The present paper focused on compare the PID-5 mean score levels across two matched community and clinical samples of Portugal and the UAE.The generalizability and universality of the Alternative Model of Personality Disorders has been thoroughly studied through the Personality Inventory for DSM-5 (PID-5) across countries and languages. However, studies comparing Western and Middle Eastern countries are still limited, in particular those who assess the PID-5 measurement invariance.We examined measurement invariance of the PID-5 scales across matched Emirati and Portuguese clinical and nonclinical groups, as well as compare and contrast the PID-5 mean score levels across both countries and samples.N = 300, 80% women and 20% men, ageM = 27.95) which were matched with Portuguese community participants , as well as clinical participants of the UAE  and Portugal . We examined measurement invariance through an unrestricted Factor Analysis based program, and mean scores levels were compared and analyzed.The Arabic and the Portuguese versions of the PID-5 was administered to Emirati community participants (Our findings supported the PID-5 measurement invariance across the Emirati and Portuguese clinical samples pointing to the universality and generalizability of the Alternative Model of Personality Disorders. The Emirati psychiatric sample exhibited somehow higher results than the Portuguese psychiatric participants, albeit the small effect size for most of the PID-5 scales.Further research is needed to examine the applicability of the PID-5 across non-clinical representative samples of Portugal and the UAE, and other Middle Eastern countries. Each culture's ethos, history and dimensions create unique contingencies that help shape the expressions of self and interpersonal functioning, as well as the promotion or suppression of specific behaviors and personality traits . ConsequPDs are not a specific mental illness of Modern Western Societies, instead they are a severe psychiatric condition deemed to affect 7.8% of the world population  and in WTraditional Islamic and patriarchal societies, such as the UAE, where religion overrides an act of faith but is embedded in every aspect of the individual’s life , 15, raiIn both classification systems, the key innovation is the conceptualization of PDs as a dyad of severity or impairment in self and interpersonal functioning, along with the presence of maladaptive personality traits that characterize the stylistic expression of PDs. This dimensional approach, led by the Alternative Model for Personality Disorders (AMPD) published in Section III (for further studies) of the DSM-5 , has achet al. [et al. [The PID-5 operationalizes Criterion B of the AMPD, or the presence of 25 maladaptive personality traits in which individuals differ (facets), subsequently organized within five high order domains: Negative Affectivity, Detachment, Antagonism, Disinhibition and Psychoticism. These traits seem to be pathological extremes of the basic dimensions of normal personality, as stated by the Five-Factor Model (FFM) , 20, supet al.  and Zimm [et al. ).With the present study, we were interested in testing if maladaptive traits measured by the PID-5, are culturally shaped in their intensity and expressions, across matched clinical and non-clinical groups from two distant countries and cultures, the Middle East culture of the United Arab Emirates and the Portuguese South European culture. In other words, can findings derived from the Portuguese and Arabic versions of the PID-5 be comparable and generalized to personality research and clinical practice?et al.’s [Nevertheless, before cross-cultural differences can be examined and interpreted it is crucial to establish measurement invariance to ensure that the same underlying pathological personality traits, measured by the Portuguese and the Arabic version of the PID-5, are being assessed in similar ways and have the same meaning, across the two countries , 24. On et al.’s  cross-cuTherefore, to examine measurement invariance (MI) of the PID-5 facets and domains, we used the IMINCE software , a free Moreover, this software is suitable for responses to items on Likert scales or dichotomic items, as it performs factor analysis using polycholic or tetrachoric correlations, and measures partial, strong, and strict invariance .Accordingly, our study goals were (1) to test MI of the PID-5 scales across matched Emirati and Portuguese clinical and nonclinical groups, and (2) to compare the PID-5 mean score levels across these countries and samples. Considering the previous studies on the Emirati and Portuguese populations, we expected that the clinical groups’ mean scores would be higher than non-clinical groups.22.1The present study was approved by the Research Ethics Committee of Zayed University Dubai (ref. ZU18_36a_F), Dubai Scientific Research Ethics Committee (ref. DSREC-02/2019_07), Ministry of Health and Prevention Research Ethics Committee (Ref. MOHAP/DXB-REC/No.10/MM/2019), Deontological Committee of Psychology Faculty of the University of Lisbon (ref. Acta n.2_CD_22Oct2020), Ethic Commission of the host Portuguese mental Health institutions. All participants were carefully informed about the study and gave written consent.2.22.2.1M = 27.95, SD = 10.19) mostly single (66.7%), had completed high school (69.3%), and at the time of the assessment, 56% were students.The study included an Emirati and a Portuguese convenience community sample. The Emirati community sample comprised 300 volunteer Emirati college students from Zayed University Dubai and Abu Dhabi, and their acquaintances (80% women and 20% men). The participants were aged 18 to 57 years , were recruited from the Psychology Faculty of Lisbon University. The majority of the participants were single (75.3%), had completed high school (59.5%), and at the time of the assessment were students (56.7%).The Portuguese community sample was matched based on the composition of gender and age with the Emirati community sample. A total of 300 Portuguese volunteer students and their acquaintances (80.3% women and 19.7% men), aged 18 to 60 years . Most of the clinical participants were single (49.3%), had completed high school (66%), and at the time of the assessment, 37.3% were unemployed, 28% were employed, 15.3% were housewives, 14.7% were student, and 4% were retired.The clinical samples were convenience samples, composed of Emirati and Portuguese psychiatric patients. The Emirati clinical sample was recruited among volunteer patients that, at the time of the assessment, were receiving treatment in mental health institutions of the UAE, namely Rashid Hospital, Al Amal Psychiatric Hospital, and National Rehabilitation Centre. The selection process was conducted by the institutions’ psychiatrists or psychologists and based on clinical records and/or clinical authority. The clinicians were asked to report the patient’s diagnosis using the DSM-5 criteria, and those who met at least one DSM-5 mental disorder were included in the study. Patients who suffered from intellectual disability, schizophrenia spectrum disorder, and major and mild neurocognitive disorders were excluded from the sample. A total of 150 inpatients and outpatients were selected, 61.3% female, 38.7% male, aged 18 to 61 years . The participants were predominantly single (50%), unemployed (51.7%), and at the time of the assessment 28.2% had completed high school, and 26.2% had a bachelor’s degree or more. The predominate diagnoses for both countries’ samples were substance related disorders, depressive disorders, obsessive-compulsive disorders, anxiety disorders and bipolar disorders.The Portuguese clinical sample comprised 150 Portuguese volunteer psychiatric patients from mental health institutions across the country, subsequently matched (based on the diagnosis), with the Emirati clinical sample. The patients were primarily men (52%), aged 18 to 68 years old Likert scale, ranging from 0  to 3 (very true or often true) that characterizes 25 empirically derived lower-level facets grouped into five major domains of maladaptive personality variation. Approximately, the instrument takes 40 minutes or less to complete and it is to be used in adults (18 years old or above). The PID-5 has shown robust psychometric properties worldwide, in clinical and non-clinical samples, such as replicable factor structure, internal consistency, convergent validity with other personality measures as well as a broad range of psychopathological constructs. The UAE sample was assessed through the Arabic version of the PID-5 [The PID-5 is a self-report measure that operationalizes the trait system of the DSM-5 Alternative Model of Personality Disorders. It is composed of 220 items, rated on a four-point he PID-5  while thhe PID-5 . Both trhe PID-5 -34.2.4et al. [The present study comprised two Emirati samples and two Portuguese samples. The Emirati community sample was recruited through email or in-person by the researchers. The sessions were held collectively at Zayed University Dubai and Abu Dhabi. Regarding the Emirati clinical sample, the selection of the patients was performed by the mental health units ‘clinicians and the participants were invited to take part in the study at the end of the follow-up appointments. The objectives of the study were explained, and confidentiality was emphasized. As the test requires approximately 40 minutes to apply, data collection sessions were scheduled dependent on the patients’ conditions and availability. All the participants signed a written informed consent form. As for the Portuguese community and clinical samples, data was made available by the authors of the Portuguese version of the PID-5, who also mentored this research project, and details regarding data collection are published in Pires et al. , 34.2.5Cronbach’s alphas and gender effect sizes were analysed. In order to explore the normality of the scales’ distributions, the following criteria were applied: skewness, kurtosis, Kolmogorov-Smirnov Goodness-of-fit test (N > 30), and Q-Q-plots. Mean score differences between the UAE and the Portuguese  samples presenting a normal distribution, were investigated by means of paired samples t test and Cohen’s d. The effect size was considered small when d ≤.20, medium when .20 ˂ d ≤ .50, large when .50 ˂ d ≤ 1.0, and very large when d > 1.0. Accordingly, the mean score differences of the scales that presented a heteroscedastic distribution were calculated by the Wilcoxon Signed-Rank test, for both countries and samples. The effect size was tested through r = z/√N, being N the number of pairs without ties, and the effect size was considered small when .10 ≤ r < .30, medium when .30 ≤ r < .50 and large when r ≥ .50.Statistical analyses were undertaken with the IBM SPSS  and the IMINCE software . In the Likert item scores, it can as well analyse sums of item scores (facets) and sets of test scores (domains). The IMINCE examines the following invariance forms: invariance of item difficulties or intercepts, factor loadings or discriminations , and residual variances (strict factor invariance). This is accomplished using 1) Cohen’s d or univariate t tests or Hotelling’s T-square and the corresponding F statistics, depending on the nature of the variables involved and the size of the samples. In our case, Cohen’s d is the most appropriate given the large size of the UAE and the Portuguese community and clinical samples. Additionally, 2) factor loadings are obtained by an Exploratory Factor Analysis method and tested by factor congruence, factor discrepancy and approximate confidence intervals. Observed congruence and discrepancy indices are compared to the critical values α and, congruence indices are considered statistically significant if they are smaller than α, while discrepancy indices are considered statistically significant if they are larger than α. Finally, 3) invariance of residual variances or strict factor invariance, is assessed through percentile intervals which are obtained from a Bootstrap resampling. Nonoverlapping intervals suggest that the residual variances of a certain item are not invariant over the two populations.Furthermore, we employed the IMINCE, a free unrestricted Factor Analysis based program that allows the assessment of MI in two populations, in this particular study, the Emirati and the Portuguese community and clinical samples. Although the program is more suitable for binary or 33.11 presents the PID-5’s facets and domains mean, SD, and Cronbach alphas of the UAE and Portuguese community and clinical samples. Overall, the PID-5 presented acceptable to good (α ≥ .70) alpha coefficients, in all domains and most of the facets, for both countries and samples. Higher reliability coefficients were obtained in the clinical samples compared to the community samples. The lowest alpha values were found in the facet Suspiciousness for the UAE community (.35) and clinical samples (.48), as well as in the Portuguese clinical (.59) sample. Also, the facet Manipulativeness presented the lowest alpha (.37) in the Portuguese community sample along with the facets Submissiveness (.52) and Restrictive affectivity (.55). On the other hand, the highest alpha values for both UAE (.91) and Portuguese (.89) clinical samples, were observed on Depressivity, whilst Callousness and Eccentricity reported the highest alphas in the Portuguese (.83) and UAE (.90) community samples, respectively. As for the PID-5 domains, the lowest alphas were found in the domain Disinhibition for the Portuguese community sample (.81) and UAE clinical sample (.80), along with Antagonism in the UAE community sample and Negative affectivity in the Portuguese clinical sample. The highest alpha values were displayed in the domain Psychoticism for both countries and samples.Table 3.2Cohen´s d was calculated to determine gender effect sizes. Our results pointed to very small effect sizes (d <.20), in some facets and domains, in both, community, and clinical samples, of the Emirati and Portuguese populations. Specifically, in the domain of Disinhibition, along with the facets of Depressivity, Distractibility and Submissiveness, in the Emirati community sample. Also, the Negative affectivity domain as well as the facets of Anxiety, Callousness, Deceitfulness, Emotional lability, Grandiosity, Irresponsibility, Restricted affectivity, and Risk taking in the Portuguese community sample. Concerning the clinical samples, the same results were found (d <.20) in the domain Negative affectivity in conjunction with the facets, Risk taking, Separation insecurity, and Submissiveness, in the Emirati clinical sample, together with the facets Deceitfulness and Callousness, in the Portuguese clinical sample.In order to prevent possible gender bias, due to uneven proportions of men and women in both, community and clinical samples, 3.32), Cohen’s d statistic identified 10 facets with large effect sizes, between the UAE and the Portuguese community populations, namely: Callousness, Cognitive and perceptive dysregulation, Deceitfulness, Grandiosity, Intimacy avoidance, Irresponsibility, Manipulativeness, Suspiciousness, Unusual beliefs and experiences, and Withdrawal. Moreover, in terms of Invariance of factor loadings or discriminations , significant differences were observed in the congruence coefficients of 6 of the PID-5 facets , and in the discrepancy coefficients of 9 facets  as presented in Table 3. As for the PID-5 domains, Detachment presented significant differences in the discrepancy coefficient along with Psychoticism in the congruence and discrepancy coefficients (Table 4). Therefore, due to these differences, the overall congruence and discrepancy indices were also compromised. Concerning strict factor invariance in the community samples, the percentile intervals of residual variances of the PID-5 facets have shown overlapping for all facets, except for Grandiosity and Suspiciousness (see Table 5). According to Lorenzo-Seva and Ferrando [Regarding Invariance of facet difficulties or intercepts  along with 2 domains (Detachment and Psychoticism) did not show partial invariance across the UAE and the Portuguese community samples.3.4Cohen’s d, no facets presented large effect sizes (Table 6). As for Invariance of factor loadings or discrimination , only the facet Separation insecurity presented significant differences in the discrepancy coefficient (Tables 7 and 8). However, the overall discrepancy index was not compromised. Likewise, concerning strict factor invariance, the percentile intervals of residual variances of the PID-5 facets, in the clinical samples, showed overlapping for all the facets, apart from Perseveration and Separation insecurity (Table 9). Taken together, as the residual variance is invariant over the UAE and the Portuguese populations, we can consider strict invariance for all facets and domains.In respect to the Invariance of facet difficulties or intercepts in the clinical populations, according to 3.510 presents the respective mean ranks score differences Wilcoxon Signed-Rank test, and effect size coefficient. Non-invariant facets are presented in grey. Most of the PID-5 facets and domains results were higher and statistically significant in the UAE community sample (p ˂.05) compared to the Portuguese community sample. Regarding the effect size, we obtained small (.10 ˂ r ≤ .30) to medium (.30 ˂ r ≤ .50) effect sizes for 16 of the 23 PID-5 facets, with the highest effect sizes displayed by Deceitfulness (.62) and Grandiosity (.64). At the domain level, except for Negative affectivity, the UAE community sample presented significant higher results (p ˂.01) than the Portuguese community sample, with the highest effect size (.66) being displayed by the domain Antagonism. In respect to the variables with normal distribution, no significant differences were reported for Anxiety, t = 1.88, p =.07, despite the Portuguese community sample presented higher mean scores  than those in the UAE sample . Conversely, Emotional lability obtained significantly higher scores in the Portuguese sample  compared to the UAE sample , t = 2.43, p =.02, albeit the small effect size.Concerning the aforementioned PID-5 scales distribution, both community and clinical samples, generally leaned to non-normality, particularly in the community samples, as only Anxiety and Emotional lability presented normal distribution. As for the clinical samples, scores were more normally distributed, specifically for 11 of the PID-5 facets and 4 of the domains. Regarding facets and domains that show non-normal distribution, in the community samples, Table 11 presents the 14 non-normal distributed facets and domains, along with the mean ranks score differences, Wilcoxon Signed-Rank test, and respective effect size coefficient. Similarly, to the community sample, also the UAE clinical sample presented higher and statistically significant results for most of the PID-5 facets and domain (p ˂.05), compared to the Portuguese sample. However, the effect size coefficients obtained were small (.10 ˂ r ≤ .30) for the most part of the facets and domains. The exceptions were the facets of Deceitfulness, Grandiosity, Irresponsibility, and the Antagonism domain, with medium effect sizes (.30 ˂ r ≤.50). As for the variables with normal distribution in the clinical samples, as displayed in Table 12, only the facets Distractibility, Hostility, Perseveration, Submissiveness and Withdrawal (p ˂.05), along with the Disinhibition and Psychoticism domains (p ˂.01) shown significant higher results in the UAE clinical sample compared to the Portuguese, though with small effect sizes (d ≤ .20). As expected, the mean score values of the Emirati and the Portuguese clinical groups were higher than the community groups with medium (20 < d ≤ .50) to very high effect sizes (d > 1.0).As for the UAE and Portuguese clinical samples, Table 4The present manuscript addressed the applicability of the PID-5 for group comparisons across the Emirati and the Portuguese clinical and community populations. To draw valid inferences regarding the mean score differences across both countries, an MI study was previously conducted, which is a prerequisite for cross-cultural comparisons , 36.Broadly, our findings have shown that all the PID-5 facets and domains can be properly compared across the Emirati and Portuguese clinical participants, but not entirely for the community participants, as just 15 of the 25 PID-5 facets, and 3 of the 5 domains proved partial invariance, from which only 12 facets have proved strict invariance. Thus, considerable caution is needed in drawing conclusions from mean trait differences on the PID-5 non-invariant scales, as they may reflect potential cultural bias -39. MoreFurthermore, it is quite common that the measurement error differs across clinical and non-clinical samples, particularly in scales assessing pathology, which impacts reliability coefficients in non-clinical groups , due to In this context, notwithstanding the importance of establishing MI, there is still little consensus on what minimum requirements would still grant practical and valid comparisons in cross-cultural personality research , 48. Altr >.50). A plausible interpretation could be related with the tendency for collectivistic cultures, such as the Emirati, in adopting an acquiescent response style, which might have biased the results [Regarding the facets that showed partial invariance in the community samples, with the exception of Anxiousness and Emotional lability, all the facets presented higher mean scores in the Emirati group, though only Attention seeking, Deceitfulness, Grandiosity, Intimacy avoidance, and Manipulativeness presented medium to high effect sizes . If we bear in mind that the Emirati and the Portuguese samples were closely matched based on diagnosis, these results may be conceptually meaningful as they reveal cultural specificities in the intensity and expression of universal maladaptive traits across the Emirati and the Portuguese population, in clinical settings. An important outcome of our findings could be related to the stigma associated with seeking professional mental health support in Arab cultures. The fear of showing weakness or lack of faith, restrain some patients from seeking early treatment, which often happens in the late stages of mental disorders [As for the psychiatric samples, the Emirati participants exhibited somehow higher results than the Portuguese participants, albeit the small effect size for most of the PID-5 scales. The noteworthy exceptions were Deceitfulness, Grandiosity, Irresponsibility, and the domain Antagonism, which could be mirroring relevant cultural differences, as they presented medium effect sizes  were found in some facets and domains in both countries and samples. Therefore, gender effect sizes did not seem to directly impact our results. However, our findings should be considered in light of some limitations, specifically, the young age of our participants, the high number of college students in the community samples, along with the predominance of substance related disorder diagnoses in the clinical samples. Finally, the community samples were not screened for psychopathology.The present study has several strengths and limitations, mostly related to our sample’s composition. The major strengths were the inclusion of closely matched community and clinical samples of both countries, as well as the exclusion of non-Emirati participants, to overcome possible cultural bias, due to the high number of expats living in the UAE. On the other hand, as community and clinical samples were composed of uneven proportions of men and women, with a strong predominance of women in the community samples, The present study supports the PID-5 measurement invariance across the Emirati and Portuguese clinical samples pointing to the universality and generalizability of the Alternative Model of Personality Disorders trait model. Nevertheless, future research should examine the applicability of the PID-5 across representative samples of the UAE and Portugal, as well as extending our study comparisons to other Arabic and Portuguese speaking countries.


Gambierdiscus excentricus and Gambierdiscus australes, were identified in the Madeira Island and Selvagens sub-archipelago, respectively. Regarding the CTX analysis (N2a cell-based assay and LC-MS) in fish, the results indicate that the Selvagens Islands are a ciguatera risk area and that fish vectoring CTX are not limited to top predator species. Nevertheless, advances and improvements in screening methods for the fast detection of toxicity in seafood along with certified reference material and sensitive and selective targeted analytical methods for the determination of CTX content are still pending. This study aims to revise the occurrence of ciguatera cases in the Madeira Archipelago since its first detection in 2008, to discuss the risk management strategy that was implemented, and to provide a summary of the available data on the bioaccumulation of CTX in marine fish throughout the marine food web, taking into consideration their ecological significance, ecosystem dynamics, and fisheries relevance.The first ciguatera fish poisoning (CFP) in Portugal dates from 2008 when 11 people reported CFP symptoms after consuming a 30 kg amberjack caught around the Selvagens Islands (Madeira Archipelago). Since then, 49 human poisonings have been reported. The emergence of a new threat challenged scientists and regulators, as methods for toxic microalgae analyses and ciguatoxin (CTX) detection were not implemented. To minimise the risk of ciguatera, the Madeira Archipelago authorities interdicted fisheries in Selvagens Islands and banned the capture of amberjacks weighing more than 10 kg in the entire region of Madeira Archipelago. The accurate identification and quantification of the benthic toxin-producing algae species spreading to new areas require efforts in terms of both microscopy and molecular techniques. Two ciguatera-causing dinoflagellates,  New toxins or toxins that are traditionally known from other regions are emerging in European waters, posing new threats to seafood safety and raising new challenges to authorities and agencies with responsibilities at the official control level. From the list of new toxins and compounds with a potential impact on seafood safety, the occurrence of ciguatoxins (CTXs) is of particular concern.Gambierdiscus and Fukuyoa [Ciguatoxins and their precursor metabolites are produced by benthic dinoflagellates of the genera  Fukuyoa ,2. Origi Fukuyoa ,4. Conse Fukuyoa ,6,7,8,9.Gambierdiscus/Fukuyoa spp. makes herbivorous fish and grazers particularly prone to accumulate the toxins. Nevertheless, potent CTX derivatives can also be found in omnivorous species and top predator fish due to toxin biotransformation in metabolic processes that are not yet fully understood [The epiphytic nature of derstood ,2,10. Seriola rivoliana from the Canary Islands in 2004 [Although ciguatoxins are recognised as highly potent neurotoxins, they are still poorly regulated, in contrast to the “classic” toxins that have a consistent EU regulation with standard operating procedures expressing the sampling frequency, reference detection methods, and safety limits ,12. Acco in 2004 . CFP epi in 2004 ,14 raiseSeriola sp.) caught around the Selvagens Islands [Sparisoma cretense), blacktail comber (Serranus atricauda), barred hogfish (Bodianus scrofa), grey triggerfish , and red porgy (Pagrus pagrus) [Seriola rivoliana) purchased in the Canary Islands but captured in the Selvagens Islands [The first reported outbreak in Portugal dates from 2008 when 11 crew members of a total of 16 persons in a fishing boat reported CFP symptoms after consuming a 30 kg amberjack . This number may be regarded as a low value, suggesting that CFP is not a common intoxication, but ciguatera is not a mandatory notifiable disease in Madeira Archipelago, which certainly leads to several unreported cases.Calonectris borealis, and other threatened seabird species in the NE Atlantic. The protection area was extended to the maritime area around the islands comprising a total of 95 km2, and delimited by the 200 m depth bathymetry line. Despite this being a protected marine area, some exceptions to the general ban on fisheries in the Selvagens Islands were initially allowed, such as spearfishing and angling; however, after the 2008 outbreak a total ban on fisheries was issued due to the risk of ciguatera poisoning. Considering that most of the CTX-contaminated fish were detected in Selvagens Islands and not in Madeira Island ,18, it i2 that includes the entire land area of the Selvagem Grande and Selvagem Pequena islands, and a series of adjacent islets, making it the largest fully protected marine area in Europe and the most intact ecosystem in the North-East Atlantic [Recently, the Madeira Government revised the legal regime of the Selvagens Islands Nature Reserve. Because of the limit of the marine protected area associated with the 200 m bathymetry line can be reached within a few hundred meters from the coastline due to the narrow and steep insular shelf, the marine-protected area was extended up to 12 nautical miles, with a total area of 2677 kmSeriola dumerili seems to be one of the most important species by weight at Selvagens Islands and S. rivoliana was found to be seven times more abundant in the Selvagens Islands compared to Madeira Island. Barred hogfish (Bodianus scrofa) was 3.4 times more abundant by weight in the Selvagens Islands compared to Madeira Island [A few studies have pointed out that the waters surrounding the Selvagens Islands are home to some of the most well-preserved oceanic habitats and some of the last remaining intact marine ecosystems in the eastern Atlantic. The Selvagens Islands have been designated by the National Geographic Society as one of most pristine ocean sites in existence  Figure . The fewa Island . In contGambierdiscus was first discovered in the late 1970s in the Gambier Islands, French Polynesia, when it was recognised as the source of ciguatoxins and linked to the recurrent ciguatera food poisoning episodes [G. toxicus, the type species [Gambierdiscus genus remained a monotypic taxon for a considerable period of time, but currently, at least 21 Gambierdiscus and 4 Fukuyoa species have been described with the aid of both morphological and molecular marker information [Gambierdiscus was first described in the NE Atlantic as Goniodoma, in Cape Verde [Gambierdiscus species, and re-described as a new species, G. silvae [The marine benthic dinoflagellate genus episodes . The iso species . The Gamormation ,27,28,29pe Verde , then prpe Verde  as a Gam. silvae . Cape VeGambierdiscus species and CFP outbreaks have increased in temperate regions [Gambierdiscus and Fukyuoa have been detected in the southern waters of the European Community, such as Canary Islands (Spain) and Madeira Archipelago , and in temperate regions of the Mediterranean Sea [The occurrence and spread of  regions ,32. Furtnean Sea ,31,33,34G. excentricus and G. australes, respectively, contrasting with the diversity of Gambierdiscus species observed in the Canary Islands, which includes G. australes, G. caribaeus, G. carolinianus, G. excentricus, and G. silvae [Gambierdiscus australes was reported in seawater, macroalgae and artificial substrate samples from Selvagens Islands [G. excentricus from Madeira Island [Gambierdiscus and other toxic benthic dinoflagellates in both Selvagem Grande and Selvagem Pequena. Again, only G. australes was retrieved at depths from 3 to 7 m. Cell concentration in the artificial substrate reached 1302 cells at 100 cm−2, which is in line with the reports made by Tester and colleagues [G. australes strains isolated from the Selvagens Islands by means of Neuro-2a (N2a) cell-based assays (CBAs). The previously determined values of up to 515 fg equiv. CTX1B. cell−1 and the former did not exceed 83 fg equiv. CTX1B. cell−1. Results from this functional assay may suggest a toxin profile composed of both CTX- and MTX-like compounds. Chemical analysis by liquid chromatography coupled to either low- or high-resolution mass spectrometry (LC-MSMS and HRMS) in six G. australes strains from the Balearic Islands (Spain) indicated the presence of maitotoxin-5 (MTX5), 44-methyl gambierone, and gambieric acids C and D, but no CTX precursors were detected [Only two species have been reported to date for the Portuguese islands of Madeira and Selvagens sub-archipelago, namely . silvae ,31,38 . On Madeira Island, rn shore . SubsequGambierdiscus strains from the Selvagens Islands were tested for cytotoxicity via the neuroblastoma 2-a (N2a) assay, no data were available for G. excentricus from Madeira Island. However, this species exhibited extremely high toxicity in strains isolated from the Canary Islands [G. excentricus strains from the Canary Islands, Brazil, and the Caribbean Sea [G. excentricus. The toxins profile of Gambierdiscus from Portuguese waters remains to be characterised by LC–MS/MS or HRMS.While  Islands . Chemicabean Sea ,44, leadFish species potentially vectoring CTXs have been captured in Madeira Island and the Selvagens sub-archipelago to assess the risk of CFP . ScreeniSeriola dumerilli and S. fasciata), and the analysis indicated extremely high levels of up to 40 µg kg−1 by CBA and up to 35 µg kg−1 by LC–MS [S. fasciata) caught in 2008 which was implicated in human poisonings [−1, with C-CTX1 the dominant compound, which reached 0.84 µg C-CTX1 kg−1 when quantified by LC–MS/MS [The first poisoning episodes were associated with greater and lesser amberjack  from the Selvagens Islands. This fish was analysed after being associated with a CFP episode in the Canary Islands [Diplodus cervinus) and barred hogfish (Bodianus scrofa), showed relevant CTX-like toxicity levels of up to 0.75 µg C-CTX1 kg−1 [Sparisoma cretense), the grey triggerfish , and the blacktail comber (Serranus atricauda), highlighting that ciguatera risk is not exclusively associated with top predators [Toxicity levels up to 0.76 µg C-CTX1 kg Islands ,48. In aTX1 kg−1 . Screeniredators . Until recently, the general consensus on ciguatera suggested that top fish predators are the most ciguateric species, either because potent CTX bioaccumulates in the fish metabolism or because of the lipophilic nature of CTX. However, evidence from both traditional knowledge and modern science, including reports from the Selvagens Islands’ nature wardens, suggests that both omnivore and piscivorous fish are toxic to ciguatera . Ophidiaster ophidianus from the southeast coast of the Madeira Island revealed the presence of mass spectrometry signals and ion fragmentation patterns associated with CTX [In addition to the pelagic environment, an opportunistic sampling of benthic organisms including the starfish with CTX , highligBodianus scrofa) by capillary LC-HRMS: 17-hydroxy-50,51-didehy-dro-C-CTX3, C-CTX3/4, 50,51-didehydro-C-CTX3, C-CTX1a, 56-methoxy-C-CTX1, and 17-hydroxy-56-methoxy-C-CTX1 [The Caribbean-CTX1 (C-CTX1) was found to be the main compound determined in fish ,17 whichy-C-CTX1 .−1) than Pacific CTX (0.01 µg P-CTX1 eq. kg−1) [The dataset of CTX analysis carried out in recent years to fish from the Portuguese coast suggests a moderate-to-high risk of CFP according to the international guidelines and recent research studies. The pioneer studies carried out by Lewis and colleagues during the 1990s investigating the in vivo lethal dose of Pacific and Caribbean CTX indicated a much lower potency compared to the later ones , and theq. kg−1) . Howeverq. kg−1) . Furtherq. kg−1) . FinallyGambierdiscus excentricus in the island´s coastal waters [Although fish caught in Madeira Island tested negative for CTXs, more extensive sampling is needed to confirm these results, especially considering the recent identification of l waters . The lowl waters . To minil waters . Howeverl waters . It may Seriola spp. ≥ 12 kg), dusky grouper , island grouper , bluefish , and the wahoo . These fish species, with these weight or above, cannot be placed on the market without a previous negative result by the N2a cell-based assay [In contrast, in the nearby Macaronesian archipelago of the Canary Islands, the government implemented an official control for ciguatoxins in certain fish species, such as amberjacks  from the Azores [Gambierdiscus/Fukuyoa into these waters.Selvagens Islands, which are the southernmost point of Portugal, are here highlighted as a key location for carrying out studies on for 24 h ,40,66. Tk of CFP . Azores  Islands ,35. Morepedition , indicate Azores , raisingGambierdiscus and/or Fukuyoa dinoflagellate species and toxic fish specimens is of high value for remote islands, such as the Selvagens Islands. A review of the state of the art of biosensors to assess the ciguatera risk has been recently published by Gaiani and colleagues [Finally, advances are needed in screening methods for the fast detection of toxicity in seafood as well as sensitive and selective targeted analytical methods for the accurate determination of Caribbean ciguatoxin contents . The devlleagues . Of critlleagues , essenti


Background: Emerging evidence has suggested a pro-oncogenic role of calponin 1 (CNN1) in the initiation of a variety of cancers. Despite this, CNN1 remains unknown in terms of its effects and mechanisms on angiogenesis, prognosis, and immunology in cancer.Materials and Methods: The expression of CNN1 was extracted and analyzed using the TIMER, UALCAN, and GEPIA databases. Meanwhile, we analyzed the diagnostic value of CNN1 by using PrognoScan and Kaplan–Meier plots. To elucidate the value of CNN1 in immunotherapy, we used the TIMER 2.0 database, TISIDB database, and Sangerbox database. Gene set enrichment analysis (GSEA) was used to analyze the expression pattern and bio-progression of CNN1 and the vascular endothelium growth factor (VEGF) in cancer. The expressions of CNN1 and VEGF in gastric cancer were confirmed using immunohistochemistry. We used Cox regression analysis to investigate the association between pathological characteristics, clinical prognosis, and CNN1 and VEGF expressions in patients with gastric cancer.Results: CNN1 expression was higher in normal tissues than it was in tumor tissues of most types of cancers. However, the expression level rebounds during the development of tumors. High levels of CNN1 indicate a poor prognosis for 11 tumors, which include stomach adenocarcinoma (STAD). There is a relationship between CNN1 and tumor-infiltrating lymphocytes (TILs), and the marker genes NRP1 and TNFRSF14 of TILs are significantly related to CNN1 expression in gastric cancers. The GSEA results confirmed the lower expression of CNN1 in tumors when compared to normal tissues. However, CNN1 again showed an increasing trend during tumor development. In addition, the results also suggest that CNN1 is involved in angiogenesis. The immunohistochemistry results validated the GSEA result (take gastric cancer as an example). Cox analysis suggested that high CNN1 expression and high VEGF expression are closely associated with poor clinical prognosis.Conclusion: Our study has shown that CNN1 expression is aberrantly elevated in various cancers and positively correlates with angiogenesis and the immune checkpoint, contributing to cancer progression and poor prognosis. These results suggest that CNN1 could serve as a promising candidate for pan-cancer immunotherapy.  The overall uptrend in the incidence and mortality of cancers remains a serious challenge globally that threatens human health and declines life quality. Over the decades, global joint projects, such as the Human Genome Project, Human Pathology Atlas, and Human Cell Atlas, have aimed to construct a series of online databases to uncover the pathogenesis of diseases and promote targeted drug developments and precision interventions. These progressions would be further promoted by the emergence of single-cell sequencing, spatial transcriptomics sequencing, and CAR-T immunotherapy. Recently, pan-cancer analysis has served as a novel hot spot to integrate multiple online databases to screen and investigate the key points and common molecular events linked to the occurrence and advancement of cancer . TherefoCNN1 is a filament-associated protein located in the cytoskeleton, which is implicated in the maintenance of blood vessel integrity and regulation of smooth muscle contraction . CNN1 waIn this study, we conducted a comprehensive pan-cancer analysis to summarize the prognostic significance of CNN1 in various types of cancer. Additionally, we explored the impact of CNN1 on mediating angiogenesis, immune checkpoint modulation, and prognostic value to assess its potential applications in targeted cancer intervention.With the TCGA tumor data collected from the TIMER database , a free The UALCAN database , a websiWe analyzed the relationship between CNN1 expression and tumor pathological stages (stages I–IV) based on the TCGA and GTEx data through the online GEPIA database . This wap < 0.05 for uniform presentation.To identify the effects of CNN1 expression on prognosis for various tumor types, we performed a prognostic analysis in the PrognoScan and Kaplan–Meier plotter databases . By searThe TISIDB platform explores tumor–immune interactions with data sets primarily derived from TCGA . Throughp < 0.05 and the false discovery rate (FDR) <0.25, the gene set was considered significantly enriched.The TCGA database was used to extract transcriptional expression profiles of 407 GC samples, and the C2 gene collection from the Molecular Signatures Database v 5.2 was used for the main analysis. Based on a gene set enrichment analysis (GSEA), we determined the function of the gene sets. When To make these findings more convincing, we included a total of 50 clinical GC samples for HE staining and immunohistochemical studies for CNN1 and VEGF expressions in clinical GC tissues. The human GC tumor specimens were obtained from the Department of Surgery II, Gaozhou Hospital of Traditional Chinese Medicine, Guangdong Province, and the Declaration of Helsinki was followed in all experimental contents of this study involving human tissues. This study was also approved by the Ethics Review Committee of Gaocheng City Hospital of Traditional Chinese Medicine (No. Y(2021)18). Before performing immunohistochemical studies, we embedded the tumor tissues in paraffin and performed HE staining. After rehydrating the tissue sections with xylene and alcohol, hematoxylin and eosin were used to label the nucleus and cytoplasm, and the pathological features of the tumor and paracancerous tissues were observed under the microscope . In the p < 0.05 was considered to be a significant difference.The Fisher’s exact test and Wilcoxon rank-sum test were used for categorical and continuous type variables, respectively, to assess the relationship between the expression levels of CNN1, VEGF, and each clinical feature. The univariate Cox risk ratio regression was used to assess the discrete hazard ratio (HR) of CNN1 expression and other pathological features, and independent variables were sought. A p < 0.01), while in PAAD tissues, the CNN1 expression level was significantly higher than that in normal tissues that were adjacent to the cancer tissues (p < 0.01). In addition, to further evaluate the expression of CNN1 in different tumor stages, the CNN1 expression levels were closely associated with the clinicopathological stages of patients with ACC, BLCA, BRCA, COAD, ESCA, KIRC, KIRP, OV, and STAD using the GEPIA database analysis , BRCA, cervical squamous cell carcinoma (CESC), COAD, esophageal adenocarcinoma (ESCA), KICH, kidney renal papillary cell carcinoma (KIRP), LUAD, lung squamous cell carcinoma (LUSC), rectum adenocarcinoma (READ), PRAD, STAD, thyroid carcinoma (THCA), and UCEC tumor tissues was significantly lower than that in para-carcinoma tissues, while the protein expression level of CNN1 in CHOL and LIHC tumor tissues was higher than that in para-carcinoma tissues . Figure versus low expression of CNN1 on tumor prognosis, we performed a pan-cancer survival analysis using the PrognoScan, Kaplan–Meier plotter, and GEPIA databases. In general, low CNN1 expression had a good prognosis in most of the tumors; high CNN1 expression also had a good prognosis in a small number of tumors. As shown in p < 0.05). In the case of CESC, ESCA, ESCC, and PAAD, patients in the CNN1 low-expression group had better RFS than those in the CNN1 high-expression group (p < 0.05). Moreover, the RFS and OS of patients in the CNN1 low-expression group in KIRP were better than those of patients in the CNN1 high-expression group (p < 0.05). It is noteworthy that in BRCA, pheochromocytoma and paraganglioma (PCPG), and SARC, patients in the CNN1 high-expression group had better OS than those in the CNN1 low-expression group (p < 0.05). Patients in the CNN1 high-expression group of uterine corpus endometrial carcinoma had better RFS than those in the CNN1 low-expression group , as detailed in p < 0.05), as shown in To explore the potential immunotherapeutic effects of CNN1, we first utilized the TISIDB website to validate the association between CNN1 expression and the immune and molecular subtypes in 37 tumors. The results showed that the CNN1 expression correlated with the immune subtypes of ACC, BLCA, BRCA, CESC, HNSC, KICH, KIRC, LGG, LIHC, LUAD, LUSC, MESO, OV, PCPG, PRAD, SARC, STAD, and TGCT   .versus advanced gastric cancer, angiogenesis-related gene set, and upregulated gene set in HUVEC cells (endothelium) treated with VEGFA, respectively. The outcomes revealed positively correlated CNN1 with all of the aforementioned gene sets: VECCHI_GASTRIC_CANCER_EARLY_DN   to perform a single gene differential analysis and visualize the results for the following four gene sets: VECCHI_GASTRIC_CANCER_EARLY_DN, VECCHI_GASTRIC_CANCER_ADVANCED_VS_EARLY_UP, WP_ANGIOGENESIS, and VEGF_A_UP.V1_UP, which are the normal tissue and early GC expression downregulated gene set, expression upregulated gene set in early GC = 0.008) , VECCHI_= 0.008) , VEGF_A_= 0.008) , and WP_= 0.008) .The HE staining results showed that the paraneoplastic tissues were lined with the nuclei. The nuclei were small, round, and located at the base. The cells were full and uniform in size, and a large number of cup-shaped cells were observed. The tumor tissue was filled with tumor cells in the submucosal lymphatic vessels, and the adenoma cells were extending and growing from the surface to the normal cells; inflammatory infiltration was present. The tumor cells were morphologically diverse, with concentrated deep-stained and crowded nuclei, severely disturbed nuclear arrangement, and an obvious degree of heterogeneity . Accordip < 0.001, HR = 13.580, 95% CI [5.806–31.740]), and high expression of VEGF was also associated with a poorer survival prognosis , lymph node metastasis , a CNN1 expression level , and the VEGF expression level  were associated with a poorer prognosis . Collectively, our findings indicate a potential and promising immunotherapeutic target for CNN1.Accumulating evidence has highlighted that immunotherapy alone or combined with chemotherapy, radiotherapy, and targeted therapy can largely and significantly improve cancer treatment . Immune During this research, we noted a significant increase in CNN1 expression in paraneoplastic tissue of gastric adenocarcinoma. Furthermore, in the comparison of tumor stages of gastric adenocarcinoma, CNN1 expression was higher in advanced gastric adenocarcinoma than in early-stage gastric adenocarcinoma. In the prognostic analysis, we also found that in gastric adenocarcinoma, the prognosis of the CNN1 low-expression group was better than that of the high-expression group. This non-linear expression of CNN1 in precancerous tissues or early tumor tissue is particularly evident in gastric adenocarcinoma. In order to understand the molecular mechanism involved in this phenomenon, we performed a single-gene differential analysis of four related gene sets using the R software. We found that CNN1 was indeed positively correlated with normal tissue, early GC expression downregulated gene sets, and the early GC and advanced GC expression upregulated gene sets, which is consistent with our description of its non-linear expression throughout tumor progression. Furthermore, in the angiogenesis-related GSEA analysis, we found that CNN1 was positively correlated with the angiogenesis-related genome and the upregulated gene composition of HUVEC cells  treated with VEGFA, which might imply that the synergistic effect of CNN1 and VEGFA drives tumor progression. Angiogenesis is an important part of maintaining the tumor microenvironment and creating conditions for tumor cell escape and distal lesion formation . CNN1, a+ T, CD8+ T, and dendritic cells and macrophages and neutrophils in STAD (p < 0.01), which not only allowed us to see CNN1 as a relevant prognostic factor in a variety of tumors, such as GC, but also showcases the promise of CNN1 as a target for immunotherapy.Based on the TIMER database, we further investigated the relevance of CNN1 to immune cell infiltration in STAD. The results showed that CNN1 expression was significantly correlated with CD4In conclusion, our results show that CNN1 plays multiple roles in tumor angiogenesis and the immune checkpoint by contributing to the progression of and poor prognosis in various cancers, thus providing a promising candidate for pan-cancer prognosis and immunotherapy. Our results also suggest that CNN1 serves as a double-edged sword between the physiological and pathological conditions, or between cancer initiation and progression, and it might also vary on the type of cancer, which might provide precautions and evidence for CNN1-targeted drug development and its clinical application.in vitro studies clearly show that phosphorylation regulates the function of CNN1 (in vitro cellular experiments or in vivo animal experiments to investigate the molecular mechanisms of CNN1 in cancer development in more detail. Fourth, although we have confirmed the great potential of CNN1 in pan-cancer immunotherapy, both inhibition and overexpression of CNN1 were found to be relevant in different tumors; therefore, more research has to be done to implement the use or targeting of CNN1 in tumor immunotherapy.Although we made some exploration of the prognostic role, immunotherapeutic potential, and possible mechanisms of action of CNN1 from a pan-cancer perspective using multiple databases and multiple analytical tools, there were still some limitations. First, due to the information collected from the various databases, the present study had some systematic biases, and we aim to overcome this in future studies by collecting clinical tumor samples and performing single-cell sequencing to obtain higher-resolution data. Second, although  of CNN1 , this st


Consumers are more than ever in search of novel and exciting beer choices, and brewers are, therefore, continuously experimenting to adapt their product portfolio. One interesting way to naturally incorporate novel flavors and tastes is by using alternative adjuncts, but this is not always an easy and straightforward process. In this study, a 40% unmalted alternative adjunct  or reference  was added to 60% barley malt, after which three different laboratory mashing processes  were performed, and their behavior during mashing and the resulting wort characteristics were investigated in detail. Overall, the extraction process of all 10 unmalted alternative adjuncts was not complete for all three laboratory mashing processes, whereby Congress mashing resulted in the highest extract and fastest filtration, whereas Evans mashing resulted in the lowest extract and slowest filtration. Pre-gelatinization of the unmalted was generally only beneficial for adjuncts with high onset starch gelatinization temperatures. This process also inactivated endogenous enzymes in the unmalted adjuncts, which had an adverse effect on the mashing process. One of the most important trends in beer consumption during the last years is the changing preference from quantity to quality. Consumers are drinking less in total volumes but increasingly ask for high-quality specialty beers and novel experiences ,2, as coThe use of unmalted adjuncts may also contribute to make the brewing process more sustainable by omitting the energy-intensive malting and kilning processes, which are also known to introduce certain undesirable compounds  into the pitching wort . AdditioIn previous research, 10 unmalted alternative cereals and pseudocereals were selected, and their characteristics relevant to the brewing process were compared with each other and with the references of barley malt and unmalted barley and wheat . The selThe aims of this study were thus to assess the brewing potential of the aforementioned 10 alternative unmalted adjuncts  relative to the aforementioned references  by performing three different laboratory-scale mashing processes  with 60% barley malt and 40% adjunct, describing the mashing and filtration processes and the resulting wort characteristics in detail. This should allow brewers to rationally select a certain uncommon adjunct for product innovation and diversification, facilitate its implementation in an actual brewing process, and indicate promising crops as alternatives for barley.Congress mashing, Congress mashing with pre-gelatinization, and Evans mashing procedures were performed in triplicate in laboratory mashing baths with 60% barley malt and 40% of each of the alternative adjuncts. These mashing procedures were also performed in triplicate for 100% barley malt, with 60% barley malt and 40% unmalted barley, or 40% unmalted wheat (as references). To minimize the influence of the different adjuncts on the mash pH, 100 mL of distilled water was substituted by 100 mL of 0.1 M acetate buffer at pH 5.2, as described before . During Commercially available barley malt , barley , wheat , einkorn , emmer , spelt , khorasan , quinoa , amaranth , buckwheat , sorghum , teff , and tritordeum  were obtained and characterized in detail, as described before . Barley The Congress mashing procedure was based on the European Brewery Convention (EBC) method 4.5. Briefly, 30 g of barley malt (60%) and 20 g of adjunct (40%) were milled at 0.2 mm with a laboratory disc mill  and added to 100 mL of a 0.1 M acetate buffer at pH 5.2 and 100 mL of distilled water to obtain a dilution of 1:4. This mash was placed in a LB Electronic mashing bath  with a continuous stirring speed of 200 rpm. The temperature was kept at 45 °C for 30 min, followed by a temperature increase of 1 °C/min until 70 °C, after which 100 mL of distilled water was added to obtain a dilution of 1:6. The mash was kept at 70 °C for 60 min, after which the mash was cooled to 20 °C and distilled water was added until 450 g to obtain a final mash dilution of 1:8.For the Congress mashing procedure with pre-gelatinization, 20 g of the adjunct (40%) was milled at 0.2 mm and added to 100 mL of a 0.1 M acetate buffer and heated at 95 °C for 20 min, after which 100 mL of cold distilled water was added and the mash was further cooled to 45 °C whereafter 30 g of barley malt milled at 0.2 mm was added, and the mashing process was continued as described above.2 in distilled water to obtain a dilution of 1:3. The temperature was kept at 65 °C for 50 min, followed by a temperature increase of 2.5 °C/min until 74 °C. The mash was kept at 74 °C for 10 min, after which 100 mL of distilled water was added, and the mash was cooled to 20 °C, whereafter distilled water was added until 450 g to obtain a final mash dilution of 1:8.The Evans mashing procedure was based on re-evaluating the small-scale Congress mash protocol . BrieflyThe time to complete saccharification was determined as described in the EBC method 4.5.1. Briefly, a drop of iodine solution was added to a drop of mash, whereby saccharification resulted in a yellow color instead of a dark blue color. For the Congress mashing procedures, a saccharification test was performed every 10 min after the temperature reached 70 °C, and for the Evans mashing procedure, a saccharification test was performed every 10 min after the temperature reached 65 °C.To assess the filtration, the cooled mash (20 °C) was brought into a funnel with a 597 ½ filter paper with 4–7 µm pore size , and the filtrate (wort) mass was weighed after 5, 10, 20, 40, 60, and 90 min. Only the filtrate mass after 60 min was used for the analysis.The extracted content and the pH of the wort samples were determined with a DMA 4500 Densimeter and Alcolyzer Plus . The content of mixed linkage [(1–3)(1–4)]-β-D-glucan content was determined using the McCleary method , as described before . The solThe color (EBC 8.5), free amino nitrogen , polyphenol content (EBC 8.12), and viscosity (EBC 8.4) of the wort samples were determined according to their appropriate EBC methods.Wort sugars  were analyzed using high-performance anion exchange chromatography coupled with a pulsed amperometric detector (HPAEC-PAD) using a Dionex ICS-3000 system , as described before . BrieflyAll experiments were performed in triplicate, and results were presented as the mean ± standard deviation. An ANOVA was performed to test for differences between the mashing process and wort characteristics (except for the saccharification time (min)) for barley malt and the 12 different unmalted adjuncts , followed by a series of post hoc pairwise comparisons using Fisher’s least significant difference (LSD) tests. These tests were performed in R 4.1.2 with a significance level of 0.05. Bivariate Spearman correlation coefficients (SCC) were calculated between the characteristics of the unmalted adjuncts as published before  and the p = 0.787) or protein  content of the adjunct (p = 0.678), which was highest with 40% unmalted buckwheat but was positively correlated with the beta-glucan content of the adjunct  and with the beta-glucan content in the wort . The latter two parameters were highly positively correlated .Filtration of wort with 40% unmalted barley, wheat, khorasan, and tritordeum was comparable to wort with 100% barley malt, while wort with 40% unmalted einkorn, emmer, quinoa, buckwheat, and sorghum filtered slower, and wort with 40% unmalted spelt, amaranth, and teff filtered the slowest ; Table 1 adjunct . The filp < 0.001), and the maltose  and maltotriose  concentrations , maltotetraose , and maltopentaose concentrations ), but not with the FAN  or wort soluble protein . The wort soluble protein was between 3 and 5 mg/L for barley malt and all unmalted adjuncts, with the highest values found for barley malt and spelt and the lowest values found for sorghum and teff  and with the diastatic power of the adjunct  and negatively correlated with the gelatinization temperature . The extracted content was not correlated with the starch  or protein  content of the adjunct. The extracted content was also positively correlated with wort viscosity . The wort viscosity itself correlated positively with the soluble protein content of the wort  but not with the FAN , beta-glucan , glucose , maltose , maltotriose , maltotetraose , and maltopentaose  content of the wort.The extracted content was highest for wort with 100% barley malt, followed by wort with 40% unmalted barley, wheat, spelt, khorasan, quinoa, buckwheat, and tritordeum, and the extracted content was lowest for wort with 40% unmalted einkorn, emmer, amaranth, sorghum, and teff ; Table 1p < 0.001), and more specifically, beta-amylase  but not alfa-amylase  of the adjunct. In contrast, the maltose concentrations were positively correlated with the diastatic power , the alfa-amylase , and the beta-amylase  content of the adjunct. The concentration of glucose correlated negatively with the concentration of maltose .The wort glucose concentration was 5–10 g/L, except for quinoa, amaranth, buckwheat, sorghum, and teff, which contained substantially higher concentrations ; Table 1p < 0.001), the protein content , and the diastatic power  of the adjunct (p = 0.027) but not with the soluble protein in the wort .The wort soluble protein was positively correlated with the TKW  and negatively correlated with adjunct protein content  and the wort soluble protein , but not with wort FAN content . Wort polyphenol content was highest for quinoa, barley, and barley malt and lowest for khorasan and amaranth.An acetate buffer was added to minimize the effect of pH, and for barley malt and most unmalted adjuncts, the pH of the wort was around 5.70 ; Table 12+ content was negatively correlated with the wort viscosity , wort soluble protein , filtrate after 60 min , and extracted content  , maltotriose , maltotetraose , and maltopentaose . However, the adjunct Ca2+ content was also negatively correlated with the adjunct starch content  and the TKW .Interestingly, the adjunct Ca< 0.001) . Ca2+ cop < 0.001), but no correlation with the beta-glucan content of the adjunct could be found  and neither with the wort FAN content   .p = 0.737) nor with the diastatic power  or starch gelatinization temperature  of the adjunct. After pre-gelatinization of the adjunct, the extracted content was positively correlated with the starch  but remained uncorrelated with the protein  content of the adjunct.The extracted content was generally lower after pre-gelatinization, except for spelt, quinoa, amaranth, sorghum, and teff ; Table 1p = 0.811) or maltose  in the wort (p < 0.001) after pre-gelatinization.After pre-gelatinization, the glucose concentrations for quinoa, amaranth, buckwheat, sorghum, and teff decreased until around 5–10 g/L, whereby the maltose concentrations for these adjuncts increased until around 40–60 g/L ; Table 1the wort . The conp = 0.185)  but not with the extracted content .The wort FAN content was lower after the pre-gelatinization of barley malt, barley, and khorasan, while it became higher after the pre-gelatinization of wheat, sorghum, teff, and tritordeum ; Table 1= 0.185) . Generalp = 0.184) nor with the beta-glucan content of the adjunct  but was correlated negatively with the FAN   .p < 0.001) and FAN of the wort  but not with the starch  or protein  content of the adjunct. The extracted content was also positively correlated with the diastatic power  of the adjunct but not with the alpha-amylase  or beta-amylase  content separately (p = 0.002).The extracted content was generally lower after Evans mash than after Congress mash, especially for amaranth and teff ; Table 1parately . Furtherp = 0.252) or the alpha-amylase  content of the adjuncts but were negatively correlated with their beta-amylase content  (p < 0.001) and the beta-amylase , but not the alpha-amylase  content of the adjunct. In contrast to the Congress mash with or without pre-gelatinization, the concentrations of glucose and maltose were not correlated after Evans mashing .The concentrations of glucose were generally lower after Evans mashing than after Congress mashing, and this effect was especially pronounced for quinoa, amaranth, buckwheat, and sorghum ; Table 1= 0.011) . In contp = 0.096)  but was negatively correlated with the extracted content .The wort FAN content after Evans mash was similar to after Congress mash, except for khorasan, where the wort contained less FAN after Evans mashing ; Table 1= 0.096) . The pH The experiments performed for this study provide insight into the behavior of einkorn, emmer, spelt, khorasan, quinoa, amaranth, buckwheat, sorghum, teff, and tritordeum during wort production on a laboratory scale. Combining this dataset with previously published results about the physical, physicochemical, and chemical properties of these adjuncts allowed a deeper understanding of their impact on the mashing process and the resulting wort characteristics .With a Congress mashing procedure, the extracted content with 40% unmalted adjuncts was always lower than the extracted content resulting from 100% barley malt. This indicated incomplete starch breakdown when unmalted alternative adjuncts were used, as barley malt contained only 55% (dm) starch while most adjuncts contained >55% starch  . Incomplgel > 65 °C) and would thus benefit most from a pre-gelatinization step. In contrast, spelt was characterized by a modest starch onset gelatinization temperature of 60.8 °C and would theoretically not benefit from a pre-gelatinization step. Care has to be taken when using starch onset gelatinization temperatures, as it was shown that small starch granules have higher onset gelatinization temperatures, which implies that the size distribution of the starch granules influences the starch gelatinization process [gel > 65 °C  resulted in especially low extracted contents with the Evans mashing procedure (isothermic mashing at 65 °C), further confirming the value of the onset starch gelatinization temperature. Pre-gelatinization promoted starch gelatinization but also inactivated endogenous enzymes. The higher diastatic power of the unmalted alternative adjunct increased the wort extracted content for the Congress mash and the Evans mash but not the extracted content for the Congress mash with pre-gelatinization. This adverse effect will probably be less pronounced for adjuncts with low diastatic power, and the effect of pre-gelatinization was indeed most beneficial for those unmalted adjuncts with the lowest diastatic power.The onset starch gelatinization temperature was indeed higher for all unmalted alternative adjuncts than for unmalted barley or wheat. When pre-gelatinization of the unmalted adjuncts was performed to eliminate the effect of different onset starch gelatinization temperatures, the extracted content generally decreased, except for spelt, amaranth, sorghum, and teff. The latter three were indeed characterized by the highest starch onset gelatinization temperatures . Alpha-amylase will probably contribute most to the starch solubilization, but the amount of this enzyme was negligible in the unmalted adjuncts. In contrast, the amount of beta-amylase in these unmalted adjuncts was substantial and differed significantly between the different adjuncts. However, diastatic power or alpha- and beta-amylase measurements may not encompass all enzymatic activity involved in starch breakdown. More specifically, proteolytic enzymes  were proven to enhance starch breakdown during mashing, both via improved starch gelatinization as well as via the degradation of inhibitors of beta-amylase and limit-dextranase .The sugar composition of the wort varied widely between Congress mash with different unmalted alternative adjuncts. The concentration of maltose in the wort increased with the beta-amylase content of the unmalted adjunct, both after Congress mash and after Evans mash, but not after Congress mash with pre-gelatinization, indicating the inactivation of endogenous enzymes during pre-gelatinization. Mashing with quinoa, amaranth, buckwheat, sorghum, and teff resulted in unusually high concentrations of glucose combined with unusually low concentrations of maltose and maltotriose. After pre-gelatinization, the glucose concentrations decreased, and the maltose and maltotriose concentrations increased, indicating the presence of substantial alpha-glucosidase activity in these unmalted alternative adjuncts, which were inactivated upon pre-gelatinization. The presence of these enzymes in these adjuncts has indeed been reported in the past ,22,23,24The filtration was not influenced by wort viscosity but proceeded faster when the extracted content of the wort was higher, indicating that more extensive macromolecule breakdown resulted in better filtration. This was probably mainly attributable to carbohydrate breakdown, not protein breakdown, as the filtration was faster with higher maltose and maltotriose concentrations in the wort but was not significantly influenced by the FAN or the soluble protein. The association between the higher extracted content and faster filtration was not present for the Evans mash, probably because (part of) the starch was only gelatinized during mashing-out at 74 °C. This starch could not contribute any more to the extracted content and might have hampered filtration. Furthermore, filtration proceeded slower when the concentration of beta-glucan in the wort was higher, as was expected . However2+ is a cofactor of amylases, promotes hot break formation during wort boiling, promotes flocculation after fermentation, prevents haze formation during beer storage, and promotes the removal of oxalates during the brewing process (which may form beer stone) [2+ at the start of the mashing process is thus common practice in the brewing industry, which is also incorporated by the Evans mash procedure [2+ on the thermostability of barley malt amylase enzymes has been known for a long time [2+ ions might decrease amylase activity in barley malt [2+ concentrations resulted in lower extracted contents  and slower filtration, together with lower wort soluble protein and lower viscosity. More Ca2+ seemed thus associated with less macromolecule (carbohydrate and protein) degradation. The adjuncts with the highest Ca2+ concentration were amaranth, teff, einkorn, and emmer, and those with the lowest Ca2+ concentration were buckwheat, khorasan, spelt, and wheat [2+ was added during the mashing process, the effect of the Ca2+ content in the adjunct on the extracted content of the wort became less pronounced. Ca2+ is known to promote the formation of macromolecule complexes, similar to the role of Ca2+ in membrane fouling [2+ reduces the dissolution of protein from malt into the wort [2+ concentration was lower when the TKW was higher, which might also have influenced the results, as unmalted adjuncts with higher TKW resulted in a better mashing process .Car stone) ,31,32. Arocedure . The posong time , but theley malt  and in rley malt . In thisnd wheat . For the fouling . These cthe wort . NeverthIn conclusion, wort production was possible with all 10 unmalted alternative cereals and pseudocereals. However, macromolecule breakdown was not complete for many unmalted alternative cereals and pseudocereals. Overall, Congress mash yielded the highest extracts and fastest filtration, followed by the Congress mash with pre-gelatinization, whereas Evans mash resulted in the lowest extracted content and filtration. The alternative adjuncts generally needed higher mashing temperatures than barley malt to obtain satisfactory extract and filtration, especially when the onset starch gelatinization temperature was >65 °C. In this case, a pre-gelatinization step could improve the extracted content and the filtration, whereas, for most adjuncts, a pre-gelatinization was not advantageous for extracted content or the filtration. This could be explained by the inactivation of endogenous enzymes during the pre-gelatinization step, which contributed substantially to the mashing process. Furthermore, these endogenous enzymes have a substantial impact on the resulting wort characteristics, especially for quinoa, amaranth, buckwheat, sorghum, or teff, whereby a wort especially rich in glucose and poor in maltose and maltotriose was obtained, probably because of the presence of endogenous alpha-glucosidases. Pre-gelatinization inactivated these enzymes, which resulted in a decrease in the glucose concentrations and a concomitant increase in the maltose and maltotriose concentrations. Most alternative unmalted adjuncts resulted in wort with lower FAN (and lower wort soluble protein) despite their higher protein content than barley malt. High wort soluble protein and wort beta-glucan were associated with high wort viscosity. However, wort with 40% unmalted buckwheat had especially high viscosity despite low beta-glucan content and moderate soluble protein in the wort. These results create novel possibilities to meet the increasing consumer demand for gluten-free and specialty beers and may contribute to the rational selection of novel and more sustainable crops for the brewing industry.


Agricultural productivity in the arid hot desert climate of UAE is limited by the unavailability of water, high temperature, and salt stresses. Growing enough food under abiotic stresses and decreasing reliance on imports in an era of global warming are a challenge. Biochar with high water and nutrient retention capacity and acid neutralization activity is an attractive soil conditioner. This study investigates the microbial community in the arid soil of Dubai under shade house conditions irrigated with saline water and the shift in the microbial community, following 1 year of amendment with indigenously prepared biochar from date palm waste.−1 of biochar, in addition to NPK for 1 year. Water holding capacity, pH, electrical conductivity, calcium, magnesium, chloride, potassium, sodium, phosphorus, total carbon, organic matter, and total nitrogen in the soil from biochar-treated and untreated controls were determined.Amplicon sequencing was used to elucidate changes in bacterial, archaeal, and fungal community structures in response to long-term biochar amendment. Samples were collected from quinoa fields receiving standard NPK doses and from fields receiving 20 and 30 tons ha−1 of biochar improved significantly over the control soil. This study strongly suggests that biochar helps retain soil fertility through the enrichment of plant growth-promoting microorganisms.The results show that soil amendment with biochar helps retain archaeal and bacterial diversity. Analysis of differentially abundant bacterial and fungal genera indicates enrichment of plant growth-promoting microorganisms. Interestingly, many of the abundant genera are known to tolerate salt stress, and some observed genera were of marine origin. Biochar application improved the mineral status and organic matter content of the soil. Various physicochemical properties of soil receiving 30 tons ha The sharp rise in human population is coupled with increased food demand and surging anthropogenic activities. The agriculture sector is facing the challenge of meeting this demand in a changed climate, triggered by global warming and shrinking area under cultivation. As the major soil type in UAE is Typic Torripsamments having an aridic moisture regime , following the amendment of agricultural soil with various doses of biochar. The focus of the study reported here, however, was to assess the impact of biochar addition on soil fertility with special reference to changes in microbial community structure. The change in soil physicochemical properties in response to biochar addition was also checked to establish whether these changes have any significant impact on microbial community structure. The biochar used in this study was produced at ICBA through pyrolysis of date palm residues, mainly dried date palm leaves at 350°C. Before the final application to the field, the biochar was crushed and passed through a 2 mm sieve , UAE, to evaluate the change in the biomass yield of quinoa , another three plots under treatment B received the same dose of NPK and 20 tons ha−1 of biochar (5% by weight), and in treatment C, three random plots received the same dose of NPK along with 30 tons ha−1 of biochar (10% by weight), as explained in +, Ca2+/Mg2+, K+, HCO3−, and Cl in a concentration of 28, 10, 0.79, 1.2, and 35.6 meq L−1, respectively) via drip irrigation system . Samples were collected in sterile polybags and were transferred to the laboratory at 4°C for further processing. Three samples from each plot were collected and mixed properly to make a composite sample. Soil samples were collected in March 2017 from the shade house trial research station at International Center for Biosaline Agriculture (ICBA), Dubai, United Arab Emirates (25°05′49“ N and 55°23′ 25” E).The soils were tested for various physicochemical properties, including electrical conductivity, soluble magnesium, calcium, chloride, potassium, and sodium content. The concentration of soluble magnesium and calcium was determined using standard titration with ethylene diamine tetraacetate  was used to extract the DNA from 10 g of homogenized soil, according to the manufacturer's protocol. The purity and concentration of DNA were checked using a Qubit fluorometer . The extracted DNA was stored at −20°C until further use.The diversity of bacterial, archaeal, and fungal communities in the samples was determined by amplifying the V3-V4 regions of bacterial and archaeal 16S ribosomal RNA (rRNA) genes and the ITS1 gene of fungi. The 16S rRNA gene amplicons were generated from 10 ng of the extracted DNA samples using a set of universal primers for the 16S rRNA gene and the ITS gene. These amplicons were checked on 1% agarose gel. To generate a V3-V4 amplicon (~460 bp), the PCR products as obtained above and a set of V3-V4 forward and reverse primers were used as described in our previous study  for retaining high-quality reads for further analysis. Reverse and forward sequences were merged into paired-end reads. Spacer and conserved regions were removed from high-quality PE reads. USEARCH was used for the dereplication of the sequences using 97% sequence similarity values. UCHIME utility of USEARCH was used to remove chimeras  under Bio-Project accession number PRJNA888571, with 18 biosamples of 16S and ITS gene-based analysis. The OTUs obtained were analyzed using various R packages, including phyloseq, microeco, and vegan . A comparison of physicochemical parameters between biochar amended and unamended samples was performed using one-way analysis of variance (ANOVA), followed by −1) and C samples (biochar 30 tons ha−1), respectively, compared to control or sample A. However, the total carbon (TC) decreased slightly with increasing doses of biochar concentration. Compared to the control, the water retention capacity of biochar-treated soil samples B and C improved significantly by 9.5 and 12.76%, respectively, supported by p-values of 0.005 and 0.0001, respectively. The pH of sample C increased slightly (4.5%) compared to the control. Electrical conductivity (EC) significantly increased in both B and C samples by 8.77 and 82.35%, respectively, compared to control soil . The biochar treatment also improved the mineral  status of the soil. In biochar-treated soil, total nitrogen (TN) levels increased significantly, in sample B, slightly higher nitrogen (11.99%) was detected compared to sample C (11.39%). When the changes with different doses of biochar were compared, it was found that 30 tons ha−1 (sample C) of biochar show a better impact than the lower dose of 20 tons ha−1 (sample B).The physicochemical properties of the biochar-treated and unamended control soil were tested. The results presented in The sequences obtained had an average read length of ~300 base pairs, and a total of 178,835 high-quality reads were used for the analysis. The sequences were assigned to OTUs based on 97% sequence similarities. A total of 16 OTUs of archaea, 1,979 OTUs of bacteria, and 24 OTUs of fungi were observed in the samples.In the taxonomic rank analysis of the OTUs, the bacteria were found to be members of 33 phyla, 122 classes, 359 orders, 687 families, and 1,148 genera. The most abundant bacterial phyla in all the samples in descending order were Bacillota (synonym Firmicutes), Pseudomonadota (synonym Proteobacteria), Actinomycetota (synonym Actinobacteria), Chloroflexota (synonym Chloroflexi), Bacteroidota (synonym Bacteroidetes), and Planctomycetota (synonym Planctomycetes), accounting for 80–95% of all OTUs . Other pBacillus, Halomonas, Planococcus, Pontibacter, Salinimicrobium, Nocardioides, Microvirga, Streptomyces, Ammoniphilus, and Kocuria. The abundant bacteria of marine origin observed in this study were Planococcus, Halomonas, Salinimicrobium, Marinicoccus, and Halobacillus. Mesorhizobium, a PGPR biocontrol bacterium Amycolatopsis with activity against plant pathogen, marine sulfur-oxidizing bacterium Sulfurifustis with PGPR activity, and Nitriliruptoria with unknown function  and biochar-treated samples was calculated in terms of the Shannon index using the vegan package. The values of the Shannon index varied considerably in control samples but were more stable in biochar-treated samples with a shift toward a higher range in samples B and C . When a .0000039 .Halorussus, followed by Haladaptatus and Halococcus. The box plot of the Shannon index shows the highest diversity in sample C, while in sample B, the Shannon index varied considerably from sample to sample, and values of the Shannon index in the same range were also observed in the control sample  increase in biodiversity . The values of the Shannon index increased with increasing values of the electrical conductivity. When the diversity of archaea in terms of the Shannon index was plotted against these physicochemical properties, no significant correlation was observed . When the bacterial diversity (Shannon Index) was plotted against the organic matter content, a slightly negative correlation between an increase in the organic matter content of soil and diversity in terms of Shannon index values was observed. The total carbon in the soil decreased slightly with increasing doses of biochar, and the values of the Shannon index also increased, indicating a significant  ratio and the gram-positive to gram-negative (G+/G-) ratio  can be found below: MK and SK: formal analysis, investigation, and writing—original draft. AS: methodology. MK: project administration. All authors contributed to the article and approved the submitted version.