Nfe2l2) induces expression of numerous detoxifying and antioxidant genes in response to oxidative stress. The cytoplasmic protein Keap1 interacts with and represses Nrf2 function. Computational approaches were developed to identify factors that modulate Nrf2 in a mouse liver gene expression compendium. Forty-eight Nrf2 biomarker genes were identified using profiles from the livers of mice in which Nrf2 was activated genetically in Keap1-null mice or chemically by a potent activator of Nrf2 signaling. The rank-based Running Fisher statistical test was used to determine the correlation between the Nrf2 biomarker genes and a test set of 81 profiles with known Nrf2 activation status demonstrating a balanced accuracy of 96%. For a large number of factors examined in the compendium, we found consistent relationships between activation of Nrf2 and feminization of the liver transcriptome through suppression of the male-specific growth hormone (GH)-regulated transcription factor STAT5b. The livers of female mice exhibited higher Nrf2 activation than male mice in untreated or chemical-treated conditions. In male mice, Nrf2 was activated by treatment with ethinyl estradiol, whereas in female mice, Nrf2 was suppressed by treatment with testosterone. Nrf2 was activated in 5 models of disrupted GH signaling containing mutations in Pit1, Prop1, Ghrh, Ghrhr, and Ghr. Out of 59 chemical treatments that activated Nrf2, 36 exhibited STAT5b suppression in the male liver. The Nrf2-STAT5b coupling was absent in in vitro comparisons of chemical treatments. Treatment of male and female mice with 11 chemicals that induce oxidative stress led to activation of Nrf2 to greater extents in females than males. The enhanced basal and inducible levels of Nrf2 activation in females relative to males provides a molecular explanation for the greater resistance often seen in females vs. males to age-dependent diseases and chemical-induced toxicity.The transcription factor Nrf2 (encoded by  Induction of oxidative stress has been observed following exposure to chemicals that activate xenobiotic receptors including the aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), and peroxisome proliferator-activated receptor α (PPARα). Chemicals that activate these receptors cause liver cancer through nongenotoxic mechanisms and generally act at the promotion stage by increasing cell proliferation [Oxidative stress reflects an imbalance between reactive oxygen species (ROS) and the ability of a biological system to detoxify reactive intermediates or to repair resulting damage. Disturbances in the normal redox state of cells through the production of ROS can damage critical cellular components leading to injury or disease. Oxidative stress plays roles in chemical-dependent and -independent cytotoxicity and tumor promotion , 2. Therferation –5.Cellular oxidants activate the nuclear factor erythroid 2-related factor 2  transcription factor  which isClinical and preclinical studies show sexually dimorphic responses of the liver to various stressors with females often exhibiting resistance to damage compared to males. Examples of heightened susceptibility of males include liver ischemia reperfusion, hemorrhagic shock-resuscitation, liver cirrhosis, and hepatocellular carcinoma. Estrogen has been suggested to be a factor responsible for this sexual dimorphism –19. One Pit1 dwarf mice have elevated mRNA levels for several Nrf2-dependent genes, as well as cellular traits, such as elevated glutathione levels, resistance to lipid peroxidation, that are known to be modulated by Nrf2 function [Prop1 and Snell dwarf mice in the absence of chemical induction [There is a growing body of evidence that Nrf2 is activated in mice with defects in GH signaling, under conditions in which the liver genome would be feminized. These mice have a number of overlapping traits including dwarfism, increased resistance to stressors, and increased longevity . Fibroblfunction . A set onduction , 37. Mornduction . Thus, eIn the present study, a gene expression biomarker was used to identify factors in a microarray database that led to Nrf2 activation or suppression. We then determined the relationship between Nrf2 activation and suppression of STAT5b using a previously characterized gene expression biomarker .https://www.illumina.com/products/by-type/informatics-products/basespace-correlation-engine.html; formally NextBio) was used as the starting point to create a compendium of mouse liver biosets. The BSCE database contains over ~21,600 highly curated, publically available, omic-scale studies across 15 species including ~134,000 lists of statistically filtered genes . Each list (bioset) is compared to all other biosets in the database using a fold-change rank-based statistical algorithm called the Running Fisher algorithm which allows an assessment of the overlap in genes and whether those genes are regulated in a similar or opposite manner. In this study, only biosets from mouse liver, mouse primary hepatocytes, or hepatocyte-derived cell lines were evaluated. Available information about each bioset was extracted from BSCE and used to populate a compendium of information about the experiments. Each bioset was further annotated using information derived from the original GEO submission and/or the original publication. Each bioset was annotated for the type of factor  and the name of the factor  examined. To assess Nrf2 activation or suppression, the Nrf2 biomarker was uploaded into the BSCE database and compared to all biosets in the database using the Running Fisher algorithm. Results of the test were exported and used to populate the annotated compendium with p-values of each comparison. We have previously used this analysis strategy to accurately identify factors that activate or suppress other transcription factors  [The methods used in this study are outlined in eptor α) –42.No animal experiments were conducted as part of this study. However, we did utilize tissues from animals that were part of published studies as discussed below.Mus musculus genome version mm9. A total of 23,238 genes were analyzed. Statistically significant genes were identified by one-way ANOVA with a false discovery rate (Benjamini-Hochberg test) of ≤ 0.01. The raw microarray files have been archived in Gene Expression Omnibus (GEO) under accession number GSE85222.Gene expression was measured in the livers of wild-type or Nrf2-null mice treated with oltipraz or vehicle in which mice were treated each day for 4 days with 75 mg/kg/day . Four biAlmost all of the statistically filtered gene lists used in our study were generated using BSCE standardized microarray analysis pipelines and are available in a searchable annotated format in the BSCE database . AdditioIndependent of the BSCE database, a database of gene expression changes was assembled by our group using comparisons from mouse liver, mouse primary hepatocytes and hepatocyte-derived cell lines. All of these experiments were conducted using Affymetrix microarrays. Affymetrix .cel files were downloaded from publicly available sources including GEO and ArrayExpress, which were first analyzed by Bioconductor SimpleAffy  to assesAnnotation has been described in our previous study . Assignmhttp://linus.nci.nih.gov/BRB-ArrayTools.html) [Analyses were performed using BRB-ArrayTools version 4.2.1 Stable Release developed by Dr. Richard Simon and BRB-ArrayTools Development Team (ls.html) . The metls.html) . Two traFA/FA) [FA/FA) compared to control wild-type mice [Probe sets that comprise the Nrf2 biomarker were derived from comparisons using livers of wild-type and Nrf2-null mice treated with CDDO-Im for 6 hrs  and untrFA/FA)  or additype mice , 49. Theype mice , 39–41. ype mice  to find ype mice , 39–41. −4 after a Benjamini Hochberg correction of α = 0.001 resulted in a balanced accuracy of 88%, 97%, 98%, and 98% for AhR, CAR, PPARα and STAT5b activation, respectively [−4) is found in Biosets from microarray experiments in which the Nrf2 activation state was known were manually curated from the following studies: GSE55001, GSE55084, GSE54597, E-MEXP-1231, E-MEXP-153, E-MEXP-347, E-MEXP-438, GSE10082, GSE10769, GSE1093, GSE11287, GSE15633, GSE15859, GSE16381, GSE16777, GSE20944, GSE23780, GSE24751, GSE25142, GSE3150, GSE33575, GSE34423, GSE35124, GSE39313, GSE40120, GSE40773, GSE4259, GSE6721, GSE867, GSE8969, GSE55002, and GSE55003. The number of biosets used to test for Nrf2 activation was 67 positives and 14 negatives. Unlike more traditional machine learning classification methods, optimal conditions for classification were not derived from gene behavior as the biomarker was fixed. In this and in our previous studies , the bioectively . The samChIP-seq data from experiments that exposed mouse cells to Nrf2-activating chemicals were obtained from GEO . Briefly, raw sequencing reads downloaded from NCBI Sequence Read Archive (SRA) were mapped to the mouse genome (mm9) using the BWA (Burrows-Wheeler Aligner) . ReplicaThe full list of genes in the Nrf2 biomarker was analyzed using the Ingenuity Pathways Analysis (IPA) canonical pathway and upstream analysis functions. The significance for canonical pathways was calculated using a right-tailed Fisher's Exact test by IPA. The p-value is the probability that the Nrf2 biomarker gene list would coincide with the IPA gene list. Upstream analysis function of potential regulators of Nrf2 biomarker genes was based on the number of differentially expressed downstream genes in the biomarker and quantified using a Z-score and p-value. The Z-score is a correlation measure of how consistent the direction of expression changes in the biomarker gene list matches the direction of change from the annotated literature for targets in the biological or regulatory group.Livers used for the RT-PCR experiments were from wild-type and Nrf2-null mice treated with the Nrf2 activator oltipraz (75 mg/kg/day for 4 days) as described above. The levels of expression of selected genes were quantified using real-time reverse transcription-PCR (RT-PCR) analysis. Briefly, total RNA was reverse transcribed with murine leukemia virus reverse transcriptase and oligo(dT) primers. The forward and reverse primers were designed using Primer Express software, version 2.0 . The SYBR green DNA PCR kit  was used for real-time PCR analysis. The relative differences in expression between groups were expressed using cycle threshold (Ct) values . The Ct values of the genes were first normalized with β–actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of the same sample. Means and S.D. (n = 3–5) for RT-PCR data were calculated by Student's or Aspin-Welch’s t-test. The level of significance was set at p ≤ 0.05.2 and F-test p-values were determined.Regression analyses were carried out in Excel. RA number of computational strategies were initially examined to identify Nrf2 modulation in genomic databases. We first examined the utility of machine learning algorithms to identify gene sets that would correctly predict Nrf2 activation status. None of the models were adequate for predicting Nrf2 .A rank-based enrichment analysis strategy called the Running Fisher algorithm  as evaluTo evaluate the Nrf2 biomarker genes for direct interactions with Nrf2 (and presumably direct transactivation), genes were compared to existing mouse Nrf2 ChIP-seq datasets. In total, 3641 genes mapped nearest to Nrf2 binding regions . Of the Nfe2l2 (Nrf2) . Other transcription factors included small Maf family members including Mafk, Mapff, and Mafg, all known heterodimeric binding partners with Nrf2.Nrf2 biomarker genes were evaluated for canonical pathway enrichment by Ingenuity Pathway Analysis (IPA) . The topThe biomarker was compared to statistically-filtered gene lists using the Running Fisher algorithm which computes a correlation direction (positive or negative) and an associated p-value of the correlation . To visuThe Nrf2 biomarker was evaluated for ability to correctly classify biosets with known Nrf2 activation status. Not surprisingly, the biosets from either the wild-type mice treated with CDDO-Im or the three Keap1-disrupted vs. wild-type comparisons exhibited statistically significant correlations , whereas the bioset from CDDO-Im treated Nrf2-null mice did not have significant positive correlation .Nqo1, Ces1g and Abcc3 which are part of the Nrf2 biomarker. The expression of these genes was confirmed by RT-PCR , but ony RT-PCR , middle.To determine the predictive behavior of the biomarker, classification was performed on a set of biosets with known Nrf2 activation status. Criteria for classification included a p-value ≤ 1E-4. The final number of biosets evaluated were 67 positives and 14 negatives. Using this relatively modest number of biosets, the biomarker had a 91% sensitivity and a 100% specificity, giving a balanced accuracy of 96% .There are published examples of sex differences in response to various stressors that may induce Nrf2 . The molThe biosets of female vs. male comparisons were analyzed for similarity to the Nrf2 biomarker using the Running Fisher algorithm as described in the Methods. Pou1fd1 (Pit1) gene which controls sex-specific GH secretion from the anterior pituitary. There was no obvious explanation to why the other bioset (mice treated for 2 wk with 5000 ppm o-nitrotoluene) did not exhibit feminization. The relationship between the Nrf2 predictions and expression of the genes in the biomarker is shown in Nqo1. Genes in the Nrf2 biomarker that exhibited significant sex differences included those that had decreased expression in females compared to males . A plot of the–logs of the Nrf2 and STAT5b predictions shows a trend toward more significant Nrf2 activation with more significant suppression of STAT5b   . All 37 Sex differences in the hepatic transcriptome are under control of sex hormones. It was hypothesized that Nrf2 activation is also under control of sex hormone levels. To test this hypothesis, biosets were evaluated in which sex hormone levels were altered; these included effects of castration, ovariectomy, and exogenous treatments with testosterone (T), dihydrotestosterone (DHT), or estradiol (E2) . Ovariectomy compared to intact mice from two biosets had no effect on Nrf2 activation, and two biosets from castrated mice exhibited Nrf2 activation, but these comparisons did not reach statistical significance  (data not shown). We next determined the dose- and time-dependent effects of exposure to the estrogen receptor agonist ethinyl estradiol (EE). Mice were exposed for 7 or 28 days to EE in the feed (described in ). Nrf2 wPou1fd1 (Pit1), Prop1), 2) prevent the synthesis and secretion of GH from the pituitary , or 3) affect the function of the GH receptor (Ghr). All of these mutations result in dwarfism and most lead to increases in longevity compared to wild-type mice through a delay in age-dependent diseases [Ghrhr and Prop1 mutants. None of the Pit1 mutant biosets reached significance. Biosets from Ghr deletion mutants exhibited different effects on Nrf2 depending on the location of the deletion. Ghr truncated at amino acid 569  had no effect on Nrf2 or STAT5b. These results were consistent with the weak to moderate effects on GH-dependent phosphorylation of STAT5b by JAK2 activated through GHR [Ghrh-null vs. wild-type mice  . Simila 2.4E-4) . Taken t2 = 0.170; p-value = 1.22E-6) (S1 File). Only one bioset caused suppression of Nrf2 and STAT5b activation .Feminization of the liver transcriptome is a common feature upon chemical treatment in male mice . We hypo1.22E-6) . Most  (S1 File). Chemicals that activated Nrf2 and caused feminization included 1,5-naphthalenediamine, malathion, oxazepam, phenobarbital, propylene glycol mono-t-butyl ether, TCDD, and TCPOBOP. Benzofuran, coumarin, and methylene chloride caused increases in Nrf2 activation and activation of STAT5b (masculinization). TCPOBOP was the only chemical that caused suppression of Nrf2 with no effects on STAT5b. Overall, these results indicate that coincident activation of Nrf2 and suppression of STAT5b often occurs in male but not female mice.In female mouse livers, there was no clear relationship between Nrf2 activation and feminization after chemical exposure (R= 0.003) . For mos2 = 0.009; p-value = 0.400). The chemicals that altered Nrf2 in vitro in the absence of effects on STAT5b are summarized in We hypothesized that if the linkage between Nrf2 and STAT5b is hormonally-driven, the observed coupling of effects would be absent in in vitro cultures. To test this hypothesis, biosets from chemically-treated primary hepatocytes or hepatocyte cell lines were examined. Given the higher basal activation of Nrf2 in females than males, we hypothesized that there would be sex differences in Nrf2 responsiveness after chemical exposure. Male and female mice were exposed under identical conditions to 11 chemicals . The livSex differences could be due to differences in levels of internal exposure to the compounds which could lead to sex differences in total number of genes altered. However, there was no clear relationship between higher Nrf2 activation and number of genes altered by the chemical in female mice. For the chemicals with the greatest sex differences, the number of genes that were differentially expressed in the livers was either about the same in each sex  or greater in males than females . These results appear to identify a set of chemicals that induce greater Nrf2 activation in females compared to males.Annotated databases of microarray data provide opportunities through novel methods of reanalysis to gain insights into the network of factors altered by chemical exposure. In the present study, we used a gene expression biomarker that can accurately predict the activation status of the oxidant-induced transcription factor Nrf2 in a microarray database and uncovered a strong linkage between increased Nrf2 activity and feminization of the male liver mediated by suppression of STAT5b. Examination of female versus male comparisons showed that Nrf2 is subtly but reproducibly activated to higher levels in females. Additional analyses showed that feminization of the liver transcriptome by genetic, dietary or chemical modulation under conditions which suppressed STAT5b consistently led to Nrf2 activation. Finally, we found that female mice generally exhibited greater induction of Nrf2-regulated genes after chemical exposure compared to male mice. These studies indicate that feminization of the liver transcriptome through many stressors leads to increased Nrf2 activity. The higher basal and chemical-induced Nrf2 activity may explain in part why female mice are often more resistant to a number of stressors compared to males.Nqo1 and Gst family members  levels and cannot sustain hepatic sex-differences in some drug metabolic genes [To screen for factors in the microarray database that lead to alterations of Nrf2 function, we compared the Nrf2 biomarker to a gene expression compendium of ~2500 annotated microarray comparisons allowing an unprecedented assessment of factors that control Nrf2 activity. The compendium contains 86 female vs. male biosets in which female and male mice were evaluated under identical experimental conditions . Almost measured . Femalesic genes . The facNfe2l2 and Hmox1 genes and decreased ROS-induced DNA damage as measured by 8-Oxo-dG and M1dG adducts in the livers of 12-wk old male offspring [Nqo1 [Sult1e1) which sulfonates and deactivates estrogens [Based on the sexual dimorphism in Nrf2 regulation and the fact that liver sexual dimorphism is under control of STAT5b, we hypothesized that hormone, genetic, dietary, and chemical treatments that disrupt the GH-regulated liver transcriptome dependent on STAT5b would lead to Nrf2 activation. Hormone treatments that activate STAT5b (testosterone or dihydrotestosterone) caused suppression of Nrf2, while treatments that suppress STAT5b  caused activation of Nrf2 . Very liffspring . Dietaryng [Nqo1 . There wstrogens . In a mostrogens –65.Prop1, Ghrhr, and Ghrh  after exposure to the oxidative stress inducer diquat [Ggt1 and Nqo1 in females compared to males [Nqo1 and Hmox1 genes as well as protein levels of Nqo1 and Gclc while there was less tissue damage in female mice [Nqo1 was found to be greater in females than males after exposure to TCDD [We found that female mice appear to mount a greater Nrf2 response to chemical exposure than male mice. After exposure to 11 chemicals identically administered to male and female mice, Nrf2 was activated to a higher level in female mice than male mice  based onr diquat . Exposurto males . There wale mice . Inducti to TCDD . Upon TC to TCDD . The dif to TCDD . These sCaenorhabditis elegans [Keap1 in male Drosophila melanogaster increases Nrf2 activity and longevity [Gsta4-null mice, in which detoxification of the lipid peroxidation product 4-hydroxynonenal is impaired, exhibit Nrf2 activation and have an extended life span [There are numerous studies which show a relationship between the activation status of Nrf2, resistance to various stressors, and longevity. Female mice live longer than males , possibl elegans . Disruptongevity . In a coongevity . Gsta4-nife span . In a coife span . The dwaife span . More reife span . When prife span .There is evidence for at least four nonexclusive mechanisms that link suppression of STAT5b and Nrf2 activation. In the first mechanism, estrogens can be metabolized by Cyp family members to 2- and 4-hydroxyestrogens which can activate Nrf2  and suppS1 FileContains 1) list of probesets and genes in the Nrf2 biomarker, 2) description of biosets from chemical treatment experiments that activate or suppress Nrf2 and STAT5b discussed in this study, and 3) genes that are mapped nearest to the location of Nrf2 binding peaks from 4 published mouse ChIP-seq datasets.(XLSX)Click here for additional data file.S2 FileContains the section “Classification analysis of Nrf2 activation using machine learning algorithms”.(PDF)Click here for additional data file.


The dynamic change and functional importance of Nrf2 signaling, as well as Nrf2 targeted intervention, are revealed in permanent, transient, and global cerebral ischemia models. In addition, key considerations, pitfalls, and future potentials for Nrf2 studies in preclinical stroke investigation are discussed.Ischemic stroke is one of the leading causes of death and long-term disability worldwide; however, effective clinical approaches are still limited. The transcriptional factor Nrf2 is a master regulator in cellular and organismal defense against endogenous and exogenous stressors by coordinating basal and stress-inducible activation of multiple cytoprotective genes. The Nrf2 network not only tightly controls redox homeostasis but also regulates multiple intermediary metabolic processes. Therefore, targeting Nrf2 has emerged as an attractive therapeutic strategy for the prevention and treatment of CNS diseases including stroke. Here, the current understanding of the Nrf2 regulatory network is critically examined to present evidence for the contribution of Nrf2 pathway in rodent ischemic stroke models. This review outlines the literature for Nrf2 studies in preclinical stroke and focuses on the  Ischemic stroke is one of the leading causes of death and long-term disability worldwide , a cis-acting regulatory element that contributes to cellular defense in eukaryotes  transcription factors. It is a modular protein composed of seven functional domains, Nrf2-ECH homology (Neh) domains 1–7, which have distinct functions  Chan et. The NehOrganisms are equipped with a defense system to maintain homeostasis against constant intrinsic and extrinsic insults that result in the damage of nucleic acids, proteins, and membrane lipids. Nrf2 is a master regulator of the inducible cell defense system by controlling a broad range of cytoprotective genes  Keap1 acts as a sensor of oxidative and electrophilic stresses for Nrf2 with a subcellular localization in the perinuclear cytoplasm   Glutathione (GSH)-based: glutamate–cysteine ligase catalytic (GCLC) subunit and glutamate-cysteine ligase modifier (GCLM) control the entry of cystine into cells; glutathione peroxidase (GPX) 2 produces oxidized glutathione (GSSG) during the reduction of peroxides; and glutathione reductase (GSR) 1 reduces GSSG for maintenance of reduced intracellular GSH levels. (2) Thioredoxin (TXN)-based: Thioredoxin (TXN) 1, thioredoxin reductase (TXNRD) 1, and sulfiredoxin (SRXN) 1 reduce oxidized protein thiols. (3) Others: G6PDH and 6PGD reduce synthesis of NADPH, antioxidant protein Trx, stress response protein heme oxygenase 1 (HO1), and other proteins. Nrf2 coordinately regulates key components in the antioxidant system that precisely controls antioxidant defense at multiple levels, thus ensuring an adequate response to oxidants in time and space  to form iron, carbon monoxide (CO), and biliverdin, which is immediately reduced to bilirubin , 6-phosphogluconate dehydrogenase (Pgd), isocitrate dehydrogenase 1 (Idh1), and malic enzyme 1  is a small non-coding RNA molecule that functions in RNA silencing and post-transcriptional regulation of gene expression. At least eight miRNAs have been identified as direct modulators of Nrf2 expression at the transcriptomic level  The prevailing view is that Keap1 is a primary repressor of Nrf2. Under basal conditions, Keap1 constantly targets Nrf2 for ubiquitination and proteasomal degradation, resulting in the disruption of Nrf2 protein stability and maintenance of Nrf2 signaling capacity at a very low level. In response to electrophiles or stressors, Nrf2 is liberated from the Keap1 repression  by inhibiting ubiquitylation and proteasomal degradation that allows newly-synthesized Nrf2 to be rapidly stabilized and activates transcriptional activation of cytoprotective genes. This view is supported by many reports of Keap1 knockout mice and Keap1 knockdown human cells that exhibited sufficient Nrf2 activity  The Nrf2 is has been described to be suppressed by b-TrCP and GSK-3; in addition to the well-studied Keap1, E3 ubiquitin ligase adaptor β-TrCP is another negative repressor of Nrf2 stabilization  Permanent cerebral ischemia (pdMCAO and pMCAO): Technically, the MCA can be selectively occluded at a distal or proximal site, referred to as permanent distal MCA occlusion (pdMCAO) or permanent  MCA occlusion (pMCAO) cerebral ischemia model, respectively. The pdMCAO model produces highly reproducible ischemic cortical lesions that are predominantly restricted to the barrel regions of cortex, inducing definable sensorimotor deficits that closely mimics ischemic stroke in humans. Thus, it is believed to be one of the most predictable and useful stroke models, allowing researchers to look at long-term recovery with high survival rates  Transient focal cerebral ischemia (tMCAO): This model is easy to perform in a controlled manner. The intraluminal suture MCAO model in rats and mice is the most frequently used model. This model exhibits reproducible MCA region infarctions that depend on the shape, size, and insertion length of the thread, allowing reperfusion by retracting the suture. MCAO generates ischemic cell death in the striatum and overlying the frontal, parietal, temporal, and portions of the occipital cortex. MCAO also precipitates variable damage in the thalamus, cervicomedullary junction, substantia nigra, and hypothalamus. Ischemic brain injury widely affects diverse brain regions and leads to complex motor, sensory, autonomic, and cognitive deficits  Global cerebral ischemia (GCI): global cerebral ischemia (GCI) during cardiac arrest results in selective and delayed neuronal death of pyramidal neurons in the hippocampal CA1 region, similar to the situation in humans, and consequent cognitive decline  Whether Nrf2 induction is protective against ischemic injury and is facilitative for recovery? Specific focus is given to the in vivo evidence in different rodent cerebral ischemia models. Then, the pitfalls and concerns of current Nrf2 experimental ischemic stroke studies are discussed, which would be valuable for future studies.In recent years, studies reported findings concerning the dynamic change of Nrf2 signaling, its functional importance, and its targeted intervention in cerebral ischemia. These findings provide insights into whether, when and how Nrf2 functions during brain injury. Accordingly, we mainly focused on the following questions. (1) What is the dynamic regulation of the Nrf2 signaling following cerebral ischemia? (2) Does the evidence from Nrf2in vivo evidence of dynamic alternation of Nrf2 expression, as well as its target genes, and cellular and subcellular distribution of Nrf2 during different stages of cerebral ischemia. These findings, utilizing focal ischemia models with or without reperfusion in addition to global ischemia models, help us to identify the role of the Nrf2 regulatory network in the context of cerebral ischemia.Normally, Nrf2 is largely localized in the cytoplasm and is maintained at a low basal level due to its binding affinity to Keap1. However, when cells are exposed to excessive oxidative stimuli during cerebral ischemia, Nrf2 is liberated from Keap1, translocates into the nucleus, and binds to the ARE sequence, thereby upregulating the expression of its target genes, which code cytoprotective proteins like anti-oxidative enzymes. In recent years, studies have provided substantial A number of studies with permanent cerebral ischemia models (pdMCAO and pMCAO) investigated the Nrf2/ARE pathway in response to ischemic insults . Severalin vivo optical signals of Nrf2 expression were not detected in the earliest stages but peaked at 24 h after ischemia. Such Nrf2 expression was mainly detected in in the penumbra area, largely localizing inside neurons and astrocytes  or knockdown animals. Reports showed that Nrf2−/− mice do not exhibit any overt abnormal phenotype regarding size, body weight, food intake, mobility, fertility, or other characteristics at baseline , which further supports the findings above  was observed before, during, or after ischemia-reperfusion between WT and Nrf2−/− at indicated time points as monitored by laser-Doppler flowmetry , and more are also depicted in Given the fundamental role of Nrf2 in redox homeostasis, many studies have presented the unique contribution of Nrf2 in neuroprotection against various diseases Ma, . For isc−/− mice, indicating that the Nrf2 pathway is required for the benefit of DMF and MMF. During the late phase (7–14 d), DMF also acted as a potent immunomodulator, reducing the infiltration of neutrophils and T cells and the number of activated microglia and macrophages in the infarct region  and its primary metabolite monomethyl fumarate (MMF) are two typical Nrf2 inducers. Several studies support their neuroprotective efficacy against ischemic brain injury through the activation of the Nrf2/HO1 pathway. DMF and MMF dramatically reduced infarct volume, brain edema, and neurological deficits over 7 days after tMCAO, along with the suppressed acute glial activation , a typical Nrf2 activator, is a naturally occurring isothiocyanate found in cruciferous vegetables. Upregulation of Nrf2 by sulforaphane pretreatment was associated with increased HO1 expression in perivascular astrocytes in the peri-infarct regions and cerebral endothelium of the infarct core. BBB disruption, lesion progression, and neurological deficits were reduced after tMCAO. This indicates that the Nrf2 defense pathway in the cerebral microvasculature provides a novel therapeutic approach for preventing BBB breakdown and neurological dysfunction in stroke victims  is a Nrf2 inducer widely used as a food additive. Nrf2 activation by tBHQ pretreatment reduced cortical damage and sensorimotor deficits from 24 h up to even 1 month after pdMCAO. Interestingly, larger infarcts were observed in Nrf2MicroRNAs (miRNAs) play vital roles in regulating neuronal survival during cerebral ischemia/reperfusion injury. MiR-93, a direct negative modulator of Nrf2 expression at the transcriptomic level, serves as a potential therapeutic target for acute ischemic stroke. MiR-93 levels in the ischemic cortex of mice increased at 24 h and 48 h after tMCAO. MiR-93 antagomir treatment reduced infarction volume, neural apoptosis and neurological deficits through the Nrf2/HO1 antioxidant pathway  is one of the most widely used herbal medicines with reported antioxidant and anti-inflammatory properties, displaying promising potential in neuroprotection. Ginseng pretreatment ameliorated short- and long-term sensorimotor deficits over 28 days, prevented the acute enlargement of lesion volume, attenuated reactive astroglial progression but not microglial activation, and enhanced the induction of Nrf2 target antioxidant proteins after pdMCAO in WT mice, an effect that was abolished in Nrf2–)-Epicatechin (EC) is especially abundant in cocoa, dark chocolate, and green tea, and it boosts antioxidant activity while supporting vascular function. EC-treated ischemic WT mice displayed a reduction of forelimb motor coordination impairments associated with reduced anatomical injury and microglia/macrophage activation in pMCAO and tMCAO models of adult WT mice, impairments which were abolished in tissues and neurons from Nrf2−/− mice  is a gaseous second messenger produced when heme oxygenase enzymes catabolize heme. At low doses  is one of the main components of Ligusticum wallichii Franchat (Chuan Xiong) that has been used to treat neurovascular and cardiovascular diseases including stroke in traditional Chinese medicine. TMP has been found to protect against pdMCAO injury and reduce inflammation and ischemia-induced neutrophils through Nrf2/HO1 activation  Nrf2 antibody: Getting potent, selective and highly specific antibodies against the various species of Nrf2 has been an ongoing serious issue. Furthermore, what is the correct molecular weight of Nrf2 protein? The biologically relevant molecular weight of Nrf2 protein has been proven to be ~95–110 KDa, but not ~55–65 KDa, based on its 2-kb open reading frame or its tertiary structure (Lau et al., This review focusses on Nrf2 activity in the context of ischemic stroke, though, over- or prolonged Nrf2 activation could potentially be problematic. For example, sustained activation of Nrf2 in drosophila would shorten its life expectancy (Tsakiri et al., in vivo evidence revealing the contribution of the Nrf2 pathway in ischemic stroke pathogenesis and neuroprotection. This review highlighted the promising potential of interventions targeting Nrf2, implying that the moderate activation of Nrf2 favors the attenuation of brain damage and long-term recovery from cerebral ischemia. Because the investigations of Nrf2 in stroke are still in the initial stages, future research is expected to elucidate the natural properties of Nrf2 in stroke leading to the development of novel drugs that target Nrf2.Emerging evidence demonstrates that the Nrf2 network plays a crucial role in cellular adaption by controlling a wide range of cytoprotective proteins, counteracting distinct endogenous and exogenous insults while providing a promising optimal therapeutic target against various diseases from cancer to brain disorders. By using various ischemic stroke rodent models, recent preclinical studies provide direct LL and SD conceived the study and designed the databases analysis. LL prepared the manuscript with input from SD. LL and LML searched databases, collected data, performed analyses, and prepared the tables and figure. All authors reviewed, discussed and approved the final 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.


Areca catechu, Polygonum minus, and Syzygium polyanthum plant extracts to compare the results of our proposed LoD method with the conventional method. Contrasted to the arduous laborious conventional method, our proposed method offer rapid analysis and simple determination of antioxidant. This proposed LoD method for antioxidant activity in plants would be a platform for the further development of antioxidant assay.Antioxidants are an important substance that can fight the deterioration of free radicals and can easily oxidize when exposed to light. There are many methods to measure the antioxidant activity in a biological sample, for example 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant activity test, which is one of the simplest methods used. Despite its simplicity, the organic solvent that has been used to dilute DPPH is easily evaporated and degraded with respect to light exposure and time. Thus, it needs to be used at the earliest convenient time prior to the experiment. To overcome this issue, a rapid and close system for antioxidant activity is required. In this paper, we introduced the Lab-on-a-Disc (LoD) method that integrates the DPPH antioxidant activity test on a microfluidic compact disc (CD). We used ascorbic acid, quercetin,  Free radicals are atoms with unpaired valence electrons that cause its chemical instability and reactivity. The valence electron of one atom accepts an electron from another molecule, which forms a new free radical. This process repeats itself, creating a free radical cascade. When free radical production becomes excessive in the body, it may cause oxidative stress, a condition whereby the body cannot counter-attack the production of free radicals, leading to cellular damage and cell death . Free raAntioxidants are molecules that can donate electrons to stabilize free radical species. They play many important roles in daily life. For example, antioxidants are hypothesized to prevent the deleterious effects of free radicals on cells . In the There are many methods to determine the antioxidant properties in a sample. Each of the methods work differently based on the chemical mechanism, among which, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant activity test is the most common and easiest method used to determine the antioxidant activity in a sample . The DPPDespite the simplicity of the DPPH antioxidant assay, DPPH itself is easily decomposed over time and is highly sensitive to changes in temperature, pH, and light exposure ,14. The Many fundamental bio-assay methods are carried out manually. The experimental steps are often time-consuming and require expert skills. The amalgamation of human-prone error, especially in the pre-analytical phase, may also cause incorrect interpretations or false positive results in the analytical and post-analytical phases . One of Lab-on-a-Disc (LoD), which are also known as centrifugal microfluidic, are a part of the microfluidic device in the form of a disc with a spinning motor. LoD is a part of the Micro Total Analysis System (µTAS), which offers miniaturization and automation of most of the chemical and biological analysis systems. LoD offers many advantages, such as simplicity of the assay procedure, fast results, and cost efficiency. LoD has automated many bioassays, such as Enzyme-linked Immunosorbent Assay (ELISA) and the loop-mediated isothermal amplification (LAMP) assay ,18. WhenJungwoo et al.  have demAreca catechu, Polygonus minus, and Syzygium polyanthum plant extract.In this paper, we present an approach of LoD method for antioxidant plant activities. We offer automation and sample miniaturization of the DPPH antioxidant activity test with parallel sequential sample loading and mixing. Our proposed LoD method has been tested on ascorbic acid, quercetin, A. catechu, P. minus and S. polyanthum were collected from Selangor and Perak, Malaysia. The plants were chosen based on their reported high antioxidant activity among local plant species [The plants  species ,21,22. TThe antioxidant activity of the samples were determined using the method described by Saha et al. , with soThe photoprotective microfluidic was designed using computer-aided design software (AutoCAD), as shown in All of the engraving of microfluidic channel features was done using a Computer Numerical Control (CNC) machine VISION 2525 by Vision Engraving and Routing Systems, USA, as shown in After preparing the plant extract (as discussed in the previous section), the plant extracts along with the two-standard reagent of antioxidant (ascorbic acid and quercetin) were then preloaded into the plant extract chambers. The plant extract chambers in the middle were designed in four different sizes in duplicate for 25, 50, 75, and 100 mg/mL plant extract. Meanwhile, as shown in There are two devices used to read the antioxidant activity absorbance, which are a microplate reader and the CD reader. The microplate reader was used to compare the antioxidant absorbance activity for both the conventional and LoD method reading over 5 min intervals up to 30 min. The final solutions obtained from the conventional and LoD reaction methods were then transferred to the 96 well plates for the absorbance measurements.Whereas, the CD reader  was usedOur proposed method has minimized human operations in the DPPH antioxidant test, the repetitive pipetting, loading, and mixing steps can be skipped by flow control mechanism of the microfluidic CD. Basically, in our microfluidic CD design, the capillary passive valves were used to control the sequential fluidic flow and the mixing of the liquids. The manipulation of the chamber position, centrifugal force and the capillary valve have determined the sequences of fluids flows to the reaction chamber, the details and analysis of the valves burst frequency is described by Thio et al.  and KazeThio et al. and Kazemzadeh et al. in their paper have been discussed extensively about the theory of the liquid flows inside the passive capillary valves toward the target chamber (in this paper is reaction chamber). In order of the liquid to move from one chamber to another, the capillary pressure need to be overcome by increasing the speed of the motor . In this paper, the location and the geometry of the plant extract chamber and the DPPH chamber on the microfluidic CD have enable the liquid flows manipulation into the reaction chamber.In this section, the comparison results of the conventional and LoD DPPH antioxidant methods are shown and discussed. All of the samples were tested for antioxidant activity by using LoD method and compared with the conventional DPPH antioxidant activity test for 30 min as this is the standard time and common procedures practises in the conventional DPPH assay . SubsequA. catechu, P. minus, and S. polyanthum activity antioxidant has been presented between conventional and LoD method at each concentration. The results showed that there was a significant difference of * p ≤ 0.05 between the conventional and LoD method at each concentration. What can be clearly observed in In Efficacious sample mixing accelerate the chemical reaction and decrease the time of assay . In a chIn A. catechu plant extract gave a result that was 16.25 min faster when compared to the conventional methods at 30 min. The analysis can be seen in Appendix C.The results in S. polyanthum plant extract gave a result that was 21.25 min faster when compared to the conventional methods. Refer Appendix E for the analysis.The results in The results has been shown in P. minus to 5 min, when compared to the standard 30 min of the conventional method. This result may be explained by the fact that the purity, compound mixtures, different form, and weight of molecules that are present in a sample did play a role for diverse antioxidant activity. In this study, the plant extract have been used to test the LoD method, the presence of compound, such as phenol, may cause interference and affect the reading when compared to the actual activity, which expound the different activity time between each plant [Referring to the ch plant ,35.The conventional DPPH method use a high concentrations of the plant extract , which proportionally need large reagent volumes. The proposed method reduced the volume usage to the final of 200 µL to provide a good range of detection as the conventional method . Figure 2 = 0.96) between the reading from the CD reader and the microplate reader, indicating a close relationship between the CD reader and the microplate reader. Unlike the conventional method, the CD reader can read the absorbance of the entire sample directly from the microfluidic CD. It is designed specially to work in complement with the microfluidic CD, and the pipetting process of transferring the solutions to the 96 well plate has be eliminated. The process of transferring the solutions to the 96 well plate risks losing volume, which can be rectified by a closed and automated system, such as that used in the LoD platform. This CD reader endpoint reading system complete and support to the development of an integrated platform for the antioxidant microfluidics CD system.The experimental work was carried out with a modified version of custom-made CD spin test system ,37. The In this study, we have integrated the conventional DPPH antioxidant activity test by proposing a microfluidics CD method for the detection of antioxidant activities in plants. The sequential sample loading and mixing in a closed LoD system minimizes human error and volume loss due to manual pipetting procedures. The “load and run concept” in the proposed LoD method has omitted the repetitive pipetting, mixing, and loading steps in the DPPH conventional method. With four parallel concentrations and duplications that able been run simultaneously on the microfluidic CD, this study has shown that the proposed LoD method for antioxidant activities of the plant extracts procedures has been automated. Using this LoD technique not only it automates the processes but also helps to reduce the incubation time to five minutes, to reach the same antioxidant activity level as the conventional method of 30 min. This approach would work as an application for antioxidant activity and will act as a platform for the determination of future antioxidant activity in a plants or another type of sample.


Asthma is the most frequent chronic disease in children, and its pathogenesis involves genetic, epigenetic, and environmental factors. The rapid rise in the prevalence of asthma registered over the last few decades has stressed the need to identify the environmental and modifiable factors associated with the development of the disease. In particular, there is increasing interest in the role of modifiable nutritional factors specific to both the prenatal and post-natal early life as, during this time, the immune system is particularly vulnerable to exogenous interferences. Several dietary factors, including maternal diet during pregnancy, the duration of breastfeeding, the use of special milk formulas, the timing of the introduction of complementary foods, and prenatal and early life supplementation with vitamins and probiotics/prebiotics, have been addressed as potential targets for the prevention of asthma. In this review, we outline recent findings on the potential role of prenatal and perinatal dietary and nutritional interventions for the primary prevention of pediatric asthma. Moreover, we addressed unmet needs and areas for future research in the prevention of childhood-onset asthma. The role of several dietary factors, including maternal diet and vitamin status, composition of the microbiome, duration of breastfeeding, the use of hydrolyzed formulas and the introduction of complementary foods, has been investigated in recent clinical trials, to identify potential targets for the prevention of childhood-onset asthma , vitamin E, and zinc had a protective effect against early life wheezing in offspring, but not on childhood-onset asthma or other atopic conditions. One recently published study, assessing the impact of pre-pregnancy diet on the risk of allergic outcomes in children  in asthma prevention is still controversial. Two early RCTs showed no significant difference between pHF and eHF in the prevention of allergic diseases in children, including asthma , 42. In In a recent birth cohort study, infants received breast milk only, pHF with or without a hypoallergenic label, or non- hydrolyzed formula. The use of the pHF-with hypoallergenic label, compared to non-hydrolyzed formula, had no protective effect on the risk of asthma up to 2 years of age and was related to a higher risk of wheezing at 1 year in high-risk infants . A recenThe hypothesis that VD status in childhood might influence the susceptibility to childhood asthma and allergy is supported by the evidence on the role of VD as a key modulator of lung growth and innate and adaptive anti-inflammatory immune responses –50. ExpeInterestingly, VD supplementation during pregnancy and infancy has been related to a reduced risk of sensitization to house dust mites at age 18 months .in-utero  or a diet-limited supplementation (control group) showed a 34% reduced risk for recurrent wheezing by 12 months in the intervention group . There iin-utero  and postin-utero , and proin-utero . InterveThere is mounting evidence showing the relationship between the composition of the early-life gut microbiome and the risk of asthma in children , 58, whiIn a 2-year follow-up RCT involving 132 infants at risk of atopy, infants that were fed with a formula containing a mixture of prebiotic oligosaccharides reported a lower incidence for recurrent wheezing compared to the placebo group . In a 1-Taken together, the evidence on the effects of oral probiotics and prebiotics for the prevention of pediatric asthma is so controversial that no definitive recommendation can be made. Differences in the probiotic strain specificity, the population treated, the timing of administration, and the duration of the intervention all contribute to the heterogeneity of the meta-analysis and of RCT outcomes.The supplementation with omega-3 long-chain polyunsaturated fatty acids (LCPUFA) during pregnancy and early life, through the administration of fish oil, has been proposed for the prevention of allergic sensitization and atopic disease, including asthma , 73. LCPConflicting results have been found in studies investigating fish oil supplementation in infants and children for the prevention of allergic sensitization and asthma –67 Tabl. A meta-Recent advances in the field of allergy prevention showed that early 2000s recommendation to delay the introduction of solid allergenic foods to the infant's diet is not an effective approach to reduce the risk of allergic sensitization and atopic diseases in children –88. MoreThe timing of the introduction of fish is of particular interest to the purpose of primary prevention of asthma, given its high content in LCPUFA. Despite the heterogeneity in the methods of analysis and outcome measures, the early introduction of fish has been associated, in many observational studies, with a reduced risk of allergic sensitization , 94. HowThe significant increase in the prevalence of asthma and allergic diseases registered in recent years has promoted research on the identification of modifiable risk factors for the prevention of such disorders. It is well-acknowledged that respiratory health is determined by a complex interaction between genetic factors and environmental drivers that occur during prenatal and early postnatal life, including dietary factors. However, it remains difficult to define the contribution of specific dietary supplements and nutritional food sources to the risk of developing pediatric asthma, due to the heterogeneous pathogenesis of this disease and the limitations of the currently available evidence, in terms of study design, type and duration of interventions and outcomes measures.Further research is needed to accurately identify dietary and nutritional modifiable risk factors for asthma and to address whether the modulation of such factors, either alone or in combination, could contribute to the primary prevention strategies of pediatric asthma.DP, GN, IT, GC, and PC made substantial contributions to conception, design, and acquisition of data. GC, IT, GN, and PC drafted the initial manuscript. DP, EV, ED'A and PC critically reviewed it for important intellectual content. All authors approved the final version of the manuscript. 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.


P< 0.001), while there was no significant improvement in the control group’s scores. Students' attitudes towards professionalism and their professional behaviors did not change significantly.Many medical schools around the world have included professionalism training in their formal curriculum. However, these efforts may not be adequate; given the exposure of students to unprofessional behaviors in the clinical settings. In the present study, we aimed to design, implement, and evaluate a longitudinal program to improve professionalism among medical students upon their transition to clinical settings. A total of 75 medical students were enrolled in the study and randomly assigned to two groups. The control group did not receive any training, while for the intervention group; a 10-hour program through 16 weeks was organized based on the Holmes' reflection approach. The effectiveness of the program was evaluated by measuring three outcomes in both groups. Data analysis was performed using paired t-test and Multiple Linear Regression. Scores of judgment of professionalism increased in the intervention group (from 7.56 to 10.17; Based on our findings, the Holmes reflection approach helps students improve their cognitive base of professionalism. Long-term follow-up and further qualitative studies will help us better understand the effects of this approach on other desirable outcomes.  Professionalism, one of the six core competencies proposed by the Accreditation Council of Graduate Medical Education , has becThe development and maintenance of professionalism are one of the challenging issues in medical education. It is important to explicitly teach professionalism and actively reinforce the desired values, throughout both the undergraduate and postgraduate curricula . Apart fReflection is described as a core element of clinical competence and a fundamental element in teaching professionalism . PhysiciThere is little evidence concerning the application and effectiveness of the Holmes' approach. Seen from a broader perspective, research in the field of medical professionalism is mostly composed of descriptive reports or qualitative studies, most of which have discussed the importance of professionalism or have elaborated on different methods of teaching professionalism , 9, 15. In this study, we tried to provide robust evidence by evaluating the educational outcomes of our longitudinal program at different levels of reaction, learning, and behavior using the Holmes' reflection approach. We chose clerks  because noticing and observing unexpected behaviors, which are in contrast with the ethical values learned in the formal curriculum, more likely occurs at the beginning of a new period of training.Study design This pre-test post-test randomized controlled study was conducted in 2017-2018 on medical students. TUMS medical students are mainly admitted to the university from high school and the total duration of their training, including the internship, is seven years. The preclinical period is an integrated organ system-based curriculum that continues for three and a half years. These medical students have to follow two unit course in ethics in the pre-clinical period. While these students have an early clinical exposure during the pre-clinical period, their first clinical experience at the patient's bedside starts in the fourth year when they are assigned to the university-affiliated hospitals for their clerkship. The internal medicine clerkship was selected for this study, as it is the longest rotation (16 weeks) and the starting point of clinical practice for many students. In the briefing session at the beginning of the internal medicine clerkship, the study objectives and procedures were described for all students, and those who were willing to participate were enrolled in the study. For data analysis, we excluded students who were absent for two or more sessions as well as those who failed to complete the evaluations. A sample size of 23 was estimated for each group as a prior; however, concerning the probability of dropouts, we considered a larger sample size. Students were randomly assigned to the control and intervention groups. The control group did not receive any training, while the intervention group participated in five sessions. To minimize the risk of contamination, participants were asked not to share program details with friends and classmates of the control group .Design and delivery of the educational interventionThe research team met regularly to discuss the details of the design and structure of the longitudinal program based on the Holmes’ approach. They made decisions on the schedule, teaching methods, content, and implementation of the intervention.We finally decided to organize the whole program as five 2-hour sessions within 16 weeks. With regard to the teaching methods, we decided to use the small group technique. Hence, we randomly allocated the students into six small groups (five groups of six and one group of eight people). As we needed facilitators to coordinate discussions in small groups, we invited a number of medical interns who were collaborating with the Educational Development Office at the Medical School. These medical interns were previously trained to play the role of facilitators; yet, before the beginning of the program, we oriented them about our study objectives, the intervention procedure, and the purpose of the sessions. We also provided them with a written guideline. The facilitators were instructed to use their own experience to encourage discussions in the small groups. With regard to the content of the program, we decided to apply the Holmes' steps in five sessions:In the first session, the program was introduced, and the importance and necessity of teaching professionalism were discussed interactively. A notebook was given to the students to record daily diaries immediately to prevent contamination of the memory. Then, the first step of Holmes' reflection approach, priming, was implemented. For this purpose, some examples of how medical students in a clinical setting might yield to external pressures to engage in inappropriate behaviors were presented. The facilitators shared their experience in this area and encouraged the students to participate in the discussion. The students were asked to pay attention to unprofessional behaviors that might occur in upcoming days during their rotation and to document their daily memories in their clinical notebooks. In the second session, according to the second step of Holmes' reflection approach, noticing, the students described the ethical issues that they were exposed to. They were encouraged to share their notes of unprofessional behaviors, to express the emotion that they had felt at those moments, and to discuss their experiences. In the third session, the third step of Holmes' reflection approach, processing, was implemented. The objective of this session was to help students develop feasible action plans to deal with similar situations in the future. In the fourth session, we repeated the processing step, so that all students would have the opportunity to reflect deeply and critically on their observations and to receive feedback. In the last session, the fourth step, choosing, was implemented. The objectives of this session were to support students in identifying and adopting behaviors aligned with the profession's values and to avoid misconducts; help students apply their action plans (an outcome of steps 3) in a way that it reinforces their progress without alienating them from their peers or seniors, and show students how to politely disagree with others. In this session, a sample of poor reflections was compared with some good reflections, and group discussions were conducted. Each facilitator was assigned to a small group and encouraged students to reflect. The facilitators provided feedback about the students' reflection and helped them distinguish a poor reflection from a good one, using a guideline . Investigation of outcomeThe assessment of outcomes was carried out at the beginning of the study and one month after the program, for both intervention and control groups. We used several tools to determine the effectiveness of the program:We evaluated the cognitive base of professionalism (level 2A of Kirkpatrick's framework) using a Situational Judgment Test (SJT), consisting of 11 clinical scenarios (seven constructed-response questions and four selected-response questions). Some examples of this test are presented in The students' attitude toward professionalism (level 2B of Kirkpatrick's framework ) was assMoreover, the students' professional behaviors were evaluated (level 3 of Kirkpatrick framework) using the Professionalism Mini-evaluation Exercise (P-MEX). The content validity assessment showed that five items  were not suitable for evaluating the TUMS clerks, and thus, were omitted. The internal consistency of the 16-item scale was found to be 0.95, based on Cronach's alpha. Since each item of the P-MEX is graded as follows: 1= unacceptable; 2= below expectations; 3= met expectations; and 4=exceeded expectations, the total score ranged from 16 to 64. Interns, residents, fellows, and faculty members used P-MEX to assess participants. One of the researchers provided the necessary instructions on the tool before conducting the assessments. Data analysis was carried out only for students whose evaluations were completed before and after the intervention, and for those who participated in three sessions and more.StatisticsThe demographics of participants were summarized using frequency, mean, and standard deviation. The paired t-test was performed to compare means of SJT, QUBPI, and P-MEX scores. Multiple Linear Regression (MLR) was performed using medical students' post-test mean scores of SJT, QUBPI, and P-MEX as the dependent variables; and the pre-test mean scores and the group as the independent variables. All data analyses were performed using SPSS version 21.The study protocol was approved by the Ethics Committee of Tehran University of Medical Sciences (TUMS) (No. IR.TUMS.MEDICINE.REC.1395.1722).Among 105 eligible students, 75 (71.4%) voluntarily participated in this study and were randomly allocated to the intervention and control groups. The mean age of students in the intervention group was 22.12 years (SD=0.95), and 44.7% (N=17) were female. The mean age of the control group was 22.77 years (SD=0.95), and 35.5% (N=11) were female. There was no significant difference between the mean age of the students in the control and intervention groups. The mean and standard deviation of pretest and posttest scores of judgment (SJT), Attitude (QUBPI), and behavior (P-MEX), before and after the program is presented for the control and intervention groups in P<0.001). Based on the results of the MLR of SJT, independent variables of “group” and “mean pre-test score” explained 64% (R2) of variance scores (dependent variable). Considering the students' attitude (QUBPI), the MLR showed that independent variables of "group" and "mean pre-test score" explained only 16% of the variance in the mean score. The MLR of students' behaviors (P-MEX) showed that independent variables of "group" and "pre-test behavior score" could explain only 0.23% of the variance in the mean score. Post-test judgment was improved in the intervention group, showing a significant difference between the control and intervention groups . Also, in line with the paired t-test results, after controlling the confounders using MLR, the students' judgment in the intervention group increased significantly in comparison with the control group (P< 0.001). Based on the results of the MLR, by a one-unit increase in the mean pre-test score of the students' attitudes, the mean score of post-test increased by 0.46 units (P< 0.001). However, after controlling the confounders using MLR, in line with the paired t-test results, there was no statistically significant difference between the two groups’ attitudes after the intervention (P= 0.34) (It should be noted that RP= 0.34) . P= 0.07). Also, with a one-unit increase in the mean pre-test score of students' behavior, the mean score of the post-test increased by 0.77 units (P< 0.001). However, after controlling the confounders using MLR in line with the paired t-test results, professional behavior in the intervention group did not show any statistically significant difference (P= 0.07).In the intervention group, in comparison with the control group, the mean score of students' behavior increased by 0.17 units, provided that the variable pre-test value was considered constant . Teaching methods included didactic lectures, journal clubs, small group discussions, and book reviews. They assessed the outcomes using four levels of the Kirkpatrick model. Given the qualitative nature of the data extracted from levels 3 and 4 in this study, outcomes were measured by the incidence of sentinel events. The results showed improvement in knowledge and professional behaviors. In addition, the participants were satisfied with the course; however, their attitude toward professionalism did not change. Since the authors did not use a randomized design, it is difficult to attribute all the changes to the training program. In addition, participants of the study included a diverse range of health professionals who were more involved in patient care, compared to the clerks in our study . Given the lack of comparable quantitative, randomized studies on teaching and assessing professionalism, our ﬁndings can be considered in a wider context including medical ethics and ethical judgement.Murrell conducted a cross-sectional study to evaluate medical students' ethical judgment. The results showed that there was no significant difference between students who participated in the professionalism course and those who did not . This fiClearly, there are many variables that could have influenced the professional behaviors of learners and their attitudes toward professionalism. Hence, we were not able to detect a significant improvement in these two outcomes. The hidden curriculum is a very powerful tool for encouraging professional values , 27; yetTo the best of our knowledge, this is the first true experimental research that evaluated a professionalism training program based on a guided reflection method. The current study had several strengths. First of all, we used a conceptual framework that facilitated guided reflection and feedback in multiple sessions. Interestingly, while the Holmes' approach is grounded in a theoretical basis, it is very practical and applicable to the real life. To make the most out of this approach, we managed to design our intervention as a longitudinal program, because standalone educational courses are considered to be less effective. Besides, to provide robust evidence on the effects of our program, we included a control group and performed pre-post comparisons. We also used previously established instruments for all of our measurements, including the cognitive base and attitude of students, as well as their actual behavior, an outcome that has often been ignored in previous studies. On the other hand, our study had also a few limitations. Despite orientating the intervention group not to share material with the control group, information exchange was possible between the two groups. In addition, while we tried to minimize the differences between small group facilitators, through training and written guidelines, their diverse experiences might have resulted in different discussions that in turn could have caused inconsistency in our program. As the TUMS clerks are not involved in direct patient care, so direct observation of their performance using P-MEX might have not been always possible. Secondly, in spite of our effort to train the raters, it is possible that some of residents, fellows, or faculty members have failed to contribute to a valid assessment due to their high workload.Finally, our study was performed on a small scale, we did not evaluate the longterm outcomes, and we did not collect qualitative data which could have provided us with a deep understanding about the impact of the program.The application of Holmes’ reflection approach improved the judgment of professionalism among medical students upon their transition to the clinical settings. It is recommended to conduct mixed-methods studies with long-term follow-ups to better evaluate the effects of reflection on the attitudes and professional behaviors of students.


We did not find any significant correlation between vitamin D concentration and body mass index. Conclusions: The burn injury has an enormous impact on the metabolism and the risk factors of the deficiency for the general population (BMI) have an effect on burned patients. Our study showed that concentration of 25-hydroxycholecalciferol is strongly correlated with serum albumin level, even more than total burn surface area and burn degrees as expected. We suspect that increased supplementation of vitamin D should be based on albumin level and last until albumin levels are balanced.Background: Burned patients have an increased need for vitamin D supply related to the maintenance of calcium–phosphate homeostasis and the regulation of cell proliferation/differentiation. This study aimed to analyze the concentration of 25-hydroxycholecalciferol and its relationship with severe condition after burn injury. Methods: 126 patients were enrolled in the study. Patients were qualified due to thermal burns—over 10% of total body surface area. On the day of admission, the following parameters were assessed: 25-hydroxycholecalciferol concentration, total protein concentration, albumin concentration, aspartate transaminase activity, alanine transaminase activity, albumin concentration, creatinine concentration, c-reactive protein concentration, procalcitonin concentration, and interleukin-6 concentration. Results: Almost all patients (92%) in the study group had an improper level of vitamin D (<30 ng/mL), with the average of 11.6 ± 10.7 ng/mL; 17.5% of patients had levels of vitamin D below the limit of determination—under 3 ng/mL. The study showed that there are several factors which correlated with vitamin D concentration during the acute phase of burn injury, including: total protein ( Burns are one of the most serious injuries, which often include multi-organ dysfunction. Every year, about 1% of Polish people (both children and adults) suffer from various types of burns . Due to The aim of our study was to analyze the level of vitamin D and its relationship with severe condition during the acute phase of burn injury.One hundred twenty-six patients with burn injuries were enrolled in the study. Participants were patients of the Western Pomeranian Center for the Treatment of Burns Injuries and Plastic Surgery in Poland. Patients were qualified due to thermal burns—over 10% of the total body surface area (TBSA); 88% of patients involved in the project meet the major burn criteria. According to the classification used in our hospital, major burn needs to include: ≥25% TBSA, or ≥20% in adults over 40 years old, or ≥10% TBSA with full-thickness burn, or all burn injuries complicated by major trauma/inhalation injury; 12% of patients met the moderate burn criteria . According to their medical history from admission to the unit, none of the included patients suffered from chronic kidney disease. The protocol used in our hospital includes no albumin administration during the first 24 h of burn injury. Instead of albumin patients were given Ringer’s lactate and fresh frozen plasma. The protocol of the study has been accepted by the local bioethical committee at the Pomeranian Medical University in Szczecin (KB-0012/143/16). Every participant signed a consent to take part in the study and was informed about its course, benefits, and potential side effects. Patient characteristics are shown in On the day of admission, the following parameters were assessed: 25-hydroxycalciferol concentration (vitamin D status predictor), total protein concentration, albumin concentration, aspartate transaminase activity, alanine transaminase (ALT) activity, albumin concentration, creatinine concentration, c-reactive protein (CRP) concentration, procalcitonin concentration, and IL-6 concentration. All measurements were performed in a commercial certificated laboratory in the Hospital. The 25-hydroxycholecalciferol measurement was based on validated automatic immunochemical method. Serum was used as basic material for all analysis.p < 0.05 were considered as statistically significant. To control type I errors, the false discovery rate (FDR) approach was used. The calculations were performed using the p.adjust function of the stats package in R 4.0.2. Multiple regression was used to assess the relationship between albumin, total protein, and vitamin D concentration. The values being at the threshold of statistical significance were established at p < 0.055 and the statistical tendency from p = 0.06 to p = 0.1. In reference to the results which were not statistically significant, the abbreviation NS (not significant) was used instead of p.The statistical analysis was performed using the “R 3.0.2” program. In order to check the normal distribution, the Shapiro–Wilk test was used. The distribution did not deviate from the norm, and parametric tests were used in the calculations. The results are presented as mean values and standard deviation (SD). In order to estimate the correlations, the Pearson’s correlation test was used. To estimate the connection between burn degree and concentration of vitamin D, the Poisson regression was used. The values of p = 0.08). The average concentration in particular subgroups is shown in Almost all patients (92%) in the study group had an improper level of 25-hydroxycholecalciferol (<30 ng/mL), with the average of 11.6 ± 10.7 ng/mL; 17.5% of patients had a level below the limit of determination—under 3 ng/mL. Poisson regression showed that there is a statistical tendency between 25-hydroxycholecalciferol concentration and burn degree (Pearson’s test showed a significant correlation between body mass index (BMI), total protein, albumin, percentage of body burns, ALT, CRP, and vitamin D concentration . The mosp < 0.0001). We also performed analysis of the correlation of percentage of body burns with albumin/protein. The results are presented in As we demonstrated that total protein and albumin concentration were significantly correlated , we decided to conduct a multiple regression analysis to demonstrate that only albumin was significantly associated with vitamin D concentration  and almost 20% of them had a level below the limit of the quantification (<3 ng/mL). Płudowski et al. revealed that 89.9% of the Polish population is vitamin D-deficient, with 18.0 ± 9.6 ng/mL of average concentration of 25-hydroxycholecalciferol [The need of supplementation in burned patients is well known and described in many medical protocols . Unfortur = 0.42), serum albumin, (r = 0.62), percentage of body burns (r = 0.36), AST (r = 0.21), and CRP (r = 0.22). However, we did not see a significant relationship between BMI and concentration of 25-hydroxycholecalciferol, which are considered to be one of the most important factors of vitamin D deficiency in the general population [There are multiple factors that are associated with low level of circulating vitamin D. Many of them are connected with poor prognosis in critically ill patients, among others: organ failure , sepsis,pulation . We can Evaluation of the vitamin D level in burned patients is a difficult issue, related to the acute phase development, which is associated with decreased levels of vitamin D binding protein (VDBP). The amount of protein that can bind 25-hydroxycholecalciferol, significantly decreases, and the tested “free amount” of the active form can be falsified. Reduced protein synthesis persists for several months after burns; therefore, the results obtained can be false . Howeverr = 0.62) and the multiple regression confirm that thesis. A majority (85–90%) of 25-hydroxycholecalciferol D is bound to VDBP and 10–15% to albumin [Our study reveals that serum protein level, mostly albumin, strongly correlated  with ser albumin . We can  albumin . However albumin .Burn injuries have an enormous impact on the metabolism in burned patients. On the other hand, the risk factors of deficiency for the general population  have a negligible effect on burned patients. Our study shows that the concentration of 25-hydroxycholecalciferol is strongly correlated with serum albumin levels, even more than TBSA and burn degrees, as expected. Albumin can be a good predictor of vitamin D status, especially since the concentration of 25-hydroxycholecalciferol is not measured in standard analysis during admission to hospital. We presume that this direction of vitamin D diagnostic should be tested in randomized clinical trials.