Individuals with autism spectrum disorders (ASD) tend to make inadequate social judgments, particularly when the nonverbal and verbal emotional expressions of other people are incongruent. Although previous behavioral studies have suggested that ASD individuals have difficulty in using nonverbal cues when presented with incongruent verbal-nonverbal information, the neural mechanisms underlying this symptom of ASD remain unclear. In the present functional magnetic resonance imaging study, we compared brain activity in 15 non-medicated adult males with high-functioning ASD to that of 17 age-, parental-background-, socioeconomic-, and intelligence-quotient-matched typically-developed (TD) male participants. Brain activity was measured while each participant made friend or foe judgments of realistic movies in which professional actors spoke with conflicting nonverbal facial expressions and voice prosody. We found that the ASD group made significantly less judgments primarily based on the nonverbal information than the TD group, and they exhibited significantly less brain activity in the right inferior frontal gyrus, bilateral anterior insula, anterior cingulate cortex/ventral medial prefrontal cortex (ACC/vmPFC), and dorsal medial prefrontal cortex (dmPFC) than the TD group. Among these five regions, the ACC/vmPFC and dmPFC were most involved in nonverbal-information-biased judgments in the TD group. Furthermore, the degree of decrease of the brain activity in these two brain regions predicted the severity of autistic communication deficits. The findings indicate that diminished activity in the ACC/vmPFC and dmPFC underlies the impaired abilities of individuals with ASD to use nonverbal content when making judgments regarding other people based on incongruent social information. Individuals with autism spectrum disorders (ASD) have difficulties in processing of both verbal and nonverbal information The characteristic behavioral responses to incongruent verbal-nonverbal information of ASD individuals have been reported in a previous study a priori language-based knowledge and novel language-based information Autistic responses to other types of incongruity have been investigated in a series of previous studies In the present functional magnetic resonance imaging (fMRI) study, therefore, we aimed to reveal the neural mechanism underlying the imbalance between the interpretation of incongruent verbal-nonverbal social information in ASD individuals using realistic movie stimuli. In each of the short movie stimuli, a different professional actor spoke a word that was related to a positive or negative emotion while producing a positive or negative emotion-related facial expression and voice prosody . To direBased on previous behavioral findings To focus on neural deficits mediating impaired social judgments in ASD individuals, we searched brain areas that are considered to be related to social judgments. Specifically, we estimated brain activity in anatomically-defined brain regions consisting of the medial prefrontal cortex (mPFC), precuneus, temporoparietal junction (TPJ), amygdala, superior temporal sulcus (STS), anterior insula (AI) and inferior frontal gyrus (IFG). According to several previous studies . After a complete explanation of the study, written informed consent was obtained from all participants. Using longitudinal clinical assessments, a trained psychiatrist (H.Y.) confirmed that all of these adult participants had no intellectual disabilities, no need for psychotropic medication, and were capable of providing informed consent. Therefore, no participant needed someone else who consented on the behalf of the participants.Fifteen male participants with ASD were recruited from the outpatient service of The University of Tokyo Hospital . FourteeThe exclusion criteria for the both groups consisted of any history of current or past neurological illness, traumatic brain injury with any known cognitive consequences or loss of consciousness for more than five minutes, and substance abuse or addiction. An additional exclusion criterion for the TD group was a history of psychiatric disease or a family history of an axis I disorder among their first-degree relatives.Handedness was determined using the Edinburgh Handedness Inventory A 3T MRI scanner (GE) was used in this experiment. The anatomical scanning sequence was a three-dimensional Fourier-transform spoiled-gradient-recalled acquisition during steady state . Gradient-echo echo-planar sequences were used for functional imaging . The first five functional images in each run were excluded from analysis to account for the equilibrium of longitudinal magnetization. Because the stimuli included human speech, we used an MRI-compatible headphone system , and confirmed before and after the experiment that the speech content and prosody in the stimuli could be heard clearly. A trained neuroradiologist (H.T. or O.A.) evaluated the MRI scans and found no gross abnormalities in any participant. Magnetic field inhomogeneities in our scanner were monitored with basic quality control conducted daily, and they were stable over the course of this study.The stimuli were 80 original monochrome movies with a length of 1,500 ms . In each movie, one of 20 professional actors  spoke a different emotional word accompanied by an emotional facial expression and expressive prosody. To exclude unnatural and unrealistic stimuli, healthy Japanese adults  examined whether the movies were natural and realistic or not. Only the movies that were rated as the most realistic and natural were used as the stimuli in the present experiment.As verbal information, words with high emotional valence and arousal were selected from the list of Affective Norms for English Words  The emotional directionality of the nonverbal information was validated in an independent behavioral experiment using a separate group of 12 TD male participants (24–34 years old) who did not undergo the subsequent fMRI experiments. To validate the nonverbal information conveyed by the actors’ facial expressions, the participants were instructed to evaluate silent movies by indicating a score on an 8-point happy–disgust scale, with one being the happiest and 8 being the most disgusted. In contrast, to validate the prosody, we had the participants perform the same evaluation of the voice stimuli without the visual component of the movie . In these evaluations of voice prosody, the participants were instructed to ignore the verbal content of the speech as much as possible. The mean scores and standard deviations for the facial expressions were 1.9±0.3 in the NV+V-, 1.8±0.2 in the NV+V+, 7.1±0.6 in the NV-V-, and 6.9±0.5 in the NV-V+ conditions. The mean scores and standard deviations for the voice prosody 3.0±0.3 in the NV+V-, 2.7±0.4 in the NV+V+, 6.8±0.5 in the NV-V-, and 6.6±0.4 in the NV-V+ conditions. There was no main effect of verbal information in a repeated-measures analysis of variance (ANOVA) comparing the positive versus negative words. All scores were significantly different from neutral facial expressions or prosody . These results indicate that the intended nonverbal information was appropriately conveyed to the participants, for both the facial expressions and voice prosody.The fMRI scanning consisted of two runs, each of which involved 10 NV-V-, 10 NV-V+, 10 NV+V-, and 10 NV+V+ stimuli, pseudo-randomly ordered. During scanning, the participants were presented with the movies sequentially, and were instructed to judge whether they believed the actor in the movie was a friend or a foe by pressing a button. They were asked to make an intuitive judgment of the actors without deep consideration. Each movie stimulus was presented immediately before a 2,000-ms response period, followed by a 2,500–4,500-ms waiting period . The meaBefore the following analysis, we needed to exclude the possibility that the friend or foe judgments were strongly biased by any exogenous feature in the stimuli. If this was the case, the stimuli may have been disproportionately judged as either friends or foes no matter who saw them. To address this issue, we applied a chi-square goodness-of-fit test to the behavioral data obtained from the TD subjects, and we found that there was no significant bias towards judging the stimuli as a friend or a foe across all of the incongruent stimuli . This suggests that the judgments cannot be explained by the exogenous features of the stimuli rather than internal processes in the participants.We classified the judgments of the incongruent stimuli into nonverbal-cue-biased and verbal-cue-biased judgments, according to the type of information that primarily affected the judgment http://www.fil.ion.ucl.ac.uk/spm/). Functional images were realigned, slice timing corrected, normalized to the default template with interpolation to a 2×2×2 mm space The fMRI data were analyzed in SPM8  × two types of response (friend and foe). Each event-related regressor had an onset at the time of stimulus presentation and a duration that corresponded to the response time. We also added six regressors for the six motion correction parameters. We then defined the brain activity during judgments biased to nonverbal cues as Bnv: [“Foe to NV-V+” and “Friend to NV+V-”] > [“Foe to NV-V-” and “Friend to NV+V+”]. This contrast was based on the assumption that, in judgments of foe to NV-V+, for example, the participants emphasized nonverbal information more explicitly than in judgments of foe to NV-V-. To make this analysis valid, we excluded types of judgments that were rarely selected . This exclusion was intended to reduce the influence of the stimuli in which the emotional content of the facial expressions and voice prosody were not exactly congruent to the verbal content of the words, though the effect cannot be completely excluded. Using the same logic, we defined the brain activity during judgments biased to verbal cues as Bv: [“Friend to NV-V+” and “Foe to NV+V-”] > [“Friend to NV+V+” and “Foe to NV-V-”].For the group level analysis, we estimated that activity in different brain regions was specifically related to nonverbal-cues-biased judgments using differences between the Bnv and Bv conditions.http://www.fmri.wfubmc.edu). To account for individual anatomical differences, we applied Gaussian smoothing  to these mask images. To investigate the involvement of other brain regions, we also conducted a whole brain analysis . Furthermore, for the regions found to exhibit activity in these analyses, we examined the effects of the affective valence of the verbal and nonverbal information  by conducting a repeated measures ANOVA of the activity  in each of the subject groups.The group level analysis was based on a random-effects model, and we first conducted within-group comparison for each of the groups. Moreover, we employed paired-t-tests and compared brain activity during the nonverbal-cue-biased judgments with that during the verbal-cue-biased judgments (Bnv > Bv). We then conducted between-group comparisons by employing a repeated-measure mixed-design two-way ANOVA of the brain activities . Using masked images, significant brain activations were examined in anatomically-defined regions described earlier, which previous studies have suggested are related to empathy, mentalizing, or uncertainty judgments To investigate which of the detected regions were more relevant to the nonverbal-cue-biased judgments, we examined Pearson’s correlation coefficients between the brain activity in the TD group during the nonverbal-cue-biased judgments and the number of behavioral judgments. This analysis is based on the assumption that activity in brain regions with relevance for certain behaviors significantly correlated with the corresponding behavioral scores across subjects Finally, to exclude the possibility that the ASD subjects may show less overall brain activity than the TD subjects, we estimated differences in the amygdala response to congruent emotional stimuli between the groups, because previous studies reported enhanced activation of the amygdala in ASD individuals A repeated-measure mixed-design two-way ANOVA of the response time data (stimulus type: incongruent and congruent stimuli × group: ASD and TD) revealed neither significant main effects of group nor any significant interaction between the group and stimulus type (P>0.39). However, there was a main effect of stimulus type  = 6.3, P = 0.01). A post-hoc paired t-test revealed that response times in both the ASD and TD individuals were longer for incongruent than congruent stimuli P<0.001, . This suFurthermore, a paired t-test did not detect any significant differences between response times to incongruent stimuli that were presented immediately following congruent stimuli compared to response times to incongruent stimuli that were presented immediately following incongruent stimuli. This finding allowed us to exclude conflict-adaptation effects in the following analysis A two-sample t-test of the number of judgments of the incongruent stimuli indicated that the ASD subjects made significantly fewer nonverbal-cue-biased judgments than the TD subjects , and left inferior parietal lobe (IPL) showed significantly greater activity in the nonverbal-cue-biased judgments (t(16) >3.5, P<0.05, FDR-corrected; To quantitatively elucidate the differences in brain activity between the ASD and TD subjects, we next conducted a repeated-measure mixed-design two-way ANOVA of the per-voxel brain activity . Voxel-level brain activity was extracted from the anatomical brain regions using the same procedures as the within-group analysis. Although there were no significant main effects, we found a significant interaction between the group and the judgment types in the anterior cingulate cortex/ventral mPFC (ACC/vmPFC), dorsal mPFC (dmPFC), bilateral AI, right IFG, left amygdala, and right superior temporal pole (STP)  >12, P<0.05, FDR-corrected). In contrast, the exploratory whole brain analysis did not find any significant activation .A post-hoc analysis of the interaction defined as (Bnv > Bv) × (TD > ASD) found that the ACC/vmPFC, dmPFC, bilateral AIs, and right IFG showed significantly greater activity in the TD group than in the ASD group (t(30) >3.5, P<0.05, FDR-corrected; We then investigated which of the 8 brain regions examined exhibited activity that was related to the nonverbal-cue-biased judgments by correlating brain activity with the number of the nonverbal-cue-biased judgments. Consequently, in the TD group, activity in only the ACC/vmPFC and dmPFC exhibited significant positive correlations with the number of nonverbal-cue-biased judgments . In both regions, the brain activity significantly and negatively correlated with ADI-R communication scores  as well as during nonverbal-cue-biased judgments , Table 2To the best of our knowledge, the present study is the first to elucidate the neural mechanisms underlying impaired social judgments of incongruent verbal-nonverbal information in ASD individuals. We found that ASD participants showed diminished activity in the bilateral AI, pIFG, ACC/vmPFC and dmPFC. Among these five brain regions, the ACC/vmPFC and dmPFC were the regions primarily involved in nonverbal-cue-biased judgments in the TD participants. In addition, the reduction in brain activity in these regions was predictive of the severity of the ASD participants’ communication deficits. These results indicate that impaired recruitment of the ACC/vmPFC and dmPFC in ASD individuals is associated with their impairments in the social judgment of incongruent verbal-nonverbal information.The current study employed short movies where a trained professional actor spoke an emotional word while displaying an emotional facial expression and voice prosody. These realistic video stimuli were chosen to maximize the similarity of the psychological task to real-life social situations. In contrast, previous studies have used nonverbal stimuli in the form of a narrator Although the present psychological task was a reaction time task, it had no correct response and allowed the participants to express what they felt in a relatively unrestrained manner. Therefore, the present task allowed the participants to respond in a more spontaneous manner than forced-choice tasks. This spontaneity not only reduced the differences between the task and real-life situations, but also enabled us to detect autistic behavioral and neural impairments, which are often difficult to capture in adult individuals with high functioning ASD The number of participants in the present study is comparable to that in previous studies By demonstrating significant contributions of the ACC/vmPFC and dmPFC in nonverbal-information-biased judgments, the present results suggest that both areas play important roles in the pathophysiology of communication in ASD individuals. Previous studies have suggested that the vmPFC is involved in self-referential mentalizing The right IFG and bilateral AI also exhibited significantly reduced activity during nonverbal-information-biased judgments in the ASD group compared with the TD group. Previous studies in TD individuals have repeatedly reported significant contributions of these brain regions to empathic processing of the emotions and sensations of other people . If the ASD participants became more habituated to the emotional stimuli during training than the TD controls, they may have exhibited less activity in the amygdala The behavioral pattern in the response times to incongruent stimuli data excluded the possibility that the ASD participants may have been unable to perceive nonverbal information. If this was the case, ASD individuals would not have been able to sense the incongruence between nonverbal and verbal information. However, as in the TD participants, the response times to incongruent stimuli were significantly prolonged compared to the response times to congruent stimuli in the ASD participants , which iFinally, by estimating the effects of the interaction among the types of judgments and the groups, we also excluded the possibility that activation in the brain regions in which the ASD subjects exhibited less activity reflected conflict monitoring rather than the effects of impaired nonverbal-cue-biased judgments. The detected activations were based on differences in brain activity during the nonverbal-cue-biased judgments compared to verbal-cue-biased judgments. Therefore, we can assume that the influence of the conflict monitoring on these activations was minimal.In conclusion, the present study demonstrated that significantly decreased brain activity in the right IFG, bilateral AI, and ventral and dorsal mPFC was associated with impaired social judgments of incongruent verbal-nonverbal information in high-functioning non-medicated adult males with ASD. In particular, the nonverbal-cue-biased judgments recruited the ACC/vmPFC and dmPFC in the TD group, and diminished activity in these two regions in the ASD subjects was predictive of the extent of their impairments in communicating with other people. Through disrupting important cognitive systems such as mentalizing, empathy, and judging uncertainty, dysfunction in these brain regions may play a role in shaping the characteristic tendencies of individuals with ASD to make impaired social judgments.Video S1(MP4)Click here for additional data file.Video S2(MP4)Click here for additional data file.Video S3(MP4)Click here for additional data file.Video S4Each video is a representative stimulus for each type of stimuli used in the present study. In a video, one of 20 professional actors  appears and speaks an emotional word (positive or negative) with emotional facial expression and prosody (happy: positive or disgust: negative). In VideoS1.mp4 and VideoS3.mp4, the actor speaks “(You look) Distressed” in Japanese with disgust or happy facial expression and prosody, respectively. In VideoS2.mp4 and VideoS4.mp4, he speaks “(You are) Friendly” in Japanese with disgust or happy facial expression and prosody, respectively.(MP4)Click here for additional data file.


A previous study of beliefs about health and illness in Zimbabweans with diabetes mellitus indicated limited knowledge about diabetes and the body, affecting self-care and health-care seeking behaviour. The aim of this study was to assess the level of diabetes knowledge in Zimbabwean adults with diabetes mellitus, to determine the main gaps in knowledge and identify the socio-demographic and diabetes-related determinants that predict diabetes awareness and self-care practices.A cross-sectional descriptive study was performed using a standardized self-report Diabetes Knowledge Test questionnaire (DKT) of 58 respondents, 32 women and 26 men. Results were analysed with descriptive and analytic statistical methods.The majority of the respondents scored average knowledge on all three sub-scales: general knowledge, insulin use and total knowledge, with an overall score of 63.1± 14, 2%. Major knowledge gaps were in areas related to diet, insulin use and glycaemic control. No significant differences in mean scores were detected in the diabetes knowledge sub-scales when comparisons were made of mean knowledge scores in relation to socio-demographic and diabetes-related characteristics. However, diabetes-related complications were significantly associated with lower total and general diabetes knowledge, and female gender was an independent determinant of low general knowledge.Knowledge gaps were evident in areas regarding insulin use, diet and glycaemic control. Low diabetes knowledge was associated with female gender and could be a risk factor for development of diabetes-related complications. Knowledge gaps need to be addressed in diabetes education to prevent development of diabetes-related complications. Diabetes mellitus affects millions of people worldwide and its related complications continue to be of great concern. The pandemic is rapidly spreading in developing countries and particularly affecting poor populations in Sub-Saharan Africa , 2.Globally, communicable diseases are slowly being replaced by non-communicable diseases (NCDs), diabetes mellitus being amongst them, associated with increased modernization, wealth and prosperity . In deveDiabetes mellitus is ranked fifth amongst the NCDs recorded in most Zimbabwean public hospitals, with a prevalence rate of 10% in the ≥ 25 age group . It was With a few exceptions –11, no oWith the increased prevalence of diabetes mellitus and ever decreasing health care resources especially in developing countries, the need for self-care management becomes paramount. It has been reported that patients with diabetes often lack sufficient knowledge about their disease and thus frequently have poor self-care management . The outHealth education by health care staff and employing new research findings and useful strategies can reduce the burden of the disease . Nurses The aim of the study was to assess the level of diabetes knowledge in Zimbabwean adults with diabetes mellitus, to determine the main gaps in knowledge and identify the socio-demographic and diabetes-related determinants that predict diabetes awareness and self-care practices.A cross-sectional descriptive study was conducted using a standardized self-report questionnaire, Diabetes Knowledge Test (DKT), to investigate knowledge about diabetes. The design enabled the investigator to gather information on the variable of interest in persons with diabetes mellitus and to make inferences about possible relationships between diabetes knowledge and the persons’ socio-demographic characteristics .The sample consisted of 58 adult respondents, 32 women and 26 men, with diabetes mellitus obtained through convenience sampling.The inclusion criteria were: diagnosis with diabetes mellitus for ≥ one year, mentally sound to give informed consent and be conversant with either English or Shona  was used to collect data on the respondents’ knowledge about diabetes, its treatment, their understanding, socio-demographic and diabetes-related background data. The participants were attending meetings at the Zimbabwe Diabetes Association. This is anaffiliated non-governmental patient organization that helps in giving both material and information support as part of continued self-management to persons with diabetes who are usually referred from the hospital-based diabetes clinics. One reason for attending the sessions was availability and lower prices of insulin than in public health care pharmacies. Another reason for attending was sharing of disease experiences and getting information about diabetes and the management based on patients’ desires on information for example about diet, physical exercises, medication, self-care etc. Meetings are held monthly for about 2 hours.A registered nurse (first author) distributed the questionnaires when the respondents met. If needed she clarified and answered questions from the participants during the completion of the questionnaires. The total time required to complete the questionnaire was about an hour.Data were collected using a standardized self-report questionnaire including the Diabetes Knowledge Test (DKT) of the University of Michigan Diabetes Research and Training Centre and socio-demographic and diabetes-related background data.The DKT has been shown to be a reliable and valid instrument  consistiThe socio-demographic and diabetes-related background data included sex, age, educational level, marital status, employment status, duration of diabetes mellitus, type of diabetes treatment, diabetes-related complications, duration of attending follow-ups and attendance of diabetes classes.The original questionnaire in English was translated into Shona by a bilingual Shona-speaking senior nurse (first author), then translated back into English by an independent Shona-speaking senior diabetes educator. Comparison of the two versions for content and meaning was done and agreed upon by the principal investigator (first author) and the co-authors. The questionnaire was then pre-tested on four persons who met the set inclusion criteria  before being used in order to test the questions and to estimate how long it took to complete the questionnaire.The study was approved by the Diabetes Association of Zimbabwe and the Medical Research Council of Zimbabwe. It was done in accordance with the Helsinki Declaration  and withData were checked regarding underlying distributional assumptions, described and analysed accordingly. For normally distributed variables, Student's t-test was used for group comparisons and one-way analysis of variance (ANOVA) used to compare > 2 groups. For the non-normally distributed variables, the non-parametric Mann-Whitney U-test was performed to compare two groups and the Kruskal-Wallis test was used to compare more than two groups. Pearson's chi-squared test was used to ascertain any association between two qualitative variables.Three multiple logistic regression analyses were performed to identify any independent association between the participants’ socio-demographic and diabetes-related characteristics and low knowledge levels . As dependent variables the third of the participants with the lowest knowledge level in the respective subscale were chosen. The analysis was performed in steps, with all independent variables included in the first step. Subsequently, step by step, the variable in the previous step with the highest p-value was excluded from the model. A p-value < 0.05 was considered statistically significant .The total knowledge score was determined by awarding one point for each correct answer and a zero for a wrong answer or no response. The total knowledge score ranged from 0-23 and was categorized as follows: < 11= poor knowledge, 11–17= average knowledge and >17 = good knowledge. The general knowledge score was categorized as: < 7= poor, 7–11= average, > 11 = good and insulin use knowledge was categorized as follows: < 5= poor, 5–7 =average and > 7 = good . KnowledOverall 63.1 ± 14.2% of the respondents correctly answered the items on the total DKT while 63.8 ± 15.2% had comparable scores on the general knowledge subscale and 62.1 ± 19.7% on the insulin use knowledge subscale (data not shown). No gender differences were found . A fifthKnowledge deficit (incorrect answers above 50%) was noted in six questions; in descending order these were related to the definition of a ‘free food’ (75.9%), the purpose of testing HbA1C (67.2%), cause of an insulin reaction (67.2%), food that should not be used to treat low blood glucose (58.6%), signs of ketoacidosis (58.6%) and what to do when one starts to have an insulin reaction (51.7%) .Comparisons of the mean knowledge scores in relation to the participants’ socio-demographic characteristics and diabetes-related data were made . No signMultiple logistic-regression analyses were used to estimate the independent associations of between poor knowledge and socio-demographic variables and diabetes-related characteristics. Female gender was an independent determinant of low general knowledge about diabetes , while no significant associations were found between insulin use knowledge, total knowledge and the respondents’ demographic and diabetes-related characteristics (data not shown).As far as we know, this is the first study investigating diabetes knowledge using a validated instrument in an African population. Overall, 63.1± 14.2% of the respondents correctly answered the DKT questionnaire with the majority scoring average knowledge in all three knowledge sub-scales: general knowledge, insulin use and total knowledge. Major knowledge gaps were noted in six questions related to insulin use, glycaemic control and diet as noted by scores below 50%. Reporting diabetes-related complications was significantly related to low total and general knowledge about diabetes, and female gender was found to be an independent determinant of low general knowledge of diabetes.Diabetes outcome depends mainly on the patient’ sound knowledge of self-care that is dependent upon their knowledge of the disease , 17, 24,The major knowledge gaps reported in this study in the areas related to diet, insulin use and glycaemic control are almost consistent with previous studies which also used the DKT, and respondents scored less than 50% in the same questions , 22. In Previous studies performed in African populations reported females being more active in self-care and information seeking than males –8, but iDiabetes-related complications was an important factor for low diabetes knowledge , which could be an indication of low knowledge as a risk factor for developing diabetes-related complications. This finding could further support the reported knowledge gaps in glycaemic control, diet and insulin use, another implication for the importance of addressing these areas through diabetes education programmes and periodically re-assessing individual patient's educational needs and barriers to ongoing diabetes education based on individual beliefs about health and illness –8, 17.The main strength of this study was the use of a validated DKT questionnaire  and simiA study sample in a general population of persons with diabetes might have given different results and possibly even lower knowledge levels.This study has some areas of knowledge deficit with regard to diet, glycaemic control and insulin use. Low diabetes knowledge was significantly associated with female gender and could be a risk factor for development of diabetes-related complications. The areas of knowledge gaps need to be addressed in diabetes education as they might influence the development of diabetes-related complications. However, the limited sample size restricts the possibility to draw conclusions from our results to the general diabetes population in Zimbabwe why further studies of a representative sample are needed.


The influences of LID and DBS on BG and M1 plasticity have been explored in animal models and in PD patients. In addition, many other factors such age, genetic factors , emotional state, time of the day, physical fitness have been documented to play role in the extent of plasticity induced by TMS in human studies. In this review, we summarize the studies that investigated M1 plasticity in PD and demonstrate how these afore-mentioned factors affect motor cortical plasticity in PD. We conclude that it is important to consider the clinical, demographic, and technical factors that influence various plasticity protocols while developing these protocols as diagnostic or prognostic tools in PD. We also discuss how the modulation of cortical excitability and the plasticity with these non-invasive brain stimulation techniques facilitate the understanding of the pathophysiology of PD and help design potential therapeutic possibilities in this disorder.In Parkinson’s disease (PD), there are alterations of the basal ganglia (BG) thalamocortical networks, primarily due to degeneration of nigrostriatal dopaminergic neurons. These changes in subcortical networks lead to plastic changes in primary motor cortex (M1), which mediates cortical motor output and is a potential target for treatment of PD. Studies investigating the motor cortical plasticity using non-invasive transcranial magnetic stimulation (TMS) have found altered plasticity in PD, but there are inconsistencies among these studies. This is likely because plasticity depends on many factors such as the extent of dopaminergic loss and disease severity, response to dopaminergic replacement therapies, development of  N-methyl d-aspartate (NMDA) receptor activation by glutamate and post-synaptic calcium influx  is a progressive neurodegenerative disorder with degeneration of nigrostriatal dopaminergic neurons in basal ganglia (BG) resulting in a movement disorder characterized by tremor, rigidity, bradykinesia, and postural instability . AccordiIn vivo, cortical activation leads to a triphasic synaptic response in SNr neurons, due to the sequential involvement of the three pathways: a first excitation, due to hyperdirect pathway activation, followed by an inhibition due to activation of the direct pathway and a late excitation from the indirect pathway , which is an important site of the synaptic plasticity (in vitro and in vivo (A commonly used rodent model in PD is prepared by injection of 6-hydroxy dopamine (6-OHDA) into the striatum. This causes degeneration of dopaminergic neurons, which leads to alterations in striatal synaptic neurotransmission and plasticity. This dopaminergic deficit leads to increased glutamatergic activity and Caasticity . MSN con in vivo  experime in vivo , 19. The in vivo . Endogen in vivo . Dopamin in vivo .Spike-timing-dependent plasticity (STDP) is another concept to explain bidirectional modulation of synaptic plasticity , 22. Accl-DOPA treatment restored LTD but not LTP types of synaptic plasticity  has been used to quantify various neurophysiologic measures in neurological and psychiatric disorders and has the potential to be used as a diagnostic and prognostic tool . VariousTranscranial direct current stimulation (tDCS) has also been used to induce M1 plasticity. Anodal cortical tDCS typically induces LTP-like changes while cathodal tDCS induces LTD-like effects . AlthougWhen two plasticity protocols are used one after another, the effect of first one modulates that of the second and this interaction has been termed as metaplasticity. Homeostatic plasticity is a concept to explain this interaction and is based on the principle that physiological systems attempt to maintain homeostasis to prevent excessive unidirectional changes. Therefore, when two LTP-like protocols applied consecutively, instead of further potentiating the effects, second protocol may bring the excitability back to the baseline. A similar concept is depotentiation. It refers to a protocol which on its own does not induce changes in the excitability but cancels the effect of a preceding potentiation protocol in order to achieve the homeostasis, which is maintained by the ratio of NR subtypes (NR1 and NR2) , 41. ThuTranscranial magnetic stimulation measures are variable and this has been widely reported –49. In al-DOPA or dopamine agonists exert a powerful effect on M1 plasticity induced by rTMS (l-DOPA converts LTP-like plasticity of M1 (induced by PAS25) to LTD whereas moderate dose of l-DOPA (100 mg) potentiates the plasticity effects in healthy subjects , the premotor area, and the cingulate motor areas project major glutamatergic fibers to the striatum, which belongs to a series of BG thalamocortical loops that project back to the motor cortex via the motor thalamic nuclei –78.l-DOPA therapy (l-DOPA-induced dyskinesia (LID) characterized by purposeless, involuntary, repetitive movements which often reduce the quality of life ” ” , comparel-DOPA . Hence, the acute (ON) effects of dopaminergic medications may be required to restore the altered PAS-induced M1 plasticity in PD in the earlier stages of the disease.Dopaminergic medications modulate the altered plasticity in PD , 52. Thil-DOPA . However a study  that comIn the later stage of PD, deficient plasticity in patients with LID was not restored by dopaminergic medications . A recenl-DOPA failed to modulate the plasticity in these patients and there was no significant difference between the more and less affected sides. Deficient iTBS induced LTP-like plasticity in PD was observed in other studies . On the other hand, in the fluctuating non-dyskinetic group, l-DOPA decreased LTP, and in the fluctuating dyskinetic group, no changes were observed following l-DOPA. There was reversal of the depression (toward LTP) induced by cTBS in all three groups following the medication. This reversal of plasticity negatively correlated with clinical effects of l-DOPA especially in patients with motor complications (fluctuating non-dyskinetics and fluctuating dyskinetics). Thus, l-DOPA modulation of cTBS induced M1 plasticity increases with disease progression and was associated with the development of LID.Several studies have shown impaired plasticity in PD patients using TBS protocols. Kishore et al.  examined studies , 60, 61. studies , inhibit studies  demonstrOn the other hand, another study  showed nSeveral studies used rTMS to the M1 and other cortical regions to modulate their excitability and to alleviate PD symptoms. Only studies that assessed the M1 excitability changes will be discussed here. A study using high-frequency (5 Hz) rTMS on M1  during ml-DOPA. This inhibitory protocol normalized the decreased intracortical inhibition of M1 tested with paired pulse protocol at 5 ms . Similar results of increasing the cortical inhibition were obtained with the first dose of l-DOPA in medication naïve patients. Thus the authors concluded that 1 Hz rTMS to PMd modulates M1 intracortical circuits, probably by induction of dopamine release. In another study -M1 connections in PD. One study  investiger study , 5 Hz rTl-DOPA decreased anodal tDCS induced potentiation and increased cathodal tDCS induced depression of cortical excitability. On the other hand, l-DOPA had inverse U-shaped dose response curve for potentiation induced by PAS protocols [Ref.  represents a breakthrough in the management of late PD with motor complications . Althougl-DOPA on plasticity in the BG have been observed in PD patients undergoing DBS implantation (l-DOPA. Since the l-DOPA dose was very low (100 mg) in this study to avoid dyskinesias during the surgical procedure, the modulation of BG plasticity by dopaminergic treatment was only partially addressed. In addition, an optogenetic study showed that modifying the activity of STN neurons was less effective than direct cortical stimulation in reversing the movement deficits following 6-OHDA lesions in mice (The effects of antation . The aut in mice . Thus moIn early stages of PD, there is decreased activity in the medial motor areas such as the SMA whereas hyperactivity was found in more lateral regions such as the M1 in more advanced stages of the disease , 101. NoThe 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.


Particular attention is devoted to the concept of ceramide as a topological cell-signaling lipid: a lipid that forms distinct structures (membrane domains and vesicles termed “sphingosome”), which confines ceramide-induced cell signaling pathways to localized and even polarized compartments.In the last two decades, the view on the function of ceramide as a sole metabolic precursor for other sphingolipids has completely changed. A plethora of studies has shown that ceramide is an important lipid cell-signaling factor regulating apoptosis in a variety of cell types. With the advent of new stem cell technologies and knockout mice for specific steps in ceramide biosynthesis, this view is about to change again. Recent studies suggest that ceramide is a critical cell-signaling factor for stem cell differentiation and cell polarity, two processes at the core of embryo development. This paper discusses studies on ceramide using  When the “father of neurochemistry”, the German pathologist Johann Ludwig Wilhelm Thudichum 1829–1901), coined the term sphingolipids at the end of the 19th century, he must have thought of the Sphinx, a mythological creature testing human wit by asking riddles (which ended deadly if not answered correctly). Indeed, defining the cell-signaling function of the sphingolipid ceramide is like trying to solve a riddle posed by the Sphinx . The discovery that ceramide has a cell-signaling function goes back by more than 20 years. Interestingly, the first studies on ceramide elevation by serum deprivation or vitamin D incubation rather focused on the role of ceramide in cell cycle arrest and differentiation 829–1901,. It was de novo biosynthesis as well as the recycling pathway is inhibited, exogenously added ceramide is not converted to other ceramide species . Exceptionally powerful is the combination of myriocin or fumonisin B1 with ceramide analogs that are not convertible to other sphingolipids. Using this combination of reagents, our laboratory has shown for the first time that specific ceramide species are vital for the formation and differentiation of embryonic epithelia [ Since then, several thousand publications have focused on ceramide-inducing apoptosis, while much fewer studies were concerned with other functions of ceramide. This was mainly due to the limited availability of genetic approaches to alter ceramide metabolism and the rather broad group specificity of ceramide analogs and biosynthesis inhibitors. In particular, studies in Dr. Alfred Merrill's laboratory have shown that although inhibitors such as myriocin  and fumonisin B1 (ceramide synthases) are specific for their target enzymes, the inhibitory effect leads to the depletion of many ceramide species and derivatives –12. Morepithelia , 17.in vitro differentiated to neural cell types undergo phenotypical changes that recapitulate embryo development. In the first differentiation step called germ layer formation, nonpolarized stem cells form embryoid bodies that are composed of a two-layer sphere: an outer primitive endoderm and an inner primitive ectoderm layer [in vitro model for the acquisition of germ layer cell polarity. Apicobasal cell polarity of primitive ectoderm cells is crucial for the organization and morphogenesis of the three embryonic germ layers: endoderm, mesoderm, and ectoderm. When embryoid bodies were depleted of ceramide by the incubation with myriocin or fumonisin B1, they did not form a primitive ectoderm layer. Instead, massive apoptosis of cells within the embryoid bodies was observed [ Embryonic stem (ES) cells rm layer . Since tobserved . Supplemin vitro complementation assays with ceramide vesicles and purified proteins, but also in living cells visualizing S18-induced polarity protein complexes by immunocytochemistry [ S18 is a special kind of ceramide analog designed and synthesized in our laboratory . By mainhemistry , 20–22. hemistry , 17, 23.β-catenin. Using the anticeramide antibody, we found that in primitive ectoderm cells of embryoid bodies, the apical membrane was tremendously enriched in ceramide and codistributed with aPKC, Cdc42, F-actin, and β-catenin [β-catenin and F-actin [ Primitive ectoderm cells encompassing the lumen of the embryoid body are polarized similar to cells in the primitive ectoderm layer of the preimplantation embryo. The apical cell membrane facing the lumen or the pro-amniotic cavity is associated with F-actin and interconnected to adjacent cells via -catenin . The api F-actin . This wa Cell polarity complexes associated with aPKC are not only involved in the acquisition of apicobasal polarity, but also in other cell-polarity-related processes such as cell adhesion and migration. Therefore, we tested if the function of ceramide can be extended to other aspects of aPKC-controlled cell polarity. So far, we have found that ceramide is critical for cell migration of neural progenitors, cell adhesion of epithelia, and ciliogenesis , 22, 24.in vitro model to study the dynamics of cell adhesion, polarity, and ciliogenesis in resting cells. MDCK cells are rapidly dividing until they reach confluence. They establish firm adhesion junctions and eventually develop a primary cilium. This cilium only appears in G0-arrested cells and disappears when cells re-enter the cell cycle. Therefore, we used confluent and serum-deprived MDCK cells to study the function of ceramide in cell adhesion and ciliogenesis. Based on our results with embryonic epithelia, we focused on the effect of ceramide on aPKC, a key factor in the assembly of polarity complexes that are critical for cell adhesion and ciliogenesis.  From the very beginning, ceramide was found to be elevated in resting cells. As a typical response to serum deprivation/withdrawal, cells go into G0 cell cycle arrest concurrent with hydrolysis of sphingomyelin to ceramide , 3. Receζ-GFP), which binds ceramide but does not associate with or phosphorylate client proteins involved in cell polarity [β-catenin. The two proteins were no longer distributed to the adherens junctions, where they participate in a larger protein complex with aPKC. Since this adherens junction complex is regulated by the association of aPKC with Par6 and Cdc42, it is likely that the dominant negative mutant competed with endogenous aPKC for binding to ceramide, but it was not associated with other proteins required to form the junction complex. Consistently, depleting MDCK cells of ceramide with fumonisin B1 also impaired transepithelial resistance, which was restored by the addition of S18 or C16 ceramide [ The role of ceramide binding to aPKC was determined by first generating a dominant negative mutant of aPKC . The ty With the advent of knockout mice for specific ceramide synthases, the focus of the ceramide field has switched from the more generalized effect of ceramide depletion or supplementation to a distinct ceramide composition. In this regard, the ceramide synthase 2 (CerS2 or lass2) knockout mouse shows a remarkable phenotype. Knockout of CerS2 results in hepatocarcinomas and myelin defects eventually leading to death of mice within two years after birth –32. The in vitro complementation assays with ceramide vesicles, coimmunoprecipitation assays, and immunocytochemistry using an antibody against ceramide developed in our laboratory, we found for the first time that ceramide induces binding of aPKC to PAR-4 [ While the previous sections were focused on the beneficial roles of ceramide in cell adhesion and migration, cell polarity of neural progenitors may also be disturbed by proapoptotic proteins competing with polarity proteins for binding to ceramide-associated aPKC. Apoptosis of neural progenitors may thus be a polarity disorder on the cellular and molecular level. We found that prostate apoptosis response 4 , a pro-apoptotic protein first described in prostate cancer cells, sensitizes neural progenitors to ceramide-induced apoptosis , 34–38. to PAR-4 , 35, 36.to PAR-4 , 40. In β-catenin) or induce migration , ceramide-associated aPKC nucleates a polarity protein complex by binding to Par6 and Cdc42. This will either stabilize cell adhesion . Interestingly, there is an enormous rate of cell death in the first 48 h of cultivation diminishing about half of the culture. The apoptotic cells show high expression level of Oct-4, a marker for pluripotency typical for primitive ectoderm cells, and PAR-4, the sensitizer for ceramide-inducible apoptosis , 35, 37. It should be noted that another group has followed up on our studies by administering liposomal ceramide to undifferentiated human ES cells . This gr Despite this translational outcome of our work showing a method to eliminate tumor stem cells via PAR-4 as a sensitizer to ceramide analogs, the physiological function of PAR-4 for neural development still remains unclear. At first, we were intrigued by the observation that PAR-4 is asymmetrically distributed during cell division of neural progenitors. We found that the PAR-4 inheriting (or expressing) daughter cell died, while the PAR-4 (-), nestin expressing daughter cell, survived and underwent further neuronal differentiation . We thouin vivo [ Neural crest-derived cells are the precursors for many neural and nonneural cell types. They originate from the roof plate of the closing neural tube  and proliferate and migrate extensively throughout the embryo. Among the tissues differentiated from neural crest-derived cells are the facial bones and many supportive cells  of the central nervous system. This shared provenance of specific skeletal and brain tissues caught our attention when we were studying the effect of ethanol on apoptosis of neural progenitor cells. In fetal alcohol syndrome, a birth defect caused by maternal consumption of alcoholic beverages during pregnancy, facial malformation, and cognitive retardation are the most obvious and severe symptoms of the newborn. Therefore, we hypothesized that neural crest-derived progenitor cells are very sensitive to ethanol, which leads to malformation of neural crest-derived tissues in fetal alcohol syndrome. Our studies showed for the first time that ethanol elevates the expression of PAR-4 and ceramide in neural crest-derived tissue culture and embryos in vivo . Elevatiin vivo . Taken tκB, which is critical for neural progenitor cell survival [β, a different cell-signaling pathway regulating the function of β-catenin [ About ten years ago, we began to investigate which cell-signaling pathway is downstream of the ceramide-aPKC apoptosis complex (CAP-AC), and what distinguishes the cell-signaling effect of this proapoptotic lipid-protein complex from that of the ceramide-aPKC-polarity complex (CAP-PC). Based on experimental evidence obtained with neuroblastoma cells and ES cell-derived neural progenitors, our very first model suggested that CAP-AC will reduce activation of Akt/PKB and MAPK/ERK2, which will lead to activation of Bad and reduced protein levels of Bcl-2  Figure . Bad andsurvival  , there are striking similarities to the apical ceramide-enriched compartment (ACEC) found in our group. It is also noteworthy that other sphingolipids have been implicated in the compartmentalization of polarized cells. Pioneering work in Kai Simmon's and Gerrit van Meer's groups demonstrated that glycosphingolipids and sphingomyelin are asymmetrically transported and distributed in polarized MDCK cells [ It should be noted that other groups have also found evidence for the ceramide-dependent compartmentalization of intracellular proteins. Yusuf Hannun's group has described a juxtanuclear recycling compartment termed “pericentrion” . FormatiCK cells . Of inteCK cells . Taken t


The majority of mammalian genes generate multiple transcript variants and protein isoforms through alternative transcription and/or alternative splicing, and the dynamic changes at the transcript/isoform level between non-oncogenic and cancer cells remain largely unexplored. We hypothesized that isoform level expression profiles would be better than gene level expression profiles at discriminating between non-oncogenic and cancer cellsgene level.We analyzed 160 Affymetrix exon-array datasets, comprising cell lines of non-oncogenic or oncogenic tissue origins. We obtained the transcript-level and gene level expression estimates, and used unsupervised and supervised clustering algorithms to study the profile similarity between the samples at both gene and isoform levels.Hierarchical clustering, based on isoform level expressions, effectively grouped the non-oncogenic and oncogenic cell lines with a virtually perfect homogeneity-grouping rate (97.5%), regardless of the tissue origin of the cell lines. However, gene levelthis rate was much lower, being 75% at best based on the gene level expressions. Statistical analyses of the difference between cancer and non-oncogenic samples identified the existence of numerous genes with differentially expressed isoforms, which otherwise were not significant at the gene level. We also found that canonical pathways of protein ubiquitination, purine metabolism, and breast-cancer regulation by stathmin1 were significantly enriched among genes thatshow differential expression at isoform level but not at gene level.In summary, cancer cell lines, regardless of their tissue of origin, can be effectively discriminated from non-cancer cell lines at isoform level, but not at gene level. This study suggests the existence of an isoform signature, rather than a gene signature, which could be used to distinguish cancer cells from normal cells. The past decade has witnessed unprecedented developments in high-throughput technologies, and their application has led to the molecular classification of many cancers . MoleculLEF1, TP63, TP73, HNF4A, RASSF1, and BCL2L1) are already known to have multiple promoters and alternative splice forms [One of the reasons for the limited success in the quest for genomic-based, personalized medicine is the assumption of a 'one gene → one protein → one functional pathway' paradigm in most of the studies investigating molecular classification or therapeutic targets for cancer . Recentlce forms -16. Morece forms . Althougce forms -20, and Microarray expression profiling remains a powerful tool for identifying different subtypes of cancers. However, almost all microarray-based studies reported to date have measured the expression of the gene at gene level in a given locus, although a few exceptions in recent years have used exon arrays to measure differences at the exon and/or at transcript variant level. The recent application of exon arrays  and the With genome-wide isoform level and gene level expression profiles in hand, it is natural to ask how the isoform level expression profiles of different oncogenic and non-oncogenic samples will cluster together, and whether isoform level expression profiles can lead to more accurate discriminators between oncogenic and non-oncogenic samples compared with gene level expression profiles. If the answer is yes, it is important to know which genes and pathways contribute to the improvement of discrimination at isoform level compared with gene level.In the present study we analyzed Affymetrix exon-array data-sets collected from the public domain, primarily the ENCODE project from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database, which comprises 160 datasets from various cell lines of either non-oncogenic or oncogenic tissue origin. These data-sets were used to test the hypothesis that isoform level expression analysis provides abetter discriminator between non-oncogenic and oncogenic cell types than gene level expression analysis.Unprocessed gene-expression datasets, generated using a whole-transcript GeneChip platform , were downloaded from the GEO public data depository, deposited mainly by the ENCODE project. The GEOloess) algorithm [The isoform level (transcript-level) and gene level expression estimates were obtained by the Multi-Mapping Bayesian Gene eXpression (MMBGX) algorithm for Affymetrix whole-transcript arrays , based olgorithm ,31, alsolimma method [P value was smaller than a cut-off value of 0.01 for all comparisons between the two conditions. Ingenuity Pathway Analysis (IPA) [P values of pathways from the IPA analysis; the P values were used as an indicator of association strength between the gene sets and pathways. For the three pairwise oncogenic/non-oncogenic comparisons , a pathway was selected and reported if it was significantly associated with the gene sets identified at isoform-level in all three pairs of comparisons, but was not significantly associated with the gene sets identified at gene-level in all three pairs of comparisons, or vice versa. The significance level was set at P < 0.05 for all comparisons. All calculations were performed using Bioconductor  and R platform  [We used the general hierarchical cluster algorithm to cluster the samples, using Euclidean distance as a measurement for dissimilarities . We alsoa method ,35. An iis (IPA)  was usedect.org) .The study protocol was approved by the institutional review board, and all data collection and analyses adhered to the protocols approved by the institutional review board. Informed consent was obtained from all participants.Women with primary operable breast cancer undergoing breast surgery at the Hospital of the University of Pennsylvania were asked to participate in our tissue-banking protocol. The study cohort included four women diagnosed with breast cancer between 2010 and 2011. Clinical characteristics, including age at diagnosis, ethnicity, histology, tumor size, tumor grade, and number of involved (positive) axilla nodes are provided . Total RNA from cells and tissues were using TRI reagent  in accordance with the manufacturer's instructions. For breast-cancer and normal breast tissues, up to 50 mg of frozen tissue was transferred to 1 ml of TRI reagent, then the tissue was immediately homogenized and RNA extraction protocol was followed. Briefly, 0.5 μg of total RNA was reverse-transcribed in a 20 μl reaction with SuperscriptII reverse transcriptase (Invitrogen Inc.) in accordance with the manufacturer's instructions. RT-qPCR was performed using a master mix  and fold expression was calculated using the 2-ΔΔCT method. RT-qPCR results were normalized based on the expression of GAPDH for TPM4 and TBP for the other transcripts. The measured isoforms and the primers used for the isoform-specific PCR are presented  all non-oncogenic and all cancer cell lines  gene uses at least nine different promoters and first exons to generate a remarkably diverse set of mRNAs and protein isoforms that differ at the N-terminus. The gene platform we used (Affymetrix GeneChip Human Exon 1.0 ST Array) has probe sets corresponding to 16 different transcript variants of this gene, based on Ensembl gene annotations. The alternative promoters of MITF reflect the tissue specificity of its isoforms, which are selectively expressed in melanocytes, macrophages, osteoclasts, heart muscle, or retinal pigmented epithelium. MITF, generally believed to play a primary role in melanocyte stem-cell proliferation and expression of a set of pigment-related genes [MITF was found by the gene level analysis. However, the MITF isoform ENST00000352241 was found to be significantly overexpressed in melanoma compared with melanocytes (FC = 3.4), whereas the transcript variants ENST00000433517 (FC = -5.6) and ENST00000472437 (FC = -3.4) were underexpressed in melanoma  was found to be strongly overexpressed (FC = 5.47), another isoform (ENST00000344824) was found to be significantly underexpressed in MCF7 samples (FC = -7.75)  were found to be significant only at isoform level. In other words, more than one isoforms of these genes displayed differential expression between non-oncogenic and cancer samples, but the gene-level expression differences were cancelled out by the combined effect of various isoforms of the same gene. These genes displayed alternate splicing between non-oncogenic and cancer cell lines. This observation strongly supports previous reports such as that by David and Manley . For exaed genes , has beeed genes . In the a Figure . Similar) Figure . These tt cancer . Our anaTPM4 gene in breast cancer as an example. The opposing expression patterns of the TPM4 isoforms were confirmed in the estrogen receptor-positive and triple-negative breast cancer sample. Although the Her2+ sample did not show the opposing pattern of expression, one isoform had the significantly highest fold change of all the samples . These results strongly support our hypothesis that isoforms of multi-transcript genes can have opposing roles in cancer.To validate the existence of the opposing expression patterns of isoforms for various genes, we measured isoform expression by RT-qPCR for two opposing isoforms of seven genes in three breastcancer cell lines relative to the non-oncogenic HMEC cell line Tables . The exps Figure . In all ® Systems, Redwood City, CA, USA) [We performed IPA   to find WNT5B, CCND2, SERPINB7, GPR176, INHBA, EFNB1, PTRF, CDH11, ZBTB16, GJA1, COL5A2, NID1, PRDM1, and TCF4. Except for CCND2 and GPR176, all other genes in our database have more than one isoform. Four genes  have two isoforms that have significantly different expression between the cancer and non-oncogenic groups. Interestingly, 12 of these 14 genes belong to the same gene network, involved in hematological system development and function, tissue morphology, and cellular development. According to the Ingenuity Pathway Knowledge Base, the network consists of a total of 27 different genes, which suggests that almost 50% of the genes belonging to this network are dysregulated either at the gene or isoform level between non-oncogenic and cancer cells  have been described. Therefore, it would be interesting to evaluate the components of this gene network in different cancers at isoform level.These 14 genes are s Figure . MoreoveTo test whether the genes that are differentially expressed at isoform level but not at gene level could reveal interesting pathways associated with cancer, we focused the pathway-enrichment analysis on two different gene sets: 1) genes that are significant at isoform level only and 2) genes that are significant at gene level only Figure Human cancer is a complex disease. It is known that most of the genes in mammalian genomes generate different transcript variants and protein isoforms, which often function in a cell/tissue type-specific and developmental stage-specific manner in non-oncogenic cells. Cancer results from the sequential acquisition of a number of genetic and epigenetic alterations, and these mutations may alter the expression of specific isoforms but not the others of a gene. Despite this growing knowledge, most biomarker and drug-discovery studies still evaluate expression differences and study gene regulatory mechanisms at gene level rather than at isoform level. In this study, we have shown that oncogenic cell lines could be more accurately discriminated from non-oncogenic cell lines, regardless of their cells of origin, by gene-expression profiling at isoform level compared with gene level. In spite of the differences in tissues of origin, the cell lines were broadly clustered into two groups, non-oncogenic and oncogenic, by isoform level gene-expression profiles. We noted that numerous genes showed differential expression at isoform level but not at gene level. For some of these genes, the differential expression of alternative transcripts occurred in the opposite direction; while some of the isoforms of the same gene were upregulated, others were downregulated, resulting in them cancelling each other out and producing insignificant expression differences at gene level between cancer and non-oncogenic groups. Our findings are in agreement with a previous study on prostate cancer that investigated the expression of 1532 splice forms for 364 prostate cancer-related genes, using data from a customized exon junction array . The autStathmin 1 (STMN1) are characteristic features of many human tumors [STMN1, PNP, RPS27A and UBA52) transcribe different transcript variants, some of which encode different protein isoforms. It is therefore necessary to evaluate the gene-expression differences and to study gene regulatory mechanisms at isoform level rather than at gene level between non-oncogenic and disease conditions, such as cancer. Recent advances in cancer genomics have shown that gene-expression signatures are useful for biomarker identification and drug discovery [The pathway-enrichment analysis of genes that are differentially expressed in cancer cell lines at isoform level but not at gene level produced three interesting pathways that have been implicated in various cancers. It is well known that protein phosphorylation and protein ubiquitination regulate most aspects of cell life, and defects in these control mechanisms cause cancer and many other diseases . Similarn tumors ,52. The iscovery . In thisTPM4 gene isoforms in non-oncogenic and oncogenic tissue samples from breast cancer patients. The present study highlights the importance of studying gene-expression signatures at isoform level rather than at gene level in characterizing the cancer transcriptome.In conclusion, we have identified a common, isoform level signature that can be used to discriminate effectively between cancer and non-cancer cell lines. We found numerous genes for which the differential expression of alternative transcripts occurred in opposing directions, with some of the isoforms of the same gene being upregulated while others were downregulated, resulting in insignificant expression differences at gene level between cancer and non-oncogenic groups. This is supported by our experimental validation of opposing expression patterns for The authors declare that they have no competing interests.ZZ designed the computational methods and performed the statistical analyses. SP designed the experimental methods and performed the RT-qPCR experiments. YB performed statistical analyses. JT participated in the design of the study, and provided non-oncogenic breast and oncogenic breast tissue samples. RD and JT formulated and directed the design of the study. ZZ, SP, and RD wrote and edited the manuscript. All authors read and approved the final manuscript.Supplementary Table S1. Summary of cell lines samples and the exon-array datasets used in this study. Excel document.Click here for fileSupplementary methods and Table S2 A and B. Table S2A Clinical characteristics of samples from patients with breast cancer used in this study. Table S2B Primer sequence used for real-time quantitative reverse transcriptase (qRT)-PCR to measure isoform expression. Word document.Click here for fileSupplementary Figure S1 and S2. Dendrograms representing hierarchical clustering of 160 (73 oncogenic and 87 non-oncogenic) datasets from cell lines using expression estimates of  transcripts and  genes at different cut-off points for coefficient of variation (CV). Powerpoint document.Click here for fileSupplementary Table S3. List of genes and isoforms differentially expressed between the oncogenic and non-oncogenic cell lines groups (Figure s Figure . Excel dClick here for fileSupplementary Table S4. List of genes and isoforms differentially expressed between non-oncogenic HMEC and MCF7 breast-cancer cell lines (Figure s Figure . Excel dClick here for fileSupplementary Table S5. List of genes and isoforms differentially expressed between the melanocyte and melanoma cell lines (Figure s Figure . Excel dClick here for fileSupplementary Table S6. List of overlapped genes and isoforms consistently upregulated or dow regulated across all three pairs of comparisons (Figure s Figure . Excel dClick here for fileSupplementary Table S7. Pathways associated with the top gene networks identified by IPA analysis of core genes. Word document.Click here for file


The use of new psychoactive substances and the new patterns of drug use can have important public health and policy implications. Few countries have monitoring systems that are sensitive to this new phenomenon in the drug field, but methodological difficulties to detect them are considerable. Nonetheless, the importance of identifying potential new threats is widely recognized. The European Union’s Early-Warning System (EWS) provides a quick-response mechanism to the emergence of new psychoactive substances on the drug scene. The European Council’s decision on new psychoactive substances establishes a mechanism for the rapid exchange of information on new drugs. It also provides the possibility to trigger a formal risk assessment process, the findings of which may lead to a political decision to place new substances under control across the European Union.AbbreviationsEWS - Early-Warning System, THC – Tetrahydrocannabinol, EMCDDA - European Monitoring Center for Drugs and Drug Addiction Most of the new synthetic substances have stimulant properties, while only a few produce hallucinogenic effects. For the first time ever, in 2008, a synthetic cannabinoid  methadone) was reported through the early-warning system. The appearance of synthetic cannabinoids marks the latest stage in the development of designer drugs: from those based on fentanyl in the 1980s to ring-substituted phenethylamines in the late 1980s and tryptamines in 1990s, to piperazines and cathinone derivatives in the 2000s [2]. There are over a hundred compounds with cannabinoid receptor activity and it can be assumed that new substances from different chemical groups will continue to appear on the drug scene. All this presents a constant challenge to public health and law enforcement agencies. Since the establishment of the new early-warning system (EWS) in 1997 [1].Synthetic cannabis products are psychoactive herbal mixtures which have similar effects to those of cannabis, when inhaled. Based on patients’ clinical medical history, smoking these substances produces cannabinoid-like effects, although they do not contain THC (delta-9-tetrahydrocannabinol - the main psychoactive substance of cannabis plant) [Fig. 1). The goal is to satisfy the users’ demand for drugs that can be obtained without prescriptions or other legal constraints [5].A large and complex variety of synthetic cannabinoids, most often cannabicyclohexanol, JWH-018, JWH-073 methanone), CP 47 497 -3-hydroxycyclohexyl]- 5-(2-methyloctan-2-yl)phenol), HU-210  are used in an attempt to avoid the laws making cannabis illegal, thus conferring synthetic cannabis the status of a designer drug. Designer drugs are pharmaceuticals, created or reformulated to bypass current laws, by modifying the molecular structures of drugs , identified Spice products in 21 out of the 30 participating countries. For the purpose of the survey, “identification” means that these types of products were available in some form in the country. JWH or CP compounds were identified by forensic/toxicological analysis in 8 out of 21 countries where Spice products were available: Austria, Germany, Finland, France, Hungary, Poland, Slovenia and the United Kingdom. The substances identified were: JWH-018; CP 47 497 - C8 homologue; CP 47 497; JWH-073  methanone) .While at least two of the ingredients — Pedicularis densiflora and Leonotis leonurus — may have some psychoactive effect, little is known about the pharmacology and toxicology of the plant materials purportedly contained in spice products . Without a legislative framework to control its distribution and insufficient detection methods, the law enforcement agencies faced a significant challenge in addressing the problem of synthetic cannabinoids. 3]. Extensive forensic investigations have been undertaken by EU Member States in order to identify the psychoactive ingredients of spice products. In December 2008, Germany and Austria detected the synthetic cannabinoid JWH-018. The chemical structure of JWH-018 differs substantially from that of tetrahydrocannabinol (THC), the main active principle in all cannabis products. In experimental animals, JWH-018 produces the same effects as THC and has been reported to be more potent. Earlier, in 2009, a second synthetic cannabinoid named CP 47 497, and three of its homologues were detected in spice samples in Europe. Some reports indicate that JWH-018 binds to the CB1 receptor (cannabinoid receptor) with even greater affinity than marijuana [7].The reported pharmacological effects of synthetic cannabinoids smoked are very similar to that of marijuana. This comes as no surprise, given that Spice and K2 are THC agonists - meaning they chemically bind to the same brain receptor (CB1) and trigger many similar responses as marijuana. The physiological effects of synthetic cannabinoids include: increased heart rate and blood pressure, altered state of consciousness, perceptual alterations (time distortion), intensification of sensory experiences, impaired short-term memory .Responding to potential health concerns, the Member States have taken diverse legal actions to ban or otherwise control spice products and related compounds . This presents an ongoing challenge, not only for their forensic and toxicological identification, but also for risk assessments and the development of possible control strategies.


Puccinia striiformis f. sp. tritici, continues to cause severe damage worldwide. Durable resistance is necessary for sustainable control of the disease. High-temperature adult-plant (HTAP) resistance, which expresses when the weather becomes warm and plants grow older, has been demonstrated to be durable. We conducted numerous studies to understand the molecular mechanisms of different types of stripe rust resistance using a transcriptomics approach. Through comparing gene expression patterns with race-specific, all-stage resistance controlled by various genes, we found that a greater diversity of genes is involved in HTAP resistance than in all-stage resistance. The genes involved in HTAP resistance are induced more slowly and their expression induction is less dramatic than genes involved in all-stage resistance. The high diversity of genes and less dramatic induction may explain durability and the incomplete expression level of HTAP resistance. Identification of transcripts may be helpful in identifying resistance controlled by different genes and in selecting better combinations of genes to combine for achieving adequate and durable resistance.Stripe rust of wheat, caused by  Four probes representing two transcripts [hydroxyproline rich glycoprotein (Ta.6952.1) and NB-ARC domain containing protein (TaAffx.103209.1)] were up-regulated when compared to their mock-inoculated checks and five probes representing 4 transcripts  were up-regulated when compared to their compatible interactions. For the HTAP resistance gene lines, two probes representing one transcript [no homology (Ta.22462.1)] were down-regulated and four probes  were up-regulated when compared to AvS. When the HTAP resistance gene lines were compared with the all-stage resistance gene lines, nine probes representing six transcripts  were down regulated and two probes representing one transcript [nonclathrin coat protein (Ta.7616.1)] were up-regulated. P <0.10) in the adult-plant tests under the high-temperature profile (10-30°C) are shown in bold in (Table 1). Seven transcripts were significant for Yr1, 10 for Yr5, 4 for Yr7, 31 for Yr8, 6 for Yr9, 13 for Yr10, 6 for Yr15, 5 for Yr17, 40 for Yr18, 4 for Yr29, 99 for Yr39 and none for Yr36. Up-regulated transcripts shared by two or more Yr genes also can be found from this table. For comparison, significant transcript values detected in previously published studies of seedling tests at the low-temperature (4-20°C) profile for all-stage resistance genes [1). In general, transcripts detected in the seedling low-temperature tests were also significant in the adult-plant high-temperature tests for race specific all-stage resistance.Transcript values with significant changes . Yr5 was more closely related to Yr17; Yr8 was more closely related to Yr10; and Yr18 was more closely related to Yr39 than to other genes. Yr29 was more distantly related to all of the other genes. Yr36 was not included in the dendrograms as none of the transcripts for other genes was significantly changed in expression levels; this could indicate that it utilizes signaling and defense pathways that are different from those identified for the other genes. Although this hypothesis needs to be tested, the results may be in agreement with the previous finding that Yr36 is a very old gene that is not present in common wheat cultivars [Based on common and unique transcripts identified in the Yr1, Yr5, Yr7, Yr9 and Yr15 and only one gene was commonly expressed in HTAP resistances mediated by different genes  (Table 2). The annotation of the five transcripts specific to all-stage resistance provided additional evidence for classic R-gene mediated pathways being involved in race-specific resistance. The five genes commonly involved in all-stage resistance included a hydroxyproline-rich glycoprotein, a NB-ARC domain containing protein, a protein kinase and two function-unknown genes. In addition to the separate analysis at the seedling stage [Yr5-mediated all-stage resistance and two were first identified in the Yr39-mediated HTAP resistance. The transcript with significant changes across all HTAP resistances is a nonclathrin coat protein. The function of nonclathrin coat protein-related resistance is not clear, but such proteins have been reported to bind to the cytoplasmic dilysine motif of membrane proteins of the early secretory pathway [Pst haustoria or hyphae. The lack of many shared transcripts in HTAP resistance compared to all-stage resistance leads to a hypothesis that diverse genes and biochemical pathways are used by HTAP resistance controlled by different genes. Together with diverse genes and pathways identified for Yr39-mediated HTAP resistance [After various comparisons, five genes were clearly identified to be involved in race-specific all-stage resistance controlled by ng stage , we founng stage . Among t pathway . The nonsistance , we conc5Yr5-mediated all-stage resistance and Yr39-mediated HTAP resistance; and common genes involved in all-stage resistance and HTAP resistance mediated by different Yr genes. The results have provided some insights for understanding the molecular mechanisms of race-specific resistance compared to race non-specific resistance. In particular, the studies have linked a large number of genes with diverse functions to race non-specificity and durability of HTAP resistance. The data of these studies lead to several hypotheses to be tested and more studies to be conducted for a better understanding of various types of resistance and how to utilize the basic information to achieve more sustainable and better control of stripe rust.The transcriptomics studies conducted so far have identified genes involved in Yr39 donor, Alpowa, using a PCR based genome walking technique . Thus far, the ABC transporter-like wheat gene has more than 75% identity in genomic sequence to a rice gene, Os01g42410, with the greatest differences occurring in intron regions. Sequence conservation of the ABC transporter gene in Alpowa (Yr39) also appears to be high across different wheat cultivars after alignment of the newly acquired sequence with Chinese Spring genomic sequences  failed to detect significant differences, supporting the high up-regulation of the gene in the Yr18 line presented above, as Chinese Spring has Yr18 [Yr18/Lr34 [We are currently conducting studies to test a hypothesis that ABC transporter proteins are involved in nonrace specific HTAP resistance. We are in the process of obtaining the full-length sequence for the identified ABC transporter gene, and so far have 5,754 bp of the genomic sequence from the has Yr18 . Comparir18/Lr34  shows thR-protein genes involved in Yr39-mediated HTAP resistance was initially a surprise to us, as R proteins are largely believed to be involved in recognition of pathogen effectors, leading to race-specific resistance. However, the high number of such types of genes leads us to believe that these genes collectively contribute to race non-specificity and therefore durability. Our hypothesis is that when up-regulated by the Yr39 master gene, these genes serve as secondary master genes regulating other defense or functionally related genes to operate the entire defense machinery against Pst infection and growth in the plant tissue. These R proteins may recognize different effectors, which may make it difficult for the fungus to change to non-recognition. In regard to the ABC transporter gene, we are currently obtaining full-length sequences of these R genes to characterize them among wheat genotypes and to determine their functions for the HTAP resistance phenotype and their roles of being regulated or regulating in the total network of defense pathways.The identification of the nine Yr5 and Yr39 studies to represent those involved in either type of resistance. However, such a cost-saving approach did not allow us to identify transcripts uniquely involved in resistance mediated by individual Yr genes. Therefore, we still do not have the majority of the genes identified for all Yr genes studied, except for Yr5 and Yr39. Using the Wheat Affymetrix GeneChip, which continues to have new genes added, is still a useful high-throughput technique to identify possible genes involved in resistance to stripe rust and other diseases in wheat. Alternately, transcriptome sequencing [With a primary goal of identifying transcripts commonly involved in all-stage resistance or HTAP resistance controlled by different genes, the custom microarray was constructed using genes identified in the quencing , 23, whiYr39 is more typical of HTAP, where the maximum expression of resistance is in flag leaves and under high-temperatures. In contrast, both Yr18 and Yr36 can express resistance even in the seedling stage when under high temperatures [HTAP resistance has two components: temperature sensitivity and developmental stage dependence. These two components are not equally required for resistance. Among cultivars with a broad-sense HTAP resistance, which can be determined by a virulent race in a seedling test under low temperatures and in a field or greenhouse test with the same race under high temperatures, resistance in some cultivars is more temperature sensitive whereas others are more plant-stage dependent. In our studies, eratures . Thus itIn comparison of a pair of two resistance genes which regulate different transcripts with another pair of two resistance genes which regulate commonly shared transcripts, the former pair of resistance genes when in combination may lead to more durable resistance than the latter pair of genes as the combined resistance conferred by the former pair is based on more diverse defense pathways. Furthermore, the correct combinations of genes may provide higher levels of resistance. In this way, transcriptomics studies of resistance genes will not only provide an understanding of the basic mechanisms of resistance, but will allow for immediate application in selecting resistance genes for precision breeding. The results may revealed which genes are more likely to be durable and which are not. It will also add to the biotechnological tool box for identification of the same or different genes. As the technology is advancing, transcriptomics testing should become less expensive and higher throughput. It will become more feasible to use transcriptomics approaches for identifying genes and developing markers for different types of resistance.


Carbohydrate is the major constituent of food that significantly influences viscosity when heated in water.Cereal-based complementary foods from non-malted ingredients form a relatively high viscous porridge. Therefore, excessive dilution, usually with water, is required to reduce the viscosity to be appropriate for infant feeding. The dilution invariably leads to To compare the sweetpotato-based complementary foods  and enriched Weanimix (maize-based formulation) regarding their 1) carbohydrate composition, 2) viscosity and water solubility index (WSI), and 3) sensory acceptance evaluated by sub-Sahara African women as model caregivers.The level of simple sugars/carbohydrates was analysed by spectrophotometry, total dietary fibre by enzymatic-gravimetric method, and total carbohydrate and starch levels estimated by calculation. A Rapid Visco™ Analyser was used to measure viscosity. WSI was determined gravimetrically. A consumer sensory evaluation was used to evaluate the product acceptance of the roller-dried ComFa, oven-toasted ComFa, and enriched Weanimix.The sweetpotato-based complementary foods were, on average, significantly higher in maltose, sucrose, free glucose and fructose, and total dietary fibre, but they were markedly lower in starch content compared with the levels in the enriched Weanimix. Consequently, the sweetpotato-based complementary foods had relatively low apparent viscosity, and high WSI, than that of enriched Weanimix. The scores of sensory liking given by the caregivers were highest for the roller-dried ComFa, followed by the oven-toasted ComFa, and, finally, the enriched Weanimix.energy and nutrient thinning.The sweetpotato-based formulations have significant advantages as complementary food due to the high level of endogenous sugars and low starch content that reduce the viscosity, increase the solubility, impart desirable sensory characteristics, and potentially avoid excessive  Therefore, a household-level sweetpotato-based complementary food has been suggested as a more suitable complementary food as it would be lower in starch resulting in a less viscous porridge compared with a maize-based infant product , which is a measure of the soluble constituents in foods, is another physical property that relates to the carbohydrate composition of foods . High WSThe level of simple carbohydrates/sugars can affect not only the physical properties (viscosity or solubility) but also the sensory properties and, consequently, food intake. Other researchers reported that infants consumed more cereal-based porridge when sweetened than unsweetened .In an effort to improve the nutritional status of infants in lower-income countries of sub-Saharan Africa, sweetpotato-based complementary foods  were proposed as alternatives to commonly used cereal–legume blends, such as Weanimix . Weanimienergy and nutrient thinning.Sucrose levels in sweetpotato roots increases during storage –20, and The objectives of this study were to compare the sweetpotato-based complementary foods  and enriched Weanimix (maize-based formulation) regarding their 1) carbohydrate composition, 2) viscosity and WSI, and 3) sensory acceptance evaluated by sub-Sahara African women as model caregivers.The choice and the proportion of the ingredients and the processing of the sweetpotato-based infant foods (ComFa formulations) have been previously reported , 16. TheCream-fleshed sweetpotato flour (72%), soyabean flour (15%), soyabean oil (6.0%), iodised salt (0.50%), sugar (0.50%), and skimmed milk powder (6.0%) were mixed, and the composite flour was either extrusion cooked or roller dried. Extrusion cooking of the composite flour was done at 120°C. For the roller-dried ComFa, slurry was prepared, cooked in a steam-jacketed pan for 10 min at 80–83°C before roller drying with steam at 107°C under 100 kPa.®, a popular proprietary dry infant cereal-based food in Africa.These are the industrial-level ComFa formulations, which could be reconstituted in the same way as Nestlé CerelacComposite flour of sweetpotato (66%), soyabean (10%), soyabean oil (6.0%), iodised salt (0.50%) sugar (0.50%), and fish powder from anchovy without the heads (17%) was toasted in a 120°C pre-heated oven for 30 min. This is the household-level sweetpotato-based formulation.A maize-based complementary food, described as Weanimix, was processed as previously described , 15, witThree samples of each complementary food were separately defatted using the Soxhlet method , and theTotal available carbohydrate was calculated as 100% minus the sum of moisture, protein, fat, ash, and total dietary fibre obtained using proximate analysis as previously reported . Levels A Rapid Visco Analyser (RVA) , interfaced with a personal computer equipped with the software Thermocline for Windows v3.0, was used to measure the pasting profile using the Newport Scientific Method Version 13. In summary, about 3 g (14% moisture basis) of non-defatted complementary foods (in triplicate) were weighed and transferred to aluminium canisters containing approximately 25 mL of distilled water (corrected to compensate for 14% moisture used for the sample). A plastic paddle was used to mix the slurry to break up any lumps before inserting the canister with the paddle into the instrument to measure the pasting profile for 20 min. The initial equilibration of the slurry was set for 2 min at 25°C, which was followed by heating to a maximum of 95°C for 5 min, and then holding at this temperature for a further 3 min. The paste was cooled to 25°C for 5 min, and held at this temperature for 3 min.consume viscosity.The maximum apparent viscosity during heating, or the heating/holding phase of the test, was reported as the peak viscosity, and the final viscosity was the viscosity recorded at the end of the test . Using tThe gravimetric method described by Jin et al.  was usedThe sensory acceptance of the products, roller-dried ComFa, oven-toasted ComFa, and enriched Weanimix, were measured using the method of Stone and Sidel . To reduEthical approval was obtained from the Massey University Human Ethics Committee (HEC: Southern A Application-09/58), PN, NZ. Before the sensory test began, written consent to take part in the study was obtained from each participant after explaining the objectives to her.Two of the participants who were feeding complementary foods to their babies at the time of the sensory evaluation testing were selected to prepare suitable food for babies from the roller-dried ComFa, oven-toasted ComFa, and enriched Weanimix, as they would do at their homes in sub-Saharan Africa. A total of 150 g of each of the three formulations were given to the selected women. These two women were informed that oven-toasted ComFa and enriched Weanimix were to be prepared as traditional household-level infant porridge while the roller-dried ComFa was to be reconstituted in the same way as Nestlé Cerelac.For sensory testing, approximately 20 g of each of the prepared samples were randomly served together to all of the participants (including the two caregivers who prepared the porridge) by the researchers in three-figure-coded plastic cups at room temperature for the product liking evaluation in a sensory booth.P <0.05). The relationship between apparent viscosity and WSI and either the sum of simple sugars, starch, or total dietary fibre was evaluated using Pearson's correlation. Minitab v15.1™  was used for the statistical analyses above.The composition of carbohydrate, viscosity, and the WSI data were subjected to Univariate Analysis of Variance (one-way ANOVA). The Tukey's studentised range test was used to compare differences between means when the ANOVA was significant  for three or more independent samples and the usual one-way ANOVA, assuming normality of data using the PASW Statistics 18, Release Version 18.0.0 .For the sweetness and saltiness intensities scoring using the just-about-right scale, the responses were categorised as ‘much too low’ (≤2), ‘too low’ , 4, justP <0.0001), sucrose , free glucose , and fructose . The roller-dried ComFa was slightly higher in maltose than the extrusion-cooked ComFa (1.1 times) and oven-toasted ComFa (1.2 times). All the other carbohydrate fractions, with the exception of free glucose, were not significantly different between the ComFa formulations. The sum of maltose, sucrose, and glucose in the sweetpotato-based complementary foods is comparable to the level of 50 g/100 g (dry matter basis) in sweetpotato roots reported by other researchers (P <0.0001), and they were higher in total dietary fibre (8–11 vs. 6 g/100 g) than the levels in enriched Weanimix. The levels of the calculated total available carbohydrate and total dietary fibre together in the ComFa formulations indicated that the sweetpotato-based formulations satisfied the estimated carbohydrate content of 60–75 g/100 g in the Codex standard  compared with the maize-based complementary food  are shown in A slurry oven-toasted ComFa and enriched Weanimix was prepared with potable water before cooking on a hot-plate for about 10 min. The roller-dried ComFa was reconstituted as ready-to-eat complementary food using potable water that was boiled and allowed to cool to 40°C. The average age of the participants who took part in the consumer sensory evaluation was 37±8 year (mean±standard deviation). All of the women also indicated that sweetpotato was readily available in their respective countries. The oven-toasted ComFa was mixed with approximately 750 mL of potable water before cooking, but the enriched Weanimix was initially mixed with about 600 mL of water, with a further addition of about 300 and 150 mL of water during cooking to reduce the viscosity. Approximately 450 mL of the warm water was used for reconstitution of the roller-dried ComFa. The amount of water used for the reconstitution was discussed and accepted by the two selected women, and the final viscosity was unanimously accepted by the other participants.Both the Kruskal–Wallis non-parametric test and the usual ANOVA gave similar results regarding the rejection of the null hypothesis . TherefoFor analysing the perceptions for sweetness , about 5The higher level of sugars in sweetpotato-based complementary foods will make them naturally sweeter than the maize-based product. Therefore, the addition of sugar would not be necessary when preparing porridge. The natural sweetness of the sweetpotato-based infant foods could offer a nutritional benefit over the maize-based complementary food as it may lead to a higher intake of food .The slightly higher level of maltose in the roller-dried ComFa than the extrusion-cooked ComFa and oven-toasted ComFa is likely due to gradual heating of the slurry that allows the endogenous β-amylase in sweetpotato  a longerThe total dietary fibre in the ComFa formulations exceeded the maximum specified content of 5 g/100 g in the Codex standard , the higThe peak viscosity measured during heating, or the heating/holding phase on the RVA, represents the maximum viscosity likely to occur during porridge preparation on a kitchen stove. For example, in Ghana, slurry is made from cereal or cereal-based dough/flour in a cooking pot, which is then cooked on a stove with frequent stirring. The porridge is usually left to cool to between 40 and 45°C before being fed to infants. The apparent viscosity at approximately 45°C (the temperature of porridge served to infants), thus represents the likely consistency of the porridge given to infants , 26. Theenergy and nutrient thinning for the ComFa formulation.The higher level of sugars and the lower starch content in the ComFa formulations partly contributed to the lower apparent viscosities of the sweetpotato-based products as reported in other studies , 35. BecThe high WSI of the sweetpotato-based complementary food in this study can be explained by the quantity of soluble molecules (e.g. sugars and possibly soluble fibre). The relatively high WSI and simple carbohydrates to starch ratio may suggest easier digestibility of the sweetpotato-based formulations than enriched Weanimix as proposed by other researchers –9. The lP <0.05) that indicated that the sweetpotato-based complementary foods were preferred by the participants above? the maize-based product for all the eight attributes evaluated, including the willingness to give the products as baby food. The just-about-right sweetness intensity score for the ComFa formulations supports our earlier suggestion that mothers would probably not sweeten the porridge for infants with sucrose. The higher consumer rating for the roller-dried ComFa compared with the oven-toasted ComFa was probably because the fish powder included in the oven-toasted ComFa imparted a flavour that was disliked by some of the participants. We have previously discussed elsewhere that the fish powder was incorporated into the household-level formulation to improve the protein quality and calcium , that caregivers taste food before serving to their babies, hence they could serve as suitable consumer sensory panellists for complementary foods as done in another study . Our samenergy and nutrient thinning compared with the maize-based formulation porridge. Also, the amount of water used for the maize-based porridge supports the relatively high peak viscosity of this product.Another important observation of nutritional significance relates to the amount of water used to prepare the same quantities of complementary food flour. Less water was used for the oven-toasted ComFa porridge compared with the enriched-Weanimix . Therefore, it is the desired viscosity of porridge that guides mothers/caregivers on the suitable proportions of complementary food flour and water to be used during preparation. The lower amount of water used for the oven-toasted ComFa porridge indicates less The sweetness score and the amount of water used for the porridge correspond to the data on the composition of carbohydrate and apparent viscosity, respectively. This therefore validates the sensory data.The sweetpotato-based formulations had a higher sugar to starch ratio than the maize-based complementary food. The levels of the total sugars and starch are the likely reasons for the lower apparent viscosity and higher WSI of the ComFa formulations compared with the enriched Weanimix resulting in requirement for less water when preparing the porridge from the ComFa formulations. The scores of the consumer acceptance given by the infant caregivers indicated highest liking for roller-dried ComFa, followed by the oven-toasted ComFa, and, finally, the enriched Weanimix. These findings, combined with the relatively low phytate and phytate: mineral ratio and high vitamin A levels , 16, 17,