Introduction: Mild cognitive impairment is often associated with affective and other neuropsychiatric symptoms (NPS). This co-occurrence might have a relevant impact on disease progression, from MCI to dementia.Objective: The aim of this study was to explore the trajectories of cognitive decline in an MCI sample from a memory clinic, taking into consideration a perspective of isolated cognitive functions and based on NPS clusters, accounting for the different comorbid symptoms collected at their baseline visit.Methods: A total of 2,137 MCI patients were monitored over a 2.4-year period. Four clusters of NPS  were used to run linear mixed models to explore the interaction of cluster with time on cognitive trajectories using a comprehensive neuropsychological battery (NBACE) administered at baseline and at the three subsequent follow-ups.Results: A significant interaction between cluster and time in cognitive decline was found when verbal learning and cued-recall were explored (p = 0.002 for both memory functions). For verbal learning, the Irritability cluster had the largest effect size (0.69), whereas the Asymptomatic cluster showed the smallest effect size (0.22). For cued-recall, the Irritability cluster had the largest effect size among groups (0.64), and Anxiety/Depression had the smallest effect size (0.21).Conclusions: In MCI patients, the Irritability and Apathy NPS clusters shared similar patterns of worsening in memory functioning, which could point to these NPS as risk factors of a faster cognitive decline, acting as early prognostic markers and helping in the diagnostic process. Biological changes bonded to impairment of cognitive functions are shown as humans age . In the Cognitive impairment is often associated with affective symptoms, such as anxiety or depression , which hstable, improved and worsened) in MCI patients, it was found that the NPS worsened class suffered the greatest cognitive and functional decline, as well as the highest conversion rate in comparison with the stable class and the improved class , a private non-profit institution focused on the diagnosis, care and research of cognitive disorders and providing services to the Catalan Public Health Service  .A total of 2,137 patients diagnosed with MCI were selected from a pool of patients evaluated at the Memory Clinic, see The Neuropsychological Battery of Fundació ACE (NBACE). The NBACE is a 50-min battery designed to assess cognitive domains especially affected in the elderly when dementia due to AD or other neurodegenerative processes is suspected ; long-term retention was the total amount free recalled words; and cued-recall was the total number of words correctly recognized among the correct items and the same amount of “interference” items. Language was measured with the 15-item version of the Boston Naming Test  . The NPIIrritability; Class 2 = Apathy; Class 3 = Anxiety/Depression; Class 4 = Asymptomatic. Then, linear mixed-effects models (LMMs) were executed to explore cognitive decline for specific domains including NPS clusters and time of assessment . Individual LMM models were calculated, one for each neuropsychological domain explored. Interaction of NPS cluster by time of assessment was considered the main effect of interest in the model, also incorporating the corresponding cluster and time main effects. For these two factors, fix and also as a random effect were analyzed, considering that assessment time points could vary among participants, and they also had different conditional probabilities of cluster belonging  for clustering participants by means of a dichotomized NPI-Q measure . Each paging see , i.e., iIrritability included 134 patients (6.3%) with high probability of irritability (0.93), together with lower probability of anxiety (0.64) and apathy (0.63). Class 2-Apathy comprised 272 patients (12.7%) and it was strongly represented by this symptom (1). Class 3-Anxiety/Depression included 1,056 patients (49.4%) who showed a high probability of depression (0.95), anxiety (0.93) and, by far, apathy (0.61). Class 4-Asymptomatic included 675 patients (31.6%) with low probabilities (<0.3) in all NPS.According to previously published findings from our group , the whoApathy patients were the oldest (mean age 76.2). In relation to gender, women were more prevalent in the Anxiety/Depression and Asymptomatic classes . Educational level attained was higher in patients in the Apathy class (8.04 years of education). In relation to MMSE score obtained, the Anxiety/Depression class had the highest results (MMSE mean 27.02). Regarding the MCI type (amnestic vs. non-amnestic), our sample was quite balanced in general terms, having percentages in the four classes ranging from approximately 51 to 66%. According to the classification of possible or probable MCI profile, the Irritability and Apathy classes had a higher percentage of patients with a diagnosis of probable amnestic . Finally, patients in the Irritability and Apathy classes showed a higher proportion of conversion to dementia.Demographic and clinical characteristics of the sample stratified by NPS cluster are shown in p < 0.001). When differences between the final follow-up and the baseline scores were calculated in terms of verbal learning, a faster decline was shown in the Irritability class, with double the difference, versus a slower decline in the Asymptomatic class, with 3.66 and 1.34, respectively. With regard to effect size (Cohen’s D), the Irritability class had the highest score (0.69), whereas for Apathy and Anxiety/Depression classes the effect sizes were medium . The Asymptomatic class showed the smallest effect size (0.22). Similar results were obtained for cued-recall, but this time the Irritability and Apathy classes had quite similar differences between follow-up measures and the baseline , and the Anxiety/Depression class had the lowest score difference from the baseline. In terms of effect sizes, the Irritability class again showed the largest effect size (0.64), followed by the Apathy and Asymptomatic classes , with the Anxiety/Depression class having the smallest effect size (0.21). Cognitive decline for memory domains  were calculated for each cluster trajectory (see Irritability class being the faster decliner (cognitive slope −0.98) and the Asymptomatic class the slower decliner (cognitive slope −0.43) in relation to verbal learning. For cued-recall, the Irritability and Apathy classes had the same cognitive slope value, and the Anxiety/Depression class showed a slower decline in this memory domain.t size 0.2. SimilaIrritability, Apathy, Anxiety/Depression, and Asymptomatic) in patients with MCI. In our sample of 2,137 MCI patients, the Irritability and Apathy NPS classes shared a similar pattern of faster cognitive decline in two memory domains , compared to the Anxiety/Depression and Asymptomatic classes, which showed a slower cognitive worsening over the stipulated follow-up period. Even though Irritability was the least prevalent neuropsychiatric condition in this sample, it proved to be the NPS class with the worst and fastest cognitive decline. Therefore, the present findings suggest that although Irritability and Apathy are less frequent NPS in MCI, these symptoms should be taken into account to improve the quality and usefulness in diagnostic and prognostic evaluation of cognitive worsening in MCI patients, especially those with an amnestic profile.The findings in our study revealed different trajectories of cognitive decline in memory domains depending on NPS clusters , compared to the Anxiety/Depression and Asymptomatic classes. The present findings emphasize the relevance of including an assessment of affective NPS when starting a diagnostic process provided that such symptoms—and in particular irritability and apathy—might act as aggravating factors. Our findings appear to open a new avenue to use NPS assessment as a clinical tool of great value when it comes to detecting in advance which patients could suffer from a marked worsening in cognition.The approach of this study explores specific cognitive decline trajectories based on affective NPS clusters in MCI patients from a memory clinic, adding some novelty with respect to previous works. Specifically, and according to our results, svalero@fundacioace.org.The data that support this study may be provided upon reasonable request. Requests to access the datasets should be directed to SV, The studies involving human participants were reviewed and approved by Hospital Clínic i Provincial of Barcelona, Spain. The patients/participants provided their written informed consent to participate in this study.NR, MP, LT, MB, and SV conceived the idea, designed the study, and wrote the protocol and methodology. MM, MA, IH, AM, MR-R, CA, EE, SM-G, JT, LV, AE, GO, AP-C, ÁS, AO, IR, SM-G, and LM acquired all the data, performed technical procedures, and managed the data set. NR, MM, and AR managed previous literature searches. NR, MP, and SV contributed to the statistical analysis, interpretation of the results and writing of the first draft of the manuscript. EA-M helped in the statistical analysis and interpretation of the results. MM, MA, AR, LT, and MB revised the manuscript critically for intellectual content. All authors contributed to the writing of the final version and approved the manuscript.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.


Uric acid serum levels correlated with negative sub-score , general sub-score , and total PANSS score  after 1 month of therapy. We have established a statistically significant positive correlation between serum concentrations of UA and IL-6 in exacerbation  and with IL-17 after treatment and in the stabilization of psychosis , suggesting potential cascades in different phases of schizophrenia that potentiate inflammation.Uric acid (UA) has been shown to have neuroprotective or neurotoxic properties, in relation to specific tissues and diseases that have been studied. Previous studies provided contradictory results on the role of UA in schizophrenia as a neurodegenerative disorder. The aim of this brief report was an additional analysis of UA sera levels in different phases of schizophrenia. Here, 86 patients with first-episode psychosis (FEP) vs. 45 patients with schizophrenia in relapse (SC in relapse) vs. 35 healthy control subjects (HC) were studied before and 1 month after antipsychotic therapy. Further, we aimed to explore the possible correlation of UA with scores presenting clinical features and with serum concentrations of the proinflammatory cytokines interleukin (IL)-6 and IL-17. When comparing the data between all three groups, we did not find significant differences in UA levels, either before or after the applied therapy. Also, comparing sera concentrations of UA in every single group, the analysis did not reveal statistically significant differences between FEP patients, but statistically, a significant difference was found in SC in relapse before and after treatment (334.71 ± 116.84 vs. 289.37 ± 109.15 μmol/L,  Uric acid (UA) is the final oxidation product of the adenine- and guanine-based purine catabolic pathway and is considered as a risk factor of numerous pathological conditions . HomeostIncreased levels of UA in the initial phase may reflect an attempt to counteract oxidative stress, suggesting its neuroprotective role , whereasThe relationship between altered UA levels and schizophrenia remains unclear, and studies have yielded conflicting results. A significant decrease in plasma UA levels was observed in the first episode of schizophrenia , and a mThis study represents an additional analysis of the database from our previous cytokine studies in different phases of schizophrenia , 20. BriA somatic examination was performed, and vital signs were measured on admission. Patients' blood samples were collected at least 8 h before the administration of antipsychotic therapy during the current psychotic episode. The control group consisted of HC subjects matched to the patients by gender and age with patients and recruited at a blood donation in the Service supply of blood and blood products, of Clinical Center Kragujevac. Serum UA levels were determined using a hematology analyzer, ABX MICROS 60-OT (UK). The reference values for UA were in the following range: 154–428 μmol/L. Serum cytokine levels were measured using conventional enzyme-linked immunosorbent assay (ELISA) kits . Immunological measurements were performed at the Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University of Kragujevac. The acute effects of treatment on UA serum levels were assessed after 4 weeks, since the first 2–4 weeks of antipsychotic treatment are crucial for a substantial reduction of positive symptoms .p-value of 0.05 was considered to be statistically significant.Statistical analyses were performed using SPSS 20.0 software . Numerical values are reported as mean, standard deviation (SD), and standard error (SE). The normal distribution of the data was tested using Shapiro–Wilk and the Kolmogorov–Smirnov test. A Paired Sample test and the Wilcoxon test were used to compare the clinical parameters in the same group before and after therapy. We used the Mann–Whitney test for non-parametric variables to assess mean differences between two groups of patients. A Kruskal–Wallis test was used to assess differences in serum UA levels between three groups . We examined the correlations between serum UA and cytokines, as well as the relationship between serum UA levels and PANSS scores, positive, negative, and general psychopathology sub-scores using Pearson's or Spearman's correlation. Multiple regression analysis was used to assess the significant demographic and clinical predictors of serum UA levels and PANSS scores. A From our previous study database , 20, onlp = 0.663) and after applied therapy , we did not detect statistically significant differences in UA values before therapy. When comparing serum UA levels in each group, the analysis did not reveal statistically significant differences between FEP patients , but statistically, a significant difference was found in SC in relapse, before and after treatment . We also analyzed serum UA levels between FEP and SC in the relapse group before treatment , and after treatment , and no statistically significant differences were observed , general sub-score , and total PANSS score  after 1 month of therapy in both patient groups, while a correlation between serum UA levels and positive sub-score was not significant   and with IL-17 after treatment and in the stabilization of psychosis  (We found a significant correlation between UA serum levels with negative sub-score (= 0.149) . In addi = 0.34) .F(3/108) = 8.027, p < 0.001, R2 = 0.187, Adjusted R = 0.163] was a significant predictor of serum UA levels, with female gender implicating an increase in UA. Uric acid serum levels  and IL-17 serum levels after treatment in all psychotic patients  are significant predictors for PANSS score (presented in Multiple regression analysis indicated that sex [In the new tendency to publish negative results, we must emphasize that we did not find statistically significant differences in serum UA levels between patients with FEP, SC in relapse, and HC subjects, neither before nor after the applied therapy. We pointed out that patients with schizophrenia had higher UA serum levels in exacerbation than in the stable phase of this psychosis. There is a significant correlation between UA serum levels and negative, general sub-score, and the total PANSS score in the stabilization phase. A statistically significant positive correlation was observed between serum concentrations of UA and IL-6 in exacerbation, and with IL-17 after treatment and in the stabilization of psychosis. Our findings suggest that in a stable phase of the illness, serum levels of UA and IL-17 are significant predictors for PANSS scores.Firstly, we would like to discuss phase specificities of oxidative and inflammatory stress in schizophrenia. Plasma UA levels were significantly lower in patients with FEP, but not in patients with bipolar or depressive disorder . In contAntipsychotics could increase the levels of UA . In a stIn addition, the immune response seems to be modulated through UA impact. We found a statistically significant positive correlation between serum concentrations of UA and IL-6 in exacerbation and with IL-17 after treatment and in the stabilization of psychosis. Interleukin-17 is a pro-inflammatory cytokine produced mainly by Th17 cells and Type3 innate lymphoid cells when stimulated by cytokines such as IL-6 or IL-1, stimulating the production of cytokines and chemokines, and facilitating inflammation . An incrYao et al. discussed the previously established links between UA and intelligence and reported the data from their research on the association between low levels of UA and greater impairment in sensory processing tasks . In a moWhen examining whether these cascades might be of clinical benefit in patients with Parkinson's disease, lower UA levels seem to imply a higher risk of transition to mild cognitive impairment . In contThis study reflects the naturalistic sampling in real clinical practice, in a specific timeframe. The diagnosis could be made only after a longer prodromal phase of the disease, sometimes to avoid stigmatization of these patients and due to inadequate family support. The limitation of this study is also that the data do not allow extrapolation in the chronic patients and required further research of a much longer duration of the illness. In addition, the fact that the decrease in FEP patients was not statistically significant could be a by-product of the higher variance in the FEP group, which also requires a larger sample size.Our results indicate a possible detrimental effect of elevated serum UA levels in certain phases of schizophrenia. No significant differences in UA levels were observed in the initial phase of the illness, but it has been shown by others that repeated acute relapses might be followed by increased UA levels in patients with a longer duration of illness. Uric acid serum levels could be associated with IL-6 elevation in exacerbation, once more showing IL-6 as a crossroads in the pathophysiology of schizophrenia. These results could be explained by the fact that a different type of immune response is activated in schizophrenia than in other neurodegenerative diseases. For example, multiple sclerosis predominates type 1 immune response, while in schizophrenia type 2 immune response overweights and type-17 immune response could be related to cognitive decline. It could be considered that the type of immune response influences the further role of UA and whether it exhibits pro/antioxidant or pro/anti-inflammatory effects. Also, it is important to consider the lower serum levels of UA after therapy in patients with chronic schizophrenia, and that this potential further elevation of UA may contribute to residual symptomatology and poorer therapeutical response. Uric acid can be considered as a marker of the disease course in the later disease cascades.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study involving human participants, was reviewed and approved by the Ethics Committee of the Clinical Centre Kragujevac and the Special Hospital for Psychiatric Diseases “Dr. Laza Lazarevic”, Belgrade, and was conducted in compliance with the ethical principles. Informed consent was obtained from all patients before any study procedures began.MB and the team of trained psychiatrists have selected the patients, performed the psychological assessment, and sampling. MB has conceptualized the manuscript together with KV. KV and MB did the statistical analysis. KV designed figures and tables. MB and SJ wrote about the psychiatric aspects of this topic. KV and NM wrote about the role of UA. IJ and NA did the cytokine measurements and wrote about immunological aspects. MB prepared an integral version of the manuscript. All authors contributed equally to the content of this manuscript and approved the final version of the manuscript.This work was supported by the Ministry of Science and Technological Development of the Republic of Serbia, No. 175069, and by the Faculty of Medical Sciences, University of Kragujevac, Nos. JP 03/16 and JP 12/09.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.


Stenotrophomonas maltophilia, a type of conditional pathogen that has shown to a gradual increase in clinical morbidity in recent years, has rarely been reported for its impact in space. In this study, S. maltophilia was exposed to a simulated microgravity (SMG) environment in high-aspect ratio rotating-wall vessel bioreactors for 14days, while the control group was exposed to the same bioreactors in a normal gravity (NG) environment. Then, combined phenotypic, genomic, transcriptomic, and proteomic analyses were conducted to compare the influence of the SMG and NG on S. maltophilia. The results showed that S. maltophilia in simulated microgravity displayed an increased growth rate, enhanced biofilm formation ability, increased swimming motility, and metabolic alterations compared with those of S. maltophilia in normal gravity and the original strain of S. maltophilia. Clusters of Orthologous Groups (COG) annotation analysis indicated that the increased growth rate might be related to the upregulation of differentially expressed genes (DEGs) involved in energy metabolism and conversion, secondary metabolite biosynthesis, transport and catabolism, intracellular trafficking, secretion, and vesicular transport. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the increased motility might be associated the upregulation of differentially expressed proteins (DEPs) involved in locomotion, localization, biological adhesion, and binding, in accordance with the upregulated DEGs in cell motility according to COG classification, including pilP, pilM, flgE, flgG, and ronN. Additionally, the increased biofilm formation ability might be associated with the upregulation of DEPs involved in biofilm formation, the bacterial secretion system, biological adhesion, and cell adhesion, which were shown to be regulated by the differentially expressed genes  through the integration of transcriptomic and proteomic analyses. These results suggested that simulated microgravity might increase the level of corresponding functional proteins by upregulating related genes to alter physiological characteristics and modulate growth rate, motility, biofilm formation, and metabolism. In conclusion, this study is the first general analysis of the phenotypic, genomic, transcriptomic, and proteomic changes in S. maltophilia under simulated microgravity and provides some suggestions for future studies of space microbiology.Many studies have shown that the space environment plays a pivotal role in changing the characteristics of conditional pathogens, especially their pathogenicity and virulence. However,  Staphylococcus, Enterococcus faecalis, Bacillus, Propionibacterium, and Corynebacterium have been identified as the most common species. Studies have showed that the space environment has some impacts on the immune system and motor system of humans, as well as on microorganisms in astronauts, such as growth rate, drug susceptibility, carbon source utilization and chemical sensitivity, biofilm formation ability, and motility  have been developed by NASA . HARVs sStenotrophomonas maltophilia , which was first isolated in 1943, is an emerging Gram-negative multidrug-resistant organism (MDRO) that is highly relevant to respiratory infections in humans , bacteremia, biliary sepsis, bone and joint infections, eye infections, endocarditis, and meningitis  liquid medium. The original strain of S. maltophilia (SMO) was cultured in a shake tube containing LB liquid medium at 37°C. Stenotrophomonas maltophilia in simulated microgravity (SMS) and S. maltophilia in normal gravity (SMN) were established by growing bacteria in continuous cultivation in high-aspect rotating vessel (HARV) bioreactors . The original Morphological changes were also observed by scanning electron microscopy (SEM), and the three groups were washed three times with sterile phosphate-buffered saline  and fixed with 2.5% glutaraldehyde at 4°C overnight. The samples were washed again with deionized water three times, and each solution was settled for 7min and then dehydrated in a gradient series of ethanol  for 15min per step. The samples were subsequently immersed in 100% ethanol (three times for 10min each) to prevent drying, and then, all the dehydrated samples were placed in a vacuum desiccator to critical point-dry. After coating the specimens with gold-palladium, the samples were observed using cold field emission scanning electron microscope /ml] were inoculated into 96-well honeycomb microtiter plates, and SMO was also prepared at the same concentration. For each group, one 96-well plate was prepared with 200μl of solution. The optical density at 600nm was measured every 2h for 24h using Bioscreen C system and BioLink software . This experiment was performed in three replicates  were used to test the ability of idimeter . A 100μl7–108CFU/ml; Drug susceptibility testing (DST) was performed using the disk diffusion method based on the CLSI M100 Ed30 document : trimethS. maltophilia. Briefly, after 2weeks, the bacterial suspension was removed from the HARV bioreactors, washed gently with sterilized water twice and then stained with 0.1% crystal violet for 15min at room temperature. Subsequently, the crystal violet dye was slowly removed through a sterile syringe, the reactor was washed with sterile deionized water three times, and then, pictures were taken  in conjunction with confocal laser-scanning microscopy . LIVE/DEAD Biofilm Viability is a culture-independent method that depends on the cell membrane integrity as a viability measure; it utilizes a mixture of the SYTO9 green fluorescent nucleic acid stain and a red fluorescent nucleic acid stain and propidium iodide. Live and dead bacteria can be distinguished without waiting for growth plate results. The working solution of the fluorescent stains was prepared by adding 3μl of the SYTO9 stain and 3μl of the propidium iodide stain to 1ml filter-sterilized water. The excitation/emission maxima for these dyes are approximately 480/500nm for SYTO9 staining and 490/635nm for propidium iodide. Three independent rounds of biofilm experiments were performed. Briefly, in each round of experiments, SMS, SMN, and SMO were inoculated in 35-mm confocal dishes , cultivated at 37°C for 24h, and then rinsed two times with sterile water to eliminate nonadherent bacteria. Then, the generated biofilms were carefully stained with 200μl working solution stain for 30min in the dark at room temperature. It was important to immediately add the stain before the biofilm dried. After removing the stain and washing two times with sterile water, images were acquired from random positions, and the number of images from each layer varied according to the thickness of the biofilm. The maximum biofilm thickness of the uniform position in the current field of view was set as the biofilm thickness .S. maltophilia, the SMS and SMN samples were taken from HARV bioreactors immediately after 14days of SMG and NG cultivation. The difference in swimming motility was assessed using agar plates with slight modifications  Bacto agar, 10g/L Tryptone (w/v), and 5g/L Yeast extract (w/v). The inoculated samples were incubated for 48h at 37°C, the plates were imaged, and the diameter of the zone of growth around the spot inoculation was measured. All the motility tests were performed in three replicates.To assess the motility of ications . BrieflyS. maltophilia was acquired using the conventional phenol–chloroform extraction method , ribosome RNA (rRNA), and tandem repeat sequences. The detailed steps were performed as follows: (a) the related coding genes were predicted by Glimmer, GeneMarks and Prodigal software; (b) tRNA genes and rRNA genes were recognized by tRNAscan-SE software (Version V2.0) and Barrnap software, respectively. The nucleotide sequence information, anticodon information, and secondary structure information of the tRNA in each sample genome were obtained; and (c) Tandem repeat annotations were retrieved by the Tandem Repeats Finder. The type, location, and sequence information of all the rRNAs in the genome of each sample were obtained .Six databases containing NR (Non-Redundant Protein Database), SwissProt, Pfam, COG (Clusters of Orthologous Groups), GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) were used for general gene function annotation. A whole-genome BLAST alignment was constructed using the six databases.The genomes of SMS and SMN were sequenced using high-throughput sequencing Illumina technology . In brieThe sequences obtained using fastq software (Version 0.20.0) were filtered by removing reads that contained adapters and poly-N as well as those with a lower sequencing quality (<Q 20). The original SMO group was used as a reference. The SMS and SMN reads were mapped to that of the reference sequence using the mem algorithm of BWA software, and the coverage was determined using Picard-tools. Then, according to the comparison bam results, the sequencing depth and relative coverage were acquired. GATK was used to realign the reads near InDels to obtain the realigned bam, which could eliminate false positive single-nucleotide polymorphisms (SNPs). Then, snippy software (Version 4.6.0) was used to detect SNPs and small InDels. Finally, snpEff was used to annotate variant sites to determine the impact of mutations on the genome .via PCR amplification and quantified by TBS380 (Picogreen), and the clusters were sequenced using the Illumunia Hiseq platform  following the manufacturer’s instructions. Around 2 μg of RNA from each sample was used for RNA sample preparation after the concentration and purity of the RNA was detected by a Nanodrop 2020. After removing rRNAs, the purified mRNAs were fragmented into small pieces (~200bp) using fragmentation buffer. The RNA fragments were used to produce first strain cDNA by reverse transcriptase PCR with random primers. Then, second strain cDNA was created using dUTP instead of dTTP. The sticky end of the double strand cDNA was linked to the adaptor and digested with the UNG enzyme. Then, the cDNA fragments were enriched platform .p<0.05 and fold change (FC) >2 in two different samples were identified as DEGs  and reads per kilobase million (FPKM) of each gene were calculated in accordance with gene length, and the regulation mechanism of genes were revealed by combining the sequence function information. According to the expression of genes in different samples, Venn, correlation and principal component analysis (PCA) analyses were performed. Venn analysis was used to display genes that were shared and uniquely expressed between samples. Correlation analysis of the biological replicate samples, on the one hand, checked whether the variation between biological replicates met the expectations of the experimental design, and on the other hand, provided a basic reference for the analysis of differentially expressed genes (DEGs). PCA could reduce the complexity of the data and determine the relationship and variation between samples. Finally, the read counts were mapped to the genes. Differential gene expression analysis was analyzed using DESeq2 software, and genes yielding a value of  as DEGs .p<0.05 were identified as significantly enriched by DEGs. R scrip was used to estimate the meaningful statistical enrichment of the DEGs in KEGG pathways , reconstituted with UPLC loading buffer and subjected to high-pH liquid fractionation with a reversed-phase C18 column . Then, the purified peptides were subjected to an EASY-nLC 1,200 instrument  and Q_Exactive HF-X  to obtain raw data. The raw data were converted into MGF format by the Thermo Scientific tool Proteome Discoverer software (Version 2.4), and the exported MGF files were searched against the established database using the Sequest and Mascot modules. Eventually, analyses of the data and bioinformatics were performed according to the previous search results . Additional protein lists were assembled based on the DEPs, including GO, KEGG, and COG functional annotation and GO and KEGG pathway enrichment analyses.According to the mass spectrometry results, all proteins and sequences were compared with information in databases, including GO, KEGG, COG, and subcellular location related databases, to obtain the annotation information of the proteins in each database and statistics on the annotations. Proteins with a fold change >1.2 or <0.83 and value of t-tests. GraphPad Prism (version 8.4.3) was used for statistical analysis. Differences with values of p<0.05 were considered significant.Differences in the growth rates among SMS, SMN, and SMO were evaluated by two-way ANOVA, followed by Tukey’s multiple comparisons test. The biofilm formation ability and motility were evaluated by unpaired Scanning electron microscopy was performed to observe the single cell morphology of SMO, SMN, and SMS. From observing the single bacteria, most of the three groups were found to be long rod shape. Under 5,000x SEM, the three groups showed entanglement and aggregation, and some shorter bacilli appeared sporadically. Under 20,000x SEM, it was found that SMO was full and smooth, but there were unequal mucus-like substances in SMN and SMS, and SMS had more mucus-like substances than SMN .p<0.0001), 8h (p=0.0004), and 10h (p=0.011), while the SMO showed a similar growth curve to that of the SMN group.Growth rate curves for the three groups are shown in Compared with SMO and SMN, SMS showed decreased carbon source utilization of D-arabitol (D3), myo-inositol (D4), D-aspartic acid (D8), L-pyroglutamic acid (E8), quinic acid (F8), and D-lactic acid methyl ester (G3), indicating that the metabolism pathways of SMS were inhibited in the SMG environment. In addition, the chemical sensitivity assay showed decreased tolerance to potassium tellurite G12; . SMS, SMS. maltophilia grown in the SMG environment were thicker than those grown in the NG environment . However, the colony diameters of SMN and SMO were 22.56±0.68mm (n=3), and 21.96±1.13mm (n=3) respectively, both of which were smaller than that of SMS, and the difference was statistically significant , pilus organizations (p=0.00012), cell adhesion (p=0.00006), protein secretion by the type II secretion system (p=0.00029), and protein transport across the cell outer membrane (p=0.00029), all of which were significantly upregulated in SMS compared with SMN . Compared with the normal gravity group, the samples after spaceflight showed a decreased size and an increased number of outer membrane vesicles. Salmonella Typhimurium cultivated in LB in HARVs showed a decrease in motility; downregulations of fliEST, flhD, and flgM; and upregulation of filC. Pseudomonas aeruginosa under SMG, including fliACDGS, fleLNP, and flgM, but there were no obvious changes in the expression of genes related to motility after spaceflight. Vibrio fischeri showed a trend of upregulation of flhA and flgM after 12 and 24h of cultivating in SMG. Interestingly, E. coli in different nutrients displayed different motility and had different expression of genes related to motility. In this study, compared with SMN and SMO, SMS showed more bacterial intercellular mucus and rougher membranes under 20,000x SEM. The swimming experiment revealed that the bacteria in the SMS group had increased motility compared with those in the SMN and SMO groups. In addition, considering that the swimming experiment was conducted under ground conditions and there would be some variables affecting the results, we will take videos of precultured bacterial cells directly from three samples to compare their motility in the subsequent experiment. Through the GO enrichment analysis performed in this research, we found that the increased motility of the SMS group may be related to some upregulated DEPs involved in locomotion, localization, biological adhesion, and binding. Moreover, according to the COG annotations, ten DEPs related to cell motility and seven DEPs related to cell wall, membrane, and envelope biogenesis were overexpressed in SMS. In addition, according to the GO enrichment analysis and KEGG enrichment analysis, DEGs, including pilP, pilM, flgE, flgG, and ronN, which are associated with cell motility, were overexpressed in SMS. These upregulated DEGs were consistent with the changes in the DEPs. In conclusion, we believe that the overlap of these changes may increase the motility of SMS in the SMG environment.Astronauts during spaceflight are in a state of reduced immunity, which accompanied by changes in microbiota. Some studies have found that some characteristics of bacteria have a certain correlation with their motility . The difS. maltophilia biofilms are related to the effects of antibiotics, hormones, or combinations of drugs. For instance, S. maltophilia, and in vitro synergistic therapeutic effect of colistin and N-acetylcysteine on S. maltophilia. However, there are few studies on the characteristics of S. maltophilia in the space environment and SMG. Therefore, we focused on microgravity as a starting point to investigate the physiological characteristics of S. maltophilia in a microgravity environment. The formation of biofilms in bacteria was first carried out under microgravity in 2001 (E. coli increased (Escherichia coli were cultured in a rotary cell culture system under microgravity and the biofilm formation ability increased in the SMG group compared with that in the NG group (S. typhimurium was also increased after the space shuttle mission STS-115 (Acinetobacter baumannii showed decreased biofilm formation after a spaceflight on the Shenzhou 11 spacecraft of China. Klebsiella pneumoniae formed different subgroups, M1 and M2, on solid medium, and an increased biofilm forming ability was found in M1 compared with that in M2 in SMG (Most of the studies on  in 2001 . After 2ncreased . EscheriNG group . The bio STS-115 . These r2 in SMG . TherefoS. maltophilia at the level of DNA or RNA are still limited. Considering the complexity of space environment and the limitation of simulated experiment condition on the ground, more relevant experiments are needed for deep research.To sum up, the changes in genome, transcriptome, and proteome can to some extent explain the phenotypic changes such as motility and biofilm formation ability. The present study might serve a basis for future studies of the complex mechanism by which bacteria adapt to the space. However, the overall changes in via the PRIDE (All the data used to draw conclusions are presented in the article. The sequence reads used for WGS and RNA-seq have been submitted to NCBI with the project accession numbers of PRJNA720482 and PRJNA720572. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium he PRIDE  partner CL and JW designed and coordinated the project. XS performed the laboratory experiments and wrote the manuscript with assistance from all the authors. YG, TF, XJ, DW, DL, PB, and BZ performed the data analysis. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Clostridium perfringens and Bacillus cereus) and non-sporulated  food-borne pathogens present in them, when they were previously added to the microbial culture media. The basil had the highest mean Ca and Mg level and showed the highest microbial growth in the food-borne pathogenic microorganisms studied (p < 0.001). For Ca, the lowest levels were measured in cloves (p < 0.001), which had the lowest capacity for microbial contamination. Ca and Mg contents in spices correlated linear and positively (p < 0.05). Ca concentrations weakly and positively correlated (p < 0.05) with microbial counts for almost all studied microorganisms, and Mg levels for B. cereus, C. perfringens, and mesophilic bacteria (p < 0.05), possibly acting as a growing factor for some sporulated and non-sporulated foodborne pathogens. These relationships are especially significant when PET vs. glass was used as a packaging material for spices.Ca and Mg levels were determined in five spices according to marketing system  containers) and correlated with microbial growth of commonly sporulated ( Spices have traditionally been used for their organoleptic properties as flavorings for sausages, meats, salads, soups, etc., as well as for their medicinal and preservative properties . This faBacillus and Clostridium genera. Therefore, they are likely to be found in foods with medium-low water activity such as dried fruits, cereal grains, milk powders and spices [Although spices are mainly sold in a dehydrated state, which ensures low microbial growth, when added to highly water-active foods that are not subjected to final heat treatment, they can be vehicles for different microorganisms that can develop rapidly, especially if consumption of these foods is not immediate . On the d spices .Listeria monocytogenes, and of sporulated microorganisms such as Bacillus cereus and Clostridium perfringens. Other authors [For this reason, different decontamination treatments are being studied for spices such as thyme, mustard, coriander, etc., and it has been indicated that ultraviolet radiation could be employed in food preservation techniques by reducing microbial counts of aerobic mesophylls, and molds and yeasts . In addi authors  have indOn the other hand, it has been indicated that the microbial counts of non-sporulated aerobic bacteria communities and other food contaminating bacteria, present in aromatic herbs, both fresh and dehydrated, such as thyme and basil, can contribute to the estimation of consumer exposure to these microorganisms from these food sources as well as the possible health risk they might pose . These rAcinetobacter , Enterobacter, Shigella and Bacillus genera being the most abundant microbial species.Other studies concluded that the hygienic quality of dried spices and herbs needs to be further controlled, as the production and growing conditions as well as the essential oil content vary considerably according to the region of production, the type of cultivation and the drying method used. This determines the differences found between different samples of oregano and thyme sold in Spanish markets . SpecifiSalmonella and Shigella in excess of 7% of the total microbial bacterial communities. Additionally, sporulated microorganisms, such as Bacillus spp. (including B. cereus) and C. perfringens, among others, were also found.According to studies conducted between 2007 and 2010, the Food and Drug Administration  publisheC. perfringens and B. cereus [In many countries, dried spices and herbs are dried in the sun and in the open air, practices that increase the probability of microbial contamination of the end product. Therefore, it is highly recommended that if these spices and herbs are part of the formulation of products that are marketed raw or ready-to-eat foods without heat treatment, they should be previously tested for total and faecal coliforms, as well as aerobic and pathogen sporulated microorganisms, such as . cereus .L. monocytogenes has been reported [Bacillus species [L. monocytogenes growth in cooked frankfurters inoculated with this microorganism [L. monocytogenes, which could be used in food packaging [2+ and Ca2+, when present in high concentrations in foods, counteract the preservative effect of enterocin LR/6, limiting its antibacterial potential. Another study [2+ content confers heat and hydrostatic high pressure with high heating resistance to spores of three strains of C. botulinum, while Mg2+ cations seemed to have a contrary effect. Finally, another study reported that when Ca and Mg ions were added to the medium, B. cereus was eradicated [C. perfringens, others reported that calcium nitrate suppressed sporulation and enterotoxin production [The role of various essential elements like Ca and Mg in microbial growth has been documented mostly by in vitro experiments regarding media formulations and partially as constituents of antibacterial active packaging. On the other hand, when an antibacterial active packaging was reinforced with zinc magnesium oxide nanoparticles, an extended shelf-life of cold-smoked salmon samples overall against reported . Similar species , and watorganism . In the organism . Moreoveackaging . Others ackaging  reporteder study  indicateadicated . For C. oduction .B. cereus and C. perfringens) and non-sporulated  food-borne pathogens present in these spices. Moreover, the influence of the commercialization system used  and glass containers) on microbial counts for referred microorganisms when the dehydrated spices are mixed with the culture media and their possible correlation with present Ca and Mg concentrations will also be studied. The aim is to determine whether the content of Ca and Mg in spices can influence their potential use as preservatives of crude foods or, on the contrary, are positively related to the microbial growth of sporulated and non-sporulated microorganisms depending on the marketing system used.Consequently, in this research we aimed to determine the Ca and Mg concentrations in five spices commonly used in Spain, namely . Additionally, we also aimed to determine whether the levels of these minerals influenced the microbial counts of commonly sporulated (n = 75) commercialized in glass (n = 25) or in PET jars (n = 25), and in bulk (n = 25), were sampled aseptically in sterile bags in different Spanish establishments. The samples were analyzed microbiologically on the collection day. Full information on the characteristic of sampling of dried spices considered in this study has been previously reported elsewhere [Studied dehydrated spices (lsewhere .3 (66%) and HClO4 (60%) of supra-pure quality  in a thermostatized multi-site mineralization block . Mineralization was carried out in two stages according to a previous developed method. The determination of total concentrations of Ca and Mg in spices was performed by an atomic absorption spectrometer (AAS) instrument . After the mineralization procedure, the samples were diluted to 10 mL with bidistillled water. Finally, additional dilution (1/500) with bidistilled water was done as a previous step to Ca and Mg measurement by flame atomic absorption spectrometry. Calibration curves were previously prepared by diluting stock solutions of 1000 mg/L in 1% HNO3 for the analyzed elements .Three hundred milligrams of spice sample were mineralized by attack with HNOn = 10) were verified by testing the certified reference standard Apple leaves powder of the National Institute for Standards and Technology (NIST) 1515 . No significant differences were found between the mean element concentrations determined in this material and the certified levels . Additionally, the accuracy of the methods was also tested on the basis of recovery experiments, after complete digestion of spiked spice samples with different amounts of elements from the standard solutions [The accuracy and precision of Ca and Mg measurement procedures  and counting procedures for sporulated microorganisms were described in detail in another study  and werefringens , ISO 483fringens , ISO 215fringens  and ISO ytogenes .p < 0.05). For data analyses, the SPSS 22.0 for Windows  program was used.The statistical study of the data was performed by the ANOVA or the Kruskal-Wallis test. Linear correlations among Ca and Mg levels and microbial counts checked in spices were also done. The significance level was set at 5% (p < 0.05). Specifically, the highest average Ca content was determined in basil, which was statistically higher than rosemary and oregano, which were statistically higher than thyme, and the latter was statistically higher than cloves (p < 0.001). For Mg, similar mean levels were determined for all spices with the exception of basil where significantly higher concentrations were measured (p < 0.001). The highest mean levels of Ca and Mg were determined in basil  and non-sporulated  food-borne pathogens considered in the study, when aromatic herbs were previously mixed with the culture media, the Spearman’s non-parametric method was employed. r) and significance levels (p) for these linear bivariate correlations. It can be observed that the microbial count of B. cereus as a sporulated microorganism and all of the non-sporulated  food-borne pathogens considered present in spices increased significantly with the Ca concentrations  and of mesophilic microorganisms (non-sporulated microorganisms) rose significantly with Mg concentrations  in all of the marketing systems used, with the exception of those found for B. cereus when these spices were marketed in glass (p > 0.05), increased significantly with the Ca content measured in the spices  for spices. However, the microbial count of B. cereus weakly but significantly increased with Mg concentrations only for spices marketed in PET , only when aromatic spices were marketed in PET. However for the other marketing systems (in bulk and glass packaging), no linear correlation (p > 0.05) between the count of this microorganism and the Ca and Mg concentrations present in the spices, was found. On the other hand, the counts of psychrophilic microorganisms were linearly and positively related to Ca concentrations in spices when they were marketed in PET (as a moderate correlation) and glass (as a weak correlation), and with the Mg concentrations when marketed in bulk (as a moderate correlation). Similarly, the Ca content and growth of mesophilic microorganisms were also positively and linearly significantly correlated (p < 0.05) when spices were marketed in bulk and in glass . In contrast, for Mg levels, we only found a positive linear correlation with the growth of these microorganisms when the herbs were marketed in PET (as a moderate correlation). For molds and yeasts, PET appeared as the marketing system where there was again a positive and linear relationship between the measured Ca and Mg concentrations  and microbial growth. Additionally, mould and yeast counts also significantly increased with Mg concentrations in dried spices when marketed in bulk and glass  (p < 0.05). Contrarily, Ca concentrations did not increase with mould and yeast growth when spices were marketed in bulk and glass (p > 0.05).In relation to non-sporulated microorganisms, the growth of In the present work, we observed that the concentrations of Ca and Mg varied depending on the spice considered A,B, withWhen comparing the results found in the spices analyzed in this research work, with those determined by other authors in other countries and locations, a great variability in the levels of these minerals (Ca and Mg) was observed. Therefore, the mean concentration of Ca and Mg present in the spices is influenced by the climatic and edaphic conditions of the particular cultivation soils of the geographical area of origin, as well as the type of spice, climate and agricultural practices .For Ca and Mg, the average levels determined in spices such as thyme, rosemary and oregano, in the present work in supermarkets and street vendors in South-East Spain, are considerably lower (between 20 and 40 times lower) than those indicated in the same dried aromatic herbs from local markets in Sicily  and Mahdia (Tunisia)  Table 1Table 1. In relation to the possible influence of the spice marketing system on the Ca and Mg concentrations present, it has been observed that the concentrations determined in spices sold in PET were lower than those sold in bulk only for Mg . This fiWe also estimated the daily intake of Ca and Mg as essential elements from the studied spices taking into consideration the present average concentration of 18.7 ± 0.99 and 6.20 ± 0.37 mg/g (dry weight: DW), respectively, and the mean amount consumed in Europe of 0.78 g/person/day . Therefor = 0.447, p < 0.001). Previous studies indicated that Ca concentrations present in different food groups  were liC. perfringens (in relation to sporulated microorganisms) and all non-sporulated microorganisms studied. Mg levels were also positively and mainly weakly correlated with microbial counts of B. cereus and C. perfringens (sporulated microorganisms) and aerobic mesophilic microorganisms (non-sporulated microorganism). Consequently, basil is the spice that suffers the greatest contamination and microbial development [In the present study, we observed how basil is the spice that has higher levels of Ca and Mg, as others have also found ,36 Tabl. In addiC. perfringens and B. cereus as sporulated bacteria as well as of L. monocytogenes among the non-sporulated ones) [For Ca, as others also found , the lowed ones) . Likewised ones) . Therefo2 than Gram (+) ones. However, in those containers in which a balanced atmosphere between O2 and CO2 concentrations is achieved during storage, the growth of aerobic and anaerobic microorganisms would not be inhibited in the expected way [2 and CO2 permeability characteristics similar to PET [When relating Ca and Mg content with microbial growth and packaging systems, we found that, in PET and for both minerals, a significant positive linear correlation between all sporulated and non-sporulated microorganisms (with the exception of mesophilic microorganisms for Ca and psychrophilic microorganisms for Mg) was found. The fact that the enhancement of Ca and Mg levels significantly increased the growth of all sporulated and a large part of the non-sporulated microorganisms for spices packaged in PET could be due to the medium permeability characteristics of this plastic in relation to gases. In this sense, this characteristic would allow the creation of a passive modified atmosphere system in a natural way that balanced oxygen and carbon dioxide inside these PET containers, favouring the growth of certain microorganisms. These results are in line with that reported by other authors  who conccted way , as the cted way , who four to PET .2 and resist high CO2 concentrations. The same situation would be true for anaerobic sporulating microorganisms such as C. perfringens. This could explain why we found a positive linear correlation between the growth of C. perfringens (as anaerobic sporulating microorganism) and the Ca and Mg content in herbs as well as between the growth of moulds and yeasts (non-sporulating anaerobic microorganisms) and the Mg levels in the samples packaged in glass, since glass is an oxygen impermeable material [p > 0.05) between Ca and Mg contents, and microbial growth for B. cereus (aerobic sporulating microorganism) and L. monocytogenes (anaerobic non-sporulating microorganism). The growth of mesophilic and psychrophilic (both non-sporulated) microorganisms in herbs was positive, linear, and significantly correlated with Ca levels in glass (p < 0.05), but not with Mg content (p > 0.05). However, the degree of growth of both sporulated and non-sporulated microorganisms was lower in herbs packaged in glass than in PET.Foods with low water activity, such as dried spices, are susceptible to attack by moulds and yeasts that can grow in the absence of Omaterial . As glas2 and CO2, and the Ca and Mg contents in herbs that could act as factors of microbial growth. Contrarily, other studies have reported that Ca and Mg added as cations or as different salts exerted an antimicrobial effect against L. monocytogenes [Bacillus species [C. perfringens [There is no scientific literature relating microbial growth in aromatic herbs to mineral content (Ca and Mg) and marketing systems. Therefore, with this work and a previous study carried out by our research group , we highytogenes ,20, fungytogenes , Bacillu species ,26 and Cfringens  when useThe Ca and Mg contents varied depending on the aromatic spice type. The Ca content differed among all dehydrated spices apart from oregano and rosemary. For Mg, only average concentrations determined in basil were different. The highest mean Ca and Mg level was determined in basil. For Ca, the lowest concentrations were measured in cloves. The Ca and Mg concentrations present in spices could stimulate growth for most of the food-borne pathogenic microorganisms studied. Ca and Mg contents in spices correlated linearly and positively. The growth of sporulated microorganisms increased strongly with Ca and moderately with Mg levels present in aromatic herbs packaged in PET. For non-sporulated microorganisms, their microbial counts also increased moderately with Ca and Mg concentrations in herbs packaged in PET, with the exception of mesophilic and psychrophilic microorganisms, respectively. This relationship between Ca and Mg levels and microbial growth for most of the studied sporulated and non-sporulated food-borne microorganisms was especially significant when using PET versus glass as a packaging material for dehydrated herbs.


When an enhanced and diverse diet was served, replacing rice with millet had only minimal growth improvement on chest circumference (p < 0.05). The quality assessment using GRADE shows that the evidence used for this systematic review and meta-analysis had moderate quality, based on eight scoring criteria. These results demonstrate the value of adding millet as the staple for undernourished communities. Further understanding of the efficacy of millets on growth in a wider range of diets is important to develop appropriate dietary programs and improve the nutritional status of various age groups across Africa and Asia.Undernutrition, such as stunting and underweight, is a major public health concern, which requires multi-sectoral attention. Diet plays a key role in growth and should optimally supply all required nutrients to support the growth. While millets (defined broadly to include sorghum) are traditional foods, and climate smart nutritious crops, which are grown across Africa and Asia, they have not been mainstreamed like rice, wheat, and maize. Diversifying staples with millets can potentially provide more macro and micro nutrients, compared to the mainstream crops. However, there is little known scientific evidence to prove millets’ efficacy on growth. Therefore, a systematic review and meta-analysis was conducted to collate evidence of the benefits of millets in improving the growth of children. Eight eligible randomized feeding trials were included in the meta-analysis. Results from the randomized effect model showed a significant effect ( Undernutrition, especially stunting, underweight, and wasting, is a major global crisis. It is estimated that there are currently 149 million children under five years who are affected by stunting, and 45 million children under five years who are affected by wasting , which iAfter birth, the first 1000 days of life are a critical stage and a window of opportunities for healthy growth, including physical, social, emotional, and cognitive development . SimilarChild malnutrition is considered as a sensitive indicator of the overall levels of food insecurity and hunger. The Sustainable Development Goal 2 (SDG 2) aims to eliminate hunger and all forms of malnutrition by 2030. Diversified diet, especially staples, are needed to address the global challenge of hidden hunger. More than 70% of the energy intake comes from Big 3; rice-, wheat-, and maize-based foods in developing countries of Africa and Asia ,6. The tMany nutrients are essential during the growth and development stages, especially for linear skeletal growth. These include adequate supply of energy, amino acids, and bone forming minerals such as calcium, phosphorus, magnesium, and zinc and other ions and vitamins, such as vitamin C, D, and K required for collagen formation and bone metabolism and/or phosphate homeostasis .Given the high nutrient content of millets, understanding their role in child growth would be instrumental in achieving sustainable nutritional security. Millets secure the sixth position in global production of cereal grains and remain a staple food in many rural marginalized regions of the world . With riIndia is one country proactively striving to bring millets back as common part of the diet. The Government of India launched the national millet mission in 2018 and celebrated that year as the ‘National Year of Millets’. The National Institution for Transforming India (NITI) Aayog announced a pilot to include millets in the Integrated Child Development Services (ICDS) and Mid-Day Meal (MDM) schemes across the country. The year 2023 has been approved by the United Nations as the International Year of Millets, which is expected to attract major attention to millets. Previous systematic reviews and meta-analyses conducted on millets’ health and nutritional benefits showed that regular consumption of millets could help manage the risk of developing type 2 diabetes , reduce Review question: Does consumption of millet-based diets help improve child growth  compared to non-millet diets?www.researchregistry.com), with the unique identification number (UIN), Reviewregistry1180, which can be accessed by browsing the registry of systematic review/meta-analyses with UIN.The study adopted a 27-item PRISMA checklist ,17 for eStudies published in English were considered. Search engines Google, Scopus, Web of Science, PubMed, and CAB abstract were used to identify relevant studies. The search key words are given in Z-score (HAZ), and weight for age Z-score (WAZ); 3. Studies conducted in populations at different stages of growth including infants (<23 months), pre-school children (<59 months) school going children (5 to 10 years), and adolescents (11 to 19 years) by feeding millet-based diets and with their anthropometry measured; and 4. Only peer reviewed journal articles were included.Inclusion criteria: 1. Randomized controlled trials conducted to test the efficacy of millets on growth; 2. Studies that assessed at least one of the following growth outcomes: height, weight, mid upper arm circumference (MUAC), chest circumference, body mass index (BMI), height for age Exclusion criteria: 1. Review articles were excluded for further consideration; 2. Animal studies were excluded; and 3. If the data were incomplete, the authors were contacted for missing and/or additional information. The minimum data required were changes in mean and standard deviation (SD) of height, weight, MUAC, and chest circumferences. In case, if the data were still incomplete (for example the change in SD was not available), even after contacting the authors then the study was excluded.change = Meanpostintervention − Meanpre-intervention. The changes in SD were calculated using the method and formula provided in the online Cochrane handbook with the topic on imputing standard deviations for changes from baseline [E and NC are sample sizes in the intervention and control groups respectively. If the t statistic was not provided, then SD was calculated using correlation formula as given below. Correlation for both groups was calculated and the mean correlation value was then obtained .Two independent reviewers (S.A and S.U) extracted data. Each study was labelled with author details and year. Trial participant characteristics such as age group and gender were recorded, along with the country in which the study was conducted, study design, sample size, duration, type and form of millet used, and outcomes assessed such as height (cm), weight (kg), MUAC (cm), and chest circumference (cm) for millet consuming group (intervention) and the regular diet or enhanced rice-based diet consuming group (control). The data were then entered into an Excel sheet as per the guidelines provided by Harrer et al. 2019) . The eff . The efCorrelation value was then used in the below formula for both groups. Again, E is replaced by C when calculating the difference for the control group).Funnel plot was used to visually assess the presence of publication bias, and other bias such as selection, detection, attrition, and reporting bias following the guidelines provided in the Cochrance handbook for systematic reviews of interventions .The quality of the evidence was assessed using eight assessment criteria as described by Cochrane author resources using the GRADE approach. The GRADE was not included during the protocol writing stage. During the drafting and review stage, the GRADE was included based on a reviewer’s comment. As per the guidance, two authors of this systematic review independently conducted the GRADE assessment. At any point, when there was disagreement on a particular item, another author of this paper was involved in the assessment for final decision. All the outcomes underwent an overall assessment rather than an independent assessment as they all originated from the same studies. The quality was assessed and rated based on the following criteria; 1. risk of bias, 2. inconsistency, 3. indirectness, 4. imprecision, 5. publication bias, 6. large magnitude of effects, 7. dose response, and 8. effects of all plausible confounding factors. Ratings were given to downgrade the first five criteria and/or upgrade the sixth to eighth criteria based on the assessment for each criterion.2). The significance of the results was determined using the fixed effect model for single source information, specifically when all the studies were conducted by one research team from one geographical location and SMD was not largely different between the fixed and random effect models, and when the heterogeneity was less than 50%. On the other hand, the random effect model was used mainly to compare finger millet-based diets vs. rice-based diets (regular and enhanced), and when heterogeneity was more than 50% to interpret the results. Results from both models were captured in each forest plot. Meta-analysis was conducted using the software R Studio version 4.1.1 (2021) to obtain forest plot along with heterogeneity (i2) and overall test effects in both models and funnel plots to determine the publication bias [The changes in mean and SD of height, weight, MUAC, and chest circumference from the baseline to the end-line for the intervention group and the control group were compared in the meta-analysis to measure Standard Mean Difference (SMD) and heterogeneity  with high heterogeneity (I2 = 94%) among studies and a pooled SMD of 2.92 (95% CI 0.23–5.62). The mean height increase was 28.2% higher in the intervention group than in the control group.p < 0.05) with high heterogeneity (I2 = 96) among the studies and a pooled SMD of 1.30   . The meap < 0.05) with high heterogeneity (I2 = 82%) among studies and SMD of 2.38 . The mean increase in MUAC was 39% higher in the intervention group than in the control group.Likewise, p < 0.05) with high heterogeneity (I2 = 91%) and SMD of 3.26 . The mean increase in chest circumference was 37% higher in the intervention group than in the control group.p = 0.26) with moderate heterogeneity (I2 = 70%) among studies and SMD of 0.16 .p = 0.11) with moderate heterogeneity (I2 = 65%) among studies and SMD of −0.21 .p = 0.19) with high heterogeneity (I2 = 84%) among studies and SMD of −19 .Likewise, p < 0.01) high heterogeneity (I2 = 82%) among studies and SMD of 0.37 . However, the mean increase in chest circumference was only slightly higher (2%) in the intervention group consuming a finger millet than in the control group consuming a rice-based diet.The publication bias was determined through the funnel plot  using thAll the outcomes were looked at for GRADE assessment. The overall effect of the interventions shows that the quality of the evidence is moderate as there is moderate confidence in the effect estimate. This implies that the true effect is likely to be close to the estimate of the effects, though there is a possibility of being substantially different.Most studies included in this meta-analysis randomized the children allocation to the treatment and control groups. However, they generally did not mention how the randomization was undertaken or whether the experiments were blinded or not. Nevertheless, the studies reported that the participants in both the intervention and control groups were similar at the baseline in terms of socio-economic status and dietary patterns. It is noteworthy that it is almost impossible to blind participants for the millet-based meals that are provided during these studies, as millets are unique in their appearance, colour, and texture, which can be identified by anyone in the study areas where millets are traditional crops. Similarly, the anthropometry measurements cannot be blinded like clinical measurements. Most clinical measurements collect biological samples, such as blood, whereas anthropometry measurements involve measuring height, weight, MUAC, and chest circumferences, which are visible to the participants. As these is not blinding, there is no risk of bias arising from blinding per se. Only allocation concealment can be considered as a small risk coming from the studies for GRADE assessment.2) value for various outcomes varied from 65% to 96%. The high heterogeneity could be due to clinical heterogeneity associated with differences among participants in different stages of growth . There is also a possibility of methodological heterogeneity as the duration of each of the studies varied from 3 months to 4.5 years. Except for one study conducted for 3 months and another for 6 months, all other studies were conducted for longer period from 8 months to 4.5 years. Therefore, the high statistical heterogeneity in the outcomes could be associated with the differences in growth stages and study duration. Subgroup analysis could not be performed due to the small number of studies in each group.The heterogeneity . Overall, two types of studies were conducted—substituting rice for millet in regular diets and substituting rice for millets in enhanced diverse diets. The studies that formulated the enhanced diverse diets (millet or rice) took into account the nutrient gaps identified in the dietary patterns of the people.Nutrient requirements generally vary by age. During adolescence, typically the weight doubles and height grew 20% in healthy individuals . During Finger millets have a threefold higher concentration of calcium than milk, and calcium retention is higher for finger millet than for any other staple . MoreoveIn the regular diets, there was a significant positive effect of replacing rice with millet on height, weight, MUAC, and chest circumference in various age groups . These positive effects of millets are attributed to the naturally high content of growth promoting nutrients , given that the rest of the diet was similar between two groups.This shows, that when the diet is enhanced, diverse, and nutritious, there is only minimal extra growth achieved from consumption of millet-based diets compared with rice-based diet. This is a reasonable result given that the control diet contains a diverse range and adequate quantities of nutritious foods. However, when the base diet was not enhanced and had lower nutritional value, substituting rice with millet had a large and significant impact on additional growth ,32,33. TThere are nine main limitations in this study as follows. 1. The studies that were eligible for meta-analysis included finger millet, sorghum, and multiple millets . The other types of millets should be studied to determine which millets have the most positive impact on growth, and hence the most important to be included in nutrition sensitive agriculture interventions and programs. 2. All the studies included in the meta-analysis were from India, which indicates a geographical limitation and a need to conduct such studies in other countries, especially across Africa where millets are traditional crops and there are serious malnutrition issues. 3. Further studies should be undertaken including all the growth measurements. Two studies measured the BMI, and other studies measured only height and weight without estimating the BMI, HAZ, and/or WAZ, which could have been included to study the stunting and underweight status. 4. The studies had similar diets and mainly only substituted the staple. However, the diets were not exactly the same and thus future studies should design the intervention by keeping all food groups exactly the same between the intervention and control groups, changing only the selected staple . 5. All major growth promoters should be measured in future studies. Methionine is an essential sulphur containing amino acid and a major growth promoter. However, none of the studies measured the level of methionine provided in the diets in both groups. Apart from methionine, total protein, calcium, and zinc are essential as growth promoters, which were also not measure. 6. Growth impacts of the different types of cooking/processing of the millets should be analysed to inform more detailed recommendations for dietary design. 7. No studies identified the varieties of millets used or the nutritional content of the grain or the total diet. Studies incorporating these factors would help identify how important the varieties are on growth outcomes. 8. Only one study was designed to analyse the impacts of millet-based diets on children’s growth during adolescence, the third critical stage of rapid growth and bone mineralization in human life, which calls for further studies. 9. Except two studies, all other studies were conducted in the 1980s by one research team, suggesting that new studies should be undertaken to corroborate the results.The developmental, economic, social, and medical impacts of the global burden of malnutrition are serious and lasting for individuals, their families, communities, and countries . Hence, These studies provide evidence that millet-based diets can be effective in improving height and weight where regular rice-based diets are currently consumed. At the same time, these studies were not aimed at making changes in dietary patterns. Policy recommendations include: 1. Nutrition intervention programs developed to diversify staples using millets. 2. School feeding programs, and mother and child programs, incorporate millet-based meals designed for different age groups, using with culturally sensitive and tasty recipes. 3. Complement these interventions with awareness and marketing campaigns to change the image of millets and create interest. Implementation of these recommendations should be studied in regards to the impact and lessons learnt from the approaches.The findings of this review paper provide scientific evidence supporting diversification of standard diets for children using millets as a solution to malnutrition. Furthermore, millets are a basket of a wide range of nutrients and have been scientifically shown to contribute to serving many of the significant nutrition and health needs globally ,13,14,15


The MetS patients produced less words in the letter K and animal categories. The analysis of descriptive statistics shows that the group of patients with metabolic syndrome generated fewer words in both the phonetic and semantic categories. Our study shows that there is an association between metabolic factors and the verbal fluency performance of MetS patients. This is true, especially for phonetic verbal fluency, which is traditionally connected with the frontal cortex. Lower switching signifies possible executive dysfunctions amongst people with MetS. Subjects with this condition generated more diverse words and created less standard associations. This further implies the existence of dysexecutive syndrome and the need for diagnosing patients in this direction and involving this group of people in therapy. The proper correction of MetS components may improve cognitive function.Metabolic syndrome (MetS) or otherwise insulin resistance (IR) is described as a cluster of several commonly occurring disorders, including abdominal obesity; lipids disorders, such as hypertriglyceridemia; and low levels of high-density-lipoprotein cholesterol (HDL-C), hypertension (≥130/85 mmHg), and carbohydrates disorders, such as impaired fasting glucose or diabetes mellitus type 2. Type 2 diabetes (T2DM) constitutes insulin resistance, which is a strong risk factor for strokes. Patients with MetS are often prone to cognitive decline. Metabolic risk factors, hypertension, and diabetes, amongst them, have been hypothesized to play a great role in the pathogenesis of Alzheimer’s disease (AD) and the development of vascular dementia. For neuropsychological diagnostic and theoretical purposes verbal fluency is defined as a cognitive function that facilitates information retrieval from memory. It engages executive control and other cognitive processes, such as selective attention, selective inhibition, mental set shifting, internal response generation, and self-monitoring, as well as imagination and psychomotor skills. A total of 90 subjects, divided into 2 groups, patients with MetS (45) and healthy controls (45), were assessed. A significant difference in performance was found between the patients and controls, both in the phonetic ( Metabolic syndrome (MetS), or otherwise insulin resistance (IR), is described as a cluster of several commonly occurring disorders, including abdominal obesity; lipids disorders, such as hypertriglyceridemia; and a low level of high-density-lipoprotein cholesterol (HDL-C), hypertension ≥130/85 mmHg), and carbohydrates disorders, such as impaired fasting glucose or diabetes mellitus type 2 0/85 mmHg,5,6. NevFor neuropsychological diagnostics and theoretical purposes, verbal fluency is defined as a cognitive function that facilitates information retrieval from memory. It engages executive control and other cognitive processes, such as selective attention, selective inhibition, mental set shifting, internal response generation, and self-monitoring, as well as imagination and psychomotor skills ,21,22,23A total of 90 subjects, divided into 2 groups, patients with MetS (45) and healthy controls (45), were assessed. All patients in the MetS group  were recruited from the Department of Endocrinology and Diabetology in Bydgoszcz, Poland. The diagnosis of the MetS group was made on the basis of the International Diabetes Federation (IDF) criteria .The control group was recruited from visiting family members of other patients with light injuries treated in the same hospital. The controls were matched to the MetS patients according to gender, age, and education. Normal subjects were excluded from the study if their medical history included any major medical, neurological or psychiatric illness, substance abuse, or history of head injury or stroke.All the subjects voluntarily provided written informed consent to participate in the testing sessions and were subjected to the same psychological examination in the form of the Mini Mental State Examination (MMSE) and verbal fluency tasks. METS patients scored lower (between 24 and 26 points) on the MMSE than the controls (full score), but still presented no symptoms of dementia. All patients included in the study showed no symptoms of increased anxiety or depression in the Hospital Anxiety and Depression Scale (HADS). Polish was the first language for all the individuals, and they were all right-handed.Procedure: the authors used tasks from the Controlled Oral Word Association Test (COWAT), which is the most popular verbal fluency test and is one part of the Halstead–Reitan Neuropsychological Battery. It measures the spontaneous production of words belonging to the same category or beginning with some designated letter. The total number of words generated in 1 min for the letter K (phonemic fluency) and animal names was obtained from all 45 participants. The subjects were instructed that proper nouns were not acceptable. The instructions included reminding them not to use proper nouns. Each individual’s conduct was recorded both on paper and on tape, which limited the chance of examiners’ mistakes.All procedures were performed after 12 h of fasting. Anthropometric measurements, including height, weight, and waist circumference (WC), were obtained for all the participants, as well as the blood pressure and body mass index (BMI) measured using the metric system. The BMI was calculated as the body weight (in kilograms) divided by the square of the body height (in meters). The WC was measured by placing a measuring tape around the waist at the upper point of the iliac crest. Finally, the following demographic factors were determined: age, sex, and obesity. The systolic and diastolic blood pressure were measured in the sitting position after 15 min of rest using an appropriately sized cuff in both upper extremities. Patients were seated quietly with their feet on the floor and the blood pressure readings were taken at 1 min intervals. An average of both measurements was calculated and used for the data analysis. Arterial hypertension was diagnosed according to the IDF definition (RR ≥ 130/85 mmHg). The levels of fasting total plasma cholesterol (TC), triglycerides (TG), high-density-lipoprotein cholesterol (HDL-C), and fasting blood glucose (FBG) were evaluated in all the patients. Low-density-lipoprotein cholesterol (LDL-C) was calculated using the Friedewald formula. Non-high-density-lipoprotein cholesterol (non-HDL-C) was figured on the base of the following formula: TC − HDL-C. In patients with abnormal fasting glycemia values and a waist circumference >80 cm in women or >94 cm in men, an oral glucose tolerance test (OGTT) was performed to determine glycemia in the fasting state and 2 h after the administration of 75 g of glucose.Venous blood samples were collected from fasting patients for biochemical analyses . Abnormal results excluded the patient from the study.Exclusion criteria: a history of heart surgery or other cardiovascular interventions, congenital defects of the heart, cardiac rhythm disorders, pregnancy, electrolyte disorders, inflammation, anemia, prostate disease, and Cushing’s syndrome. A self-reported history of medical and psychiatric problems, including a list of all currently prescribed medications, was obtained from each participant. Additionally, subjects after stroke or with dementia were excluded, as were those presenting other conditions that compromise cognition, such as depression, anxiety, taking psychotropic drugs, psychiatric diseases, and history of alcohol or chemical addiction or uncorrected visual or hearing disorders.n = 14) were diagnosed with 3 components, 40% (n = 18) with 4, and 29% (n = 13) with all 5. All the patients were diagnosed with obesity, 86.67% with HT, 66.67% with high pre-meal blood glucose levels, 60% with TG, and 70% with HDL-C. The characteristics of the study group are presented in In order to diagnose a patient with MetS, at least three out of five components must be present. Amongst the subjects that constituted our study group, 31% .The analysis of the descriptive statistics shows that the group of patients with metabolic syndrome generate fewer words in both the phonetic and semantic categories .t-test by entering the basic scores in the form of the number of produced words in each task. A significant difference in the performances was found between the patients and controls, both in the phonetic (p < 0.01) and semantic fluency trials (p < 0.001). The MetS patients produced less words in the letter K and animal categories. In addition to estimating the apparent differences in verbal fluency between those two samples, we were interested in comparing the profile of verbal fluency conduct. In order to achieve that, we entered the difference between the phonemic and semantic fluency calculated by subtracting the number of produced words in one trial from the number of words produced in the other. Next, we performed the Student’s t-test to compare the verbal fluency general profile. We did not observe any differences between the METS patients and controls, as both produced less words in the phonetic fluency task.In order to identify the major differences between the groups’ performances in fluency tasks, we initially carried out a first exploratory analysis. We performed a Student’s t-test procedure to compare the performances of both samples. The METS patients produced significantly less clusters in the phonemic fluency task (p < 0.1), but there was no such difference in the semantic fluency task. What is interesting though, is that the METS patients produced more phonetic clusters in the semantic animal category trial (p < 0.5) than the healthy subjects. We observed no significant difference between those two samples in the number of semantic clusters produced in the animal category task.As we were interested in a detailed analysis of verbal fluency conduct, we entered the cluster numbers produced by the patients and controls in both fluency trials, and used the Student’s Further analysis involved the use of the Spearman’s correlation test to measure the strength and direction of the monotonic association between the verbal fluency results, in both the semantic and phonetic tasks, and each key medical factor contributing to the MetS condition .The gathered results indicate a significant association between fasting glucose levels and the patient’s performance in the phonetic verbal fluency task. Higher TGs are associated with a worse performance in phonetic fluency tasks. However, lower HDLs correlate with worse semantic and phonetic fluency tasks.The complexity of verbal fluency neuronal bases was confirmed by many studies on brain activity, for which extent and intensity are dependent on the fluency task type  ,26, the Furthermore, studies show the differences in verbal fluency realization amongst subjects with various brain dysfunctions ,28. In p1c, which is a measure of average glucose concentrations over 2 months, is associated with a lower cognitive function in T2DM [There are data that point to the adverse effects of MetS on cognitive function . Unfortu in T2DM . Limited in T2DM  compared in T2DM ,30. Our  in T2DM ,32. Hype in T2DM . Some li in T2DM . Similar in T2DM . Age canOur study shows that there is an association between metabolic factors and the verbal fluency performance of MetS patients. It is true, especially for phonetic verbal fluency, which is traditionally connected with the frontal cortex, that lower switching signifies possible executive dysfunctions amongst people with MetS. The subjects with this condition generated more diverse words and created less standard associations. This further implies the existence of dysexecutive syndrome and the need for diagnosing patients in this direction and involving this group of people in therapy. The proper correction of MetS components may improve cognitive function.