Background: High levels of xanthophyll carotenoids lutein (L) and zeaxanthin (Z) in the central nervous system have been previously correlated with improved cognitive function in community-dwelling older adults. In this study, we tested the effects of supplementing L and Z on older men and women with a range of baseline cognitive abilities.Objective: The purpose of this study was to determine whether or not supplementation with L+Z could improve cognitive function in community-dwelling, older adults.Design: Double-masked, randomized, placebo-controlled trial. A total of 62 older adults were randomized into groups receiving either 12 mg L+Z or a visually identical placebo. Data from 51 participants (M = 73.7 years) were available for analysis. Retinal L+Z levels  were measured psychophysically using heterochromatic flicker photometry as a biomarker of cortical L+Z levels. Cognitive function was measured using the CNS Vital Signs computerized test platform.Results: Participants receiving the active L+Z supplement had statistically significant increases in MPOD (p < 0.03) and improvements in complex attention (p < 0.02) and cognitive flexibility domains (p < 0.04), relative to participants taking the placebo. A trend was also seen for the executive function domain (p = 0.073). In male participants only, supplementation yielded improved composite memory (p = 0.04).Conclusions: Supplementation with L+Z improved cognitive function in community-dwelling, older men and women. It has long been understood that cognitive function, along with its manifestation as behavior and subjective experience, is a product of the activity of the brain. Hebb , for insIf, however, these neural assemblies are the physical basis of cognition and its age-related change, then if follows that the physical factors involved in forming and maintaining those physical structures would, inevitably, influence the end-product itself. Such factors are often systemic. For example, interferon-Y, a component of the immune system, has recently been shown to regulate neural connectivity and social behavior in mice  are under higher oxidative and inflammatory stress. Late stage intervention could possible lower such stressors helping to retard the cascade that ultimately accelerates the degenerative process ; no previous history of stroke, dementia, Parkinson's disease, or any other neurological condition known to impair cognitive function, with the exception of affective disorders such as anxiety or depression; absence of gastric conditions known to impair absorption of nutritional supplements, such as gastric bypass or gastric ulcer. Inclusion criteria were verified as follows: all participants were given a medical examination by a qualified physician at the University of Georgia Health Center; self-reported health information was obtained; and all participants participated in a structured clinical interview, administered by qualified neuropsychological staff (see below).Of the 80 participants that were screened for enrollment, a total of 62 participants met inclusion criteria and were randomized into one of two groups: the active supplement group, or the placebo group. Simple randomization was conducted by the clinical coordinator, who had no data collection responsibilities. A set of numerical codes was generated that corresponded with either the active supplement or the placebo. The codes were placed in an opaque envelope, and a unique code was drawn for each participant. Of the 62 participants who were randomized, 20 participants were randomized into the placebo group, and 42 participants were randomized into the active supplement group. In the placebo group, two were lost to follow-up (completed baseline but failed to attend subsequent testing sessions) and three were withdrawn due to non-compliance. In the intervention group, four were lost to follow-up and one was withdrawn.The active supplement contained 10 mg L and 2 mg Z. The placebo was visually identical to the active supplement. Supplements and placebos  were contained in identical opaque, sealed bottles with labels that were visually identical, with the exception of the randomization code on the label, and contained instructions for one tablet to be taken from the bottle, daily, with a meal. Compliance to the intervention was monitored by bi-monthly telephone calls and pill counts from bottles returned by the participants during study visits.N = 51) are presented in Table p > 0.48), or education level (p > 0.89).Of the 62 participants who were randomized, a total of two participants from the placebo group and four participants from the active supplement group were lost to follow-up over the year of intervention. Three participants from the placebo group and 1 participant from the active supplement group were withdrawn by study personnel for either failure to maintain inclusion criteria, or because of non-compliance with the study regimen, determined by self-reported failure to take study supplements on a minimum of four of the compliance telephone calls. Baseline characteristics of the analyzable sample  and female  participants in the composite memory domain (p < 0.05). No other significant differences were present between male and female participants at baseline.With respect to cognitive function, significant differences were detected at baseline between male , participants and collaterals were given the Clinical Dementia Rating Scale (CDR) as part of the larger interview Morris, . PotentiRetinal L+Z levels, as macular pigment optical density (MPOD) were measured psychophysically using customized heterochromatic photometry (cHFP)  at four different time points: baseline, and after 4-, 8-, and 12-months of taking the study intervention. Participants were tested in a low distraction environment, in full ambient room lighting, in the presence of a trained research assistant who could answer questions about the test procedures should they arise. Each participant completed a practice session prior to each individual test.Raw scores from individual functional tests taken during the test session were used to compute performance on the following larger cognitive domains: Verbal Memory (VeM), Visual Memory (ViM), Reasoning Ability (R), Executive Function (EF), Psychomotor Speed (PmS), Cognitive Flexibility (CF), and the Neurocognitive Index (NCI). For example, in order to gauge performance in CF, errors on the shifting attention test and commission errors on the Stroop task were subtracted from correct responses on the shifting attention test. For a complete list of cognitive tests administered during the battery, as well as functional domains computed and analyzed, see Table a priori hypotheses were directional in nature . Prior to enrolling subjects, a power analysis was conducted to determine what sample size would yield 1−β = 0.80 for a difference of 0.10 log units of MPOD. The current sample size yielded statistical power of 85.7%. A statistically significant increase in MPOD in the supplementation group was analyzed as the primary outcome variable in this study, and improvements in cognitive function in the supplemented group relative to the placebo group were analyzed as secondary variables.Statistical analyses were performed using SPSS version 23 (IBM), with α = 0.05. Tests of cognitive function were one-tailed, as M = 0.58, SD = 0.23; p < 0.03) in the group that received the active supplement. The placebo group did not change significantly over the course of the year  was comparable to other published data on a different sample with approximately the same average age, recruited from the same geographic region (0.47) . Given the relatively low sample size present in this study, the lack of statistical significance is not surprising.At baseline, participants in the active supplement group were not significantly different from participants who were randomized into the placebo group on any of the cognitive domain scores analyzed or on global cognitive health, as measured by the CDR , and a trend was seen for errors of attention in the complex attention domain . A trend for relation between MPOD and verbal memory  was also present for those participants whose MPOD improved from the baseline to the 12-month time point, regardless of whether or not they received the supplement. Within the supplementation group, participants with the greatest changes between the baseline and 12-month time points in cognitive function in the reasoning  and complex attention  domains also tended to have the highest MPOD at the 12-month time point. Trends for the relationship between magnitude of cognitive change and 12-month MPOD were also seen for the visual memory  and cognitive flexibility  domains.When correlations between MPOD and cognitive function were analyzed at the 12-month time point, MPOD was significantly related to performance in the reasoning domain  and cognitive flexibility  domains, relative to participants taking the placebo. A trend was also present  for the executive function domain .Given the fact that participants were tested four times throughout the course of the study, and given the fact that participants were given practice sessions prior to each active test session, practice effects were anticipated. In order to determine whether or not change was reliable and meaningful, the Reliable Change Index (RCI) was computed for each cognitive index and group, with a standard criterion of 1.96. Participants who took the active supplement had significantly improved performance in complex attention  in a sample of older adults.This study was designed as a year-long intervention with the dietary carotenoids L and Z. These plant pigments, long known for their effects on systemic and ocular health, have been identified in brain , well-nourished and educated, group such as was sampled in our study. As with any dietary experiment, there were no true placebos, as the term is typically used in pharmaceutical studies: subjects have been exposed to the “intervention” all of their life . Even during the intervention year, the control group maintained their normal diet which contains L and Z. Hence, any effect of L and Z on the treatment group would therefore have to be simply additive. Using this framing, the research question becomes: does adding LZ in supplement form to relatively well-nourished subjects with normal LZ intake improve cognition in a sample that was already well-educated? Education tends to attenuate any relation between diet and cognition (Akbaraly et al., Authors LR, BH, and LM contributed to research study design; LR, BH, LM, CL, and CM collected study data; MB served as clinical coordinator; LR, BH, and LM contributed to data analysis; LR and BH drafted the initial manuscript; LH, BH, and LM primarily edited the manuscript; LR, BH, LM, CL, CM, and MB assume responsibility for the final content of the manuscript.ClinicalTrials.gov number, NCT02023645.During a portion of data collection, author LR was employed by Abbott Nutrition. LR is now solely employed by the University of Georgia.Authors LR and BH have received honoraria from Abbott Nutrition for presentation of research findings. The other 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.


Anthropometric data collected in clinics and surveys are often inaccurate and unreliable due to measurement error. The Body Imaging for Nutritional Assessment Study (BINA) evaluated the ability of 3D imaging to correctly measure stature, head circumference (HC) and arm circumference (MUAC) for children under five years of age. This paper describes the protocol for and the quality of manual anthropometric measurements in BINA, a study conducted in 2016–17 in Atlanta, USA. Quality was evaluated by examining digit preference, biological plausibility of z-scores, z-score standard deviations, and reliability. We calculated z-scores and analyzed plausibility based on the 2006 WHO Child Growth Standards (CGS). For reliability, we calculated intra- and inter-observer Technical Error of Measurement (TEM) and Intraclass Correlation Coefficient (ICC). We found low digit preference; 99.6% of z-scores were biologically plausible, with z-score standard deviations ranging from 0.92 to 1.07. Total TEM was 0.40 for stature, 0.28 for HC, and 0.25 for MUAC in centimeters. ICC ranged from 0.99 to 1.00. The quality of manual measurements in BINA was high and similar to that of the anthropometric data used to develop the WHO CGS. We attributed high quality to vigorous training, motivated and competent field staff, reduction of non-measurement error through the use of technology, and reduction of measurement error through adequate monitoring and supervision. Our anthropometry measurement protocol, which builds on and improves upon the protocol used for the WHO CGS, can be used to improve anthropometric data quality. The discussion illustrates the need to standardize anthropometric data quality assessment, and we conclude that BINA can provide a valuable evaluation of 3D imaging for child anthropometry because there is comparison to gold-standard, manual measurements. The Multicenter Growth Reference Study (MGRS) and subsequent development of World Health Organization (WHO) Child Growth Standards (CGS) in 2006 provided a single set of reference measurements for children around the globe . The WHOThe non-digital, manual measurements currently in use are susceptible to human error , and theThere is recognition that the quality of child anthropometry should be assessed before using and disseminating the data. DHS, MICS and Standardized Monitoring and Assessment of Relief and Transitions (SMART) Survey methodologies include assessment of anthropometric data quality. The assessments are all loosely based on recommendations from a WHO expert committee convened in 1995 , but theThe data we analyze is part of the Body Imaging for Nutritional Assessment Study (BINA), which compared a 3D imaging system to currently recommended non-digital, manual measurements of stature (length and height), arm circumference (MUAC) and head circumference (HC) in 474 children under five years of age in Atlanta, USA. BINA used the AutoAnthro 3D imaging system, a system comprised of an off-the-shelf, low-cost, handheld scanner  and custom software for scanning children developed by Body Surface Translations, Inc. (BST) . In order to draw conclusions on the ability of 3D imaging to replicate manual anthropometry, the study needed to collect gold-standard manual anthropometry. A thorough analysis of manual anthropometry quality was particularly important for BINA because, unlike previous research on 3D imaging for anthropometry, the study sample consisted of children under five years of age, an age group that can be difficult to measure with manual methods because of noncooperation. This paper describes the training, standardization and data collection methods for manual anthropometry in BINA; evaluates the quality of BINA manual anthropometry; and provides some recommendations for achieving gold standard manual anthropometry and standardizing data quality assessment to guide clinicians, researchers and public health program managers.Primary caregivers of all children participating in BINA gave written, informed consent and the study was approved by the Emory Institutional Review Board.All five field staff selected for the BINA Study held college degrees, with three of the five holding a master’s degree at the time of the study. In August 2016 field staff completed a three-week training led by trainers from Emory University who had extensive experience with anthropometry in clinic, survey, and research settings; including experience in the study used to develop the 2006 WHO CGS. Training consisted of theoretical and practical sessions on 3D imaging and manual measurements, and field staff were trained to function as both anthropometrists and assistants. Emory University faculty and field staff developed training materials and a study manual.Training culminated with a three-day standardization test for manual anthropometry at a local daycare center, which consisted of a lead anthropometrist from Emory University and all field staff taking repeated measurements of ten children under five years of age. The lead anthropometrist was considered an expert anthropometrist based on previous experience and a reliability test carried out before BINA. As anthropometrists, field staff were assessed on all measurements except weight, and data was analyzed using ENA Software 2011  and a MiUtilizing convenience sampling we recruited and measured children in facilities, primarily daycare centers, in Atlanta, GA, USA from September 2016 to February 2017. For manual anthropometry we followed measurement techniques used to develop the 2006 WHO Growth Standards , includiDuring data collection sessions, the five field staff split into two teams of two, with the fifth person acting as a floater. The techniques for scans and manual measurements required an assistant, and teammates alternated as the anthropometrist and assistant. Anthropometrists also took turns as a floater, and the main roles of the floater were to prepare children for measurement and to act as a second assistant for younger children. Anthropometry procedures designed for household surveys often contain a role for the child’s parent. However, since we obtained data primarily in daycare centers where the caregivers were not present, a second assistant was needed to hold and position the child.After taking time to acclimate the child and establish rapport, and after undressing the child (to their diaper or to skin-tight shorts/leotards provided by the study), the field team started with 3D scans which was then followed by manual measurements. The first anthropometrist completed one session, measuring head circumference, MUAC, and length or height. Instead of one anthropometrist completing two sessions concurrently, the field team reversed roles and a new anthropometrist completed her first session of manual measurements. After both team members completed their first session, the process was repeated for the second session. We staggered manual measurements to reduce the likelihood of anthropometrists remembering their first measurement. To further minimize bias in inter-observer error, anthropometrists entered their own measurement results and did not inform the assistant of the number, which is different from the typical anthropometry procedure of the assistant recording results on behalf of the anthropometrist.Our custom software for electronic data capture included range checks for non-digital measurements to catch data entry errors. If a measurement was below the 0.01 percentile or above the 99.9 percentile, a pop-up box appeared on the screen and the anthropometrist could either re-enter or accept the value. The software also included automatic triggering of a third measurement for non-digital measurements. The triggers were programmed based on the MGRS standards for the maximum allowable difference  , but ourFor manual anthropometry, field staff received ongoing monitoring and supervision. This included examining digit preference, the percent of intra-observer measurements exceeding the maximum allowable difference, inter-observer TEM, and estimates of bias by comparing to an expert anthropometrist who measured a subsample of children. In addition to comparing to an expert anthropometrist, field staff also periodically compared with each other. Field staff regularly received data quality reports covering all measures of quality and supervision from expert anthropometrists.digdis package for analysis. We tested for any digit preference with Pearson’s Chi Squared Test. We calculated the difference between the expected and observed percentages and tested for preference of each individual digit using a two-sided Binomial Test. To determine how close our observed proportions were to a uniform distribution, we summed the difference between expected and observed for positive differences only. The sum gives the percentage of digits that would need to be reclassified (moved from an overrepresented digit to an underrepresented digit) to achieve a uniform distribution, which is similar to the Myer’s Blended Index [For digit preference, we examined the proportion of non-digital, manual measurements  with each of the possible digits (0–9) in the tenths place (mm). We analyzed the first measurement from each anthropometrist. Since each child was measured by two different anthropometrists, we included 948 observations in the analysis. We used the Stata SE 13  ed Index  and the ed Index . We calcWe used the 2006 WHO Growth Standards to calculate z-scores for weight-for-length/height (WHZ), length/height-for-age (HAZ), weight-for-age (WAZ), head circumference-for-age (HCZ), and arm circumference-for-age (ACZ). We assessed the plausibility of z-scores by determining the proportion of measurements flagged as falling outside of the plausible range as defined in WHO macros  . We alsoMi1Mi2 are the closest repeated manual measures for one child by one observer. For inter-observer TEM we compared the average measurement of one observer to the average measurement of another observer for the same child, changing the numerator in the equation above:We analyzed both intra- and inter-observer error for height, HC and MUAC using repeated measurements. Intra-observer technical error of measurement (TEM) was calculated with the following formula:g by 100 . Total T effects  and an aSample characteristics in All measurements  showed evidence of terminal digit preference when we tested the child’s first measurement with Pearson’s Chi-Squared Test . For all measurements, the terminal digit four was significantly overrepresented. In addition, eight was overrepresented for HC, and six was overrepresented for MUAC . The sumUsing means of repeated measures and cutoffs defined by WHO for biologically plausible z-scores one child (0.2%) was flagged, falling above the range for WHZ, and ACZ. Also, the length of one child (0.2%) was lower than the plausible stature range. With single measures, the length of one additional child was lower than the plausible stature range, and no additional children had implausible z-scores.For means of repeated measures, the standard deviation for all z-scores  was close to 1.0, ranging from 0.92 for WHZ to 1.07 for HAZ. Standard deviations using single measures were slightly higher, with the largest differences in ACZ (0.03) and WHZ (0.02) . With inrds of repeated measurements were within ±0.22 cm and 95% of replicate measurements were within 2*TEM, or ±0.44 cm. Inter-observer TEM was higher than intra-observer for all measurements: stature TEM 0.34 cm, %TEM 0.42%; HC TEM 0.25 cm, %TEM 0.54%; and MUAC TEM 0.19 cm, %TEM 1.22% (The correlation between an anthropometrist’s first and second measurement of a child (intra-observer), and between two anthropometrists’ average measurements of a child (inter-observer) was near perfect. The Intraclass Correlation Coefficient for all intra- and inter-observer measurements was exactly 1.00  except for inter-observer arm circumference, with ICC of 0.99 . Intra-observer TEM in centimeters was 0.22, 0.13, and 0.16 for stature, HC, and MUAC respectively. Intra-observer TEM values corresponded to relative TEMs of 0.26%, 0.29%, and 1.04% respectively . As an iEM 1.22% . AlthougOur data shows that manual anthropometry in BINA was of excellent quality. Only 0.4% of the sample had biologically implausible z-scores or measurements, which is well below the 1.0% cutoff recommended by a WHO expert committee as an indicator of data quality problems . Our z-sIn our study, we achieved high quality manual anthropometry through following established protocols and adding additional quality control components. We followed advice from MGRS protocol authors to develop a study specific training manual, train and test staff with standardization, and provide adequate supervision during data collection . For meaThere have been suggestions to take repeated measures in surveys . Our resThere is some consensus on what to assess for anthropometric data quality. A 1995 WHO expert committee recommended looking at accuracy, reliability, biological plausibility, z-score standard deviation, and digit preference ; and theWe can consider z-score standard deviation to illustrate the importance of reaching consensus on interpretation and action. WHO and the US CDC promote the use of normative ranges of SD to determine if survey quality is acceptable , 26, butWe designed BINA as a validation study for 3D imaging and did not set out to evaluate how to improve the quality of manual anthropometry. The study design did not allow us to determine the effect of individual protocol components, such as repeated measurements, on data quality. Future studies designed specifically to evaluate quality improvement could help to determine the components of the protocol that are the most cost-effective for quality anthropometry. An additional limitation to our study was that convenience sampling resulted in a sample that did not have normally distributed age, and our findings cannot be extrapolated to a specific age group. Also, while we tried to follow every aspect of the WHO MGRS measuring protocol [The high quality manual measurements in BINA can likely be attributed to highly motivated and competent field staff, reduction of non-measurement error through the use of technology, and reduction of measurement error through adequate monitoring and supervision. We made slight improvements to training materials and added to the MGRS protocol for quality control by utilizing electronic data capture and including field staff in the development of the study manual. BINA methodology can be followed to improve anthropometric data quality, but will need to be adapted for use in some contexts. For example, BINA field staff were highly educated and reported child age is accurate in the US; in a different context different strategies may be needed to allow field staff to contribute to manual development, and double data entry may not be sufficient to ensure quality age data. Furthermore, for systematic improvement of anthropometric data in surveys the availability of adequate methodology may not be enough. The lack of consensus on how to interpret and act on analysis of anthropometric data quality leads to survey results being published without reproach, which limits the institutional need and motivation to improve quality. In addition, it may not be feasible to follow the full anthropometry protocol in a clinical setting. Ultimately, while high quality anthropometry is possible with available manual equipment, improved technology may be the most efficient driver of widespread quality improvement; and BINA can provide a good evaluation of 3D imaging technology for child anthropometry because manual measurements were of excellent quality.


Setaria viridis is an emerging model for C4 species and it is an important model to validate some genes for further C4 crops transformation, such as sugarcane, maize, and wheat. In the present work, Setaria viridis was used as a model plant to overexpress a newly identified MATE gene from Brachypodium distachyon (BdMATE), closely related to SbMATE, for aluminum tolerance assays. Transgenic S. viridis plants overexpressing a BdMATE presented an improved Al tolerance phenotype, characterized by sustained root growth and exclusion of aluminum from the root apex in transgenic plants, as confirmed by hematoxylin assay. In addition, transgenic plants showed higher root citrate exudation into the rhizosphere, suggesting that Al tolerance improvement in these plants could be related to the chelation of the metal by the organic acid anion. These results suggest that BdMATE gene can be used to transform C4 crops of economic importance with improved aluminum tolerance.Acidic soils are distributed worldwide, predominantly in tropical and subtropical areas, reaching around 50% of the arable soil. This type of soil strongly reduces crop production, mainly because of the presence of aluminum, which has its solubility increased at low pH levels. A well-known physiological mechanism used by plants to cope with Al stress involves activation of membrane transporters responsible for organic acid anions secretion from the root apex to the rhizosphere, which chelate Al, preventing its absorption by roots. In sorghum, a membrane transporter gene belonging to multidrug and toxic compound extrusion (MATE) family was identified and characterized as an aluminum-activated citrate transporter gene responsible for Al tolerance in this crop.  Aluminum is extremely rhizotoxic, affecting root growth and function and therefore limiting crop production and its toxicity, along with drought, is a primary food security concern , a membrane transporter gene belonging to the MATE family was identified and characterized as an aluminum-activated citrate transporter gene responsible for the Al tolerance in this crop, codifying for a proton-dependent transporter protein  demonstrated an Al tolerant phenotype and increased root citrate exudation when compared to NT plants (MATE family from S. bicolor (SbMATE) is an Al-activated citrate secretion transporter conferring aluminum tolerance. In addition, it was demonstrated that MATE homologs are root citrate transporters in other monocot species such as maize , when expressed in tomato, was able to increase Al resistance, accompanied by enhanced citrate efflux , Brachypodium distachyon MATE (BdMATE), under the control of the maize ubiquitin constitutive promoter (ZmUbi-1) for aluminum tolerance. The BdMATE gene was chosen because it is up-regulated under Al conditions . The rationale was to use a close homolog of SbMATE and a patent-free gene, in order to use as proof of concept to further transform C4 crops of economic importance such as sugarcane, wheat, and maize, which are difficult and time-consuming to transform. S. viridis plants overexpressing BdMATE showed an Al tolerance phenotype, characterized by sustained root growth under {20} μM Al3+, whereas NT plants showed RGI. Moreover, exclusion of aluminum from the root apex in transgenic plants was inferred based on results from hematoxylin assays. In addition, transgenic plants constitutively overexpressing BdMATE presented higher citrate secretion levels when compared to NT plants under Al stress, suggesting that the mechanism of organic acid anion exudation and consequent Al chelation by these compounds are involved in the Al toxicity amelioration in these plants. The expression of aluminum tolerance-related genes was evaluated, and the results obtained are discussed based on the known Al tolerance. Our results demonstrated that, in the future, the overexpression of BdMATE could be used to develop Al-tolerant crops closely related to S. viridis, such as maize, wheat, and sugarcane. studies . It is a studies , making protocol . In the nditions , whereasSorghum bicolor MATE (SbMATE) gene in the Phytozome database were performed in order to find orthologs in the genome of B. distachyon. Based on the different sequences found, the alignment of these sequences was generated using the Clustal Omega  gene that confers aluminum tolerance in different species , driven by ZmUbi1 constitutive promoter and the hpt gene selection marker (which confers hygromycin resistance), was synthetized by DNA Cloning Service, Germany , at different concentrations, . Transgenic and NT plants were evaluated for liquid root growth, and the percentage of RGI in treatments in the presence or absence of aluminum was calculated as described in A protocol was established in order to evaluate the phenotype of Hoagland , CamargoHoagland , and MagHoagland  , according to the manufacturer’s instructions. RNA from roots was extracted using a LiCl method (TM First Strand cDNA Synthesis kit (Thermo Fisher Scientific). These steps were all performed according to the manufacturer’s instructions. The qPCR was carried out using Platinum® SYBR® Green PCR SuperMix-UDG with ROX  with synthesized single-stranded cDNA as template, using the protocol recommended by the StepOnePlusReal-Time PCR Systems (Applied Biosystems). The primers used in the qPCR are described in Supplementary Table After 7-days-old plantlets had been submitted to 500 μM CaCll method . The samSiEF1-α; SiSUI, SiCAC, and SiCUL were used as reference genes  was composed of 40 plantlets bulked together for extraction . The extraction was normalized to the root biomass for each line. The solutions were lyophilized for organic acids analysis on a gas chromatography–mass spectrometry (GC-MS) system (Varian GC 3800 and MS 2000). The lyophilized material were washed in pure methanol and dried under vacuum. To the dried samples were added 100 μL of pyridine and 50 μL of BSTFA trifluoroacetamide) + 1% TMCS (trimethylchlorosilane). The analysis of GC/MS was performed according to -1. The analysis was performed under the following temperature program: 5 min of isothermal heating at 70°C, followed by a 5°C min-1 oven temperature ramp to 310°C, and a final 1 min of heating at 310°C. Mass spectra were recorded at 2 scan s-1 with a scanning range of 50–600 m/z. Both chromatograms and mass spectra were evaluated using the OpenChrom software . Samples for all analysis were collected at days 1, 3, and 5. Differences among treatments per sample were analyzed using t-test, considering p < 0.05 as significant.Experimental data were analyzed using randomized block design (RBD) with replications for each treatment  gene showed a close similarity to the SbMATE gene . The selected amino acid sequence of B. distachyon MATE protein (BdMATE – Bradi1g69770.1) were aligned using the Geneious software . The sequence Bradi1g69770.1 was chosen for the present study due to its close similarity to the S. bicolor MATE sequence.In order to find orthologous sequences of software  with thesoftware  . In the absence of the metal, both NT plants and the transgenic line 29.2 showed a similar root growth pattern, while the line 28.1 demonstrated a lower root growth even in control conditions .The most common criterion used to measure Al toxicity is the comparison of the root length of Al-affected plants with control plants grown in the absence of aluminum . In the Figure 3A) and the percentage of the RGI  confirmed the Al tolerance phenotype demonstrated by BdMATE transgenic plants. It is worth to mention that the morphology of the roots was not affected from the constitutive expression of the BdMATE gene. A RGI above 90% was observed in NT plants after 5 days in {20} μM Al3+ . Under the same conditions, transgenic plants overexpressing BdMATE demonstrated from 30 to 65% of RGI for the events 28.1 and 29.2. According to BdMATE overexpression did not fully eliminate the toxic effects of Al, but ameliorates such effect.Quantification of the relative net root growth , while in the transgenic plants in the same conditions the blue-purplish color representing Al accumulation is absent. The staining of the plants is not observed in plants not submitted to Al . These results suggest that NT plants are accumulating higher concentrations of Al than transgenic plants, indicating that tolerant plants are avoiding the absorption of Al by the roots.Hematoxylin assay was performed after 24 h of plants exposed to {0} or {20} μM AlBdMATE, when overexpressed in S. viridis plants, was able to increase the levels of this anion in transgenic plants when compared to NT plants . In the presence of {20} μM Al3+, the transgenic lines presented a threefold increase of citrate exudation in comparison to the NT plants, suggesting that BdMATE plants are conferring Al tolerance through the mechanism of citrate release into the rhizosphere.The measurements of root citrate secretion demonstrated that a new MATE homolog, Figure 4 demonstrated that no difference of CS (SiCS) and MD (SiMD) gene expression levels were observed between transgenic and NT plants, in the absence or presence of Al. The signaling of Al tolerance in plants is still a matter of debate and therefore more studies are necessary in order to elucidate the relationship between the levels of citrate produced by the TCA cycle with Al tolerance in plants. The results obtained on gene expression levels will be discussed in the latter section.The S. viridis MATE gene (Sevir.9g493200.2) were verified to demonstrate the involvement of SvMATE in Al stress responses. The Supplementary Figure SvMATE expression levels did not change in plants submitted to Al when compared to plants in the absence of the cation, indicating that the endogenous SvMATE does not participate in aluminum stress responses in S. viridis. Although the similarity of the MATE sequence of S. viridis and B. distachyon is 83.2%  was constitutively overexpressed in Setaria viridis (BdMATE gene was chosen instead of the S. viridis MATE gene (SvMATE) because BdMATE is a close homolog of SbMATE expressed under Al conditions . The results obtained with the overexpression of BdMATE in S. viridis plants clearly showed an Al tolerance phenotype in these plants . The main symptom of aluminum toxicity is inhibition of root growth, which is observed consistently in plants sensitive to this metal  and RNRG  when compared to NT plants. On the other hand, the percentage of RGI of transgenic plants under Al stress was lower when compared to the control . However, it is worth to mention that the root growth of transgenic lines was lower in the presence of Al than in the absence of this metal, indicating that BdMATE ameliorates but do not completely eliminates the phytotoxicity effect of this cation.Sugarcane is a very important economic crop for Brazil nowadays. Brazil is the largest sugarcane producer worldwide, and its production is intended mainly for ethanol and sugar production. In addition, the biomass released from the sugarcane process can be used as lignocellulosic material to be degraded by microorganisms to generate renewable fuels and added-value products. Despite of its great importance, the regions for sugarcane growth in Brazil are becoming scarce. An alternative for growing sugarcane could be the Cerrado region, characterized by its soil with acidic pH. Therefore, a great effort has been made by the scientific community in order to improve sugarcane traits for its growth in poor soils. ormation . In the  viridis , in ordenditions , while Sis metal . In the BdMATE plants did not accumulate Al in the root apex, a phenotype demonstrated by NT plants . The primary site of aluminum accumulation and damage is still a matter of debate. In many species, the root apex was identified as the primary site of perception of aluminum toxicity, and consequently the expression of tolerance . Thus, it is worth to verify the gene expression profile of target genes related to Al tolerance in different portions of S. viridis roots in non-transformed or transgenic plants submitted to Al stress, an approach that is currently being performed by our group.Furthermore, hematoxylin staining, a common method used to monitor the location and aluminum accumulation in plants , demonstolerance . Sivagurolerance , maize . Organic acids are metabolically active solutes with a diverse array of functions in many organisms. These acids have a potential role in the osmotic adjustment and in the balance of cation concentration of the cells, for instance. As described above, OAs anions are secreted by the root apex as an aluminum resistance mechanism in most plants. Citrate, malate, and oxalate are the main OAs anions secreted to the rhizosphere in many plant species .As described above, the exudation of organic acid anions by the root apex is a common mechanism to confer Al tolerance in plants, as these compounds are able to chelate this metal in the rhizosphere, avoiding root absorption. It was demonstrated that MATE homologs were root citrate transporters in different monocot species such as maize and rice . Here, insporter  in S. vi species . Organic species . The acc species . RecentlTriticum aestivum L. (wheat) and Secale cereale L. (rye), the evaluation of CS production in aluminum stress conditions have been performed aluminum . Thus, it could be very difficult to observe drastic changes in the expression levels of a gene that is involved in a fine-tune regulation of pivotal metabolic processes in the cell. Therefore, more studies are necessary in order to elucidate the relationship between the levels of citrate produced by the TCA cycle with Al tolerance in plants. We were not able to measure if the transformed plants had a carbon loss penalty compared to NT plants, especially because the experiments were performed in hydroponic conditions during a short time. We are currently establishing Al stress experiments in soil to further study the biomass of plants transformed with BdMATE. In addition, root-specific and/or Al-induced promoters are being currently studied by our group, to minimize the putative carbon loss caused by the constitutive expression of BdMATE.The relationship between increased CS activity and Al tolerance is still inconclusive. In some species such as aluminum . Plantlealuminum . Our resegulated . The CS B. distachyon MATE transporter gene (BdMATE) was more tolerant to aluminum stress than wild plants through the exudation of citrate into the rhizosphere, with subsequent avoidance of Al absorption and accumulation in the DTZ of the roots. As consequence, the transgenic plants demonstrated sustainable root growth in the presence of Al when compared to NT plants. Our results suggested that, in the future, the overexpression of BdMATE could be used to develop Al tolerant crops closely related to S. viridis such as maize, wheat, and sugarcane.Altogether, our results suggest that a C4 plant overexpressing a in silico analysis. AR, WRS, FV, and KD performed the abiotic stress assays. AR, BC, and FV carried out the RT-qPCR assays and analyzed the data. AR, PM, WRS, and AK performed hematoxylin assay. PAO, RC, DC, WRS, MB, and HM performed and analyzed the organic acids. WRS and AR wrote the manuscript. AR, WRS, GC, JVM, CS, MB, and AA contributed to the discussion of the results. HM, AK, and GC provided intellectual input and revised the manuscript. All authors read and approved the final manuscript.HM, AK, AR, PM, WRS, and AA conceived and designed the experiments. PM and AR performed the 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.


To investigate orbital vascular resistance by Doppler velocimetry in patientswith steady-state sickle cell anemia, as well as to characterize itsrelationship with biomarkers of hemolysis.This was a cross-sectional study of two groups: 71 outpatients with sicklecell anemia; and 32 age- and gendermatched, healthy subjects (controlgroup). All participants underwent Doppler velocimetry of the orbitalarteries and laboratory tests.p < 0.0001 vs. controls). In the patientgroup, Doppler velocimetry revealed increased flow velocities in theophthalmic artery and reduced flow velocities in the central retinal artery,as well as high values for the resistance index (RI) and pulsatility index(PI) in both arteries (p < 0.0001 vs. controls).Biomarkers of hemolysis were found to correlate significantly with the RIand PI indices. In the ophthalmic artery, the reticulocyte count showed amoderate direct correlation with RI and with PI. In the central retinalartery, hemoglobin showed a strong inverse correlation with RI and withPI.All biochemical laboratory markers were abnormal in the sickle cell anemiapatients (Orbital vascular resistance, as evaluated by Doppler velocimetry, is elevatedin patients with steady-state sickle cell anemia and shows a significantcorrelation with biomarkers of hemolysis. Estimates suggestthat 250,000 children across the globe are born annually with HbSS. Brought to theAmericas through the slave trade, HbSS is most common in communities whereindividuals of African descent predominate. It is estimated that approximately 2,500children are born annually with HbSS in Brazil. Non-whites were estimated at 50.74%of the population in the 2010 Census, and 1–6% of such individuals reportedly haveHbSS.Sickle cell anemia, also known as homozygous sickle cell disease (HbSS), is the mostcommon hereditary hemoglobinopathy,5. Hemolysis alsoplays a significant role in the pathogenesis of the disease, possibly by reducingthe bioavailability of the nitric oxide produced by vascular endothelialcells. The clinicalmanifestations of this multisystemic disease stem from chronic hemolytic anemia andfrom the vaso-occlusive effects of the sickle cells, which affect nearly all tissuesin the body, including those of the eyes.Sickle cell disease is characterized by the abnormal production of hemoglobin, alsoknown as hemoglobin S. Sickle-shaped erythrocytes are formed during thepolymerization and intracellular precipitation of hemoglobin S in its deoxygenatedstate, creating cellular rigidity that reduces the microvascular blood flow, leadingto tissue ischemia and cardiac arrest,and can be grouped into proliferative and nonproliferative forms. In the retina,various pathological processes may occur  than in those with HbSS. Earlierstudies have described Doppler velocimetry findings in intracranial, pulmonary,renal, and peripheral arteries in patients with HbSS,10-14. To our knowledge, however,little is known about the orbital vascular changes in patients with HbSS, or aboutthe relationships such changes have with biomarkers of hemolysis. The objective ofthis study was to investigate the state of arterial vascular resistance in andaround the eye by Doppler velocimetry in patients with HbSS and to determine itsrelationship to biomarkers of hemolysis.Doppler ultrasound of the eye permits the noninvasive evaluation of arterial vascularresistance through the calculation of two indices-the resistance index (RI) and thepulsatility index (PI)-especially in the study of blood flow in oculardiseasesThis was an analytical cross-sectional study conducted from September 2012 to October2013 at in the Department of Diagnostic Imaging and the PediatricHematology/Oncology Outpatient Clinic at the University Hospital of the FederalUniversity of Sergipe.n = (Zα/2 *σ/E)2,where: Zα/2 represents the critical value of Z for aconfidence interval of 95%; σ is the standard deviation in thepopulation; and E is the margin of error. Because the value of s is unknown, itwas replaced with the standard deviation in the sample (s). Based on apreliminary pilot study involving 39 patients, the values s =13.05 and E = 3.0 were calculated, whereas a value ofZα/2 = 1.96 was obtained from the statistics table,resulting in an estimated sample size of 73 patients.To calculate the sample size (n), we employed the following formula:Two groups  were examined. One group composed of 73patients with HbSS (confirmed by hemoglobin electrophoresis), of whom 38 weremale. Ages ranged from 3 to 31 years. Those 73 patients were under regularfollow-up treatment at an outpatient clinic, were in a compensated state, andhad not had a severe pain episode or required a blood transfusion in the lastthree months. "Compensated" here means the patient is in a basal state withoutacute pain requiring hospitalization. The other group was composed of 32apparently healthy controls, matched for age and gender, of whom 16 were male.Ages ranged from 3 to 27 years. The patients in the control group had noclinical history of pathological changes. The controls were recruited by randomselection of patients undergoing routine ultrasound examination of unrelatedorgans and systems in the Department of Diagnostic Imaging. None of the patientsin either group showed objective signs of ocular involvement.Of the group of 73 patients with HbSS, 2 were excluded for lack of cooperation.None of the remaining participants were lost to follow-up. The final sampletherefore consisted of 71 patients and 32 controls. The study was approved bythe Research Ethics Committee of the University Hospital, and all participantsgave written informed consent.,16. The patients were examinedin the supine position, looking straight ahead but with their eyes closed. Adrop of gel was applied to the transducer, which was then positionedtransversely on the upper eyelid. The examination was performed undercomfortable conditions with a non-compressive technique to avoid pressure fromthe transducer on the eyelid interfering with the results of the Dopplervelocimetry. The ophthalmic and central arteries of the retina were identifiedwith the aid of the color mode, which helped in the positioning of the samplevolume, which was adjusted between 2 mm and 4 mm at the smallest possible angleof insonation, for the measurement of the Doppler velocimetry variables. Thefollowing Doppler velocimetry parameters were evaluated: peak systolic velocity(PSV), final diastolic velocity (FDV), mean velocity (MV), RI, and PI. Eachparameter was measured only after at least three consecutive images of similarappearance had been observed in the spectral mode. Measurements were obtainedwith the resources available in the ultrasound system software. The RI and PIwere calculated in a conventional way with the following formulas: RI =(PSV – FDV)/PSV and PI = (PSV – FDV)/MV. Theaverage of three readings of each parameter for each artery in both orbits wasobtained and recorded for each participant. The average duration of theultrasound examination was 10–15 min per patient, depending on subjectcooperation in keeping their eyes immobile. Heart rate and blood pressure weremeasured prior to the procedure. At the time of the examination, all of theparticipants had a normal heart rate and were normotensive. All examinationswere performed by the same radiologist (with seven years of experience), who wasblinded to the allocation of the subjects.The ultrasound evaluation was conducted with a Logiq P6 system , which has color and pulsed Doppler capability, with alinear (11L), multifrequency (3.4–10.8 MHz) transducer, according to astandardized technique, we considered hemoglobin levels, reticulocytecounts, lactate dehydrogenase levels, and  bilirubinlevels.Blood samples were collected for complete blood counts, obtained with anautomated analyzer , and for erythrocytecounts, as well as for the determination of hemoglobin, hematocrit, meancorpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobinconcentration, and the reticulocyte count. The biochemical investigation wascarried out in an automated hematology analyzer , with the determination of the total and indirect levelsof bilirubin, as well as that of the level of lactate dehydrogenase. Asbiomarkers of chronic intravascular hemolysist-test orMann-Whitney test. Multiple linear regression was used in order to determine therelationship between biomarkers of hemolysis and vascular resistance indices (RIand PI). Spearman's test was applied after the regression analysis to measurethe correlation strength of the variables with statistical significance, asdetermined by calculating the linear correlation coefficient(r). The strength of the correlation was categorized, on thebasis of the absolute value of r, as follows: 0 ≤ r <0.2, very weak; 0.2 ≤ r < 0.4, weak; 0.4 ≤r < 0.6, moderate; 0.6 ≤ r <0.8, strong; 0.8 ≤ r < 1.0, very strong. Allstatistical tests were two-tailed, and values of p < 0.05were considered significant. The statistical analysis was conducted using theBioStat program, version 5.3 .The distribution of patient groups and controls was tested for normality of datadistribution (by the D'Agostino-Pearson test). The mean and standard deviationwere calculated for age of the subject, biochemical variables and Dopplervelocimetry variables . For the comparison of groups,the values of p were calculated by unpaired p < 0.0001 for all comparisons).The groups did not differ in age or heart rate . All biop < 0.0001 vs. thecontrol group). The PSV of the central retinal artery was lower in the HbSS group,although the difference between the two groups was not statistically significant. r = 0.62), as wellas between the hemoglobin level and PI (r = 0.63).Because the multivariate analysis showed the RI and PI to correlate significantlywith the reticulocyte count and hemoglobin level, we calculated the value of. Filled with aqueous humor, the anterior chamber is anenvironment with low pH and low oxygen concentration. In the setting of hemorrhage,erythrocytes and leukocytes consume oxygen and liberate carbonic gas and lactate,thus causing acidosis and sickling. The sickle cells obstruct the trabecularmeshwork (responsible for the drainage of the aqueous humor), leading to an increasein intraocular pressure, also known as acute glaucoma, which constitutes anophthalmologic emergency. In the posterior segment, manifestations include retinalhemorrhage and exudate, as well as angioid streaks, chorioretinal infarction,vitreous hemorrhage, occlusion of the central retinal artery or its branches, andproliferative retinopathy. Arteriolar occlusion and loss of capillary perfusion inthe periphery of the retina are the most striking features of sickle cellretinopathy, being more common in HbSC hemoglobinopathy than in the HbSS form andoccurring predominantly in the superior temporal region; ischemic areas releasesubstances that stimulate angiogenesis, and the initial vascular remodeling at thejunction between the nonperfused periphery and the perfused central region promotesthe formation of arteriovenous anastomoses,17.The ophthalmologic complications of HbSS can affect the orbit, conjunctiva, anteriorchamber, and posterior segment. In the orbit, it manifests as orbital compressionsyndrome, caused by infarction of the sphenoid bone with subperiosteal hematomaformation and an inflammatory response. In the conjunctiva, vascular segmentsundergo transient saccular dilation, with a "sausage" or "comma" aspect, notably inthe lower bulbar portion; the "comma sign" is more common in HbSS than in HbSC.Endothelial proliferation and aggregation of erythrocytes in the distal portion ofthe capillaries, together with dilation and thinning of proximal vessel segments,are the predominant histopathological findings; low levels of hemoglobin andhematocrit predispose to conjunctival changes. We observed highresistance to flow in both vessels. However, Aikimbaev et al. detected increased RI and PI onlyin the central retinal artery, without significant changes in the ophthalmic artery.Tantawy et al. found thatophthalmic artery velocities were higher in children and adolescents with sicklecell disease than in healthy controls, although the difference was not statisticallysignificant. The authors followed a protocol involving the use of transcranialDoppler ultrasound, with a standard 2.0-MHz transducer, which provides lower spatialresolution than does the 10.8-MHz probe used in the present study. The higherresolution facilitates the study of superficial structures, with greater sharpnessand sensitivity in the Doppler velocimetry evaluation. That may have contributed tothe detection of statistical significance in our sample.Our data show increased velocities in the ophthalmic artery and reduced velocities inthe central retinal artery, in agreement with the findings of Aikimbaev etal. stated that theretinal artery RI, as measured by Doppler ultrasound, does not correspond to trueretinal vascular resistance, when measured by a much more complex method . The authors found that, after the use of 100% oxygen as aretinal vasoconstrictor agent, the RI and the true resistance both demonstrated anincrease in vascular resistance, although the effect detected by RI was lower. Thoseauthors used transducers with a lower frequency than that of those employed in thepresent study (5.0 MHz vs. 10.8 MHz), and the software accompanying our equipmentwas more advanced than was that available 13 years ago, when Polska etal. published theirdata. Technological advances have allowed the improvement of diagnostic imagingtools in all areas, including Doppler ultrasound, with an increase in the accuracyof the measurements. Calculation of the RI currently allows better estimation ofretinal vascular resistance than in the past decade. In addition, its use is morepractical, available, and reproducible than the complex methodology proposed byPolska et al., as well asbeing much more affordable and not compromising the clinical evaluation of thepatient.Polska et al., compared with 163 ± 17µm for the central retinal artery; that is, the diameter of the central retinal artery isapproximately ten times smaller than is that of the ophthalmic artery. The expectedeffect of vasoconstriction on the ophthalmic artery is increased flow velocity, asoccurs in other arteries during pathological processes that promote luminalnarrowing, such as atheromatous plaque stenosis in the internal carotid, renal, andfemoral arteries. In contrast, the effect of vasoconstriction on an artery of verysmall diameter, such as the central retinal artery, could promote a reduction inflow velocity, because vasoconstriction of an already anatomically narrow lumenwould have what amounts to a subocclusive effect. According to Poiseuille's Law, thevolume of flow in a vessel is directly proportional to the fourth power of theradius of that vessel. The central retinal artery has a radius ten times smallerthan that of the ophthalmic artery and would therefore have a flow volume tenthousand times lower. Any vasoconstrictive event, however mild, would have ahemodynamically significant repercussion, which would explain the finding of reducedvelocity. Similar behavior is observed in the small vessels of the bulbarconjunctiva, when evaluated by computer-assisted intravital microscopy, a methodused in order to characterize the microcirculation. In the presence ofvasoconstriction, flow velocity decreases instead of increases.The mean diameter of the ophthalmic artery is 1430 ± 260 µm demonstrated a strong association between reduced levelsof hemoglobin and increased retinal vascular resistance , although we did not identify a statisticallysignificant relationship between such resistance and reticulocyte counts. However,Cheung et al. did not find thehemoglobin level or the reticulocyte count to correlate significantly with the meancerebral artery flow velocities, as measured by transcranial Doppler ultrasound, orwith the velocities in the microcirculation of the bulbar conjunctiva, as evaluatedby computer-assisted intravital microscopy. Neither of the studies cited aboveevaluated the other two biomarkers of hemolysis (lactate dehydrogenase andbilirubin).Regarding biomarkers of hemolysis, our study revealed a strong inverse correlationbetween the hemoglobin level and RI in the central retinal artery, and we found thatthe RI and PI showed a moderate direct correlation with the reticulocyte count inthe ophthalmic artery. Aikimbaev et al.,22,23: vasculopathy/endothelial dysfunction/hemolysis ; andviscosity/vaso-occlusion . The rate of chronic intravascular hemolysis can beestimated by identifying biomarkers, which are characterized as follows,5: low hemoglobin levels; high lactate dehydrogenase levels; highbilirubin levels; and high reticulocyte counts. The literature does not mention thesubphenotype to which sickle cell retinopathy belongs. Some evidence points to thefirst subphenotype: vascular fibroproliferation, which occurs both in proliferativeretinopathy and in pulmonary hypertension-involving nitric oxide depletion and ametabolic shift from arginine and nitric oxide synthesis to proline and collagensynthesis,17,22-andcorrelation with the biomarkers of hemolysis characterized in the present study(hemoglobin levels and reticulocyte counts). The vaso-occlusive manifestations inthe conjunctiva bulbar and retina, however, suggest the secondsubphenotype,21.Further studies, includingmolecular studies, are needed in order to improve our understanding of thesesubphenotypes.Recent studies have classified the clinical complications of HbSS into two distinctsubphenotypesTo our knowledge, there have been no studies explaining why RI and PI correlate onlywith some biomarkers of hemolysis. This is an interesting starting point foradditional investigations. Our study has certain limitations. For example, having asecond radiologist would have allowed us to evaluate interobserver agreement throughdetermination of the kappa index. In addition, for lack of an appropriate diagnostictool, we did not study the retinal microcirculation.The present study demonstrated that arterial vascular resistance in the orbitalarteries, as measured by Doppler ultrasound, is high in patients with HbSS, showingmoderate to strong correlations with some biomarkers of hemolysis.


Although a decrease in the body mass index was seen in the melatonin group, the difference was not significant in the random-effects analysis model. To conclude, in patients on atypical antipsychotics, melatonin at a dose of up to 5 mg/day for a treatment duration of up to 12 weeks attenuated the rise in diastolic blood pressure compared with placebo but had no significant effects on other metabolic parameters.The objective of our study was to determine the effect of melatonin administration on atypical antipsychotic-induced metabolic adverse effects in patients with psychiatric disorders. A systematic search was performed in PUBMED, Cochrane Library, Scopus, Web of Science, and EBSCOhost electronic databases. Randomized controlled trials studying the effect of melatonin on antipsychotic-induced metabolic adverse effects were identified and subjected to meta-analysis. Four studies were included in the meta-analysis, including 57 patients on melatonin and 61 patients on placebo. Melatonin produced a significant decrease in the diastolic blood pressure compared with placebo (mean difference = −4.44 ;  Atypical antipsychotics are a widely prescribed group of psychotropic drugs. While having distinct advantages over the conventional antipsychotics, they are associated with metabolic adverse effects such as obesity, hyperglycemia, and dyslipidemia –3. SinceOwing to the long-term consequences of the metabolic adverse effects, even a partial attenuation of these by administration of melatonin may confer significant benefits to the patients. Wang et al.  have carThe meta-analysis was carried out in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines.Both the authors searched the following electronic databases: PUBMED, Cochrane Library, Scopus, Web of Science, and EBSCOhost. We developed a detailed search strategy for the PUBMED electronic database, and the same was adapted, with suitable modifications, for searching other databases. The MeSH terms and the keywords used for the PUBMED search included, but were not limited to, melatonin, schizophrenia, bipolar disorder, psychosis, antipsychotics, body mass index, body weight, cholesterol, triglycerides, dyslipidemias, LDL, HDL, and fasting glucose. We searched for gray literature, conference abstracts, and related articles in Google Scholar. We also searched the references listed in the selected articles for any relevant citations. There were no language or time limitations included in the search. The search included all articles published in the respective database from inception until May 20, 2017.We initially screened the title and the abstract of the potentially eligible articles. Full-text of the articles considered suitable was obtained and read to confirm their eligibility for the review and meta-analysis. We included only randomized controlled trials which studied the effects of melatonin on atypical antipsychotic-induced metabolic adverse effects. Studies including patients of any age, of both genders, and with a diagnosis of a psychiatric disorder were eligible. We excluded animal studies and clinical studies assessing the effect of melatonin receptor agonists. Any discrepancy or difference of opinion between the authors with regard to the inclusion of a study for the review was resolved by consensus.Both the authors independently extracted the data using a predesigned data extraction form. In the case of missing or inadequate data, the study authors were contacted for obtaining the complete data. The extracted data included the sample size in each group, age, psychiatric diagnosis, dose and duration of treatment of melatonin, atypical antipsychotic and concomitant medications used, body mass index (BMI), body weight, total cholesterol and triglyceride level, fasting blood glucose, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) levels, systolic and diastolic blood pressure (SBP and DBP), and fasting insulin levels.Both the authors independently assessed the quality of the included studies using the Cochrane Collaboration's tool for assessing risk of bias . Any disp values. Since all the included randomized trials compared melatonin with placebo and reported the outcomes as the mean difference from baseline values, a fixed-effect model was used for the meta-analysis. Wherever statistically significant estimates were obtained for the outcomes, we performed a random-effects analysis to determine whether it would influence the outcome estimate. Heterogeneity between trials was determined using the I2 statistic. I2 > 50% was interpreted as being indicative of substantial heterogeneity among studies.We used the reported mean difference and standard deviations (SD) for each group to determine the treatment effect. Statistical analysis was performed using Review Manager (RevMan) computer program . Wherever SD was not reported, the same was imputed using the reported standard error of mean or The primary outcome of interest was the effect of melatonin on the body mass index, fasting glucose, total cholesterol and triglyceride levels, and blood pressure. Secondary outcomes included its effect on waist circumference, LDL and HDL cholesterol, and fasting insulin level.We included four eligible randomized controlled studies with a total of 118 patients, 57 patients on melatonin and 61 on placebo, in the meta-analysis. The details of the search results and selection of studies for the meta-analysis are shown in  Z = 2.08; p = 0.04). The treatment effect in the individual trials and overall is depicted in the forest plot in Q = 5.14; df = 3; p = 0.16; I2 = 42%). We also carried out an analysis based on the random-effects model to determine the sensitivity of the analysis. The random-effects model provided a statistically nonsignificant mean difference of −0.43 in favor of melatonin . Since changes in body weight were reported by all the studies, we used this data to analyze the effect of the intervention. Fixed-effect model analysis showed a mean difference in the body weight of −1.27 in favor of melatonin. However, this estimate did not reach statistical significance . I2 statistic showed presence of significant heterogeneity between the trials .The meta-analysis showed that melatonin treatment attenuated the rise in BMI. When compared with placebo, the mean BMI in the subjects receiving melatonin was less by 0.48 . There was no heterogeneity between the trials .The data on fasting blood glucose levels were reported by two studies  , 19. Met Z = 0.74; p = 0.46). There was no significant heterogeneity between the trials . Analysis using a random-effects model did not alter the outcome .Meta-analysis did not reveal any beneficial effect of melatonin on the total cholesterol levels when compared with placebo . There was no heterogeneity between the trials .Meta-analysis did not reveal any beneficial effect of melatonin on the triglyceride levels when compared with placebo . The treatment effect in the individual trials and overall is depicted in the forest plot in Q = 3.67; df = 3; p = 0.30; I2 = 18%). Analysis using a random-effects model did not alter the outcome .There was a nonsignificant attenuation of the rise in SBP in the melatonin group. When compared with placebo, the mean SBP in the subjects receiving melatonin was lower by −2.70 mmHg . The treatment effect in the individual trials and overall is depicted in the forest plot in Q = 2.30; df = 2; p = 0.32; I2 = 13%). Analysis using a random-effects model did not alter the outcome .The data on DBP was reported by two studies  , 19. Mos Z = 0.44; p = 0.66). There was considerable heterogeneity between the trials .The data on waist circumference was reported by two studies  , 19. Met Z = 0.54; p = 0.59) and HDL  cholesterol levels.Two studies  , 19 repoData on fasting insulin levels was reported by only Modabbernia et al. They did not find any significant intervention effect. Z = 2.10; p = 0.04) but not BMI  or SBP .Since the study by Romo-Nava et al.  includedOur meta-analysis of randomized controlled trials of the use of melatonin in patients with a psychiatric disorder on atypical antipsychotics showed a beneficial effect of melatonin on blood pressure. The melatonin group showed a significantly lesser increase in DBP, but not SBP, following initiation of antipsychotic therapy compared with the placebo group. However, melatonin treatment did not show a significant beneficial effect on other components of MS. While the meta-analysis showed a lesser rise of BMI in patients who received melatonin, this small effect was not significant in the random-effects model. Moreover, analysis of data on body weight and waist circumference (data reported by only two studies) did not show a beneficial effect of melatonin. Also, no difference was seen between the groups with regard to total cholesterol, LDL, HDL, and triglyceride levels. Hence, the only component of MS which was beneficially modified by melatonin was DBP. Although SBP was lower in the melatonin group following treatment, it failed to reach statistical significance.A beneficial effect of melatonin supplementation on blood pressure has been shown in several clinical studies. A meta-analysis involving 221 healthy participants or those with cardiovascular disease showed that melatonin, particularly the controlled-release formulation, was effective in ameliorating nocturnal hypertension, and the magnitude of blood pressure reduction differed between the fast-release and controlled-release melatonin formulations . Severalβ-cells, enhance insulin receptor signaling, and improve glucose tolerance [While a number of studies have described the potential role of melatonin in obesity, the evidence largely comes from preclinical research , 27. Redolerance . Also, iolerance . Howeverolerance . Similarolerance –33.Despite the lack of conclusive evidence, the presence of melatonin activity imbalance in psychiatric disorders and the worsening of its effect by the added adverse metabolic effects of atypical antipsychotics make melatonin an attractive therapeutic option. Considering the lack of apparent benefit of melatonin in the current meta-analysis, except for its effect on DBP, three issues need to be considered. Firstly, the dose of melatonin used in the included randomized trials was 3–5 mg/day, and the duration of treatment was 6−12 weeks. These may not be adequate to bring about the desired changes in patients on antipsychotics . SecondlOur meta-analysis showed that administration of melatonin in patients with a psychiatric disorder on atypical antipsychotics was effective in counteracting the increase in DBP but not SBP or the other components of MS. Future studies will need to take into consideration the dose and formulation of melatonin used, the duration of treatment, and the need for combining it with other treatment strategies to determine its effectiveness in attenuating the metabolic adverse effects of atypical antipsychotics.


Background: In the current study, we investigated the effect of physical activity (PA) on the risk of coronary heart disease (CHD). Methods: In total, 146 cases of CHD and 157 matched controls were included in the study. Data on sociodemographics, lifestyle, and medical history factors were collected using an interviewer-administered questionnaire. A standard World Health Organization (WHO)-based lifestyle questionnaire was used to assess PA. The risk of CHD was analyzed in relation to PA patterns using logistic regression. Results: Vigorous-intensity leisure PA was not associated with a lower risk of CHD. Subjects in the highest tertile of moderate occupational PA had a significantly lower risk of CHD compared to the lowest tertile (adjusted odds ratio (OR) 0.31, 95% confidence intervals (CI) 0.17–0.56). Subjects in the highest tertile of walking hasd an adjusted OR of 0.37 (95% CI 0.20–0.70). Subjects in the medium and highest tertiles of sedentary behavior had adjusted ORs of 2.01 (95% CI 1.06–3.79) and 3.88 (95% CI 2.14–7.02), respectively . Conclusion: The current results showed that both moderate occupational PA and walking protected against CHD. Sedentary behavior increased the risk of CHD. Cardiovascular disease (CVD) is the leading cause of mortality and morbidity worldwide, measured by disability-adjusted life years ,2. AccorIn the Arab world, ischemic heart disease was the leading cause of mortality in 2010, contributing to 14.3% of all deaths . This haNumerous studies have shown a strong positive association between lifestyle factors—such as unhealthy dietary pattern ,7, insufA few studies have been conducted in the Arab world ,19,20 orOur study is an ongoing cross-sectional study to investigate the prevalence and effect of lifestyle factors (mainly PA) on the risk of CVD. The study also examines other risk factors such as dietary pattern, prevalence of exposure to outdoor and indoor air pollution, and smoking. In the current analyses, participants include cases of coronary heart disease (CHD) and control subjects from Prince Sultan Medical Center, also known as Riyadh Armed Forces Hospital, located in Riyadh City, Saudi Arabia. Cases of CHD were defined as patients who were diagnosed with the disease in the past year. Eligible cases were identified among those admitted to the coronary care unit with a doctor’s diagnosis confirmed by a cardiologist based on clinical presentation and laboratory examinations with any of the following: ST/non-ST elevation myocardial infarction, stable/unstable angina, or heart failure. Initially, we identified 207 cases of CVD. Several cases either refused to participate or were excluded for a variety of reasons, including being too sick to complete the questionnaires, living outside Saudi Arabia, or having been diagnosed with the disease more than one-year ago. At least one control for each case was recruited from the same hospital, matched by age (±5 years) and gender. The controls have never been diagnosed with CVD and were admitted for reasons other than diabetes, hypertension, dyslipidemia, and respiratory problems, and were not obese or overweight. Controls were recruited from non-CVD-related clinics, such as ear, nose and throat (ENT), orthopedic, and radiology, and were selected based on the matching criteria of gender and age. In total, 235 subjects were approached or recruited as controls. Exclusion was based on several criteria, including obesity. Some of the controls that refused to participate indicated that the main reason was that they did not have time. The remaining control subjects showed no interest in participating in the study.Ethical approval was obtained from the Internal Review Board of King Abdulla International Medical Research Centre, King Saud Bin Saud University for Health Sciences (grant SP15-086). Each participant provided written informed consent.Trained researchers administered the structured questionnaire in a uniform and standardized manner. The questionnaire included questions about demographic variables  and family history of CVD, diabetes mellitus, hypertension, and hyperlipidemia.http://www.who.int/chp/steps/GPAQ/en/). The part of the survey on PA contained questions about average frequency (days/week) and duration (hours/day) of moderate- and vigorous-intensity occupational PA and leisure time PA, along with frequency and duration of walking. According to the questionnaire, “vigorous-intensity activities” are activities that require hard physical effort and cause large increases in breathing or heart rate, whereas “moderate-intensity activities” are those that require moderate physical effort and cause small increases in breathing or heart rate. Subjects were also asked about the number of hours per day spent in sedentary behavior apart from sleeping . Hours per week of moderate- and vigorous-intensity PA and walking were converted to metabolic equivalents (METs)/week based on the following values: 3.6 METs for moderate PA and walking and 7.1 METs for vigorous-intensity PA. Total METs/week for each participant were computed by adding all METs for each type of PA. Values for METs were derived from the studies of Gong et al. (2013) [To assess PA, we used the Arabic version of the WHO Global Physical Activity Questionnaire (GPAQ) for PA surveillance (. (2013)  and Jett. (2013) . Variablhttp://www.who.int/chp/steps/instrument/en/). This included questions about the frequency (number of days/week) and amount (number of servings/day) of consumption of fruits, vegetables, dairy products, meat products, seafood, and traditional diet.Smoking status was evaluated according to history of cigarette and waterpipe smoking . Information on the number of years of smoking and packs per week was also obtained from the subjects. In addition, exposure to environmental tobacco smoke at home and at work was evaluated . Weight and height measurements were taken to obtain body mass index (BMI). Dietary pattern was assessed using the dietary assessment section of the WHO STEPwise approach to surveillance of noncommunicable diseases  and 95% confidence intervals (CIs) were calculated for the different variables. The multivariate logistic regression model was adjusted for age, gender, education, and smoking, as well as other variables that resulted in p < 0.1 in the univariate model. Continuous variables of different types of PA and dietary patterns were converted into categorical variables of tertiles and were entered into the multivariate models. The median value of the lowest tertile was used as the reference in these analyses. To further examine the relationship between sedentary behavior and the risk of CHD, we used a semi-parametric regression model with natural cubic splines. These methods are based on smooth polynomial functions to examine nonlinear relationships of risk and outcome [Data were analyzed using SAS statistical software version 9.4 . Chi-square tests and paired  outcome . Effect A total of 303 subjects (146 CHD cases and 157 controls) were recruited in the current study. Selected characteristics of the study participants are shown in p-value for trend 0.007). Walking was negatively associated with CHD risk . Subjects in the highest tertile of walking time had an adjusted OR of 0.37 (95% CI 0.20–0.70). With regard to leisure time PA, vigorous exercise was not associated with increased risk of CHD . Participants engaging in more than 1 h/week of moderate PA (highest tertile) had a lower risk of CHD compared to those in the lowest tertile , but the OR increased to 1.10 (95% CI 0.65–1.89) after adjusting for confounding variables . Sedentary behavior, which was measured by time participants were sedentary, was associated with an increased risk of CHD. The adjusted ORs for subjects in the medium and highest tertiles of sedentary behavior time were 2.01 (95% CI 1.06–3.79) and 3.88 (95% CI 2.14–7.02), respectively . Overall, PA was associated with a lower risk of CHD; subjects in the highest tertile for total METs had a lower risk of CHD .Prevalence and effects of different types of PA among cases and controls are shown in To further explore the relationship between sedentary behavior and the risk of CHD, natural cubic splines were fitted. As shown in The risk of CHD increased at a slower rate for 4–6 h of sedentary behavior time, and then increased more steeply with longer sedentary behavior time (>6 h). Further, we explored the interaction between intensity of PA both at work and during leisure time with other variables. PA was classified into three levels based on whether the subject engaged in vigorous- or moderate-intensity PA or was mainly sedentary .p-value 0.007) and hypertension  affected the levels of work-related PA to modify the risk of CHD. Among cases, there was higher proportion of hypertensive subjects (55%) who were engaged in vigorous-intensity PA than among controls (20%).The results showed that gender  and women . By contrast, moderate PA was associated with a significantly lower CHD risk in both genders; however, this was more pronounced in men  than in women . Furthermore, when moderate occupational PA was categorized into tertiles, the gender difference between CHD risk was more defined, whereas a 75% reduction in risk  was observed in the highest tertile of moderate PA among male subjects, a non-significant lower risk  was found among female subjects.In the current study, we examined the effects of engaging in PA and sedentary behavior on the risk of CHD. The results showed that moderate-intensity occupational PA as well as walking protected against CHD. Leisure PA did not have a protective effect against CHD. Our results also highlighted the strong adverse effect of sedentary behavior on the risk of CHD. This behavior is very common in the Gulf region; the prevalence was estimated to be as high as 73% among women in Saudi women, for example .The rapid sociodemographic transition in the Arabian Gulf introduced substantial changes in lifestyle that was largely characterized by replacement of traditional diets with diets higher in fat and refined and processed foods, and more comfortable lifestyle that discourages PA. These changes contributed to an increased prevalence of non-communicable diseases, such as CVD, diabetes, and cancer ,28,29,30The current finding of a low prevalence of PA was consistent with results from previous studies, which showed that Gulf countries have one of the world’s highest rates of insufficient PA; for example, 68.8% in Saudi Arabia, 64.5% in Kuwait, and 45.9% in Qatar . A numbeThe current finding of a lack of protective effect of vigorous-intensity PA, especially at work, is consistent with results from previous studies. A number of studies investigating the association between work-related PA and the risk of CVD reported increased risk ,37,38,39The lack of a protective effect against CHD with moderate- and vigorous-intensity PA during leisure time is puzzling considering the ample evidence from the literature of an inverse association. This may be attributed to residual confounding, the very low prevalence of this type of PA in the current population, or measurement errors. Another possible explanation is that the current population may have only engaged in leisure PA sporadically, which does not offer protection against CHD. Some studies reported that oxidative stress, which has a harmful effect on CHD, might increase with acute vigorous exercise, especially among non-athletics .Moderate work PA as well as walking had protective effects, with evidence of a dose–response relationship. This is consistent with previous studies showing that moderate PA—such as walking, stair climbing, bending, or other light indoor activities—have beneficial effects on overall cardiovascular health ,41. WalkThe current results showing a strong negative association between sedentary behavior and the risk of CHD have important implications, especially among today’s population with a low prevalence of PA ,32. LackThe results of differential effects of moderate work PA on CHD between male and female subjects are noteworthy. The relationship was stronger for men, especially in the highest tertile. By contrast, Cheng et al.  found thThe current study has several limitations. Because the sample was drawn from a hospital, it might not represent a balanced distribution of the overall population with regard to risk factors. However, to reduce the effect of this selection bias, we selected controls from the same hospital. Self-reporting of most variables in the current study may introduce measurement errors as evidenced by the relatively large standard deviation of some of the PA measurements. Differential recall bias, which is common in case–control studies, is a factor that may either bias the estimates toward null or overestimate the effects of PA, depending on the extent of differential over- or under-reporting of PA by cases and controls. However, our results on total activity-related energy expenditure are consistent with those from previous studies. Thus, recall bias is less likely to play a role in our study. Another limitation is the relatively small sample size, which to some extent may limit the generalizability of the results. We are expanding the study by recruiting more subjects.The main strength of our study is that we explored the dose–response effect of sedentary behavior, which is very common in the Gulf society. Careful examination of the association between sedentary time and CHD risk revealed a significant and rapid increased CHD risk for more than 5 h of sedentary behavior. We also collected detailed information about dietary patterns and smoking, which allowed adjustment for these important confounding variables. The important finding in this study of the possible harmful effects of vigorous-intensity occupational PA highlights the need to address this issue in occupational settings.The current results showed that moderate-intensity occupational PA protected against CHD, and an increased risk of CHD was observed with sedentary behavior. Walking was also inversely associated with CHD risk. Future research should focus on understanding the specific types of PA that reduce the risk of CVD. In addition, attention must be paid to the distinct effects of occupational PA and PA during leisure time. Similarly, examining the proportion of time spent in sedentary behavior at work compared with at home is important to delineate strategies to encourage occupational PA.It is important to implement educational programs that enhance PA at all levels, including in schools and at the workplace. Awareness should be raised of the important health benefits of sufficient PA; the WHO recommends that adults should engage in moderate-intensity aerobic PA for 300 min per week or vigorous-intensity aerobic PA for 150 min per week, or an equivalent combination of moderate- and vigorous-intensity PA.