Basal lipid and gene expression profiles, measured by LC/MS-MS-based targeted lipidomics and qPCR transcriptomics, respectively, highlighted a differentially altered intestinal eCBome and oxylipin tone, possibly linked to increased mRNA levels of inflammatory markers in db/db mice. In particular, the duodenal levels of several 2-monoacylglycerols and N-acylethanolamines were increased and decreased, respectively, in db/db mice, which displayed more pronounced intestinal inflammation. To a little extent, these differences were explained by changes in the expression of the corresponding metabolic enzymes. Correlation analyses suggested possible interactions between eCBome/oxylipin mediators, cytokines, and bacterial components and bacterial taxa closely related to intestinal inflammation. Collectively, this study reveals that db/db mice present a higher inflammatory state in the intestine as compared to ob/ob mice, and that this difference is associated with profound and potentially adaptive or maladaptive, and partly intestinal segment-specific alterations in eCBome and oxylipin signaling. This study opens the way to future investigations on the biological role of several poorly investigated eCBome mediators and oxylipins in the context of obesity and diabetes-induced gut dysbiosis and inflammation.Obesity is associated with a cluster of metabolic disorders, chronic low-grade inflammation, altered gut microbiota, increased intestinal permeability, and alterations of the lipid mediators of the expanded endocannabinoid (eCB) signaling system, or endocannabinoidome (eCBome). In the present study, we characterized the profile of the eCBome and related oxylipins in the small and large intestines of genetically obese ( Overweight and obesity have reached epidemic proportions globally and are one of the leading public health issues of the 21st century ,4. In faN-arachidonoyl-ethanolamine, AEA, and 2-arachidonoylglycerol, 2-AG), their congeners , and other eCB-like bioactive lipid mediators, their receptors, and metabolic enzymes [N-acylphosphatidylethanolamine phospholipase D, Napepld, in white adipocytes or intestinal epithelial cells, induces a shift in gut microbiota composition and related dysmetabolism [N-acylserotonins, were modified in the small intestine [Among the different systems implicated in regulating the intestinal barrier alongside the gut microbiota and bacterial metabolites is the expanded endocannabinoid (eCB) system—also known as the endocannabinoidome (eCBome)—which includes the eCBs  and diabetic (db/db) mice, despite exhibiting the same body weight and fat mass, are characterized by a divergent eCBome signaling in two different sites  that could be related to the etiology or consequences of the different inflammatory tone observed in the two mutant strains [db/db mice reflect an increased intestinal permeability [ob/ob and db/db mice using targeted LC/MS-MS-based lipidomics and qPCR array-based transcriptomics. Additionally, by performing correlative analyses, we sought to determine to what extent specific bacterial components and bacterial taxa could be related to either inflammatory markers or eCBome components. Deciphering the role that the lipidome targeted here plays in intestinal inflammation, per se or in relation to gut microbiota composition, might open up new possibilities for the treatment of metabolic diseases.In the context of obesity, type 2 diabetes (T2D), and inflammation we have previously shown that genetically obese  and db/db mice (BKS-Lepr/db/db/JOrlRj) and their respective control lean littermates  were used and followed for seven weeks. All mouse experiments were approved by and performed in accordance with the guideline of the local ethics committee . Housing conditions were specified by the Belgian Law of 29 May 2013, regarding the protection of laboratory animals (agreement number LA1230314).As previously described , six-weeThe duodenum, jejunum, ileum and distal colon used in this study to explore the eCBome tone originated from the same mice extensively phenotyped in Suriano et al., . BrieflyLipids were extracted from tissue samples according to the Bligh and Dyer method , as prevThe quantification of eCBome-related mediators , was car−ΔΔCt method and are represented as fold change with respect to baseline within each tissue. Rps13 was used as a reference gene for the eCBome-related receptors and metabolic enzymes, whereas Rpl19 was chosen as the housekeeping gene for the intestinal inflammatory markers. cDNA was prepared by reverse transcription of 1 µg total RNA using the Goscript RT Mix OligoDT kit  as previously described [Total RNA and cDNA were obtained, and their quantity and quality were assessed as previously described . Sixty-fAs previously described , a correob/ob and db/db mice were reported. The differences between experimental groups were considered statistically significant with a p ≤ 0.05 and were represented as follows: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.005, **** p ≤ 0.001. Data were analyzed using GraphPad Prism version 8.00 for Windows . The presence of outliers was assessed using the Grubbs test.Data are presented as the mean ± standard error of the mean (S.E.M), as specified in the individual tables and figures. The differences between the groups were determined using a One-Way ANOVA followed by Tukey’s post hoc test on ΔΔCt and on fmol/mg tissue for gene expression levels and mediator levels, respectively. Only statistically significant differences between db/db mice had a significant increase in the full intestinal length as compared to ob/ob mice  in three segments of the small intestine  and in one segment of the large intestine . Although no significant changes were observed in the mRNA expression of Il6, Il1b, Tnfa (data not shown) in any gut segments, a common signature was found in the duodenum, jejunum, ileum, and distal colon of db/db mice as compared to ob/ob mice, which were characterized by a significant increase of the expression of Rorgt2, a transcription factor that directs the differentiation of inflammatory T helper (Th)17 cells [Il17a , one of the major pro-inflammatory cytokines secreted by the Th17 cells, was also observed in the db/db mice , 2-linoleoylglycerol (2-LG), 2-arachidonoylglycerol (2-AG), 2-eicosapentaenoylglycerol (2-EPG), 2-docosapentaenoylglycerol (2-DPG) and 2-docosahexaenoylglycerol (2-DHG). The levels of docosapentaenoic acid (DPA), an omega-3 fatty acid which is the precursor of 2-DPG, were also augmented. Conversely, as shown in , N-oleoylethanolamine (OEA), N-linoleylethanolamine (LEA) and anandamide (AEA). The levels of an endogenous putative metabolite of AEA, N-arachidonoyl glycine (NAGly), were also significantly decreased in the db/db group  did not differ between the ob/ob and db/db groups (data not shown), the levels of their bioactive derivatives 13(S)-HOTre, 12- and 15-HEPE, and 14 and 17-HDHA, respectively, were significantly higher in db/db than ob/ob mice , the same trend was observed for Trpa1, a proposed target for the eCB, AEA. Differences in gene expression were also observed at the level of eCBome-related metabolic enzymes  and Pla1a, and for the catabolic Abhd16a (p = 0.06) and Ces1d whose transcripts were less expressed in db/db compared with ob/ob mice. The transcript levels of Dgke, which is an intracellular lipid kinase that phosphorylates diacylglycerol (DAG) to phosphatidic acid, were also decreased in db/db mice. Concerning NAE metabolic enzymes, there were decreases, in db/db compared to ob/ob mice, of the transcript levels of NAE biosynthetic enzymes Gde1 and Napepld, the latter showing only a trend (p = 0.06) toward reduction, which could explain the decreased levels of several NAEs, as described above. However, an increased expression was observed for the anabolic Pla2g5. With regard to NAE hydrolyzing enzymes, the expression of Faah was reduced in db/db compared to ob/ob mice. We also observed an increase in the transcript levels of the lipoxygenase Alox15 and a decrease of Ptgs2 (Cox2) in the db/db mice. The reduction of the expression of Ptgs2, which is the rate-limiting enzyme primarily responsible for the conversion of cell membrane-derived arachidonic acid into prostaglandins, could explain the decrease of PGF2α and PGD2 levels, but also the increase of 2-AG, which is a good substrate for this enzyme.Specifically, for the 2-monoacylglycerol metabolic enzymes, the most relevant results were found for the biosynthetic db/db as compared to ob/ob mice. Accordingly, the levels of DPA were also augmented in the db/db group. As for the NAEs, only the eCB and AEA, showed a decreasing trend (p = 0.09). Modifications were also observed for the omega 3 derivative molecules; particularly 15-HEPE, 17-HDHA, and 13(S)-HOTre, which are the metabolites of EPA, DHA and ALA, respectively, which were increased in the db/db compared to the ob/ob group, although no changes were observed in the free omega 3 fatty acid levels. Regarding the eCBome receptors, we observed, a significant increase in the gene expression of Cacna1b and Cacna1h, Gpr18, and Trpv4 in db/db mice  and eCBome mediator tissue concentrations or metabolic enzyme and receptor mRNA expression levels of the respective two gut segments. An analysis of the Pearson’s rank correlation matrix confirmed the existence of potential links between certain eCBome lipids and genes and either inflammatory markers or bacterial metabolites. In detail, we found that the Gram-negative bacterial component LPS was significantly correlated with the duodenal levels of the PPARα agonist, and hence the anti-lipogenic and anti-inflammatory mediator, 2-PG (Abhd16a (Rorgt2 and Il17a) were positively correlated with 13S-HoTre and the mRNA expression levels of Trpv4, Mgll, Abhd16a, Inpp5d, and Alox 15 17 cells [Il17a , one of the major pro-inflammatory cytokines secreted by Th17 cells. This observation confirms a previous finding of ours [Il6, Il1b and Tnfa, did not differ between the two mice strains, suggesting a selectively impaired Th17 cell balance and a greater inflammatory tone in the intestine of db/db mice. Moreover, the increase in the total intestinal length observed in db/db mice might reflect impaired intestinal epithelial proliferation, apoptosis, and mucosal hyperplasia, as previously described in db/db mice with the same body weight as ob/ob mice [The eCBome modulates several physiological functions, including GI tract function and energy metabolism . It is a/ob mice . The fir17 cells , and, ac of ours  that the/ob mice . db/db mice was accompanied by significant alterations of eCBome mediators and receptors, as well as of other oxylipins and eicosanoids, as compared to ob/ob mice. Common to all intestinal segments were the increases of 2-DPG and 13-HOTrE, which, given their biosynthetic origin from omega-3 fatty acids, are expected to have anti-inflammatory properties. These changes could represent protective responses to inflammation, but studies on the pharmacological activity of these compounds are needed to substantiate this hypothesis. Indeed, pro-inflammatory prostaglandins were, instead, decreased in both the duodenum and ileum and/or jejunum of db/db mice, and so was the potentially pro-inflammatory oxylipin, 11-HETE. On the other hand, the levels of the TRPV1 agonist, 13-HODE-G [db/db as compared to ob/ob mice. These observations, also substantiated by several other, more segment-specific differences in the levels of other eCBome mediators and oxylipins with proposed opposing functions in inflammation, suggest that the observed increased intestinal inflammatory state of db/db mice engenders a series of adaptive and maladaptive responses at the level of these lipid signals, whose effects might also depend on the concomitant, and again segment-dependent, changes of their receptors. In support of this hypothesis are two other novel findings of the present study: (1) the levels of two well established anti-inflammatory mediators, PEA [db/db compared to ob/ob mice, and (2) while no alteration in the expression of the proposed receptors of the former compound  was detected, two of the proposed targets of NAGly, the anti-inflammatory Gpr18 and the pro-inflammatory Cana1h, for which this mediator is an agonist and an inhibitor, respectively [Trpv4, another emerging pro-inflammatory receptor [db/db and ob/ob mice in the levels of the lipid mediators investigated here and the expression of their several known receptors, the assessment of the significance of each of these changes in determining the increased intestinal inflammatory state of diabetic mice will require several further investigations.More relevant to the aims of this study, we observed that the increased inflammatory response in the intestine of 3-HODE-G , were elors, PEA  and NAGlors, PEA , were siectively ,28, alsoreceptor , which hreceptor , was insdb/db and ob/ob mice could explain, at least in part, the different levels of the mediators. In the duodenum, the main enzymes for both the biosynthesis and inactivation of both 2-MAGs and NAEs were less expressed in db/db compared with ob/ob mice, offering no explanation as to why the levels of the former mediators were mostly increased, whereas those of the NAEs were mostly decreased. This discrepancy was even stronger in the distal colon (and jejunum), where the expression levels of the catabolic enzymes for 2-MAGs, but not those of their anabolic enzymes, were increased, and yet the concentrations of 2-DPG were also counterintuitively increased. The effect of the availability of the ultimate biosynthetic precursors for these fatty acid derivatives may in part have prevailed on the expression of metabolic enzymes, since we found that in all intestinal segments the levels of DPA, but not those of other measured fatty acids, were increased in db/db compared with ob/ob mice, thus reflecting the increased concentrations of 2-DPG. On the other hand, the relative levels of expression, between db/db and ob/ob mice, of Alox15 and Ptgs2 (respectively increased and decreased in the former genotype) in the duodenum, and of the former enzyme also in the jejunum and distal colon (again increased in the former genotype in both segments), explained the increased and decreased levels of several 15-LO and COX-2 derivatives in intestinal sections of db/db mice. The increased levels of some 15-LO derivatives of linoleic acid were also possibly sustained by the observed concomitant increase of this fatty acid precursor, at least in the jejunum and ileum.We also studied the segment-specific mRNA expression of enzymes catalyzing the biosynthesis and degradation of the lipid mediators analyzed here to gain a preliminary understanding as to whether or not putative differences between db/db mice. In particular, the pro-inflammatory bacterial component LPS was significantly correlated with the duodenal levels of 2-PG, a PPARα agonist with likely anti-inflammatory action, whereas, in the distal colon, the two inflammation markers found to be up-regulated in db/db mice, i.e., Rorgt2 and Il17a, were positively correlated with the potentially anti-inflammatory mediator, 13S-HoTre, and with the mRNA expression levels, respectively, of the pro-inflammatory channel Trpv4, and of the inactivating enzymes of mostly anti-inflammatory 2-MAGs, i.e. Mgll and Abhd16a, as well with Alox 15, which produces both pro-and anti-inflammatory mediators. These correlations suggest that alterations in the signaling of eCBome mediators and oxylipins may depict segment-specific adaptive or maladaptive responses to the increased intestinal inflammation observed in db/db mice. Likewise, the correlations observed between the absolute abundance of fecal bacterial taxa involved in gut inflammation [Clostridium_sensu_stricto_1, Dubosiella, Bacteroides and Turicibacter, with both duodenal and colonic concentrations of several 15-LO derivatives and Alox15 and Gpr18 expression levels, reinforced this hypothesis. This is also supported by the fact that the quantity of these bacterial taxa, which was significantly, or tended to be, affected by either the ob/ob or the db/db genotype or both, was also either positively or negatively correlated with several metabolic and inflammatory markers in other body sites  [Finally, the observed correlations among eCBome and other lipid mediators and the levels of inflammatory markers or the relative abundance of some gut microbiota taxa reinforce the hypothesis that alterations in the intestinal tone of the eCBome and oxylipin signaling are related to higher inflammation and dysbiosis in ammation ,32,33,34 tissue) , furtherdb/db mice display a higher inflammatory state in the intestine when compared to ob/ob mice. This difference is associated with profound and potentially adaptive or maladaptive, and partly intestinal segment-specific, alterations in eCBome and oxylipin signaling. These alterations: (1) are only to a minor extent explained by changes in the expression of the corresponding metabolic enzymes, and (2) correlate with both inflammatory markers and fecal microbiota taxa that have been previously found to be altered during conditions of intestinal inflammation in mice, or following the correction thereof. This study opens the way to future investigations on the biological role of several poorly investigated eCBome mediators and oxylipins in the context of obesity and T2D-induced gut dysbiosis and inflammation.In summary, we discovered that


E-cigarette use among middle and high school youth increased from 2. 5 million in 2014 to 9.2 million in 2019, becoming the most common tobacco product used among youth. Hispanic youth, the largest ethnic minority in the United States, have higher rates of tobacco use, including e-cigarettes, than non-Hispanics. Identifying factors that put youth at risk for future e-cigarette use is vital to focusing prevention efforts. Informed by social cognitive theory, this study identifies predictors of e-cigarette uptake among e-cigarette naïve youth in a predominantly low-income Hispanic community.n = 862). Outcome measures were e-cigarette initiation and current use at follow-up. Logistic regression models tested six hypotheses about predictors of e-cigarette initiation and current use: (1) intention, (2) outcome expectations, (3) knowledge, (4) friendship network exposure, (5) normative beliefs, and (6) social acceptability.1,249 students (6–8th grades) from two middle schools in El Paso, Texas consented to participate in this longitudinal survey during the 2016–2017 school year. The study sample for analysis was restricted to e-cigarette naïve students (n = 71) reported initiation at follow-up; of these, 3% (n = 23) reported current use. Significant predictors of initiation were intention , outcome expectations , friendship network exposure , normative beliefs , and social acceptability . Significant predictors of current use were intention  and friendship network exposure .Among e-cigarette naïve students at baseline, 8% (With the increasing popularity of e-cigarettes, age appropriate and culturally sensitive prevention strategies tailored at altering these predictive factors are essential in preventing future e-cigarette use. Use of electronic cigarettes (e-cigarettes) among middle and high school youth increased from 2.5 million in 2014 to 9.2 million in 2019 . Much isExamining the beliefs that underlie e-cigarette use behavior among adolescents will be beneficial to address this health problem and successfully guide prevention efforts. Thus, this study is informed by social cognitive theory (SCT), which posits that learning occurs in a social context with a three-way reciprocal interaction of the individual, social environment, and behavior and has previously been used to study tobacco use among adolescents , 7. GrowIf I were to use an e-cigarette, I would enjoy it—are important determinants of tobacco initiation and subsequent steady use  and their impact on youth tobacco use , 21. ButHispanic youth report greater intention to use tobacco and e-cigarettes compared to non-Hispanic peers  and greaThe purpose of this study is to examine SCT-based predictors of e-cigarette initiation and current use among 6, 7, and 8th grade youth who had never tried e-cigarettes at baseline in a low-income Hispanic community. We hypothesized that significant predictors of e-cigarette initiation and current use at the individual level were (1) intention, (2) outcome expectations, and (3) knowledge; and at the social environmental level, (4) friendship network exposure, (5) normative beliefs, and (6) social acceptability. We examined the hypothesized predictors to identify their unique influence beyond demographic characteristics  identified as factors related to e-cigarette use among youth , 11, as To identify predictors of e-cigarette initiation and current use over time, we analyzed data collected at baseline and again at follow-up during the 2016–2017 school year as part of an evaluation of Teens Against Tobacco Use (TATU), a school-based peer-led tobacco prevention intervention . Two TitAmong the 1,695 eligible students, 1,249 (73.7%) returned signed parental consent forms; 1,166 (93.3%) of these students completed surveys at baseline and follow-up. The sample for this analysis, however, was limited to e-cigarette naive students at baseline, defined as never tried an e-cigarette at the time of the baseline survey, with 862 (73.9%) students included in the final sample.The anonymous survey consisted of Likert scale questions drawn primarily from the Pierce et al. measure of smoking susceptibility  and the Have you ever tried or experimented with electronic cigarettes, even one or two puffs?” .E-cigarette initiation was assessed by asking “ puffs?” . StudentPredictors of e-cigarette initiation and current use were assessed at baseline through a composite of SCT constructs of intention, outcome expectations, and knowledge in the individual domain; and friendship network exposure, normative beliefs, and social acceptability in the social environmental domain.r = 0.61), e.g. Do you think you will use any kind of tobacco product or electronic cigarette in the next year?; outcome expectations through five items (α = 0.61), e.g. Do you think you might enjoy smoking an electronic or regular cigarette?; knowledge through three items (α = 0.56), e.g. Do you think electronic cigarettes contain harmful chemicals?; normative beliefs through three items (α = 0.42), e.g. Do you think smoking looks cool?; and social acceptability of tobacco use among peers through three items (α = 0.91) asking “Do you think it is okay for people your age to: Smoke cigarettes? Use e-cigarettes? Smoke hookah?”. Response options ranged from 1 = “Definitely NOT!”, 2 = “Probably not”, 3 = “Neutral”, 4 = “Probably yes”, and 5 = “Definitely YES!”. Responses for two items under outcome expectations  and one item under normative beliefs (Smoking cigarettes or e-cigarettes is a good way to make friends.) were on a different 5-point scale, where 1 = “Strongly disagree”, 2 = “Disagree”, 3 = “Neutral”, 4 = “Agree”, and 5 = “Strongly agree”. Friendship network exposure was measured with three items (α = 0.87) asking “How many of your friends: Smoke cigarettes? Use e-cigarettes? Smoke hookah?” with response options ranging from 1 = “none” to 5 = “all”.We assessed each construct on a 5-point Likert scale using multiple questions and calculated the scale mean to create a continuous variable. Intention was assessed through two items , school performance , and school . For thep < 0.05) to assess their combined influence on e-cigarette initiation at follow-up. We replicated these models with current use of e-cigarettes at follow-up as the dependent variable, rather than initiation. The amount of missing data was minimal, ranging from 0.006 to 3.5%; therefore, we used listwise deletion, with sample sizes differing minimally across models. We analyzed the data using STATA version 15.1 statistical software.Descriptive statistics characterized non-initiators and initiators. Sampling was not classroom dependent and we therefore did not apply multilevel modeling at the classroom level. We instead used logistic regression to test the relation between each hypothesized predictor and e-cigarette initiation at follow-up, controlling for gender, ethnicity, grade, school performance, and school. It should be noted that initial testing for multicollinearity did not produce problematic results. We then fit an additional model with the significant independent predictors (n = 71) reported e-cigarette initiation at follow-up (n = 23) reported current use. E-cigarette initiation was made up of slightly more females  than males . Grade was the only covariate that significantly differed across participants (p < 0.05). Initiation was reported by more 8th grade students  than 7th  and 6th grade students . Most non-initiators  and initiators  reported school performance of mostly A's, mostly B's, and mostly C's. The number of initiators was slightly higher at middle school 2  versus middle school 1 .Among the 862 e-cigarette naïve respondents at baseline, 8.2% . A one unit increase in baseline scores for outcome expectations—e.g., from Agree to Strongly Agree—predicted 1.73 times higher odds of initiation at follow-up . Knowledge was not a significant predictor of initiation .Logistic regression models separately examined the hypothesized predictor of e-cigarette initiation at follow-up , controlAOR = 1.53; 95% CI = 1.11–2.11; p = 0.01). Students with higher baseline scores on normative beliefs had 2.12 times higher odds of initiating e-cigarettes at follow-up . Those with higher social acceptability of e-cigarette use at baseline had 1.91 times greater odds of initiation at follow-up .In the social environmental domain, normative beliefs, friendship network exposure, and social acceptability were significant predictors of initiation . RespondAOR = 1.86; 95% CI = 1.13–3.07; p = 0.02). Likewise, a one unit increase in friendship network exposure scores—e.g., from Some to Most—predicted 1.52 times increased odds of initiation . McFadden's R2 ranged from 0.03 to 0.06 in the separate models. When we entered the significant predictors into a combined model, McFadden's R2 was 0.08.When entering only the significant predictors of e-cigarette initiation from the separate models into a combined model , intentiAOR = 1.98; 95% CI = 1.07–3.69; p = 0.03). Outcome expectations at baseline were not a significant predictor of current use at follow-up , nor was knowledge .Following the same analytic process used to predict initiation, we entered the hypothesized predictors into separate logistic regression models to predict e-cigarette current use at follow-up , with geAOR = 1.69; 95% CI = 1.06–2.70; p = 0.03). Normative beliefs at baseline were not a significant predictor of current use at follow-up , nor was social acceptability .In the social environmental domain, friendship network exposure was a significant predictor of e-cigarette current use at follow-up. Students with higher baseline friendship network exposure to tobacco products had 1.69 times increased odds of current use at the end of the school year . Intentions remained associated with a higher risk of current use in the multivariate model (p > 0.05). McFadden's R2 ranged from 0.02 to 0.03 in the separate models and was 0.05 in the combined model.Upon entering only the significant predictors from the separate models into a combined model, friendship network exposure significantly predicted e-cigarette current use. A one unit increase in baseline friendship network exposure scores predicted 1.68 times higher odds of current use at follow-up .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Screening tests for body posture defects and abnormalities conducted over the past several decades have revealed a significant and constantly increasing problem of health risks in children. A sedentary lifestyle, which is considered to be the primary cause, can result in proprioceptive disorders leading to postural instability. The aim of the study was to find the correlation between the level of proprioceptive control and the number of postural disorders. The study involved a representative group of 1090 children aged 8–10 years, from randomly selected primary schools. Subjects who scored from 1 to 5 points in a prior postural screening test were qualified for the examination of the postural control system. The examination of the postural control system was carried out using an electronic station enabling assessment of postural stability and proprioception. A single leg stance test showed that the number of postural disorders does not significantly impact proprioceptive control. Proprioceptive control was found to significantly increase with the age of the children, and girls presented significantly better proprioceptive control in relation to the boys in each age group. The process of posturogenesis is accompanied by a continuous, conditioned, and encoded development of postural reflex and locomotion. A normal process of body posture development depends on numerous exogenous and endogenous factors . DecreasScreening tests designed to detect postural defects and abnormalities, conducted over the past several decades, have shown there is a significant and constantly growing problem of health risks in children ,4,5,6. EA group of 1090 individuals, 564 girls and 526 boys aged 8–10 years from selected primary schools of the city and municipality of Zielona Góra, were enrolled in the study. The examinations were carried out between 2009 and 2010 as a part of a program entitled “Prosto do zdrowia”, focusing on postural defects prevention, as well as diagnostics, and aimed toward prophylaxis and therapy of postural defects, and at improving the overall level of health and physical fitness in children and adolescents. The specific objective of the program was to also shift the emphasis from treatment to prevention in accordance with current guidelines for health care development. The characteristics of the study group are presented in Individuals were assessed for the presence of body posture defects. The individuals who were diagnosed with at least one postural defect were qualified for the specialist examination performed using a Delos Postural System Professional 4.0 device . The tests were carried out by a specialist in physiotherapy with a doctoral degree in medical sciences.Disturbed alignment of the spine in the frontal plane;Disturbed alignment of the spine in the sagittal plane;Scoliosis (positive result in the Adam’s test);Knee malalignment ;Foot malalignment .Points were awarded depending on the occurrence of:One posture defect scored one point. Thus, the respondents could obtain a result from 0 (no posture defect) to 5 points .The inclusion criteria were obtaining from 1 to 5 points in the body posture examination by visual assessment and Adam’s test.-physical disability;-mental disability;-developmental anomalies of the feet;-true shortening of a lower limb;-sensory integration disorders;-neurological conditions;-nystagmus;-attention deficit disorder;-feeling unwell on the day of the examination.The exclusion criteria were as follows:Parents/legal guardians of the subjects expressed their written consent for the examination.The use of the research for this paper was approved by the Independent Bioethics Committee of the Medical University of Gdańsk, dated 19 September 2011, resolution number: NKEBN/359/2011, and accepted in writing by the coordinator of “Prosto do zdrowia” program.The test was performed under similar lighting and time conditions by the same person.The stability test was performed by means of electronic postural proprioceptive station (DPPS). The station, connected to a personal computer with specific software DPPS 4.0), consisted of an electronic postural reader, an infrared sensor bar, and a display . In case.0, consiThe data from the postural reader were a stream of acceleration samples taken by converting the sensor outputs into the digital domain at a rate of 100 Hz. These raw data were initially averaged with a 4-tap sliding window; therefore, the 3 dB bandwidth was narrowed to approximately 11 Hz. Scaling with the calibration data of the instruments was then performed to convert the raw data into angles. The equations involved in the management of data from the postural reader have been described in a previous article [The stability index with closed eyes, SI CE (%), was used to assess proprioceptive control. This index allows researchers to grade all tests and to compare individuals with high stability scores obtained during the test  and those with a lower level of stability (very low autonomy of the system) .Static Riva Test was used to assess the functional status of the postural control system. It was performed on an electronic station for postural stability and proprioception tests, Delos Postural System Professional 4.0.Static Riva Test was performed using Delos Vertical Controller (DVC), which was attached to the subject’s sternum, and Delos Postural Assistant (DPA), which the subjects were asked to use as hand support in case of imbalance. The DPA was adjusted to each person’s height to provide adequate support. The device was connected to a computer with specialized software (PSM 4.0) designed to receive and analyze information from the specific parts of the device.Static Riva Test consisted of 20 s trial of one-leg stance on the left leg with eyes open, then 20 s on the right leg with eyes open, followed by the same amount of time on the left leg with eyes closed, and on the right leg with eyes closed. Every 20 s trial was followed by a 15 s interval.All calculations were performed using Microsoft Excel version 2007 and Statistica version 8.5 statistical package. In the statistical description of the quantitative data, classic position measurements were used, such as arithmetic mean, median, and mode, as well as the standard deviation and the definition of minima and maxima as general measures of variation.p < 0.05 was assumed as the level of statistical significance of differences.When comparing two groups for quantitative and stepwise data, the Mann-Whitney U test (comparison of two groups) was used, and the Wilcoxon test (comparison of two measurements with the same people). In the comparative analysis of a larger number of nonparametric data groups, the Kruskal-Wallis test was used together with the post hoc multiple test (Dunn test) as a non-parametric equivalent of ANOVA, the parametrically evaluated method of analysis of variance (ANOVA) using the Fischer test, and the post hoc tests Scheffegi and RIR Tukey. In order to assess the strength, direction, and significance of linking variables, Pearson’s linear correlation coefficient was used. In all statistical tests, Statistical analysis was carried out in cooperation with the School of Medical Informatics and Biostatics at the Medical Faculty of the Medical University of Gdańsk.The influence of the number of postural disorders on the level of proprioceptive control index in the children aged 8 years.p = 1)  . The Mann-Whitney U test showed that the level of stability index of the girls was significantly higher than the level of stability index of the boys .The influence of the number of postural disorders on the level of proprioceptive control in the children aged 9 years.p = 0.4)  . p < 0.0001). The level of the stability index for the girls was significantly higher than the level of the stability index for the boys   . p < 0.0001). The Mann-Whitney U test showed that the level of the stability index for the girls was significantly higher than the level of the stability index for the boys representing this age group . A multiple comparison test was performed to identify the age group with the highest differences. The test showed that all the age groups differed from one another in a statistically significant manner, and the level of the stability index with closed eyes visibly increased with age. The relationships are shown in detail in The Kruskal-Wallis test showed statistically significant differences between the level of the stability index with closed eyes and the age of the subjects (According to Riva, decreased physical activity results in reduced proprioceptive information supplied, which consequently leads to poorer neuromuscular control causing functional instability of the joints and decreased balance capacity, which may eventually lead to postural instability ,5. DynamThe current study showed significant age-related differences in the scores identified during one leg stance trials. Posture stability level constantly increased from 8 to 10 years of age. This is consistent with results reported by Morioka, who believed that the ability to maintain a standing position on one leg with eyes open rapidly improves from late pre-school to early school age, and then slows down during later school age. Statistically significant differences were also observed between girls and boys. The results indicate that girls have a better ability to maintain a stable posture in a one leg stance, compared to boys in every group from 8 to 10 years of age.On the other hand, the present study has shown that the number of postural defects does not significantly impair posture stability in any of the age groups from 8 to 10 years. In some age groups it is possible to only observe a tendency towards a decrease in the stability index associated with a larger number of postural defects. In the 8-year-old group, the downward trend was observed only if there were more than two defects in body posture. In the group of children aged 9 years, a similar tendency was observed in cases with up to three postural defects. In 10-year-olds, poorer results were observed only in the group of children with three defects in comparison to the children with two defects. The lack of a downward trend in the stability index in the groups with more than three disorders in body posture may be linked to the small size of the groups with four or five defects, which is reflected by a relatively large range of standard error. It should also be considered that postural defects were not rated by the authors for the quality of the disorder, but only in terms of their presence. Further research should also consider the degree of the disorder, as it is possible that the stability index will deteriorate with increasing defects. Studies by Herdea et al. have shown that some cases of idiopathic scoliosis occur and progress due to insufficient levels of vitamin D, calcium, and melatonin . TherefoProprioceptive control significantly increases with age in children with diagnosed postural defects.Girls aged 8 to 10 years had statistically significantly better results in proprioceptive control compared to boys, in all age groups.The number of postural defects does not significantly impair posture stability in any of the age groups from 8 to 10 years.The number of postural defects has no significant effect on proprioceptive control.


A cohort study was conducted to examine the association of an increased body mass index (BMI) with late adverse outcomes after a carotid endarterectomy (CEA). It comprised 1597 CEAs, performed in 1533 patients at the Vascular Surgery Clinic in Belgrade, from 1 January 2012 to 31 December 2017. The follow-up lasted four years after CEA. Data for late myocardial infarction and stroke were available for 1223 CEAs, data for death for 1305 CEAs, and data for restenosis for 1162 CEAs. Logistic and Cox regressions were used in the analysis. The CEAs in patients who were overweight and obese were separately compared with the CEAs in patients with a normal weight. Out of 1223 CEAs, 413 (33.8%) were performed in patients with a normal weight, 583 (47.7%) in patients who were overweight, and 220 (18.0%) in patients who were obese. According to the logistic regression analysis, the compared groups did not significantly differ in the frequency of myocardial infarction, stroke, and death, as late major adverse outcomes (MAOs), or in the frequency of restenosis. According to the Cox and logistic regression analyses, BMI was neither a predictor for late MAOs, analyzed separately or all together, nor for restenosis. In conclusion, being overweight and being obese were not related to the occurrence of late adverse outcomes after a carotid endarterectomy. According to the World Health Organization (WHO), in 2016, almost 60% of adults in the WHO European Region were overweight and obese ,4,5,6. TExtracranial carotid disease accounts for approximately 18–25% of ischemic strokes ,12. CaroData on the association between obesity and CEA adverse outcomes are inconsistent. In some of the investigations ,14, therThe aim of the present study was to examine the association of increased BMI (overweight and obesity) with late adverse outcomes after CEA.As previously described in detail , this co2. For the purpose of the present study, patients were categorized into four categories: underweight (<18.5), normal weight (BMI = 18.5–24.9), overweight (BMI = 25.0–29.9), and obese (BMI ≥ 30) . Moreover, there was no significant correlation between the independent variables included in the study. None of the correlation coefficients was higher than 0.75, taken as the limit value significant for multicollinearity. The follow-up data concerning stroke, myocardial infarction, and death, separately and together, did not show any difference between those with a normal weight, those who were overweight, and those with obesity, analyzed by the Kaplan–Meier test. The curves almost overlapped with p values ranging from 0.973 to 0.764.  had significantly higher perioperative cardiac complications, while bleeding was significantly more frequent in patients who were underweight; however, neither of these two BMI groups was analyzed separately in our study. However, while we did not find an association between being overweight and having obesity with any of the late adverse outcomes, Volkers et al. [2 was one of the predictors of five-year mortality after CEA for asymptomatic carotid stenosis. In the present investigation, we analyzed symptomatic and asymptomatic patients together, and we did not include patients who were underweight in the analysis due to the very small number. Jeong et al. [The literature data on the association of BMI with late adverse outcomes after CEA are scarce. In fact, we found only a few articles ,20,21,22s et al.  found ths et al. , also prs et al. . In the Although being overweight and having obesity were not significantly related to late complications after CEA, they were, significantly and independently of other variables, associated with some factors found to be predictors of late adverse outcomes, such as non-insulin-dependent diabetes mellitus ,26,27,28In the present study, non-insulin-dependent diabetes mellitus was significantly more frequent in both patients who were overweight and patients with obesity, compared to those with a normal weight. It is well known that an increased BMI is strongly correlated with type 2 diabetes mellitus (T2DM) ,34. AccoBoth obesity and atherosclerosis are lipid storage disorders, with triglyceride accumulation in the fat tissue and cholesterol esters in atherosclerotic plaques . In our It is also known that obesity contributes directly to incident cardiovascular risk factors, including dyslipidemia, type 2 diabetes, hypertension, and sleep disorders, but it can lead to the development of some cardiovascular diseases and cardiovascular disease mortality, independent from other risk factors ,44. The There were some limitations to our investigation. Due to the small numbers, patients who were underweight were not included in the present study. Moreover, class II and III obesity were put together with class I obesity, and all these BMI subgroups could be, according to the results of other investigations, associated with complications after CEA. There is also the question of whether BMI is the best measure of adiposity, or whether it would be better to use measures such as the waist circumference, waist-to-height ratio, waist-to-thigh ratio, or the InBody Test ,47,48,49Compared to the patients with a normal weight, the patients who were overweight were significantly more frequently males, with non-insulin-dependent diabetes mellitus, and a more frequent use of ACEI in hospital discharge therapy. The patients with obesity were significantly younger, with myocardial infarction and non-insulin-dependent diabetes mellitus in their personal history, with more frequently increased triglyceride levels, more frequent usage of OAC in the therapy before the operation, and a shorter clump duration.However, being overweight or having obesity was not significantly associated with the occurrence of myocardial infarction, stroke, death, and restenosis, as the late adverse outcomes after a carotid endarterectomy.


Mycobacterium smegmatis mc2155. McGee has a genome 156,008 bp long, containing 237 protein-coding genes, 31 tRNA genes, and 1 transfer-messenger RNA (tmRNA) gene. McGee shares high gene content similarity to phages in actinobacteriophage cluster C1.Mycobacteriophage McGee is a myovirus isolated from a wet soil sample collected at Manassas, VA, using  Mycobacterium smegmatis mc2 155 have been isolated and characterized, providing important insights into viral diversity and population structure . The soil sample was washed with 7H9 liquid medium, the wash filtered , and the filtrate plated in soft agar overlay containing Mycobacterium smegmatis mc2 155. McGee was purified with multiple rounds of plating. Negative-stain transmission electron microscopy revealed McGee to possess a Myoviridae morphology , and sequenced on an Illumina MiSeq instrument v3 reagents), resulting in 77,248 single-end 150-bp reads providing 73× coverage. The raw reads were assembled and assessed using the programs Newbler v2.9 ( reagentshttp://cobamide2.bio.pitt.edu/) (http://phages.wustl.edu/starterator), whereas gene functional assignments were determined using BLASTp searches against the NCBI nonredundant v2.13.0 (13Genes were identified and annotated using DNA Master v5.23.6 (tt.edu/) , GLIMMER v2.13.0  and acti v2.13.0 , HHpred  v2.13.0 , and Pha v2.13.0 . Aragorn v2.13.0  and tRNA v2.13.0  were use2.13.0 1315 and TM2.13.0 13, 17. AllA total of 269 putative genes were identified in McGee, including 237 protein-coding genes, 31 tRNAs, and 1 tmRNA. Among those that could be assigned a function, the virion structure and assembly genes include a major capsid protein (gp101), capsid decoration protein , tail sheath protein (gp129), tail assembly chaperones , tape measure protein (gp134), minor tail proteins , and baseplate wedge protein . The genes involved in McGee’s DNA metabolism include DNA helicase (gp188), thymidylate kinase (gp197), DnaC-like helicase loader (gp201), DnaB-like double-stranded DNA (dsDNA) helicase (gp202), DNA primase (gp204), DnaJ-like chaperonin (gp206), single-stranded DNA (ssDNA) binding protein (gp207), DnaE-like DNA polymerase III α (gp208), RF-1 peptide chain release factor (gp209), RecA-like DNA recombinase (gp210), Holliday junction resolvase (gp210), RusA-like resolvase (gp214), Ro-like RNA binding protein (gp233), and multiple proteins with a helix-turn-helix DNA binding domain . Genes involved in lysis, lysin A, holin, and lysin B are encoded by gp252, gp253, and gp254, respectively. The largest gene in McGee is a hypothetical protein gp98  and the smallest gp64 (75 bp long).https://phagesdb.org/), McGee was assigned to phage cluster C1 (Based on its gene content similarity (GCS) of at least 35% to phages in the Actinobacteriophage database (uster C1 , 18, 19.ON637764, while its raw reads have been deposited under Sequence Read Archive accession number SRX14483217.The complete genome sequence for McGee can be found at GenBank under accession number


The application of different approaches calculating the anthropogenic carbon net flux from land, leads to estimates that vary considerably. One reason for these variations is the extent to which approaches consider forest land to be “managed” by humans, and thus contributing to the net anthropogenic flux. Global Earth Observation (EO) datasets characterising spatio-temporal changes in land cover and carbon stocks provide an independent and consistent approach to estimate forest carbon fluxes. These can be compared against results reported in National Greenhouse Gas Inventories (NGHGIs) to support accurate and timely measuring, reporting and verification (MRV). Using Brazil as a primary case study, with additional analysis in Indonesia and Malaysia, we compare a Global EO-based dataset of forest carbon fluxes to results reported in NGHGIs.2yr−1), while Brazil’s NGHGI reported a net carbon source (+ 0.8 GtCO2yr−1). After adjusting the EO estimate to use the Brazilian NGHGI definition of managed forest and other assumptions used in the inventory’s methodology, the EO net flux became a source of + 0.6 GtCO2yr−1, comparable to the NGHGI. Remaining discrepancies are due largely to differing carbon removal factors and forest types applied in the two datasets. In Indonesia, the EO and NGHGI net flux estimates were similar (+ 0.6 GtCO2 yr−1), but in Malaysia, they differed in both magnitude and sign . Spatially explicit datasets on forest types were not publicly available for analysis from either NGHGI, limiting the possibility of detailed adjustments.Between 2001 and 2020, the EO-derived estimates of all forest-related emissions and removals indicate that Brazil was a net sink of carbon (− 0.2 GtCOBy adjusting the EO dataset to improve comparability with carbon fluxes estimated for managed forests in the Brazilian NGHGI, initially diverging estimates were largely reconciled and remaining differences can be explained. Despite limited spatial data available for Indonesia and Malaysia, our comparison indicated specific aspects where differing approaches may explain divergence, including uncertainties and inaccuracies. Our study highlights the importance of enhanced transparency, as set out by the Paris Agreement, to enable alignment between different approaches for independent measuring and verification.The online version contains supplementary material available at 10.1186/s13021-023-00240-2. The intde Table  35, 54], 54−1) a2) in 2019 [The large difference between the gross emissions in Malaysia may be linked to the fact that most reported emissions and area change (> 99%) in the Malaysian NGHGI are associated with “forest land converted to settlement”  had limited capacity and (b) had less historical responsibility in terms of emissions contribution. Under the Enhanced Transparency Framework, there is now greater requirement for transparency and all countries will be required to provide a Biennial Transparency Report (BTR) from 2024  to improTransparent methods used in each of Brazil’s datasets made it possible to distinguish and quantify methodological and definitional differences such as forest categories. This transparent information enabled adjustments to be made to improve comparability and thus credibility of the estimates. Unfortunately, such a geospatial analysis was not possible in other countries, such as Indonesia and Malaysia, where digitised versions of the LULC maps were unavailable for open-access analysis. While it may not always be possible to fully reconcile differences between datasets as each has their own motivations and assumptions, the differences should be traceable so they can be understood. For greater transparency, countries could consider clearly documenting the emissions and removal factors used in their submissions to the UNFCCC, as was demonstrated in the Malaysian BUR4.All the NGHGIs considered in our study clearly stated that tree crops such as palm oil and rubber are classified as perennial agricultural crops. Associated emissions and removals are, therefore, not necessarily forest-related Tables , 2. The Variations in definitions are not limited to our analysis and vary between global models and other internationally applied flux datasets such as the FAO , emphasiFor completeness, there is a need to consider all forest types, which might be GHG sources or sinks. EO data can help capture spatial and temporal heterogeneity of forest types and associated fluxes, to improve accuracy.Our analysis has shown spatial differences in the forest cover between the Brazilian NGHGI and Global EO approach, especially outside the Amazonia biome, with divergences in the savannah and dry forest biomes Fig. . These dAcross the Brazilian biomes, fire and logging disturbances can cause forest degradation that often occurs at smaller-scales, and thus goes undetected by moderate resolution (> 30 m) remote sensing products . Due to Our analysis highlighted the differences in the removal factors used by different datasets as an important discrepancy, demonstrating the ongoing uncertainty with regards to applying removal factors for different forest types. Per unit area, the difference was particularly evident in young secondary forests in Brazil (“other land converted to secondary forests”) Table . HoweverAs many countries and organisations aim to reduce their net carbon emissions through forest conservation and restoration, accurate and representative removal factors for all forest types , 45, 56 In this study, we highlighted the differences between the flux datasets regarding the forest classification assigned to a given pixel in space and time, which has implications for the emission/removal factor applied . The impIndependent data, such as that from the Global EO model included here, have the potential to provide key sources of information for the MRV of NGHGIs . Convers2 yr−1) and the NGHGI flux (0.8 GtCO2 yr−1) from 2001 to 2020 was 1.0 GtCO2 yr−1 and estimates were opposite in sign. Adjusting the Global EO flux by applying the Brazilian NGHGI definition of managed forest area and only considering the assumptions in their inventory (in terms of land transition and flux categories) resulted in the adjusted Global EO net flux to be 0.6 GtCO2 yr−1, now just 0.2 GtCO2 yr−1 lower compared to the NGHGI. The various adjustments also highlight the impact of considering all land as managed versus different applications of the managed land proxy. The analysis was made possible due to the availability of the geospatial data and transparency in methodologies from all flux datasets.Using Brazil as a primary case study, we could reconcile the difference between GHG fluxes from a Global EO dataset and the NGHGI. With no adjustment to the Global EO dataset, considering all forest fluxes in the country boundary, the difference between the Global EO flux -country scales. For completeness and information purposes, NGHGIs could voluntarily consider providing fluxes associated to unmanaged land or consider all land to be managed.Full transparency is crucial for understanding differences and making effective comparisons between datasets. This includes ensuring transparency in the methodology and open access to the data used by all approaches.Further disaggregation in the forest types and associated fluxes in all datasets will facilitate greater comparability and completeness. Independent datasets could disaggregate the fluxes by the land use categories and forest types used in NGHGIs, to allow greater comparability. All flux datasets could explicitly consider disturbance type and intensity, and associated recovery.The adjustments outlined in this study can also be used to aid comparisons with and benchmarking of other approaches, such as global models or in other regions. Below we outline some key lessons learned in this study:As more information and datasets become available, including EO, countries’ requirements and capacity to report is expected to increase in transparency, comparability, consistency, completeness, and accuracy. Additionally, region specific information compiled for use in NGHGIs can also be used to improve local applicability of Global EO assessments. Given the approval of Article 6.4 of the Paris Agreement, and the numerous pledges and commitments that have been made by business organisations, cities, nations, and international agreements to protect and restore forests, the need to measure, report and especially verify estimates becomes increasingly important. If this is not achieved in accurate and credible ways, we risk mis-representing the carbon fluxes of the world’s forests, one of our most important allies on land, to tackle the climate emergency.Additional file 1: Figure S1. Comparison of the area of Primary and non-Primaryforest land according to the Global EO  in the year 2000, for which they used dataset byTurubanova et al. (2018)and the Unmanaged forest land and Managed forest land according to the NGHGI in 1994 and 2002. Figure S2. Percentage contribution of different plantation types to the total plantation area in different biomes of Brazil. Colouring denotes if the plantation is a forest plantation (shades of blue) or a Tree crop plantation (yellow-oranges). Theareas considered for analysis only included those that were also classified as plantation area in the National Greenhouse gas inventory of Brazil in 2016. Figure S3. Bar graphs representing the area contribution of different forest and non-forest cover types that make up the six biomes of Brazil according to three different approachesfor the year 2020. The three approachesare the Global Earth Observation (EO), the National Greenhouse Gas Inventory (NGHGI) and the independent estimate (SEEG). As the NGHGI is only available up to 2016, area numbersfor theNGHGIhave been adjusted by multiplying by the fractional difference in the area of each forest type in 2016 and 2020 according to SEEG (Mapbiomas).Note differences in the Y axis scale. Figure S4. Bar graphs representing theaveragegross removals contribution of different forest types within the six biomes of Brazil according to three different approachesover the period 2001 to 2020.The three approachesare the Global Earth Observation (EO), the National Greenhouse Gas Inventory (NGHGI) and the independent estimate (SEEG). As the NGHGI is only available up to 2016, numbersfor the NGHGI have been adjusted by multiplying by the fractional difference in the removalsof each forest type in 2016 and 2020 according to SEEG.Note differences in the Y axis scale. Table S1. Summary of the key United Nations Framework Convention on Climate Change (UNFCCC) principles as outlined in the 5th Conference of the Parties (COP5) in 1999. Table S2. Modifications made to the NGHGI of Brazil to account for the shorter time-period available. The fractional difference of the average fluxes were calculated for the period 2002 to 2016 (period of the NGHGI) and for 2001 to 2020 . Table S3. The total number of pixels per biome which are considered as non-primary forests in the Global Earth Observation dataset and overlap pixels classified as Managed forests  pixels in the National Greenhouse Gas Inventory (NGHGI) of Brazil. Table S4. The categories of forest-based transitions used within this study and how they are referred to in the National Greenhouse Gas Inventory (NGHGI) of the case study countries Indonesia and Malaysia.


Schizophrenia is a chronic neuropsychiatric syndrome that significantly impacts daily function and quality of life. All of the available guidelines suggest a combined treatment approach with pharmacologic agents and psychological interventions. However, one in three patients is a non-responder, the effect on negative and cognitive symptoms is limited, and many drug-related adverse effects complicate clinical management. As a result, discovering novel drugs for schizophrenia presents a significant challenge for psychopharmacology. This selective review of the literature aims to outline the current knowledge on the aetiopathogenesis of schizophrenia and to present the recently approved and newly discovered pharmacological substances in treating schizophrenia. We discuss ten novel drugs, three of which have been approved by the FDA . The rest are under clinical trial investigation . However, additional basic and clinical research is required not only to improve our understanding of the neurobiology and the potential novel targets in the treatment of schizophrenia, but also to establish more effective therapeutical interventions for the syndrome, including the attenuation of negative and cognitive symptoms and avoiding dopamine blockade-related adverse effects. Schizophrenia is a neuropsychiatric disorder, most likely due to neurodevelopmental reasons arising from abnormalities in different circuits and neurotransmitter systems. This developmental process includes uncontrolled synaptic pruning, deriving from gene–environment interaction . NumerouThe dopamine (DA) hypothesis of psychosis is one of the most enduring ideas in psychopharmacology. Undeniably, one central dysfunction stems from dopaminergic abnormalities, but in a different way than initially thought  . MoreoveIncreasing evidence implicates glutamate, cholinergic, and serotonin neuronal networks, trace amines, and their TAAR receptors. Focusing on these alternative neurotransmitters/receptors is critical to fully understand the underlying pathophysiology of the psychoses, in addition to developing drugs with novel mechanisms of action other than DA receptor antagonism; the latter results from the necessity for safer, more tolerable, and more effective medications . ImproveIn this review, we aim to present an update on the current understanding of the aetiopathogenesis of schizophrenia, along with the most promising novel agents in the treatment of this disorder, namely olanzapine/samidorphan, lumateperone, brilaroxazine, xanomeline/trospium, emraclidine, ulotaront, pimavanserin, sodium benzoate, other DAAO inhibitors, D-serine, and iclepertin.www.clinicaltrials.gov database. We considered all clinical trials evaluating patients strictly with schizophrenia-type psychosis. We also checked the references in publications found to acquire any additional data, as well as the official sites of the pharmaceutical companies developing the novel compounds in question.MEDLINE was searched for articles written in the English language from inception to 31/7/23 that matched any combination of the following keywords: ((olanzapine/samidorphan[tiab] OR lumateperone[tiab] OR (RP5063[tiab] OR brilaroxazine[tiab]) OR (xanomeline[tiab] OR Kar-XT[tiab]) OR (emraclidine[tiab] OR CVL-231[tiab]) OR (ulotaront[tiab] OR SEP363856[tiab]) OR pimavanserin[tiab] OR sodium benzoate[MeSH] OR DAAO inhibitor*[tiab] OR D-serine[tiab] OR (iclepertin[tiab] OR OR voltage-gated sodium channel blocker[tiab]) AND (schizophr*[MeSH] OR schiz*[tiab]) AND ). Using the same keywords, the search was repeated for each drug separately in the www.clinicaltrials.gov search yielded ongoing trials for each compound under investigation  articles on the olanzapine/samidorphan combination, eight (8) on lumateperone, one (1) on brilaroxazine (RP5063), six (6) on xanomeline/tropsium, one (1) on emraclidine, four (4) on ulotaront, four (4) on pimavanserin, nine (9) on sodium benzoate, one (1) on the DAAO inhibitor luvadaxistat, and fifteen (15) on D-serine. Our tigation . The eliThe classic concept of striatal functioning proposes that the dorsal striatum regulates motor movement, and the ventral striatum regulates emotions via the nigrostriatal and the mesolimbic pathway, respectively . HoweverAccording to the dopamine synthesizing capacity (DSC) theory, dopaminergic dysfunction predates the subsequent onset of frank psychotic illness in people with symptoms that are truly prodromal to a psychotic disorder . In refrAt present, it is widely accepted that the DA receptors in the prefrontal cortex (PFC) maintain a balance of excitation/inhibition (E/I) by modulating glutamate neurotransmission and GABAergic interneuron function . In schiThe neurodevelopmental theory of schizophrenia suggests that DA has pruning properties, mostly notable during the early stages of neuronal development. Excessive pruning is considered the cause of decreased brain volume, which is the most consistent brain abnormality in schizophrenia. Evidence indicates that DA induces a decrease in neurite extensions, mainly via the activation of D1 receptors , and neuHallucinogens, including the naturally occurring mescaline and psilocybin and the synthetic lysergic acid diethylamide (LSD), act via the serotonin 5-HT2A receptors (5-HT2AR) and enhance glutamatergic transmission to induce their profound changes in human consciousness, emotions, and cognition. In the prefrontal cortex, 5-HT2AR stimulation increases the release of glutamate . The serAntagonism of the 5-HT2AR receptors on glutamatergic neurons is a common feature of second-generation, or atypical, antipsychotics. 5-HT2ARs are excitatory, leading to excitatory glutamate release at downstream targets, and thus to the hyperactivation of the mesostriatal pathway and hypoactivation of the mesocortical pathway. The serotonin hyperfunction hypothesis of psychosis suggests that psychosis may be caused by an imbalance in excitatory 5-HT2A receptor stimulation on the glutamate pyramidal neurons, which directly innervate mesostriatal DA neurons and visual cortex neurons, inducing delusions and hallucinations. This theory explains the psychotic symptoms in PD dementia and the psychotic effect of 5-HT2A agonists such as psilocybin and LSD. Moreover, it indicates the promising role of pure 5-HT2A antagonists in psychosis .The inhibitory 5-HT1A receptors are located on the glutamate neurons projecting to the VTA or the substantia nigra (SN). When activated by serotonin or a 5-HT1A agonist, they inhibit glutamatergic neurons to enhance mesocortical and nigrostriatal DA pathway neurotransmission . The 5-HBeyond the 5-HT1A and 5HT2A receptors, other serotonin receptors, including the 5-HT2C, 5-HT3, 5-HT6, and 5-HT7 receptors, continue to represent promising drug targets for the discovery of novel multi-receptor antipsychotic agents aiming to address cognitive and negative symptoms .Moreover, serotonin antagonists improve the extrapyramidal side-effects of antipsychotics ,28, and Glutamate is the main and most common excitatory neurotransmitter in the mammalian brain . DysfuncDisturbances in glutamatergic neurotransmission may influence synaptic plasticity and cortical microcircuitry, especially NMDA receptor functioning . NMDA reThe key NMDA receptors are in the indirect cortico-cortical glutamate pathways . ThroughDisturbances in glutamate signaling may be an attractive drug target for schizophrenia due to its key role in the aetiopathogenesis of the disorder regarding cognitive impairment and negative symptoms . AntipsyHowever, classical agonists at the NMDA did not prove to be useful as the excessive stimulation of NMDA receptors results in excitotoxicity and neuron damage. The glycine modulatory binding site on the NMDA receptor is considered a more promising target, and direct or indirect glycine modulatory site modulators, including glycine, D-cycloserine, D-serine, glycine transporter 1 (GlyT1) inhibitors, and D-amino acid oxidase (DAAO) inhibitors, have been actively researched in clinical trials and, as discussed in more detail below, show promise in the treatment of schizophrenia following the glutamatergic hypothesis of schizophrenia .We have now discussed the dopaminergic, serotoninergic, and glutamatergic hypotheses of schizophrenia. There is also growing evidence that inhibitory GABA signaling is dysregulated in schizophrenia, particularly in the cortex. Dopaminergic, GABAergic, and glutamatergic signaling are all modulated by the cholinergic system; therefore, the modulation of ACh receptors represents an exciting new target. Muscarinic ACh receptors (mAChR) appear to be suitable for interventions. The M1, M4, and M5 mAChR subtypes can all modulate schizophrenia-related circuitry and are intriguing targets for novel schizophrenia treatments .Using traditional therapies that lack specificity among mAChR subtypes requires drug tailoring to hit a therapeutic window between beneficial effects  and classical cholinergic side-effects. These side-effects are mediated by peripherally expressed M2 and M3 receptors, making the selective targeting of M1, M4, and M5 therapeutically desirable. ACh binds on the mAChRs’ orthosteric binding pocket, which is a specific area on each subtype of mAChR, making it challenging to create molecules that specifically target this site with high subtype selectivity. However, some agonists have been developed that, while lacking complete subtype-selectivity, exhibit preferential activation for certain subtypes over others. Allosteric agents have proved easier in targeting binding pockets outside the orthosteric binding pocket binding ACh. In particular, selective positive allosteric modulators (PAMs) have significantly advanced subtype-selectivity .Substantial evidence supports that the cholinergic system robustly modulates striatal DA signaling through the activation of both mAChRs and nicotinic ACh receptors (nAChRs) via cholinergic projections from the brainstem to the midbrain, where they stimulate DA neurons in SN and the VTA .The M4 receptor is among the best-studied muscarinic targets for psychosis. In the striatum, the M4 subtype is primarily expressed postsynaptically on direct pathway spiny projection neurons , on cholinergic interneurons, where it acts as an autoreceptor, and on glutamatergic inputs, where it acts as a heteroreceptor .The M4 receptors on cholinergic interneurons and spiny projection neurons modulate DA signaling through different mechanisms. The activation of M4 autoreceptors on cholinergic interneurons reduces ACh release. Typically, ACh induces striatal DA release by activating nAchR on the DA axonal projection, arriving from the VTA/SN complex. Consequently, an M4-mediated decrease in ACh secretion leads to a reduction in DA output. Furthermore, the activation of the M4 receptors on spiny projection neurons leads to an endocannabinoid-dependent blockade of DA release independent of nAChRs. Beyond its role at the striatum, the M4 receptor regulates the hippocampal circuitry involved in cognitive processes. Recent findings suggest that M4 activation (by PAMs) alleviates cognitive deficits and positive symptom reduction .The M1 receptor is another well-studied muscarinic receptor. There is robust evidence that M1 activation leads to pro-cognitive effects and alleviates negative-type symptoms . This efMoreover, deficits in M1 receptor expression are brain region-specific. In schizophrenia, M1 receptor-expressing neurons are reduced in the cortex but not the thalamus or the hippocampus. As for the M1-selective compounds, pure PAMs seem superior in their pro-cognitive efficacy and exhibit fewer adverse side effects. Although M1 is primarily considered a target for enhancing cognition, there are indications that M1 activation is associated with an antipsychotic effect. Such implications derive from animal studies where M1 PAMs have demonstrated efficacy in positive symptoms . The norThe M5 receptors need to be better studied, and data are scarce. However, they modulate DA signaling via actions at the DA terminals and DA cell bodies, making them an interesting target for the future . The advTrace amines are endogenous substances found in trace levels in the body. They are formed from amino acids inside CNS during the synthesis of monoamines when certain enzymatic steps are omitted. They are commonly found in foodstuffs and can be produced and degraded by microbiota. These molecules do not act as typical neurotransmitters; namely, they are not released upon nerve firing. There are five main human trace amines: β-Phenylethylamine, p-Tyramine, Tryptamine, p-Octopamine, and p-Synephrine .Trace amine-associated receptors (TAARs) are G-protein receptors. There are six isoforms in humans, and TAAR1 is the most studied. Except for TAAR1, all other TAAR subtypes are expressed only in olfactory neurons. TAAR1 are present in multiple CNS areas, including several monoamine brainstem centers and projection areas. They are also expressed in the periphery, where they may have a role in nutrient-induced hormone secretion . Cross-tTAAR1 are predominantly located intracellularly. The putative mechanism of DA rheostasis is the following. When the TAARs are occupied by an endogenous or an exogenous agonist, the derivative complex is translocated to the cell surface, where it couples with D2. This heterodimerization directs the second-messenger pathway to move preferentially towards the inhibitory G (Gi) protein signal transduction cascade, inhibiting the synthesis and release of DA, rather than towards the β-arrestin-2 excitatory pathway (production of GSK-3 and overstimulation). Presynaptically, the amplification of the Gi pathway leads to the inhibition of DA release, while postsynaptically, the amplification of the Gi pathway can lead to the reduced production of GSK-3 and, therefore, to less stimulation. Consequently, drugs that target TAAR1 constitute a promising field of indirect dopamine modulation ,61.μ-opioid receptor.The Olanzapine/Samidorphan (OLZ/SAM) combination was approved by the FDA for the treatment of adults with schizophrenia or bipolar disorder type I in June 2021. Strictly speaking, this is a novel combination of a well-established antipsychotic, olanzapine, with samidorphan. This treatment can be used for maintenance monotherapy or the acute treatment of psychotic, manic, or mixed episodes. Olanzapine, acting primarily via the DA and 5-HT receptors, is a second-generation  antipsychotic (SGA) approved for treating schizophrenia and bipolar disorder type I. Samidorphan is a 3-carboxamido-4-hydroxynaltrexone, acting as an opioid antagonist, preferentially on the Enlighten-1 was a 4-week, phase III, randomized, double-blind, placebo- and olanzapine-controlled study conducted in patients with schizophrenia in acute exacerbation . It aimep = 0.003). The combination was well-tolerated, with the most common adverse events being weight gain, somnolence, and dry mouth.Enlighten-2 evaluated the weight gain profile of OLZ/SAM versus olanzapine over 6 months in 561 patients with stable schizophrenia . CompareA systematic review of the literature in 2022  highlighLumateperone is a butyrophenone atypical antipsychotic approved by the FDA in 2019 for the treatment of schizophrenia. It is a potent antagonist at 5-HT2A, a presynaptic partial agonist and a postsynaptic antagonist at the dopamine D2 receptors, and a dopamine receptor phosphoprotein modulator. It is also a D1 receptor-dependent indirect modulator of glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate (NMDA) GluN2B receptors and a blocker of the serotonin transporter (SERT) . Thus, lLumateperone is thus claimed to be an effective antipsychotic agent, appropriate as a first-line treatment for patients with schizophrenia, claiming a possible long-term positive impact on cognitive and negative symptoms . MoreoveBrilaroxazine is an antipsychotic agent under development with a unique binding profile ,76. It aIn 2018, Cantillon and colleagues  publisheA phase II, 4-week, double-blind, placebo- and aripiprazole-controlled clinical trial evaluated the safety and efficacy of brilaroxazine  in 234 patients with schizophrenia or schizoaffective disorder in acute episodes. It demonstrated that brilaroxazine at 15 mg and 50 mg significantly improved the PANSS total score compared to the placebo . The mosXanomeline is a muscarinic ACh receptor agonist with reasonable selectivity for the M1 and M4 subtypes and an M5 receptor antagonist studied for the treatment of both Alzheimer’s disease and schizophrenia, particularly for the cognitive and negative symptoms ,80,81,82post-hoc analysis of the EMERGENT-1 study, KarXT was associated with a low overall adverse event burden [A recent phase II, 5-week, double-blind, randomized, placebo-controlled clinical trial evaluated the combination of xanomeline/trospium in 182 patients with schizophrenia in acute episodes . The xant burden . The majp < 0.0001). In the xanomeline/trospium group, the improvement in negative symptoms was slight but significant. Xanomeline/trospium was well-tolerated, with the most prevalent side-effects being cholinergic. There were no reports of any side-effects commonly associated with atypical antipsychotics. Furthermore, using the PANSS to assess the effectiveness, response was demonstrated as early as 2 weeks relative to the placebo. KarXT demonstrated improvements vs. the placebo in all five PANSS factors  [The results of a phase III, randomized, double-blind, placebo-controlled clinical trial in 252 in-patients with schizophrenia have been presented as a poster ,88. Patiression) . There aEmraclidine is an M4-selective positive allosteric modulator (PAM) currently in development as a novel antipsychotic drug for the treatment of schizophrenia . PAMs inp = 0.023; emraclidine 20 mg bid, p = 0.047). At present, three phase II clinical trials are ongoing to confirm the efficacy, safety, and tolerability of emraclidine . Furthermore, it showed a favorable safety and tolerability profile. A six-month extension study followed up the recruited patients who completed the initial study to evaluate the safety and efficacy of long-term ulotaront treatment. Ulotaront use was associated with continued improvement in the PANSS total and BNSS total scores [The results of a phase II, 4-week, randomized, double-blind, flexible-dose (50 mg/day or 75 mg/day) aimed to evaluate the efficacy and safety of SEP 363,856 in adults with schizophrenia in acute relapse were published in 2020 . Ulotarol scores . The mosl scores .A population analysis  of the pUlotaront is in phase III clinical development for further evaluation of the efficacy, safety, and tolerability . Two mulPimavanserin primarily functions as an inverse agonist and a partial inverse agonist at the 5-HT2A receptors. It spares the dopamine post-synaptic receptors, including the D2. Pimavanserin binds with high affinity to 5-HT2A, with lower affinity to 5-HT2C, and demonstrates negligible binding at the 5-HT2B, dopaminergic (D3), muscarinic (M5), and opioid (sigma 1) receptors . PimavanADVANCE-II, a phase II, 26-week, randomized, double-blind, placebo-controlled, multi-center, international study, assessed the efficacy and safety of pimavanserin in 403 stable patients with predominantly negative symptoms of schizophrenia while being on stable antipsychotic therapy . PimavanENHANCE-III, a phase III, 6-week, randomized, double-blind, placebo-controlled clinical trial, assessed pimavanserin as an adjunctive treatment in resistant-to-treatment out-patients with schizophrenia . The stuIn the brain, D-amino acid oxidase (DAAO) is a peroxisomal flavoenzyme. Through oxidative deamination by DAAO, D-serine—the main co-agonist of synaptic N-methyl-D-aspartate (NMDA) receptors (NMDARs) —is degraIt has been hypothesized that excessive DAAO activity may cause reduced D-serine levels, possibly contributing to the NMDAR hypofunction in schizophrenia . DAAO isSodium benzoate , an accessible food preservative, is a D-amino acid oxidase (DAAO) inhibitor, initially shown to increase synaptic D-serine levels . Howeverp < 0.001 for all; ES  =  1.16–1.69). Additionally, a neurocognitive assessment revealed the superiority of the sodium benzoate treatment compared to the placebo, especially in terms of processing speed and visual memory. Sodium benzoate exhibited a good safety profile without significant adverse effects.In 2013, Lane and colleagues presented the results of a 6-week, randomized, double-blind, placebo-controlled trial that examined the efficacy and safety of adjunctive sodium benzoate at 1 g/day, in 52 patients with stable schizophrenia for at least three months . The stuIn a 6-week , double-In contrast, a 12-week, double-masked, placebo-controlled, randomized clinical trial from Australia  examininA recent meta-analysis from India  evaluateAs the hypofunction of the NMDA receptor is implicated in the pathophysiology, particularly cognitive impairment, of schizophrenia, in a randomized, double-blind, placebo-controlled trial in patients with chronic schizophrenia, add-on sodium benzoate was given in combination with sarcosine, a glycine transporter I (GlyT-1) inhibitor, to enhance NMDA receptor-mediated neurotransmission (see below). The investigators concluded that the combination of these two NMDA-enhancing agents, but not sarcosine alone, can improve cognitive function, despite the non-improvement in the clinical symptoms of patients with chronic schizophrenia .® as an add-on treatment for schizophrenia, one in adolescents and one in adults  failed to address the negative symptoms of schizophrenia but resulted in cognitive symptom enhancement , and a pCurrently, the most reliable way to assess NMDAR-related function in humans is through mismatch negativity (MMN), which relies on the NMDAR’s dual voltage- and ligand-sensitivity. MMN is an event-related brain potential that is sensitive to stimulus deviation from a repetitive pattern, thought to reflect the activity of sensory memory, with, at most, moderate influences of higher-level cognitive processes, such as attention . MMN is An early meta-analysis including 29 trials and a total of 1253 cases aiming to evaluate the potential of NMDA receptor modulators as adjunctive therapy for schizophrenia found D-serine, N-acetyl-cysteine (NAC), and sarcosine to have therapeutic benefit in the treatment of total and negative symptoms as adjuncts to non-clozapine antipsychotics. However, while glycine improved the positive and total symptoms as an adjuvant to non-clozapine antipsychotics, it worsened them when added to clozapine . A more In addition to its powerful role as an inhibitory neurotransmitter through its binding to the Glycine Receptor (GlyR) in the spinal cord and the brainstem, glycine, a non-essential amino acid, is an important co-agonist of the NMDAR in excitatory glutamatergic transmission . VariousIn the synaptic cleft, glycine’s reuptake is regulated by glycine transporters, namely type I (GlyT1) and type II (GlyT2). GlyT1 is considered a highly potential target for psychosis therapy. A phase II study conducted on bitopertin, a selective and non-competitive GlyT1 inhibitor, demonstrated benefits for negative symptoms in a per-protocol population . HoweverIclepertin  is a potent and selective GlyT1 that showed significant (d  =  0.34) improvements in cognition over 12 weeks of treatment in patients with schizophrenia during a phase II study . AnotherVoltage-gated sodium channel blockers, in particular the antiepileptic drugs carbamazepine, oxcarbazepine, phenytoin, and lamotrigine, increase the motor threshold, and the corticospinal system becomes less excitable . HoweverEvenamide  is a voltage-gated sodium channel blocker and inhibits the synaptic release of glutamate. It thus reduces hyperexcitability in the PFC and the hippocampus. Unlike other antipsychotics, it does not interact with other neurotransmitter systems , acts preferentially on sodium channels, and modulates glutamate release. It was shown to reverse the NMDR antagonist ketamine- and phencyclidine-induced reduction in prepulse inhibition  and has Indeed, the results from a pilot, 6-week, randomized, open-label, rater-blinded study with a 46-week extension indicate very good tolerability with an exceptional, clinically important, increase in the efficacy of evenamide  as an add-on treatment to antipsychotics in treatment-resistant schizophrenia patients .p = 0.023), but there were no significant differences in the negative symptoms between groups. In conclusion, it has been suggested that a dosage of 800 mg of L-Carnosine was promising for improving executive functions in schizophrenia [L-Carnosine is an antioxidant reported to improve negative and cognitive symptoms in schizophrenia . A randoophrenia . The treatment of schizophrenia remains a significant challenge in clinical practice, despite the enormous progress in psychopharmacological research. The complexity of its aetiopathogenesis leads to high variability in the medication response across patients with schizophrenia and schizoaffective disorder. While several psychotropic drugs are currently available, one out of three patients remains a non-responder , the effNovel compounds in the pipeline for use in schizophrenia appear to work beyond the dopamine system .Future drug discovery should focus on the optimal implementation of the available treatments, emphasizing improvements in medication adherence due to improved side-effect profiles, and hence adherence. Long-acting or dual preparations are a good example of such strategies. Most importantly, based on novel mechanisms of action, prospective medications should address the debilitating negative and cognitive symptoms, in addition to treating the positive symptoms, whilst avoiding the burden of post-synaptic DA blockade-induced side-effects.