical and apparent angular discoloration characteristics was successfully developed. Detailed investigation found that the polymer networks developed by different methods had different effects on the mechanical properties: the polymer dispersed network by thermal-polymerization contributed more for peel strength improvement, and the polymer dispersed network by photo-polymerization contributed more for toughness enhancement. It is believed that the ChLC anti-fake film will have practical application in the negotiable instrument field such as for currencies, bank documents, and so on.


To build a representation of what we see, the human brain recruits regions throughout the visual cortex in cascading sequence. Recently, an approach was proposed to evaluate the dynamics of visual perception in high spatiotemporal resolution at the scale of the whole brain. This method combined functional magnetic resonance imaging (fMRI) data with magnetoencephalography (MEG) data using representational similarity analysis and revealed a hierarchical progression from primary visual cortex through the dorsal and ventral streams. To assess the replicability of this method, we here present the results of a visual recognition neuro-imaging fusion experiment and compare them within and across experimental settings. We evaluated the reliability of this method by assessing the consistency of the results under similar test conditions, showing high agreement within participants. We then generalized these results to a separate group of individuals and visual input by comparing them to the fMRI-MEG fusion data of Cichy et al (2016), revealing a highly similar temporal progression recruiting both the dorsal and ventral streams. Together these results are a testament to the reproducibility of the fMRI-MEG fusion approach and allows for the interpretation of these spatiotemporal dynamic in a broader context. To solve visual object recognition, the human brain has developed a particular cortical topology within the ventral and dorsal streams, recruiting regions in cascading sequence, to quickly build a representation of what we see i.e., ,6,7,8,9),7,8,96,7In order to reveal the complex neural dynamics underlying visual object recognition, neural representations must be resolved in both space and time simultaneously ,13,14,15reliability of the fusion method at capturing the spatiotemporal dynamics of visual perception by assessing the neural agreement of visually similar experiences within individuals, asking: ‘Do similar visual experiences obey similar spatiotemporal stages in the brain?’ Given the known variation across brain regions for separate visual category input  and Experiment 2  from the LaMem dataset [In Experiment 1, the stimulus set consisted of  dataset . Twin-se dataset ,38,39  based analysis along ventral and dorsal streams  the conventional ROI-based analysis: in this analysis, the condition-specific voxel responses within an ROI are extracted, and the pairwise condition-specific response dissimilarities are computed to create an ROI RDM which is then compared with the time-resolved MEG RDMs resulting in correlation time series .We first assessed the reliability of the fMRI-MEG similarity-based data fusion method. For this we applied fMRI-MEG fusion separately to the two sets of images making up the Twins-set  and compared the results.P < 0.01, and cluster size threshold of P < 0.01). This demonstrates the reliability of the fusion method in reproducing similar patterns of neural activity across similar visual experiences.To quantitatively compare the spatiotemporal fusion maps between the Twin-sets we performed spatially restricted voxel-wise fMRI-MEG fusion on five regions-of-interest (ROIs) , ventral occipital area (VO), parahippocampal cortex (PHC), and inferior parietal sulci (IPS0 and IPS1). We averaged the correlation values over the voxels within each ROI resulting in one correlation time series per individual , around 140 ms in ventral and dorsal ROIs. The peak latency and onset times with their corresponding 95% confidence intervals are reported in Next, we performed an ROI-based fMRI-MEG fusion following the method described in ,9 see Fa. StudieP < 0.01, and cluster size threshold of P < 0.01). This revealed significant clusters in the reliability map across both the ventral and the dorsal stream  between Twin set 1 and 2, for each participant separately, we compared the voxel-wise fMRI-MEG correlation time series of Twin set 1 with Twin set 2. This resulted in one 3D reliability (correlation) map per participant see . We assel stream .P < 0.01 cluster-definition threshold, P < 0.01 cluster threshold).We evaluated the generalizability of the fMRI-MEG similarity-based data fusion method by comparing results across participant groups presented with different images (Twins-Set and ImageNet-Set from Experiment 2 of ). We appP < 0.01, and cluster size threshold of P < 0.01).Qualitatively, P < 0.01 cluster-definition threshold, P < 0.01 cluster threshold). We observed that in both datasets, correlation time series peak significantly earlier in EVC compared to high level regions in ventral (VO and PHC) and dorsal  pathways  reflecting the established structure of the visual hierarchy. Peak and onset latencies of curves in To further investigate the similarities and dissimilarities between these two sets of data quantitatively, we performed ROI-based analyses. For each dataset, we correlated ROI-specific fMRI RDMs with time-resolved MEG RDMs resulting in correlation time courses shown in One of the central tenants of science is reproducibility ,44,45. Treliably consistent spatiotemporal dynamics, with each subset showing responses first in the occipital pole, and then signals in the anterior direction, with similar path in both ventral and dorsal streams. The strong agreement between the full brain spatiotemporal maps of the Twin-sets suggests that the signals detected by the fMRI-MEG data fusion method is likely to reliably reflect the representations of the stimulus. This sequence of representational signals followed the established spatial [The first analysis compared neural data within the same subject groups across Twin image sets, with equalized low-level visual features, and sharing highly similar semantic concepts . We observed that the analysis yielded  spatial ,48 and t spatial ,49 patteThis high degree of reliability, within subjects, across these separate but matched image-sets has pragmatic consequences in reinforcing the power and confidence of this method. The agreement within results of unimodal neuro-imaging have been shown have low test-retest reliability . This regeneralizes across subjects and natural images. The results of both experiments illustrate the spatiotemporal progression associated with hierarchical visual processing [Across the two experiments, findings from the full brain correlation maps and spatially restricted ROI analysis revealed that the fMRI-MEG data fusion captures the spatiotemporal dynamic patterns common to visual processing in a manner that ocessing ,52,53, wWe found that overall this generalized response had similar expected spatiotemporal response patterns, but with greater variability in high-level visual areas. Previous work examining the replicability of neuroimaging data has shown that low-level brain functions (motor and sensory tasks), show generally less variance than high level cognitive tasks . MoreoveCognitive neuroscientists have many methodological options for studying how experimental variables are systematically related to the spatiotemporal dynamics underlying a cortical process of interest. To date, much of our understanding has been advanced by methods that yield high spatial or temporal resolution, but not both. Separately, the limitations of these modalities in their application to understanding cognition are well known. Here we show that combining these methods through representational similarity analysis provides a reliable and generalizable tool for studying visual perception. This confirmatory replication within and between image-sets and subject-groups demonstrates the replicability of the method in the study of visual processing. By establishing replicability our study increases the trustworthiness of the method, and we hope that this motivates further researchers to use the method in the study of human brain function.


RNA interference (RNAi) is a potent and specific post-transcriptional gene silencing process. Since its discovery, tremendous efforts have been made to translate RNAi technology into therapeutic applications for the treatment of different human diseases including respiratory diseases, by manipulating the expression of disease-associated gene(s). Similar to other nucleic acid-based therapeutics, the major hurdle of RNAi therapy is delivery. Pulmonary delivery is a promising approach of delivering RNAi therapeutics directly to the airways for treating local conditions and minimizing systemic side effects. It is a non-invasive route of administration that is generally well accepted by patients. However, pulmonary drug delivery is a challenge as the lungs pose a series of anatomical, physiological and immunological barriers to drug delivery. Understanding these barriers is essential for the development an effective RNA delivery system. In this review, the different barriers to pulmonary drug delivery are introduced. The potential of RNAi molecules as new class of therapeutics, and the latest preclinical and clinical studies of using RNAi therapeutics in different respiratory conditions are discussed in details. We hope this review can provide some useful insights for moving inhaled RNAi therapeutics from bench to bedside. Lung diseases are among the leading causes of death worldwide. Current treatment of lung diseases such as lung cancer , respiraLong dsRNAs (around 500–1000 nucleotides in length) have been employed to study gene functions. After the exogenous dsRNA is introduced into the cytoplasm of the cells, it is cleaved by RNase III enzyme Dicer into short dsRNA called siRNA. The siRNA which is 21–23 nucleotides in length is loaded into a protein complex called the RNA-induced silencing complex (RISC). The siRNA is then unwound, and the sense strand  of the siRNA is degraded, whereas the remaining antisense strand  guides the activated RICS to the target messenger RNA (mRNA) through full complementary binding. The mRNA is then cleaved by Argonaute 2 (Ago2) in the RISC, leading to the silencing of the target gene ,15. SincshRNA is a sequence of RNA transcribed in the nucleus of the cells from a DNA vector by either RNA polymerase II or III. The primary transcript is called primary shRNA (pri-shRNA), which contains a hairpin like stem-loop structure. The pri-shRNA is processed into a 50–70 nucleotides long loop-stem precursor shRNA (pre-shRNA) by a protein complex containing the RNase III nuclease Drosha and the dsRNA binding domain protein DGCR8. It is then transported to the cytoplasm through a specialized nuclear membrane protein, exportin-5 (Exp5). The loop sequence of the pre-shRNA is cleaved by the Dicer to form a double-stranded siRNA. This endogenously produced siRNA is loaded into RISC and induce RNAi through the similar process as the synthetic siRNA ,19. SincmiRNA is a naturally occurring non-coding RNA that plays a key role in regulating gene expression. Primary miRNA (pri-miRNA) is transcribed by RNA polymerase II from endogenous miRNA gene in the nucleus. The hairpin-containing pri-miRNA is structurally similar to the pri-shRNA, therefore the maturation pathway of miRNA is also similar to shRNA . The priRNAi therapeutics offer several advantages over the traditional small molecules and protein-based drugs. They could virtually target any genes with high selectivity, including those “undruggable” targets. The design and synthesis of RNAi molecules are relatively simple because they do not need a cellular expression system, complex protein purification, or refolding schemes . CompareHuman respiratory tract is divided into the conducting region  and the respiratory region . The former is the pathway for gases conduction whereas the latter is the site of gaseous exchange . The extThe presence of fluid layers in the airways, including the mucus and the pulmonary surfactant, also creates a barrier to the delivery of drug molecules to the lungs. Mucus is a gel of three-dimensional network structure. It acts as a sieve that filters out large molecules (>500 nm) . Mucins,Mycobacterium tuberculosis which is the causative agent of tuberculosis that typically reside in the alveolar macrophages [Apart from the physical barriers, the RNAi molecules may undergo enzymatic degradation following pulmonary delivery. RNA is extremely susceptible to nuclease activity. Naked siRNA has a short half-life of less than 15 min in serum . Due to rophages , otherwiOnce the RNAi molecules overcome the aforementioned extracellular barriers and reach the target cells, they need to overcome a set of intracellular barriers. The ultimate site of actions depends on the types of RNAi molecules. For synthetic siRNA and miRNA mimic, they need to reach the cytoplasm where the RISC locates, whereas DNA encoding shRNA or miRNA need to enter the nucleus in order for transcription to take place. Surface adhesion is the first step of cellular entry. There are various endocytic mechanisms involved in the cellular uptake of macromolecules, which subsequently influence their intracellular trafficking and delivery efficiency ,53,54,55A delivery vector or carrier is generally necessary to overcome the aforementioned barriers by promoting cellular uptake and offering protection to the RNAi molecules. RNAi delivery system can be categorized into viral and non-viral vectors according to their nature, and these delivery systems have been extensively reviewed ,65,66,67The viral vectors refer to the of use of viruses to deliver genetic materials to the cells. They are extremely efficient in transducing cells and providing either transient or long-term gene expression, depending on the type of viruses employed. Most of the in vivo studies used the adenoviruses, adeno-associated virus (AAV) and retroviruses to express the plasmid DNA encoding shRNA or miRNA to induce RNAi . The mosl-lysine) (PLL), cell penetrating peptides (CPPs) and pH-responsive peptides have been investigated for the delivery of the RNAi molecules [Compared to viral vectors, non-viral vectors generally have better safety profile and lower production cost. They have been investigated for the pulmonary delivery of the RNAi molecules. Lipid-based, polymer-based and peptide-based are three major non-viral delivery systems . The lipolecules ,88,89,90olecules ,92. HoweThe therapeutic potential of RNAi-based therapy targeting respiratory disorders has been widely explored in the past decade. In the following sections, the pre-clinical and clinical studies that involved the delivery of RNAi molecules to the airways for the treatment of lung diseases are discussed.Lung cancer is a leading cause of cancer death in the world. The mainstay of treatment is chemotherapy, which is indicated for around 70% of newly diagnosed patients with local and advanced metastatic disease . Other tGenes that are essential for tumor growth and progression, such as Akt1  and c-myc, are evaluated as RNAi targets. Akt1 is an important mediator of cell growth, proliferation and survival of non-small cell lung cancer (NSCLC). Activation of Akt1 pathway is observed in most NSCLC patients. It is found to reduce chemo- and radiation-induced apoptosis to extend the survival of tumor cells ,101. SorC-myc oncogene is a downstream conduit for most oncogenic signals. It is necessary for the growth control, differentiation and apoptosis. Abnormal expression of c-myc is associated with many tumors. De-activation of myc is efficacious for lung tumor therapy . Conde eGenes associated with MDR were also evaluated as target of RNAi, such as ribophorin II (RPN2) , B-cell Besides the inhibition of MDR associated proteins alone, the co-delivery of anticancer drugs with RNAi therapeutics is as an alternative approach to tackle MDR in various types of cancers ,116,117.Respiratory infection can be caused by a wide range of microorganisms in the airways including bacteria and viruses . It is oSince RNAi was first reported to inhibit respiratory syncytial virus (RSV) in 2001 , an incrRSV infection is one of the most common infections in children ,135. In In 2005, Bitko et al. reported the inhibition of RSV and parainfluenza virus (PIV) replication by siRNA targeting viral phosphoprotein (P protein), a crucial subunit of the viral RNA-dependent RNA polymerase. Since RSV replicate primarily in the superficial layer of the respiratory epithelium, local delivery of RNAi molecules to the lungs is a rational approach to inhibit RSV replication . In the P protein may not be an ideal target as it is limited by its specificity to a particular viral strain . Anotherp = 0.058), inhaled ALN-RSV01 treatment induced a clinically meaningful reduction in the incidence of BOS, reinforcing the potential of inhaled RNAi as new antiviral therapeutics.The phase I clinical study of ALN-RSV01 was reported in 2008 . Intrana2 ion-channel protein inhibitors (adamantanes) and neuraminidase inhibitors (zanamivir and oseltamivir). However, some influenza A virus strains have shown to be resistant to these drugs [Seasonal influenza viruses, especially influenza type A virus, cause annual epidemics that led to millions of death worldwide . It is ose drugs ,144, makse drugs . The first animal study applying RNAi against influenza was reported by Ge et al. . siRNA tAnother study reported the administration of siRNA targeting NP and/or PA in lipid carrier to influenza-infected mice by hydrodynamic injection and intranasal administration. The combined siRNA delivery method effectively protected animals against lethal challenge of highly pathogenic avian influenza A viruses, including the H5N1, H7N7 and H9N2 subtypes . Since tMycobacterium tuberculosis (Mtb). Because of the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB, the slow development of new anti-TB agents and the inefficiency of vaccination, TB is still a major global health problem. WHO reported that 9.6 million people suffered from TB in 2014 and 1.5 million die annually because of the disease [Tuberculosis (TB) is a bacterial lung infection caused by  disease . A new aThe approach of RNAi-based therapeutics against TB aims to modulate the gene expression of the host instead of the bacillus because bacteria do not contain the requisite machinery for RNAi. Currently, RNAi has been investigated for TB treatment in two directions: (i) targeting the host factors that are essential for the survival of mycobacteria; and (ii) modulating the host immune response to facilitate the elimination of mycobacterium . Autopha+ T cells during chronic TB infection. It was suggested that XCL1 regulates IFN-γ production by CD4+ T cells and contribute to the stability of the granuloma [Another strategy is to modulate host immune response to improve the antimicrobial ability of the host . One of ranuloma . When a ranuloma . Although1) response and macrophage activation, leading to substantial reduction of their antimicrobial activity [Other targets of RNAi-based immunotherapy include the transforming growth factor β (TGF-β) and interleukin 10 (IL-10) . Both ofactivity . In thatRespiratory inflammatory diseases impose substantial healthcare burden worldwide as many of the patients are inadequately controlled by current therapies ,5,162. T2-adrenergic receptor agonists and leukotriene receptor antagonists, either alone or in combination. Although these medications can manage the conditions in most of the patients, there are still challenges of poor patient compliance, intolerability and long term adverse effects [Asthma is characterized by the variable and reversible airflow obstruction, airway hyperresponsiveness (AHR), mucus hypersecretion and chronic inflammation . The mos effects . For per effects . Alterna effects , transcr effects , tyrosin effects  and co-s effects .+ T helper 2 (Th2) cell-driven inflammatory response [h2 cytokine expression are increased in the airways of atopic asthma patients after exposure to allergen [h2-type cytokines, interleukin-4 (IL-4), IL-5 and IL-13, are the main players in the pathology of allergic airway inflammation, contributing to allergic sensitization, IgE production, eosinophil infiltration and AHR respectively [In allergic asthma, there is predominant activation of CD4response . T cell allergen . Th2-typectively ,184. Cliectively , IL-13 [ectively ,187 and ectively  or targeectively ,181. h2 cells mediated allergic response via controlling the balance between Th2 and Th1 cells [h2 cell associated cytokines, IL-4, IL-5 and IL-13, leading to substantial reduction of airway inflammation, AHR as well as IgE production. Reduction of mucus secretion and collagen deposition was also detected in the airways. Besides Th2 cell, other T cells subsets, such as Th9 and Th17 cells, also contribute the inflammatory response in asthma by releasing IL-9 and IL-17A. These molecules may also be the potential targets for RNAi therapy [Targeting suppressor of cytokine signaling (SOCS) 3 is an alternative approach . SOCS prh1 cells . The exph1 cells , the sil therapy . Further therapy . The the therapy . The inth2 cytokine in the airways of patients with asthma. They were investigated as targets for RNAi in the management of asthmatic inflammation. NF-κB plays a key role in the expression of many pro-inflammatory proteins [h2 cytokines. These studies support the hypothesis of using RNAi molecules targeting transcription factors to manage airway inflammation and AHR of asthma. Many transcription factors including nuclear factor-κB (NF-κB) , signal proteins . Inhibitproteins ,192. Oneproteins . Consistproteins  and GATAproteins ,193 in mTyrosine kinases, which are broadly classified into receptor and non-receptor types, play an important role in activating the transcription factors for inflammatory gene expression upon activation by the inflammatory signals ,195. Theh2 cells and hence contribute to the progress of airway inflammation [h2 cells [Targeting dendritic cells (DCs) in the airways by RNAi is another strategy for the treatment of asthma. DCs are antigen presenting cells that could be found in the airways ,201. Theammation . DCs exph2 cells . Blockinh2 cells . Likewish2 cells . 2-adrenergic receptor and anticholinergic drugs), corticosteroids and phosphodiesterase-4 inhibitor. They are effective in relieving the symptoms of COPD, but none of them can reverse the progressive deterioration in lung function or cure the disease [COPD is a persistent lung disease characterized by irreversible chronic airflow limitation. The disease is usually progressive and associated with an increased chronic inflammatory response in the lungs ,206. It  disease . It is dDespite similar clinical features, the mechanisms of underlying airway inflammation in asthma and COPD are different with distinct patterns of inflammatory cells and mediators being involved . The mecCurrent research work on RNAi therapy for COPD remains mainly in the in vitro stage, likely due to the insufficient understanding of the highly complex mechanisms underlying the pathology of the disease . MoreoveALI is a severe form of diffuse lung disease that is commonly caused by acute systemic inflammatory disorders, such as sepsis, pneumonia, trauma and pancreatitis ,212. It RNAi targeting the essential inflammatory mediators could offer protection against the progression of the disease. In an in vivo study, systemic delivery of siRNA targeting TNF-α significantly reduced the expression of TNF-α in the lungs and lung injury in a shock-induced ALI mouse model . The finIncrease microvascular permeability is a major cause of amplification of the inflammatory responses in ALI, leading to the progression to severe pathological condition . The inth2 lymphocytes [Although several in vivo studies demonstrated promising results of using RNAi therapeutics for the management of respiratory inflammatory diseases, there are several concerns that need to be taken into consideration with this approach. The distribution of naked siRNA following intranasal and intratracheal administration was investigated. It was shown that the siRNA molecules were predominantly accumulated in the peribronchial epithelial cells within 24 h post-administration . Since tphocytes , it is hphocytes . Effectiphocytes , an apprphocytes . The ratMost of the key signaling molecules involved in the pathogenesis of respiratory inflammatory disorders have multiple functions and participate in normal physiological activities to maintain homeostasis. Short-term regimen (up to four days) was employed in the majority of the animal studies. The duration of treatment may not be sufficient to observe any long-term effect. Since many inflammatory diseases are chronic disorders, long-term therapy is often required to control the disease. Therefore, it is crucial to evaluate the long-term effect of any RNAi therapeutics.Inhaled corticosteroid is commonly used to control the symptoms of lung inflammatory disorders. Steroid treatment reduces airway inflammation, partially through down-regulation of the key effectors molecules, such as c-kit  and GATAPulmonary fibrosis is a respiratory disorder characterized by the progressive and irreversible destruction of lung architecture, which ultimately leads to organ malfunction, disruption of gas exchange and even death . It is iRNAi has been exploited to suppress the production of key molecules involved in the fibrotic process, such as TGF-β ,224, ampOne complication of using TGF-β as the molecular target of RNAi is related to its physiological role in maintaining immune and cellular homeostasis. It has a role in tumor suppression as suggested by the finding that specific inhibition of TGF-β in mouse stromal fibroblasts results in spontaneous carcinoma formation in the adjacent epithelium . As a reAnother downstream mediator of TGF-β is amphiregulin (AR), which is an epidermal growth factor receptor (EGFR) ligand primarily induced by TGF-β1 . siRNAs RNAi technology has been under rapid development for the last decade. The therapeutic potential of RNAi molecules in airway diseases has been demonstrated in several clinical studies , and theAmong the different types of RNAi molecules for therapeutic development, siRNA has made significant progress, with a couple of clinical studies investigate the pulmonary delivery of siRNA to treat respiratory diseases. There are also a growing number of clinical pipelines involving the use of siRNA and other RNAi molecules. Several key questions are raised that need to be answered. For infectious diseases, as demonstrated in several in vivo studies, it appears that RNAi is more effective in prophylactic regimen than in therapeutic regimen. How can we improve the therapeutic efficacy of RNAi in combating infections to make it clinically relevant? For inflammatory diseases, long-term therapy is expected. How can we minimize the undesirable effects when cytokines/chemokines are being targeted? When the respiratory functions are compromised in patients with lung disease, is pulmonary delivery a suitable route for administration? As complicated signaling pathway is involved in the pathogenesis of different lung disorders, a thorough investigation of the underlying pathological mechanisms of the disease is a prerequisite for identifying the target of RNAi. Despite the challenges and obstacles, we believe that the pulmonary delivery of RNAi molecules targeting airway diseases is promising.


Autonomous robots that operate in the field can enhance their security and efficiency by accurate terrain classification, which can be realized by means of robot-terrain interaction-generated vibration signals. In this paper, we explore the vibration-based terrain classification (VTC), in particular for a wheeled robot with shock absorbers. Because the vibration sensors are usually mounted on the main body of the robot, the vibration signals are dampened significantly, which results in the vibration signals collected on different terrains being more difficult to discriminate. Hence, the existing VTC methods applied to a robot with shock absorbers may degrade. The contributions are two-fold: (1) Several experiments are conducted to exhibit the performance of the existing feature-engineering and feature-learning classification methods; and (2) According to the long short-term memory (LSTM) network, we propose a one-dimensional convolutional LSTM (1DCL)-based VTC method to learn both spatial and temporal characteristics of the dampened vibration signals. The experiment results demonstrate that: (1) The feature-engineering methods, which are efficient in VTC of the robot without shock absorbers, are not so accurate in our project; meanwhile, the feature-learning methods are better choices; and (2) The 1DCL-based VTC method outperforms the conventional methods with an accuracy of 80.18%, which exceeds the second method (LSTM) by 8.23%. The current decade has witnessed an increasing application of autonomous vehicles and mobile robots in industrial transportation, ground reconnaissance, planetary exploration, etc. ,2. TheseRobotic terrain classification refers to the process of a mobile robot identifying the current or forthcoming terrain, such as gravel, grass, etc., using its on-board sensors. The terrain classification methods can be categorized into two parts: the non-interactive and the interactive methods. The non-interactive methods are usually realized by optical sensors  ,10,11,12All the existing work concerning VTC concentrates on the robots with relatively hard tires and without shock absorbers, because the vibration signal could reflect the truth of ground surfaces. However, in real-world applications, to guarantee the stability and security of the robot itself, and protect the equipment or cargo on the robot, the robot is often equipped with elastic tires and shock absorbers. Hence, it is quite practical to study the VTC under the effect of shock absorbers. The difference between the vibration signals with and without shock absorbers can be seen in A large body of comparative experiments are conducted to demonstrate the performance of the existing feature-engineering and feature-learning classification methods.According to the long short-term memory (LSTM) network, we propose a one-dimensional convolutional LSTM (1DCL)-based VTC method to learn both spatial and temporal characteristics of the dampened vibration signals.In this paper, we explore the VTC, in particular for wheeled robots with shock absorbers. Because the vibration sensors are usually mounted on the main body of the robot, the vibration magnitude are dampened significantly, which results in the vibration signals collected on different terrains being more difficult to discriminate. Hence, the existing VTC methods applied to a robot with shock absorbers degrades. The contributions are twofold:In feature-engineering approaches, the combination of power spectral density (PSD) and random forest (RF) achieves the highest accuracy of 69.88%.The conventional feature-learning approaches outperforms the feature-engineering ones slightly by about 2%, though they are computationally intensive.The proposed 1DCL-based VTC method outperforms the conventional methods in an accuracy of 80.18%, which exceeds the second method (LSTM) by 8.23%.The real-world experiment on eight different terrains demonstrates that:The remainder of this paper is organized as follows. A large body of studies about non-interactive methods using exteroceptive modalities have been done. Image-based methods usually use digital cameras or infrared cameras. Digital image-based methods mainly extract visual features from terrain images using local binary patterns (LBP), bag of visual words (BOVW) or speeded up robust features (SURF) ,11,25,26Interactive methods using proprioceptive modalities has not been explored in the same depth as non-interactive methods. The existing interactive methods are usually realized by acoustics, haptics, and vibration. Acoustic terrain classification is motivated by vehicle-terrain interaction sounds which is produced when the robotic locomotion mechanism  is pressing the ground. This idea is first proposed by . In theiThe most popular proprioceptive modality in interactive method is vibration. Vibration signals are easily collected in contrast to haptic signals, since the classifier can perform well even with a consumer-grade accelerometer The early work that involves the VTC uses power spectrum as the feature . In lateMulti-modality methods combine several modalities based on their complementary characteristics, thus possessing a higher accuracy and robustness against environmental interference ,39. In , three-aIn the following comparative study, we refer to some of the feature extraction methods and classification methods of the aforementioned literature.The dampened VTC is illustrated in In this section, we introduce the feature engineering approaches.We collected vibration data by an accelerometer mounted perpendicularly on the main body of a wheeled mobile robot with shock absorbers. In our experiment, we recover the vibration signal from the accelerometer readings by subtracting the gravitational acceleration, and then split the vibration signal into short segments. Each segment contains 50% overlap of successive segments. In Now we are in the position to extract features from each segment. Feature extraction is performed in the time domain and in the frequency domain. Time-domain vibration feature extraction method aims to obtain a simple and compact representation of vibration signal. In VTC, mean, standard deviation, norm, autocorrelation, RMS, maximum and minimum are usually selected as statistical features ,42. In vm is the number of samples. For each sample n is the number of acceleration values in a segment, the time-domain features are the followings. Please note that we will omit subscript j later for brief. That is to say, symbol ith value of sample (1)Zero crossing rate (ZCR). The ZCR counts the number of times that the signal crosses the zero axis, which is an approximate estimation of the frequency of (2)Mean. The mean expresses the average roughness of ground surface.(3)Standard deviation. Intuitively, the standard deviation is greater with a rougher ground surface.(4)Norm. Usually the (5)r is a measure of the non-randomness as defined byr gets larger with the dependence of successive vibration values.Autocorrelation. The autocorrelation (6)Maximum. Find the maximum of the degree of bump.(7)Minimum. Find the minimum of the degree of bump.(8)Skewness. The skewness (9)Excess kurtosis. The excess kurtosis Consider the training sample set n, the N-point DFT of Frequency-domain representation is widely used in signal processing field as it simplifies the mathematical analysis and helps analyze the components of signals. The most common tool for frequency-domain transformation is Fast Fourier Transform (FFT) that is an extensive application in both VTC ,45 and aN is specified as a power of 2. Hence, the training vectors of different lengths in our experiments implement the corresponding points FFT. PSD is defined as the Fourier transform of the autocorrelation function. For limited discrete values, the PSD of Usually Various classifiers have been used for VTC and other fields. Among them, SVM is used the most widely ,34,39,45The classification performance of feature-engineering approaches relies on handcrafted feature extraction heavily. In recent years, there has been a considerable effort on the development of end-to-end learning methods . InsteadIn this section, we first introduce two widely used end-to-end feature-learning methods: LSTM and CNN. Next, we propose a novel neural network architecture which is called 1DCL.A typical convolutional neural network consists of alternating convolution and subsampling layers after the input layer and a generic multilayer network (fully connected layers) at the last stage of the architecture. At a convolution layer, the input feature maps from the previous layer are convolved with kernels which should be learned, and then the convolved results are put through the activation function to form the output feature map. After feature extraction in the convolution layers, the output feature map is transferred to subsampling layers  for feature selection and information filtering. The last few layers are fully connected layers that are identical to a multilayer perceptron (MLP) for estimating the decision (classification) vector. In CNNs, each convolutional neuron is only connected to some of the nodes in the previous layer called receptive field, which is a main characteristic of CNNs named sparse connection. Another characteristic of CNNs is parameter sharing that all the units in a feature map share the same parameters. These parameters constituted matrix refers to as kernel.Recurrent neural networks (RNNs) can process and predict sequence data due to their recurrent structure . Howevert) called forget gate W and b are weight vectors and bias vectors. The LSTM contains special units called memory blocks (mentioned as LSTM cells in the following) in the recurrent hidden layer. The LSTM cells contain a memory cell To our knowledge, the CNN-based and LSTM-based VTC have rarely been investigated. Many existing CNN and LSTM models are often general-purpose, thus not suitable for our application. In our study, the dampened vibration signals increase the difficulty of terrain classification, so we design a dedicated neural network model called 1DCL by modifying and integrating CNN and LSTM. Such a neural network model can learn both spatial and temporal characteristics of the raw vibration signals.The proposed 1DCL shown in We employ a 1D convolutional layer in the 1DCL to learn preliminary spatial features. Structural differences between the traditional 2D and the proposed 1D CNN are visible, that is, the usage of 1-D arrays replace 2-D matrices for both kernels and feature maps. In the 1DCL, convolution kernel of the 1D convolutional layer is of size 3. Convolutional stride is fixed as 1. A max pooling layer with a kernel of size 2 follows the 1D convolutional layer. Max pooling layer provides some invariance and reduces parameters, thereby being able to avoid overfitting. The posed in  can chanfunction .Rather than constructing multiple convolutional layers to learn deep spatial features, we only build the above two convolutional layers because we pay more attention to learn temporal dynamics from the vibration sequence. As shown in Equation ) are useAs for the hyperparameters, we adopt the Spearmint Bayesian optimization library  to tune To compare different classification methods applied in terrain classification, we conduct the experiment with a four-wheeled mobile robot on 8 different terrains.The experimental robot is shown in We collect vibration data by controlling the four-wheeled mobile robot to wander on eight terrain types which are different in rigidity, roughness, and flatness. Some of them are artificial terrains , while some are natural ones . In our experiment, we recover the vibration signal from the accelerometer readings by subtracting the gravitational acceleration, and then split the vibration signal into short segments. The length of each segment is set as 200 points initially, and each segment contains 50% overlap of successive segments; consequently, the dataset contains 11,224 samples in total . In this section, we first study the performance of different classifiers in combination with handcrafted vibration features, i.e., feature-engineering approaches. Each pair of handcrafted feature and classifier is evaluated to find out the best combination. We also analyze the performance of 1DCL and compare it with feature-engineering approaches and other feature-learning approaches. Finally, experiments on different segment lengths are done.The 10-fold cross-validation is used to evaluate the classification performance by three parameters, namely, accuracy, True Positive Rate (TPR) and F1-score. The accuracy TPTP+FP,Rc=TPTP+FWe haveThe calculation of chnology , i.e., aConsidering the ability to distinguish each terrain type, as illustrated in 1D-CNN. A one-dimensional convolutional neural network with alternating convolutional and pooling layers learns spatial features of a segment, and is followed by two fully connected layers and a SoftMax layer.LSTM. A single layer LSTM model directly handles 200 points length segments with 10 LSTM cells in series, which helps to learn temporal dynamics.1DCL. The proposed model is a spatiotemporal architecture.1DCL-FC. A variant of our 1DCL that we use a fully connected layer to replace the 1DCL-3Conv. Another variant of our 1DCL model that we consider increasing the number of convolutional layers to learn better spatial features. Here, we build two normal convolutional layers and a Now we are in the position to evaluate the performance of feature-learning approaches. The experiments are conducted on a system with a NVIDIA GeForce GTX 1080 Ti GPU and an Intel i7-7700K processor. We perform the experiments using different neural network model configurations to gain insight on the effect of learning spatiotemporal relationships. We consider the following five model configurations:Results from the comparison are shown in The two variants of our 1DCL, 1DCL-FC, and 1DCL-3Conv, do not perform as well as the 1DCL seen from Since wheeled robot with a terrain-dependent control system works in outdoor environments, terrain classification must be implemented in real time to ensure the safety of the robot and make the robot adapt its driving style to the current terrain. As depicted in At the end of this section, we perform experiments using varying vibration signal segment length on two representative approaches: FFT-SVM and the proposed 1DCL. Please note that splitting raw vibration signal into segments with different lengths will result in different number of instances. Therefore, we randomly select the same number of instances for each experiment to ensure the comparability of the experiment results, which may correspondingly cause a slight drop for the accuracy due to the reduction in training samples.Evaluating the segment sizes presents a resolution-efficiency trade-off. That is, a longer segment length contains more information of ground surface thus yielding increased accuracy requires more execution time, while a shorter segment length with faster real-time response leads to worse classification results. For applications of terrain classification, fast response and execution rates are essential for traversability evaluating. In this paper, we conducted an extensive comparative study of VTC with shock absorbers. We presented a large body of experiments on feature-engineering approaches including 21 combinations of 3 kinds of features and 7 different classifiers, and feature-learning approaches including 3 neural networks. Referring to this study, one can find the most appropriate classification method according to their demands. If there are no limits on computational complexity, the proposed 1DCL could be the best solution. In future work, we will enhance the proposed 1DCL to implement real-time computation, which is indispensable for outdoor mobile robots, referring to literature . Additio