Lannea coromandelica (Houtt.) Merr. are used in the Garo, Pahan, and Teli tribal communities of Bangladesh as a traditional medicinal plant to treat hepatitis, diabetes, ulcers, heart disease, and dysentery. However, there have been limited phytochemical and biological studies on the bark of L. coromandelica. This study aimed to investigate the antioxidant activities of L. coromandelica bark extract (LCBE) and the underlying mechanism using RAW 264.7 cells. The LCBE was analysed by high-pressure liquid chromatography (HPLC) to detect its key polyphenolic compounds. Various in vitro antioxidant assays were performed using RAW 264.7 cells to assess the antioxidant effects of the LCBE and to understand the underlying molecular mechanism. HPLC revealed the presence of gallic acid, (−)-epigallocatechin-3-gallate, catechin, chlorogenic acid, and caffeic acid in the LCBE. The extract showed a very potent capacity to scavenge numerous free radicals through hydrogen atom transfer and/or electron donation and also quenched cellular reactive oxygen species (ROS) generation without showing any toxicity. The LCBE was found to combat the oxidative stress by enhancing the expression, at both transcriptional and translational levels, of primary antioxidant enzymes as well as phase II detoxifying enzymes, especially heme oxygenase 1, through the upregulation of the nuclear factor erythroid 2-related factor 2 (NRF2)-mediated pathway in RAW 264.7 cells via the phosphorylation of p38 kinase and c-Jun N-terminal kinase (JNK). The LCBE exhibited strong antioxidant activities and mitigated the cellular ROS production. These results provide scientific evidence of its potential as an ideal applicant for a cost-effective, readily available, and natural phytochemical, as well as a strategy for preventing diseases associated with oxidative stress and attenuating disease progress.The leaves of  The role of oxidative stress has been confirmed in the development of neurodegenerative disorders , autoimmune pathologies, diabetes, cancer, and digestive diseases such as gastrointestinal inflammation and ulcerative disorders [450) and phase II (detoxifying and antioxidant proteins) enzymes to detoxify potentially detrimental substances and sustain cellular redox homeostasis [Oxidative stress, induced by the production of reactive oxygen species (ROS), including free radical compounds such as the hydroxyl radical , and γ-glutamyl cysteine synthetase catalytic subunit (GCLc), which are associated with oxidative stress response, cytoprotection, and drug metabolism [The ubiquitously expressed transcription factor nuclear transcription factor erythroid 2-related factor 2 (NRF2) serves as a master regulator of cellular defence against oxidative stress by modulating the basal and inducible expression of various detoxifying and antioxidant enzymes, such as heme oxygenase 1 (HO-1), NAD(P)H:quinone oxidoreductase (NQO1), glutathione tabolism . Under ptabolism . A confotabolism . In additabolism .Antioxidants, which modulate oxidative processes in the body, are believed to be important for health maintenance. Even though many synthetic antioxidants are available and frequently used, the safety and toxicity of these antioxidants have raised imperative concerns . HoweverLannea belongs to the family Anacardiaceae and includes tropical deciduous trees widely distributed in India, Bangladesh, and other tropical countries. Traditionally, various parts of the plants have been used as a medicine for various diseases. The stem bark is used by the Garo, Pahan, and Teli tribal communities in Bangladesh to treat hepatitis, diabetes, ulcers, heart disease, and dysentery [Lannea spp. against various diseases. It has been shown that twigs induce apoptosis of human liver cancer cells [Lannea coromandelica (Houtt.) Merr. bark extract (LCBE), and the mechanisms behind the LCBE regulation of the expression of these enzymes are yet to be investigated.The genus ysentery . Leaf juysentery , while tysentery . The barysentery , eruptioysentery . Based oer cells  and leaver cells , antioxier cells , and anter cells  activitier cells , hypotener cells , antihyper cells , antimicer cells , analgeser cells , and theer cells . HoweverTherefore, we hypothesised that components of LCBE are functionally associated with NRF2 to stimulate the expression of certain antioxidant enzymes. The current study focused on the role of LCBE in cell antioxidant processes by using various in vitro antioxidant assays and measuring the messenger RNA/protein expressions of antioxidant enzymes in RAW 264.7 cells. We also studied the underlying mechanisms by determining the activation of NRF2 functions by LCBE in RAW 264.7 cells. The results revealed that LCBE controls antioxidant enzymes by moderating the NRF2 function in RAW 264.7 cells. To detect the active components of the LCBE, HPLC analysis was performed using five representative antioxidant standard materials. Interestingly, the LCBE exhibited peaks with the same retention times as the following standard compounds: gallic acid (13.07 min), epigallocatechin-3-gallate , catechin (55.21 min), chlorogenic acid (57.52 min), and caffeic acid (59.69 min) .•-, ABTS•+-, superoxide radical-, and hydroxyl radical-scavenging activities were measured. The LCBE scavenged DPPH•, a steady organic nitrogen radical, dose-dependently  are extremely sensitive free radicals created in biological systems and capable of damaging almost every molecule found in cells [Depending on their mechanisms of action, antioxidants may inhibit cellular damage caused by oxidative stress directly or indirectly. Scavenging of free radicals, ROS, and/or reactive nitrogen species (RNS) by an antioxidant through the donation of hydrogen or electron is considered a direct pathway. In contrast, indirect antioxidant pathways induce the expression of phase II detoxifying and antioxidant genes . To inveendently A. Furthed ABTS•+ B. The suin cells . In thisBecause the LCBE showed strong radical-scavenging activities, it was tested for scavenging activity against cellular ROS induced by 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), with gallic acid (a positive control). AAPH is a water-soluble azo small molecule, and upon decomposition, AAPH generates one mole of nitrogen and two moles of carbon radicals. The carbon radicals can either bind to generate secure products or react with molecular oxygen to produce peroxyl radicals, thus continuing a constant rate of free radical production in solution . LCBE trSod1, Sod2, Cat, and Gpx1 (To investigate the effects of LCBE on the induction of the antioxidant enzymes superoxide dismutase (SOD)1/SOD2, catalase (CAT), and glutathione peroxidase 1 (GPX1), and the phase II detoxifying enzymes HO-1, NQO1, GCLc, GCLm, and GSTpi), RAW 264.7 cells were treated with the LCBE at various concentrations for 24 h. Reverse transcription (RT)-PCR analysis indicated a dose-dependent increase in the mRNA expression of and Gpx1 A, as weland Gpx1 C. Increaand Gpx1 B, and HOand Gpx1 D were fuand Gpx1 E. These Nrf2, whereas that of Keap1 was mitigated dose-dependently , hydrogen peroxide (H2O2), and the hydroxyl radical (HO•) are partially produced by the reduction of oxygen during cellular metabolic reactions and served as sever intracellular toxic species. There is considerable evidence that strong, chemically reactive ROS are considered to function entirely as cellular damaging agents, indiscriminately reacting with proteins, lipids, and DNA [Oxidative stress can be viewed as an imbalance between the generation of free radicals and their elimination via cellular defence mechanisms. ROS such as the superoxide anion radical (O and DNA . In currp) and linear regression analysis (data not shown). The results exhibited very strong correlations with 2,2-diphenyl-1-picrylhydrazyl (DPPH), ABTS, superoxide, and hydroxyl radical-scavenging activities . These data are in accordance with those obtained by other researchers who have displayed that a high total phenol content boosts the antioxidant activities [Mounting evidence suggests that phenolic compounds have diverse biological effects, including antioxidant activity. The antioxidant capacity of phenolic compounds is mainly due to their redox capacities, which can play a pivotal role in adsorbing and neutralising free radicals, scavenging singlet and triplet oxygen, and decomposing peroxides . Flavonotivities  and thattivities .L. coromandelica is rich in phenol and flavonoid compounds such as ellagic acid, quercetin-3-arabinoside, quercetin [R,3S)-(+)-3′,5-dihydroxy-4,7-dimethoxydihydroflavonol, physcion, -(+)-4′,5,7-trimethoxydihydroflavonol, -(+)-4′,7-di-O-methyldihydroquercetin, -(+)-4′,7-di-O-methyldihydrokaempferol, -(+)-4′-O-methyldihydroquercetin, leucocyanidin, leucodelphinidin [L. coromandelica for gastrointestinal disorders and diabetes and suggests the identified major compounds as contributors to production of ethnopharmacological effects. Additionally, PASS predicted antioxidant and free radical scavenging properties of EGCG, catechin and chlorogenic acid  which were supported by previous studies [The identification of active components in extracts from traditionally used plants is a crucial goal for the development of nutraceuticals. Numerous scientific reports have suggested that uercetin , phinidin , β-sitosphinidin . Based ophinidin . Moreove studies ,35. Furt•-, ABTS•+-, superoxide radical-, and hydroxyl radical-scavenging assays as well as FRAP, CUPRAC, and ORAC assays, were performed to estimate the antioxidant activities of LCBE. The results confirmed that LCBE has a broad range of antioxidant properties. The radical-scavenging activities of the LCBE are summarised in • and ABTS•+ assays are thought to be the most common spectrophotometric methods for the determination of the antioxidant capacity of any extract. The magnitude of free radical quenching in both DPPH and ABTS assays was dose-dependent and steadily increased with the increase of sample concentration  is generated by the one-electron reduction of O2 during the mitochondrial electron transport chain reaction. It is well known that O2− is rapidly converted into H2O2 by SOD, and H2O2 can be detoxified into H2O by the scavenging enzymes GPX and CAT. These enzymes act together in the metabolic pathway of free radicals [The quenching of free radicals, ROS, and RNS through donation of either hydrogen or electrons demonstrates the direct cellular antioxidant capacity of an antioxidant, whereas phase II detoxifying and antioxidant enzymes are believed to provide indirect defensive mechanism against oxidative stress . Free raradicals ,41. In tHmox1 gene, and HO-1 levels have been found to decrease with increasing oxidative stress [Hmox1 expression due to the presence of gallic acid, EGCG and catechin. Therefore, it is assumed that the induction of phase II enzymes may be another mechanism accounting for the activity of LCBE against oxidative stress. To further investigate this mechanism, the mRNA and protein levels of NRF2, the key regulator of phase II enzyme activation, were examined. NRF2 is sequestered by KEAP1 in the cytosol and constitutively targeted for polyubiquitination under normal conditions. Cell exposure to electrophilic and oxidative stress, however, releases NRF2 from the KEAP1 complex, which enables NRF2 to translocate to the nucleus and activate a battery of phase II cytoprotective genes by binding to the ARE [Ginkgo biloba extract; however, details of the likely interaction between LCBE and KEAP1 require further investigation. G. biloba extract was found to stimulate NRF2 and its translocation into the nucleus, with a subsequent increase in the expression of phase II genes, such as Nqo1, Hmox1, and Gclc through the KEAP1–NRF2–ARE signalling pathway in Hepa-1c1c7 and HepG2 cells [O-caffeoylquinic acid modulates the NRF2 nuclear translocation and ARE-dependent gene expression, such as Hmox1, Nqo1, and Gst in HT29 cells [Following the treatment of RAW 264.7 cells with LCBE for various times and at different doses, significant increases in phase II enzymes were observed at both mRNA and protein levels C–E. GCLce stress . Severale stress  and our  the ARE . In thisG2 cells . An acti29 cells . Other s29 cells ,49. In t29 cells , EGCG [329 cells , and chl29 cells , have thL. coromandelica bark was collected from the adjoining area of the Jahangirnagar University campus, Bangladesh, during August 2015. The plant material was taxonomically identified by the National Herbarium of Bangladesh, whose voucher specimen (DACB: 37549) has been deposited in our laboratory for future reference. Dried and crudely pulverized bark (100 g) was extracted with methanol under reflux for 3 h (three times). The extract was then filtered through filter paper . The filtrate was collected and subjected to lyophilisation to give 3.44 g of extract (yield: 3.44%). Finally, the lyophilised sample was dissolved in deionised water at a concentration of 10 mg/mL.S-triazine, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS), 3--2,5-diphenyltetrazolium bromide (MTT), dimethyl sulphoxide (DMSO), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2′,7′-dichlorofluorescin diacetate (DCFH-DA), and phosphate-buffered saline  were obtained from Sigma-Aldrich . Dulbecco’s modified Eagle’s medium (DMEM), foetal bovine serum (FBS), penicillin-streptomycin mixture, and 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) were purchased from Gibco BRL Life Technologies . Anti-superoxide dismutase 1 (SOD1), anti-HO-1, anti-catalase (CAT), anti-glutathione peroxidase 1 (GPX1), anti-NRF2, and anti-β-actin were obtained from Santa Cruz Biotechnology . Anti-phospho-JNK, anti-p44/42 MAPK (ERK1/2), anti-phospho-p44/42 MAPK (ERK1/2), anti-phospho-p38, and anti-p38 antibodies were also used for this experiment after purchasing from Cell Signaling Technology .2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), phenazine methosulphate (PMS), neocaprione, 2,4,6-tris(2-pyridyl)-Phytochemical characteristics of the LCBE were studied by high-pressure liquid chromatography (HPLC) using standard compounds, including gallic acid, (−)-epigallocatechin gallate (EGCG), catechin, caffeic acid, and chlorogenic acid. HPLC analysis was carried out using an Agilent 1200 chromatographic system  with a UV-Vis diode-array detector and the ChemStation software . A 0.45-μm nylon filter  was used to filter the sample and polyphenolic compounds were analysed according to the method described previously . A Zorbacontrol is the absorbance of the control and Abssample is the absorbance of the sample. All samples were analysed in three independent experiments.The DPPH radical-scavenging assay used for the evaluation of free radical-scavenging activity of the LCBE was operated following the protocol described elsewhere . AscorbiThe method of Re et al.  was adop2•−) was generated by a non-enzymatic PMS/NADH system, which reduces nitro blue tetrazolium to purple-coloured formazan, and the O2•−-scavenging activity of the LCBE was measured according to a previously reported method [The superoxide radical  generated by the Fenton reaction using a Fe3+-ascorbate–EDTA–H2O2 system. Equation 1 was used to determine the percent inhibition activity of the LCBE.A previously described method  was adopThe ferric reducing antioxidant power (FRAP) assay was performed to measure the reducing power capacity of LCBE as described previously  in whichThe cupric reducing antioxidant capacity (CUPRAC) of the LCBE was determined according to the method described previously . The ascThe oxygen radical absorbance capacity (ORAC) assay was performed as previously reported . Trolox,http://preadmet.bmdrc.kr/) were employed. The toxic risks assessed were interpreted and expressed in a flexible manner: (+) low potential, (++) medium risk, (+++) high risk and non-detected risk (N) [http://www.pharmaexpert.ru/PASSonline/predict.php) was used. Prediction results were expressed in percentage of probable activity (Pa) and probable inactivity (Pi). Pa and Pi values vary from 0.000 to 1.000, thus, here, we considered only activities with Pa > Pi and Pa > 0.700.To estimate possible toxicity risks  and the possible biological activities of the major compounds of LCBE, a computational simulation study was performed. For toxic risk prediction, four online computer programs: ACD/Labs , admetSAR server, pkCSM platform, and the PreADMET web-based application (risk (N) . For bio2. The tetrazolium dye colorimetric test (MTT) was used to determine the viability of RAW 264.7 cells. RAW 264.7 cells (5 × 105 cells/mL) and BJ cells (1 × 105 cells/mL) were first cultured in 96-well plates for 24 h and treated with different concentrations of test samples, followed by removal of the media and addition of the MTT reagent to each well and incubation at 37 °C for 1 h. Then the media were also removed, and the wells were washed twice with PBS (pH 7.4). Then, 100% DMSO was used to dissolve the formazan and the absorbance in each well was measured at 570 nm using a microplate reader , and the percentage of viability was calculated.RAW 264.7 cells and Skin Fibroblast cells (BJ cells)  were cultured at 37 °C in DMEM supplemented with 10% FBS, streptomycin–penicillin  in a humidified atmosphere of 5% CO5 cells/mL) with DMEM for 24 h. Then, the cells were pretreated with various concentrations of the LCB extract. After 1 h, the cells were stimulated with AAPH (600 µM) and incubated for an additional 30 min. Consequently, they were washed with PBS twice and 25 µM DCFH-DA was added into the cells incubated for 30 min at 37 °C. The fluorescence intensity was calculated at an excitation wavelength of 485 nm and an emission wavelength of 528 nm using a Victor3 fluorescence microplate reader (PerkinElmer). Cellular oxidative stress due to ROS production by 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) was measured spectrofluorometrically using the DCFH-DA method . RAW 264TRIzol  was used to extract the total RNA using manufacturer’s protocol. Total RNA (2 µg) was reverse transcribed using reverse transcriptase  and oligo(dT) primers. cDNA was synthesized using a thermal cycler Dice TP600 . Polymerase chain reaction (PCR) products were visualised by ethidium bromide staining after electrophoresis. Specific oligonucleotide primers for mouse transcripts were shown in Standard protocol was used to prepare the RAW 264.7 cell lysates and then mixed with sodium dodecyl sulphate (SDS) sample buffer , followed by denaturing at 100 °C for 5 min. For nuclear protein extraction, a nuclear/cytosolic fractionation kit  was used. SDS–polyacrylamide gel electrophoresis (10%) was performed to separate the sample protein (20 µg/lane). Following electrotransfer to nitrocellulose membranes , the membranes were incubated overnight with 5% skim milk used to block the protein, followed by incubation of the membranes incubated overnight with primary antibodies such as anti-goat IgG–horse radish peroxidase , and anti-rabbit IgG–HRP (Santa Cruz Biotechnology) applied as secondary antibodies. The antigen–antibody reaction was exposed using an enhanced chemiluminescence system (PerkinElmer).p < 0.05 or p < 0.01. All analyses were executed using SPSS for Windows, version 10.07 .All data are denoted as the mean ± standard deviation. Data were analysed using one-way analysis of variance. Differences were considered significant at


This review will first present the role of H2S in the renal system and provide evidence for the links between H2S signaling and the underlying mechanisms of renal programming, including the renin–angiotensin system, oxidative stress, nutrient-sensing signals, sodium transporters, and epigenetic regulation. This will be followed by potential H2S treatment modalities that may serve as reprogramming strategies to prevent hypertension and kidney disease of developmental origins. These H2S treatment modalities include precursors for H2S synthesis, H2S donors, and natural plant-derived compounds. Despite emerging evidence from experimental studies in support of reprogramming strategies targeting the H2S signaling pathway to protect against hypertension and kidney disease of developmental origins, these results need further clinical translation.Adverse environments occurring during kidney development may produce long-term programming effects, namely renal programming, to create increased vulnerability to the development of later-life hypertension and kidney disease. Conversely, reprogramming is a strategy aimed at reversing the programming processes in early life, even before the onset of clinical symptoms, which may counter the rising epidemic of hypertension and kidney disease. Hydrogen sulfide (H One in three adults worldwide has high blood pressure (BP). The prevalence of hypertension is even higher among patients with kidney disease. Conversely, kidney disease is the most common form of secondary hypertension. In addition to sharing many common risk factors, hypertension and kidney disease could be the cause and consequence of each other. Hypertension as well as kidney disease, when found early, can be treated to prevent more related disorders and serious complications. Environmental factors in fetal and infantile life can be important early origins of adult disorders due to fetal programming that permanently shapes the body’s morphology and function and contributes to adult disease. This idea is framed as the developmental origins of health and disease (DOHaD) . Notably2S) plays a crucial role in the normal physiology and pathogenesis of many disorders, and many therapeutic targets exist for H2S therapy [2S signaling that may serve as a reprogramming strategy to counter the growing epidemic of hypertension and kidney disease.Kidneys play a decisive role in blood pressure (BP) regulation. Adverse environments occurring during nephrogenesis may produce long-term programming effects on the structure and/or function of the kidney, formally known as renal programming , which m therapy ,8. Based2S, the third gasotransmitter, next to nitric oxide (NO) and carbon monoxide (CO), plays a key role in BP control and renal physiology [2S can occur via two pathways, enzymatic and nonenzymatic. Enzymatic synthesis of H2S from l-cysteine occurs through three enzymes, cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulphurtransferase (3MST), which work together with cysteine amino transferase (CAT) [l-cysteine serves as a substrate for both CBS and CSE to form H2S. CBS and CSE can generate H2S using many different substrates. CBS catalyzes homocysteine and serine to generate cystathionine. CSE catalyzes l-cysteine to produce pyruvate, H2S, and ammonia. CSE also breaks down cystathionine into l-cysteine, α-ketobutyrate, and ammonia. In an alternative pathway, CAT and D-amino acid oxidase (DAO) provide 3-mercaptopyruvate (3MP) for 3MST to produce H2S. Additionally, d-cysteine can be metabolized by DAO to generate H2S in the kidney [l-cysteine to form 3MP, whereas DAO produces 3MP from d-cysteine. All of these H2S-generating enzymes are expressed abundantly in the kidney. Interestingly, renal production of H2S from d-cysteine is approximately 80 times greater than that from l-cysteine [2S occurs through glucose, glutathione, inorganic and organic polysulfides, and elemental sulfur. The biochemical pathways related to the H2S synthesis are illustrated in Hysiology ,8,9. These (CAT) . l-cystee kidney . In the cysteine . Alterna2S has diverse and widespread biological functions, including vasodilatation, mitochondria bioenergetics, metabolic modulation, renal excretory function, angiogenesis, anti-inflammation, antioxidant, and antiapoptosis [2S signaling have been incompletely understood. One pathway for H2S signaling is through protein sulfhydration by the formation of a persulfide bond to the reactive cysteine residues of target proteins [2S signaling regulates physiological processes via sulfhydration, but the mechanisms involved are still not well characterized. Hpoptosis ,8,9. Untproteins . There i2S deficiency appears prior to the development of hypertension, whereas the administration of exogenous H2S donor (NaHS) protects SHRs against hypertension [2S deficiency in hypertension has been studied in other models of hypertension, including the renovascular hypertensive model [2S and hypertension [2S signaling deficiencies have also been associated with various kidney diseases, such as renal ischemia/reperfusion injury, obstructive nephropathy, diabetic nephropathy, hypertensive nephropathy, and the 5/6 nephrectomy rat model of chronic kidney disease. These models have been reviewed elsewhere [2S on hypertension and kidney disease, and this has also been reviewed elsewhere [In the spontaneously hypertensive rat (SHR), a commonly used genetic hypertensive model, Hrtension . The conve model , NO-defive model , and Dahve model . In agrertension . Howeverrtension . H2S siglsewhere ,19. Convlsewhere ,21,22. H2S and the abovementioned mechanisms. Each of these examples will be discussed in turn.Though a variety of organ systems can be programmed in response to early-life adverse environmental insults, renal programming is considered crucial in the development of hypertension and kidney disease ,5,6. Des2S levels or downregulation of H2S-generating enzymes have been found in some hypertensive models with activation of the RAS [2S administration has been shown to suppress renin release [Ren, Atp6ap2, Agt, Ace, and Agtr1a [2S contributing to the protection of programmed kidney disease need to be further investigated. The RAS is a well-known hormonal cascade controlling BP and kidney development ,25. Curr the RAS ,32. Exogd Agtr1a , and decd Agtr1a , resulti2S is a Janus-faced molecule that acts either as a pro-oxidant or antioxidant [2S has an antioxidant property, by which it modulates the concentration of glutathione, an important intracellular thiolic antioxidant, and acts as a free radical scavenger [2S causes vasodilatation and there is crosstalk between these two molecules to regulate BP [2S. However, whether the antioxidative ability of H2S in itself is important in reducing the BP in concert with other BP-lowering actions remain to be clarified.The development of the embryo occurs in a relatively low-oxygen environment, and the developing fetus is highly vulnerable to oxidant injury due to its low antioxidant capacity . Oxidatiioxidant . It can cavenger . Like NOulate BP ,8. Thus,2S is the activation of AMPK and promotion of autophagy [2S on nutrient-sensing signals and their relationships to hypertension and developmental kidney disease.Imbalanced maternal nutrition and metabolic insults can disturb nutrient-sensing signals, which play a key role in fetal metabolism and development, resulting in renal programming and developmental hypertension ,40. A nuutophagy . Further+/Cl− cotransporter (NCC), type 3 sodium hydrogen exchanger (NHE3), and the Na+/K+ATPase α1 subunit (NaKATPase). In addition to prenatal insults, our previous study has shown that a maternal high-fructose diet plus postnatal high-salt diet increased renal levels of NKCC2, NHE3, and NCC in a two-hit model of programmed hypertension [2S donor (NaHS) has been reported to inhibit expression and/or activity of several sodium transporters, which may account for its protective effects on renal function and hypertension [2S affects sodium transporters from animal models of programmed hypertension and kidney disease. It will be of great value to reveal the reprogramming effect of H2S on sodium transporters in different models of programmed hypertension and kidney disease.Fetal programming induced by early-life environmental insults that contribute to the development of hypertension is related to enhanced sodium reabsorption attributed to increased expression of sodium transporters . These irtension . Therefortension ,53. Neve2S has epigenetic effects by mediating DNA methylation [2S signaling and renal programming.Epigenetic regulation such as histone modifications, DNA methylation, and noncoding RNAs are involved in mediating the effects of early-life environmental influences on later-life health . Epigenehylation , histonehylation , and michylation . These f2S and other important mechanisms involved in renal programming , and D-cysteine are precursors for endogenous H2S synthesis. H2S donors, including donor molecules and H2S-releasing drugs, can be divided into three types: inorganic sulfide salts , organic compounds , and agonists of H2S-synthesized enzymes [2S treatment modality as they can stimulate the production of H2S [Although excess Httention . As ment enzymes ,61. Addi2S signaling pathway. Of note, pharmacotherapies will be narrowly restricted to key periods during early development. These H2S treatment modalities are listed in Here, we summarize studies which document reprogramming in animal models of hypertension and kidney disease, focusing on interventions aimed at the Hl-cysteine supplementation is widely used to create endogenous H2S in experimental studies. l-cysteine has been known to lower BP directly or through its storage form, glutathione, by decreasing oxidative stress, improving insulin resistance and glucose metabolism, and modulating NO [l-cysteine is not a good H2S precursor due to its multiple metabolic fates. Indeed, the reported antihypertensive and renoprotective effects may not be directly related to H2S signaling. Another H2S precursor, d-cysteine, is nutritionally antagonistic [d-cysteine supplementation has received less attention. There is only one report demonstrating that d-cysteine supplementation protects the mouse kidney against ischemia/reperfusion injury [ating NO . N-acetyating NO ,69, whicgonistic ; therefon injury .2S precursors in the kidney in response to a variety of early-life insults. We first showed that early supplementation of d- or l-cysteine between 4 and 6 weeks of age  can protect SHRs against high-salt-enhanced elevation of BP and kidney injury [d-cysteine pathway is 80-fold greater in H2S-producing activity in the kidney compared to the l-cysteine pathway [G-nitro-l-arginine-methyl-ester (L-NAME)-induced pre-eclampsia [2S precursors in response to early-life insults that have been studied in experimental animal models, there remains a lack of data regarding their clinical translation. So far, very few studies have addressed DOHaD research to explore the protective role of Hy injury . Althoug pathway , our dat pathway . As such pathway , suramin pathway , NG-nitrclampsia , and matclampsia . Despite2S) are used as H2S donors to generate exogenous H2S [2S derived from inorganic sulfide salts in different experimental models of kidney disease have been reviewed elsewhere [2S to supraphysiological concentrations [2S compounds have been synthesized [2S compounds, hybrids of H2S and other drugs are receiving attention. Although H2S-releasing hybrids such as SG1002 (H2S-NO hybrid) and ATB-346 (H2S-NSAID hybrid) are now being demonstrated in clinical trials, there remains a need to better understand the underlying mechanisms for the treatment of a wide range of disorders, especially hypertension and kidney disease [In most studies, inorganic sulfide salts such as sodium hydrosulfide (NaHS) and sodium sulfide (Nanous H2S . NaHS hanous H2S , Ang II-nous H2S , NO-definous H2S , and Dahnous H2S . Additiolsewhere ,22. Howetrations . To overthesized . Among tthesized ,76. Addithesized . In addi disease . 2S donors in hypertension and developmental kidney disease. NaHS therapy between four and six weeks of age has been reported to prevent hypertension in adult SHRs [2S compounds and H2S-releasing hybrids have been examined in terms of their beneficial effects on programmed hypertension and kidney disease.In contrast to the multitude of studies in adults, there are very few DOHaD studies on the role of Hult SHRs . Anotherult SHRs . However2S. A growing body of reports have shown that garlic-derived polysulfide compounds have the potential for ameliorating hypertension and associated morbidity [Garlic-derived polysulfide compounds have drawn attention as natural precursors of Horbidity ,79. Polyorbidity , acetamiorbidity , and genorbidity . However2S concentrations, such as ramipril, atorvastatin, amlodipine, vitamin D, digoxin, aspirin, paracetamol, carvediol, testosterone, 17β-estradiol, and metformin [2S field, very few studies have targeted their potential in reprogramming. With a better understanding on the interplay between the H2S signaling pathway and other mechanisms underlying renal programming, the application of reprogramming strategies targeting the abovementioned mechanisms is feasible for early intervention. A number of currently used drugs have been reported to increase Hetformin . Despite2S downstream signal [2S-releasing drugs are still in preclinical experiments, targeting the H2S downstream signal-related mechanisms would appear to be a practical approach for further clinical translation.For example, there is a growing body of evidence that AMPK activators possess therapeutic potential in treating hypertension and kidney disease ,84. Sincm signal , it woul2S signaling pathway may provide potential therapeutic targets for treating hypertension and kidney disease. Targeting the H2S signaling pathway as a reprogramming strategy against hypertension and developmental kidney disease is a flourishing field and will become even more important in light of the growing epidemic of hypertension and kidney disease. Advances in experimental research indicate that the H2S signaling pathway, including precursors for H2S synthesis, H2S donors, and natural plant-derived compounds. Also, reprogramming interventions may be achieved by targeting the H2S downstream signal, which mediates particular mechanisms of renal programming. Although emerging evidence from animal studies supports H2S as a reprogramming strategy for long-term protection against hypertension and developmental kidney disease, these results await further clinical translation.This review has provided an overview on the various reprogramming strategies that are relevant to the H


NAC genes were identified from the woodland strawberry genome and were classified into 13 groups based on phylogenetic analysis. And further analyses of gene structure and conserved motifs showed closer relationship of them in every subgroup. Quantitative real-time PCR evaluation different tissues revealed distinct spatial expression profiles of the FvNAC genes. The comprehensive expression of FvNAC genes revealed under abiotic stress , signal molecule treatments , biotic stress . Expression profiles derived from quantitative real-time PCR suggested that 5 FvNAC genes responded dramatically to the various abiotic and biotic stresses, indicating their contribution to abiotic and biotic stresses resistance in woodland strawberry. Interestingly, FvNAC genes showed greater extent responded to the cold treatment than other abiotic stress, and H2O2 exhibited a greater response than ABA, melatonin, and rapamycin. For biotic stresses, 3 FvNAC genes were up-regulated during infection with C. gloeosporioides, while 6 FvNAC genes were down-regulated during infection with R. solanacearum. In conclusion, this study identified candidate FvNAC genes to be used for the genetic improvement of abiotic and biotic stress tolerance in woodland strawberry.The NAC transcription factors involved plant development and response to various stress stimuli. However, little information is available concerning the NAC family in the woodland strawberry. Herein, 37  Magnaporthe oryzae [Phytophthora infestans [Sclerotinia sclerotiorum [Colletotrichum graminicola [Transcription factors containing the highly conserved NAC domain NAM-ATAF1/2-CUC) in the N-terminal region compromise is one of the largest transcription factors families in plants /2-CUC in. The C-te oryzae ,34, Phytnfestans , Sclerotrotiorum , and Colminicola , to nameNAC genes in the model plant Arabidopsis thaliana [NAC genes in Oryza sativa [NAC genes in Nicotiana tabacum [NAC genes in Populus trichocarpa [NAC genes in Vitis vinifera [NAC genes in Setaria italic [NAC genes in Gossypium raimondii [NAC genes in Musa acuminate [NAC genes in Cicer arietinum [NAC genes in Manihot esculenta [NAC genes in Zea mays [NAC genes in Morus notabilis [NAC genes in Cucumis melo [NAC genes in Solanum lycopersicum [NAC family in the rosaceous fruit crop, woodland strawberry (Fragaria vesca L.).To date, genome-wide analyses have identified a large number of NAC family members in several species, including 117  tabacum , 163 NACchocarpa , 74 NAC vinifera , 147 puta italic , 145 NACaimondii , 167 NACcuminate , 71 NAC rietinum , 96 NAC sculenta , 148 NACotabilis , 82 NAC NAC genes induced by abiotic stresses play critical roles in the regulation of plant tolerance to abiotic stress. Three Arabidopsis NAC genes  showed up-regulation after drought, high salinity, and abscisic acid (ABA) treatments, and positively regulate drought tolerance in the overexpression plants [ATAF1 in Arabidopsis enhanced plant tolerance to drought, ABA, salt, and oxidative stress [ATAF1 was negatively regulate the defense response to necrotrophic fungi (Alternaria brassicicola and Botrytis cinerea) and bacterial pathogens (Pseudomonas syringae pv. tomato) [NAC genes, in increasing the tolerance of plants to abiotic stress, was also found in rice. SNAC1, an NAC gene from O. sativa, can improve drought and salt tolerance in rice, and transgenic plants were found to produce a 22–34% higher yield than the control in the field under severe drought stress conditions [OsNAC10-overexpressing rice plants showed increased grain yield in comparison to the controls under both normal and drought conditions [NAC genes  increased plant tolerance to drought and salt stresses [OsNAC6 led to increased resistance towards rice blast [ZmNAC41 and ZmNAC100 were found to be induced in Z. mays during infection with C. graminicola [BnNAC1-1, BnNAC5-1, and BnNAC5-7 were induced in Brassica campestris during infection with S. sclerotiorum. NAC family genes thus appear to be crucial regulators of plant tolerance to abiotic and biotic stress, as well as crop yield.Notably, accumulated evidence has confirmed that a large number of n plants . Similar tomato) . The samnditions . Accordinditions . Overexpstresses ,49, and minicola . SimilarFragaria × ananassa Duch.) is one of the most important fruit crops in the world. The woodland strawberry  has a smaller genome (~ 240 Mb) that is highly congenic with the cultivated strawberry [in vitro regeneration and transformation systems have been established for studying the counterparts of many important genes in rosaceous fruit crops [The cultivated strawberry  seedlings were cultivated in a growth chamber at 24±2°C under a 14 h/10 h light/dark photoperiod with 80% relative humidity. The F. vesca seeds collected from Beimu Experimental Station, Yumin County, Xinjiang Province of China. Hundred-day-old woodland strawberry tissues  were collected from the greenhouse for the treatments. The cold and heat stress treatments was performed by transferring the plants to a low temperature (4°C) for 48 h following recovery, or to a high temperature (40°C) for 4 h following recovery. The leaves of the plants treated with cold and heat were then collected at 2 h, 6 h, 12 h, 2 d, 7 d, 14 d post-treatment (dpt). Drought or salt stresses were simulated by irrigating potted strawberry plants with 200 mM mannitol, 100 mM NaCl, respectively. The leaves of the plants treated with drought and salt were collected at 2 h, 6 h, 3 d, 14 d, 18 d, 24 d post-treatment. Signal molecule treatments were performed by spraying the woodland strawberry leaves with a solution containing 10 mM H2O2, 0.1 mM ABA, 0.5 mM melatonin, 0.01 mM rapamycin, and the leaves were collected at 2 h, 6 h, 12 h, 1 d, 2 d, 3 d post-treatment. The control woodland strawberry seedlings were similarly irrigating or spray with distilled water. 1×106 conidiospores/mL C. gloeosporioides inoculation leaves were collected at 12 h, 22 h, 40 h, 60 h, 96 h, 120 h post-inoculation (hpi), 1×108 CFU R. solanacearum irrigating woodland strawberry seedlings were collected at 2 h, 6 h, 12 h, 24 h,48 h, 72 h post-inoculation (hpi), and uninfected leaves served as a negative control. All materials harvested from each treatment were immediately frozen in liquid nitrogen and stored at -70°C before for RNA isolation.Woodland strawberry (http://planttfdb.cbi.pku.edu.cn/) [https://phytozome.jgi.doe.gov/pz/portal.html) and Pfam databases (http://pfam.xfam.org/) [Arabidopsis  NAC amino acid sequences were acquired from PlantTFDB version 4.0. The rice (Oryza sativa subsp. japonica) NAC amino acid sequences were acquired from PhantTFDB and Nuruzzaman et al. [Arabidopsis and rice NACs were entered as queries in BLAST and were used to identify the predicted NACs in the woodland strawberry database. The full-length amino acid sequences of the NAC proteins from Arabidopsis, rice and woodland strawberry were used to generate a phylogenetic tree based on ClustalX 2.0 alignment [Whole protein sequences of woodland strawberry were obtained from the Plant Transcription Factor Database version 4.0  , Phytozoam.org/) . The Aran et al. . Additiolignment  and the lignment .http://web.expasy.org/compute_pi/)[FvNAC was retrieved from the F. vesca version 1.1 database. The FvNAC genomic sequences and CDS sequences extracted from PlantTFDB were compared using the online GSDS 2.0 software (http://gsds.cbi.pku.edu.cn/) [FvNAC genes. The conserved motifs of the FvNAC genes were obtained from the online Multiple Expectation Maximization for Motif Elicitation (MEME) .The molecular weight and isoelectric points (pI) of the presumed FvNACs were predicted using the online ExPASy proteomics server database (pute_pi/). Informaedu.cn/)  to inferFvNAC genes by quantitative real-time PCR (qRT-PCR) analysis, performed in an Applied Biosystems QuantStudioTM 6 Flex Real-Time PCR System using the UltraSYBR mixture from Beijing CoWin Biotech (Cat. CW2601M). The reaction mixture was as follows: 1 μL cDNA template, 10μL 2 × PCR Mix, 0.5μL forward and reverse primers each (10μM), and 8μL ddH2O. PCR reactions were carried out with a denaturing step (95°C 10 min) and 40 cycles of denaturing at 95°C for 10 s, followed by annealing at 56°C for 30 s, and elongation at 72°C for 32 s. Primers with high specificity and efficient amplification on the basis of a dissociation curve analysis and agarose gel electrophoresis were used to conduct the quantification analysis. The relative expressions of the target genes were determined using the 2–ΔΔCt method [Actin-7 gene (LOC101313051) was used as an internal reference gene for all the quantitative real-time PCR analyses in this study [Total RNA was isolated using an RNAprep Pure Plant Kit  and the concentration and purity were evaluated by NanoDrop 2000c . Reverse transcription was implemented using 1 μg total RNA of each sample with a FastQuant RT Kit . Expression of  Cat. CW21M. The ris study . The priFvNAC genes were identified in the woodland strawberry genome as putative members of the NAC family from PlantTFDB v4.0 and Phytozome v12, and were designated as FvNAC01-FvNAC37. The characteristic parameters of all predicted FvNAC proteins are listed in A total of 37 Arabidopsis and rice, an unrooted neighbor-joining phylogenetic tree was constructed with the amino acid sequences of the NAC family proteins from the woodland strawberry, Arabidopsis, and rice. The results indicated that 37 FvNACs could be divided into 13 groups (A to M) together with their orthologs of Arabidopsis and rice  and between the woodland strawberry and rice , suggesting that FvNACs are closely associated with the proteins from Arabidopsis.To study the evolutionary relationships between woodland strawberry NAC proteins and known NACs from and rice . PhylogeFvNAC genes, intron/exon distribution and conserved motifs were analyzed according to their phylogenetic relationships  to 16 (FvNAC18), and it was found that 16 of the 37 FvNAC genes had 2 introns, while 7 of the 37 FvNAC genes had 3 introns. Most of the FvNAC members in the same group exhibited similar exon-intron structure. The conserved intron numbers in each subfamily supports their close evolutionary relationship and group classification.The diversification of gene structure during the evolution of multigene families facilitated the evolutionary co-option of genes for new functions in order to adapt to changes in the environment. To further examine the structural features of ionships . Gene stionships  indicateFvNACs identified in this study possessed conserved features of the NAC family. Interestingly, it was found that most of the conserved motifs were located in the N-terminal of the NAC proteins that are highly conserved for DNA-binding, indicating that these motifs may be essential for the functioning of NAC proteins. However, the FvNAC18 motifs were located in the C-terminal. FvNAC02 and FvNAC14, lacking motif, and FvNAC34 and FvNAC15, with only a domain, were excluded from further analysis as they cannot be used to construct acceptable phylogenies. In general, the NAC proteins clustered into the same groups and shared similar motif compositions, indicating functional similarities among members of the same group.To further examine the structural diversity of FvNACs, 17 conserved motifs were predicted using Multiple Em for Motif Elicitation (MEME) and subsequent annotation with InterPro , S1 Fig.NACs in the woodland strawberry, the distribution of the 37 FvNACs transcripts were surveyed in 5 major organs  under non-stress conditions. As shown in FvNAC genes  showed lower transcript abundance in leaves than in the stems, roots, flowers, and full reddening fruit. All 37 FvNAC genes showed transcripts in different tissues, in which 24 (64.9%), 16 (43.2%), 29 (78.4%), and 23 (62.1%) genes exhibited high transcriptional abundance  in the stems, roots, flowers, and full reddening fruit tissues, respectively. FvNAC13 and FvNAC18 did not show any transcripts in the roots , heat (40°C), drought, and salt stresses at the transcriptional level, the transcripts of FvNAC genes under these treatments were determined using quantitative real-time PCR. As shown in FvNAC genes under cold stress at all treated time points was observed, with FvNAC03 and FvNAC31 being up-regulated, and FvNAC02, FvNAC06, FvNAC09, and FvNAC32 being down-regulated. As shown in FvNAC genes  were down-regulated under heat stress, while no FvNAC genes were up-regulated. As shown in FvNAC genes  were down-regulated under conditions of drought stress. Additionally, as shown in FvNAC04 and FvNAC29 were mostly up-regulated under salt stress, while 20 FvNAC genes  were mostly down-regulated.To examine the response of FvNAC genes to H2O2, ABA, melatonin, and rapamycin stress at the transcriptional level, the transcripts of the FvNAC genes under these treatments were determined by quantitative real-time PCR. As shown in FvNAC genes was observed under H2O2 stress, with FvNAC03, FvNAC04 and FvNAC29 being up-regulated, and FvNAC02, FvNAC14, FvNAC17 and FvNAC20 being down-regulated. As shown in FvNAC genes was documented under ABA stress, with FvNAC30 being up-regulated, and FvNAC18, FvNAC20 and FvNAC23 being down-regulated. As shown in FvNAC genes  were down-regulated under melatonin stress, while no FvNAC genes were up-regulated. Moreover, as shown in FvNAC genes was observed under rapamycin stress, with FvNAC04 being up-regulated, and FvNAC15, FvNAC18, FvNAC19, FvNAC22, FvNAC23, FvNAC32 and FvNAC34 being down-regulated. Notably, FvNAC18 tended to be down-regulated under all 4 stress treatments.To examine the response of FvNAC genes in plant-pathogen interactions, quantitative real-time PCR was used to analyze the response of woodland strawberry leaf infected with C. gloeosporioides and R. solanacearum in comparison to a control. As shown in FvNAC genes occurred under C. gloeosporioides infection, with FvNAC04, FvNAC28 and FvNAC29 being up-regulated, and FvNAC09, FvNAC18 and FvNAC20 being down-regulated. As shown in FvNAC genes  were steadily expressed under R. solanacearum infection and were found to be down-regulated, while no FvNAC genes were steadily up-regulated. However, FvNAC01, -03, -04, -18, -28, -29, -34, 36 and -37 tended to be up-regulated  at 1 d post-infection.To investigate the possible role of C. gloeosporioides and R. solanacearum) during the growth and developmental stages. In order to address this, farmers and researchers have been increasing the cultivation area and improving cultivated techniques. However, the effects of this appear to be very limited [FvNACs were chosen as candidate genes for further investigation for their potential use in genetic breeding in woodland strawberries. Overall, 37 FvNACs were identified from across the genome and the phylogenetic evolution of these genes was also revealed. Using quantitative real-time PCR analysis, the comprehensive expression profiles of the 37 FvNACs were assessed. To our knowledge, this is the first study to extend our understanding of the FvNAC gene family. Generally, the expression profiles could be divided into 2 sections. The first involves the expression of 37 FvNACs at different developmental stages, or in different tissues, and comprises the basic information of this gene family. We discovered that the transcripts of some FvNACs have strong expression levels for specific tissue and developmental stage, indicating their possible roles in specific growth or developmental stages, such as in the flowers or fruit. The other section involves gene expression in response to various abiotic and biotic stresses, which intends to identify several candidate genes commonly regulated by various stresses for stress-related genetic breeding.Woodland strawberries face severe destruction from various abiotic and biotic stresses  and biotic (necrotrophic pathogen B. cinerea) stress responses [sistance ,32. FvNAArabidopsis, ANAC053 and ANAC017 were found to be induced by cold stress [ONAC007, -010, -015, -027, -028, -039, -045, -059, -067, -068, -073, -074, -085, -103, -122 and -132) showed up-regulation under cold treatment [FvNAC03 and FvNAC31 were up-regulated at all treated time points, and constituted the most highly induced genes  plants, 7 CsNAC genes  showed up-regulation under heat (40°C) stress at 24 h [FvNAC15 and FvNAC31 were up-regulated at 2 h, and FvNAC36 between 2 d and 14 d, constituting the most highly induced genes . On the contrary, 18 FvNAC genes under melatonin stress were found to be down-regulated  at 24 h Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S6 Table(XLS)Click here for additional data file.S7 Table(XLS)Click here for additional data file.S8 Table(XLS)Click here for additional data file.S9 Table(XLS)Click here for additional data file.S10 Table(XLS)Click here for additional data file.S11 Table(XLS)Click here for additional data file.S12 Table(XLS)Click here for additional data file.S13 Table(XLS)Click here for additional data file.S14 Table(XLS)Click here for additional data file.S1 FigConserved motifs and the sequence logos were generated using the MEME search tool. Numbers on the horizontal axis represent the sequence positions in the motifs and the vertical axis represent the information content measured in bits.(TIF)Click here for additional data file.


MYCN amplification, 17q gain, and others, suggesting the involvement of epigenetic regulation . Thereafter, NCCs migrate and give rise to a variety of tissues. In the sympathoadrenal lineage, NCCs reach their destinations close to the dorsal aorta at E9.5-10.0, where they proliferate, differentiate into neurons and glia, and finally form the sympathetic ganglia and adrenal medulla. Furlan et al. revisited this dogma . UnexpecMYCN mice, we developed a spheroid culture method that well represents the in vivo status of neuroblasts at the molecular level. Passageable spheres could be established from sympathetic ganglia of E13.5 TH-MYCN mice, but not wild-type mice. Surprisingly, spheres from E13.5 TH-MYCN mice formed allograft tumors, suggesting that early molecular events of tumorigenesis occur no later than E13.5. Copy number alterations were late events, as they were found in advanced tumors, but not in E13.5 spheres, of TH-MYCN mice. Importantly, E13.5 spheres from TH-MYCN mice exhibited upregulation of MYC target genes and downregulation of polycomb repressive complex 2 (PRC2) target genes. Consistent with this, the H3K27me3 level was increased around transcription start sites of PRC2 targets in E13.5 spheres from TH-MYCN mice. Knockdown or inhibition of Ezh2, a methyltransferase in PRC2 catalyzing H3K27me3, led to a striking decrease in sphere formation and tumor formation in TH-MYCN mice. Moreover, expression of PRC2 targets reversely correlated with that of MYC targets in human neuroblastoma. Consistently, lower and higher expression of PRC2 targets and MYC targets, respectively, predicted a poor prognosis. Our study highlighted the importance of epigenetic regulation of MYCN-driven neuroblastoma, and revealed that tumorigenesis is initiated in unexpectedly early embryonic days (no later than E13.5) in this mouse model.Our group addressed the long-standing question of why neuroblastoma develops without driver gene mutations . Using tMYCN. MYCN-expressed NCCs developed subcutaneous tumors that exhibited chromosomal alterations and gene expression similar to those of MYCN-amplified human neuroblastoma. We also demonstrated that MYCN could transform spheres from E13.5 wild-type mice to passageable spheres [MYCN alone often induces apoptosis rather than transformation. These results collectively suggest that embryonic cells at mid-gestation, such as NCCs and SCPs, have a competency to be transformed if an appropriate insult, such as epigenetic dysregulation, occurs. In this context, a hypothesis proposed by van Noesel for the origin of stage 4S neuroblastoma is interesting [Olsen et al. provided another piece of evidence that NCCs could be a target of transformation . Using Eeresting . NCCs arThe idea that neuroblastoma is a developmental disease of the neural crest will always be useful when we conduct further studies on this disease.


Transcription factors regulate gene expression through binding to specific enhancer sequences. Pancreas/duodenum homeobox protein 1 (PDX1), Neurogenin-3 (NEUROG3), and V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA) are transcription factors critical for beta cell development and maturation. NEUROG3 is expressed in endocrine progenitor cells and controls islet differentiation and regeneration. PDX1 is essential for the development of pancreatic exocrine and endocrine cells including beta cells. PDX1 also binds to the regulatory elements and increases insulin gene transcription. Likewise, MAFA binds to the enhancer/promoter region of the insulin gene and drives insulin expression in response to glucose. In addition to those natural roles in beta cell development and maturation, ectopic expression of PDX1, NEUROG3, and/or MAFA has been successfully used to reprogram various cell types into insulin-producing cells in vitro and in vivo, such as pancreatic exocrine cells, hepatocytes, and pluripotent stem cells. Here, we review biological properties of PDX1, NEUROG3, and MAFA, and their applications and limitations for beta cell regenerative approaches. The primary source literature for this review was acquired using a PubMed search for articles published between 1990 and 2017. Search terms include diabetes, insulin, trans-differentiation, stem cells, and regenerative medicine. Type 1 diabetes mellitus (T1D) is an autoimmune-mediated disease in which pancreatic beta cells are destroyed by the immune system, thus causing life-long dependence on exogenous insulin therapy [Transcription factors play critical roles in regulating gene expression. Ectopic expression of selected transcription factors can change, or trans-differentiate, the fate of somatic cells. This process is called cellular reprogramming. Reprogramming nonbeta cells into insulin-producing cells potentially offers novel regenerative approaches for T1D therapy. There are several transcription factors involved in early pancreatic progenitor formation, including pancreas/duodenum homeobox protein 1 (PDX1), forkhead box A2 (FOXA2), and sex determining region Y-box 17 (SOX17). Some transcription factors are critical for endocrine lineage specification and differentiation, such as Neurogenin 3 (NEUROG3) and neurogenic differentiation 1 (NEUROD1). Late maturation of beta cells is shown to be regulated by factors including V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA), V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB), paired box gene 6 (PAX6), and estrogen-related receptor gamma [PDX1 gene [PDX1 gene. In nonobese diabetic (NOD) mice and human T1D patients, PDX1 autoantibodies are detected, suggesting PDX1 could be an autoantigen for T1D [PDX1, also known as insulin promoter factor 1, is a homeodomain transcription factor. PDX1 expression is observed as early as embryonic day 8.5 (E8.5) at the 5–6 somite stages in mouse  and arouDX1 gene . In matuDX1 gene . This no for T1D . These dNeurog3 gene in pancreatic precursor cells in mouse embryos, under the control of Pdx1 promoter, gives rise to endocrine cell differentiation, primarily alpha cells, and blocks exocrine development. Conversely, in NEUROG3-deficient mice, four islet cell types  and endocrine precursor cells are not generated, and neonates die postnatally from diabetes [Neurog3+/− heterozygous islets show no notable difference from Neurog3+/+ islets in expression of PDX1, NKX6.1, GLUT2, MAFA, and MAFB on a per cell basis [NEUROG3 is a member of the basic helix–loop–helix transcription factor family involved in the central nervous system and embryonic pancreas development. During the embryonic development of mouse pancreas, expression of NEUROG3 is first observed in the dorsal pancreatic epithelium at E9, increases between E9.5 and E15.5, and then decreases to a very low level in neonatal pancreas . In humadiabetes . Intrigull basis , suggestNEUROG3 have functional endocrine cells capable of releasing C-peptide despite severe enteric anendocrinosis from childhood [db/db mice, the NEUROG3 expression level is increased markedly [NEUROG3 [Despite these observations, it remains unclear whether NEUROG3 is absolutely required for beta cell development in humans. In contrast to mouse studies, all reported patients with biallelic mutations in hildhood . All of markedly . In cont[NEUROG3 . In NOD [NEUROG3 . Similar[NEUROG3 .MAFA, also known as RIPE3b1, is member of the MAF family of basic leucine zipper. It is identified as a transcription factor that specifically binds to a conserved insulin enhancer element RIPE3b/C1-A2 and activates insulin gene expression. In mice, MAFA is initially detected at E13.5 only in insulin-producing cells, and expresses exclusively in beta cells in the adult pancreas . SimilarPdx1 premature truncation mutation in the C terminus, which is dispensable for pancreas organogenesis, demonstrate a reduced number of NEUROG3-positive cells from E13.5 to P1. Additionally, transcript levels of Neurog3 as well as other transcription factors regulating Neurog3 gene expression, including Sox9, Hnf6, Hnf1b, and Foxa2, decrease in Pdx1 mutated mice, suggesting that PDX1 regulates NEUROG3 directly but not solely through its role in the formation of pancreatic progenitor cells. PDX1 also regulates the expression of MAFA [Mafa gene expression in Pdx1 knockout mice is downregulated in islets compared with wildtype mice. NEUROG3 appears to have minimal effects on PDX1, as NEUROG3 disruption in hESCs only marginally reduced PDX1-positive cells compared with wildtype hESCs [In addition to their independent roles in beta cell development and maturation, PDX1, NEUROG3, and MAFA are also mutually interacted/regulated during the pancreatic developmental process. Because of the limited availability of human beta cells, the interaction among the three factors is better understood in rodent models. PDX1 regulates the expression of NEUROG3 . Mice wi of MAFA , since Mpe hESCs . Other bpe hESCs . In collpe hESCs , and FOXpe hESCs . MAFA anpe hESCs  Reprogramming adult somatic cells into therapeutic cell types is an attractive approach for novel cell-based therapies for degenerative diseases. Recently, transcription factor-mediated “reprogramming”, or “trans-differentiation”, approaches have been used to regenerate beta-like cells or insulin-producing cells from nonbeta cell sources in vitro and in vivo. Notably, studies have demonstrated that overexpression of combinations of the three transcription factors, PDX1, NEUROG3, and/or MAFA, can reprogram the fate of various nonbeta cell types into insulin-producing cells. In the following paragraphs, we will summarize the progress of this unique approach.One of the first proof-of-concept studies was performed by Ferber et al., which demonstrates PDX1-mediated induction of insulin genes in the liver and amelioration of drug-induced hyperglycemia in diabetic mice upon hepatic PDX1 overexpression by an adenoviral vector . NEUROG3Screenings of different combinations of critical transcription factors expressed in beta cells or their precursors in vivo, including NKX2.2, NKX6.1, PAX4, PAX6, NEUROG3, NEUROD1, PDX1, ISL1, and MAFA, have identified the combination of PDX1, NEUROG3, and MAFA (PNM) as a critical reprogramming factor to trans-differentiate pancreatic exocrine cells into insulin-producing beta-like cells . OverexpIn addition to adult pancreatic exocrine cells, other adult cell types are also utilized for beta cell regeneration. Plasmid-based PNM gene delivery into the inferior vena cava segment has facilitated transient induction of insulin transcripts in rat livers . Upon syThese observations clearly indicate the ability of the PNM cocktail for beta cell regeneration from liver cells. However, it does not rule out the contributions of generation of insulin-producing cells from nonliver cell types. To address this, Chen et al. performed an in-vivo screening study of a wide spectrum of tissues and found that duodenum and jejunum tissues are more “susceptible” to the PNM-mediated trans-differentiation than other tissues , althougIns1 and Ins2 gene expression better. However, somatostatin gene expression levels are much lower in PNM, suggesting that PNM-induced cells may be more lineage specific.Pluripotent stem cells, such as hESCs and induced pluripotent stem cells (iPSCs), are characterized by pluripotency and infinite propagation. Their successful differentiation into insulin-producing progeny could provide unlimited cell sources for islet regeneration. For translational purposes, stem cells are a preferable cell source for beta cell regeneration compared with somatic cells, as adult somatic cell reprogramming appears to require developmentally closely related cell types  for high reprogramming efficiency , 42. Xu The major impediments in utilizing hESCs are ethical issues and immunological intolerance. Human iPSCs are ideal cell sources for beta cell regeneration given that they are similar to hESCs and free from ethic constraints associated with the use of embryo-derived cells. Saxena et al. have shown that human iPSC-derived pancreatic progenitor cells can be differentiated into glucose-sensitive beta-like cells using a synthetic lineage-control network to express PNM in a dynamic way, mimicking the intrinsic expressing timeline as introduced previously . This prN-acetyl cysteine, Alk5 inhibitor II, and thyroid hormone [Based on the critical roles of PNM for beta cell development and function, pharmacological approaches to induce endogenous PNM genes, an alternative to exogenous genetic manipulations, have been studied extensively in terms of a therapeutic perspective. Growth factors/small molecules such as retinoid acid, fibroblast growth factor 7 and 10, sonic hedgehog signaling inhibitors , and protein kinase C signaling pathway activators  can upregulate PDX1 expression . TGF-β t hormone . The appRegenerative medicine has opened a new era for T1D cell therapy approaches, which may have greater effectiveness, safety, and versatility over exogenous insulin therapy. Key beta cell transcription factors, PDX1, NEUROG3, and/or MAFA, have been studied extensively for their roles in beta cell development and function, as well as their potential applications for trans-differentiating nonbeta cells into insulin-producing cells. However, it is of note that the PNM gene combination generally does not reprogram nonbeta cells into genuine beta cells, and that successful reprogramming into insulin-producing cells typically needs developmentally related cell types rather than unrelated types . For suc


These findings were supported by clinical studies indicating that low content of n-3 PUFAs in diet is linked to an increased susceptibility to psychiatric disorders. PUFAs regulate membrane fluidity and exert their central action by modulating synaptogenesis and neurotrophic factor expression, neurogenesis, and neurotransmission. Moreover, they are precursors of molecules implicated in modulating immune and inflammatory processes in the brain. Importantly, their tissue concentrations are closely related to diet intake, especially to maternal consumption during embryonal life, considering that their synthesis from essential precursors has been shown to be inefficient in mammals. The scope of this review is to highlight the possible mechanisms of PUFA functions in the brain during pre- and post-natal period and to evaluate their role in the pathogenesis of psychiatric diseases.Evidence from human studies indicates that maternal metabolic state and malnutrition dramatically influence the risk for developing psychiatric complications in later adulthood. In this regard, the central role of polyunsaturated fatty acids (PUFAs), and particularly  Maternal malnutrition has been linked to the development of different neuropsychiatric disorders in infancy (Autism Spectrum Disorder (ASD)) , in chiln-3, n-6, and n-9. PUFAs with a relevant biological function belong to the n-3 or n-6 family. The most relevant molecules are docosahexaenoic acid , n-3 family, and arachidonic acid , for n-6 family, representing 40% and 50% of brain PUFAs, respectively [n-3 family is eicosapentaenoic acid  [n-6 or n-3, also appear relevant considering that have been shown to partially substitute DHA in condition of scarce n-3 PUFA intake [n-6/n-3 has dramatically increased during recent decades because of profound changes in lifestyle and changes in food sources. Such ratio, that during early 20th century was estimated around a value of 1, is now ranging in Western countries around 15 or even more [n-6), is found in soybean, canola, corn, safflower, sunflower and cottonseed, while AA is present principally in meat or products derived from animal fed with high n-6 content diets [n-3), the precursor of DHA and EPA, is highly concentrated in walnut, rapeseed, flax seeds and chia [n-6/n-3 PUFA ratio. Importantly, the high ratio n-6/n-3 is particularly unfavorable for proper central nervous system (CNS) functioning. LA and ALA compete with each other for enzymatic convention and, although n-3 in healthy conditions are favored [PUFAs have been shown to be crucially involved in normal brain development and functioning . These mectively ,9,10,11.20:5n-3) ,13,14,1520:5n-3) ,17 consi20:5n-3) . IsomersA intake ,20,21. Icid DHA, :6n-3, n-ven more ,24. AA pcid DHA, :6n-3, n-nt diets . On the and chia . DHA andand chia . Main so favored ,29, high favored . Importa favored . In the n-3 PUFAs in mammalian brain and particularly in gray matter. It has been shown that DHA accounts for around 10%–20% of total fatty acid composition of prefrontal cortex (PFC) in adulthood [Mammalian brain is very rich in lipids (60%/65%) comprising saturated, mono- and polyunsaturated fatty acid. DHA is the major dulthood ,33.DHA and AA source can be derived directly from diet or through metabolic conversion of their essential precursors ALA and LA, respectively. The enzymatic conversion relies on the action of Δ5 and Δ6 desaturases and on the elongase, enzymes responsible of converting LA into AA e ALA into EPA and DHA. Although in mammals these enzymatic reactions are possibly occurring, in humans this conversion is quite inefficient, thus tissue concentrations of these lipids reflect diet intake . ConfirmIn humans, during the prenatal period DHA accumulates rapidly particularly during the third trimester of gestation, when synaptogenesis, differentiation, neurogenesis and expansion of gray matter occur ,42. At en-3 PUFAs have been reported to exert their central action by modulating synaptogenesis and neurotrophic factor expression [n-3 availability, leads to higher lipoperoxidation levels and lower cell viability in cortex, along with reduced catalase activity in striatum and hippocampus, and increased generation of reactive species in striatum [n-3 PUFAs may influence central redox homeostasis by increasing the levels of neuroprotectin D1, a protectin produced in neurological tissues and derived by DHA enzymatic conversion. Neuroprotectin D1 can in turn regulate pro-survival and repair activity through Bcl-2-related molecule activation and BAD, BAX, and Bid inhibition [n-3 PUFA dietary supplementation was shown to reduce placental levels of F2-isoprostanes, a highly reliable marker of oxidative damage associated with enhanced fetal and placental growth [Childhood and adolescence are critical periods for brain maturation, and particularly for the creation of connections between brain areas involved in mediating attention and executive functions, such as frontal lobe regions, and brain areas implicated in the regulation of emotions and mood, such as limbic areas ,46,47. Spression . Indeed,pression . Furtherpression . This phpression . Figure pression , thus repression . In thispression . Furtherpression  and prodpression ,56. Earlpression  and thenpression . Furtherpression . Interesstriatum . In addihibition . Intereshibition . In thisl growth .n-6 PUFAs in brain maturation and development is much less studied. However, the main n-6 derivative, AA, plays crucial physiological functions considering that it is an important precursor of bioactive mediators and it has been shown to activate protein kinases and ion channels leading to enhanced synaptic transmission [The role smission . Importasmission  and to bsmission . Furthersmission . Endocansmission ,66 and ismission ,68.n-6/n-3 is particularly unfavorable for proper central nervous system (CNS) functioning. Indeed, when diets are particularly poor in n-3 PUFAs, endoreticulum, as well as peroxisomes, metabolically produce n-6 derivatives, namely n-6 docosapentaenoic acid (22:5n-6), whose insertion into biological membranes, in place of DHA, is considered the main process responsible of CNS dysfunction [n-6 PUFA consumption. Indeed, when DHA or in general n-3 PUFA intake is limited in presence of a diet rich in LA, infant brain accumulates n-6 PUFAs and less n-3 PUFAs [n-6 PUFAs, and then AA, are precursors of important molecules, the eicosanoids, involved in inflammation and immune system regulation. In this regard, high n-6 PUFA consumption during early development and during infancy has been associated with an increased risk of allergic syndromes indicating that these lipids may play a role in programming of immune system [On the other hand, a high ratio function . Interes-3 PUFAs . This eve system .n-3 PUFA may have in offspring, particularly in relation to psychiatric disorders.The effect of maternal PUFA intake in fetal programming is becoming evident, although the exact mechanisms still need to be fully elucidated. Many studies, either epidemiological or preclinical, are now indicating the deleterious effects that low maternal consumption of A large body of recent literature has focused the attention on the study of how the exposure to environmental factors during pre-, peri- and post-natal period can influence health status and disease occurrence later in life. The effects of this early exposure can persist later in life, thus this phenomenon has been called “early life programming” (ELP). The fetal origins of adult disease model has its roots on Barker’s observations that associated undernutrition of the fetus, and thus low body weight at birth, with an increased risk of cardiovascular disease, diabetes and metabolic syndrome later in life . ELP hasIn animal models, ELP has been studied by using several paradigms, such as maternal protein restriction, maternal caloric restriction, maternal iron restriction, maternal glucocorticoid exposure, maternal hypoxia, uterine placental ligation and many others . All theStudies on animal models based upon maternal dietary restriction, such as protein , caloricMaternal glucocorticoid exposure is considered a model of prenatal stress . ChronicHsd11b2, a gene encoding for the enzyme 11β-hydroxysteroid dehydrogenase type 2 responsible of converting cortisol into an inactive metabolite and thus providing fetal protection from cortisol [Gr noncoding exon17 and in the Crf promoter in hypothalamus [n-3 PUFAs [In trying to understand mechanisms that predispose to disease development later in life, epigenetic studies provided interesting results. In particular, prenatal stress has been shown to induce hypermethylation in the placenta and hypomethylation in the fetal hypothalamus, respectively, of cortisol . Such gecortisol ,95,96. Fthalamus . These r-3 PUFAs ,99. Sign-3 PUFAs  and psyc-3 PUFAs .n-3 PUFAs during pregnancy has been reported to modulate global methylation levels and to imbalance the ratio of T helper cell 1 and 2 in infants, thus altering cytokine production. Neuroinflammation has been indicated in this regard as a possible bridge between neuropsychiatric disorders and increased stress response [Furthermore, supplementation with response . Therefon-3 PUFA content in diet was inversely correlated to the development of depressive symptoms [n-3 PUFAs in diet is linked to an increased susceptibility to psychiatric disorders like depression supported these findings [n-3 supplementation alone or in adjunctive therapies showed positive results in the treatment of this mental disorder [n-3 PUFA treatment in depressive disorders such beneficial effects were further endorsed [n-3 PUFAs, followed by a post-natal diet with the same deficiency, led to increased anxiety- and depressive-like behavior in the offspring. Moreover, such behavioral impairment was associated with ablation of endocannabinoid-mediated long-term synaptic depression in the prelimbic PFC and accumbens, and alterations in cannabinoid receptor signaling [Recent evidences have pointed out that the prevalence of depression has reached epidemic proportions in last decades. Such an increase has been linked to many environmental factors; among these, the influence of dietary factors has gained great attention. In particular, it has been reported the symptoms ,104,105,symptoms . Clinicafindings ,108. n-3disorder . In a reendorsed , as alsoendorsed . Howeverendorsed ,112. Aniendorsed  demonstrignaling ,114. In ignaling .n-3 PUFA intake, especially in perinatal period, is linked to decreased brain derived neurotrophic factor (BDNF) content [n-3 PUFA deficiency can influence neuronal cortical morphology and depressive-like behavior through corticosterone secretion [n-3 diet intake induces a phenotype comparable to one induced by chronic social defeat stress and high corticosterone levels were also described [n-3 PUFA diet from conception, further supporting the major role of these lipids in cortical maturation [On the other hand, deficiency in  content  and low  content . Glucoco content ,118. In  content  and Larrecretion . Furtherescribed . Moreoveturation .n-3 PUFAs, from gestation until 8 weeks of life in rats, leads to a consistent depressive-like and anxiety-like profile. Furthermore, these behaviors were associated with a hyper-activation of HPA axis, with increased plasma corticosterone levels and high hypothalamic CRF. In addition, only in PFC reduced serotonin levels were found, thus supporting behavioral with neurochemical outcomes [n-3 PUFA deficiency by increasing Aβ levels and perturbing HPA axis functioning can predispose to alteration in emotional profile. In good agreement, a dramatic increase also in Alzheimer’s disease (AD) prevalence has been documented either in developing or in industrialized eastern countries, such as Japan, after Westernization of national diets, and thus after shifting n-3 in favor of high n-6 PUFA intake [n-3 PUFAs, given in adulthood to rats, were shown to reduce the production of interleukin 1β and interferon γ induced by central injection of Aβ [n-3 supplementation was shown to be potentially beneficial in preventing and/or treating several chronic inflammatory conditions and neurodegenerative diseases such as AD [n-3 supplementation has been shown to improve cognitive and depressive symptoms in AD [n-3 deficiency in maternal diet and later development of degenerative symptoms. Future studies are surely warranted.In this regard, we have recently reported that lifelong nutritional deficiency in outcomes . In furtoutcomes ,123,124.outcomes . In addioutcomes ,127,128.A intake . A bridgA intake ,131, andA intake . Moreoveon of Aβ , moleculon of Aβ ,135,136.ch as AD ,138. Morch as AD . Althougms in AD , no datapostmortem studies in schizophrenic patients indicated modification in the expression of genes responsible for regulating the biosynthesis of fatty acids at PFC levels [n-3 PUFAs [Schizophrenia Spectrum Disorders are now accepted as a neurodevelopmental disorders. Some etiopathogenic origins of these disorders have been found as early as the prenatal period . Early lC levels . MoreoveC levels ,156. LowC levels  along wi-3 PUFAs ,158,159.-3 PUFAs ,161.n-3 PUFAs was associated with lower verbal IQ, diminished prosocial behavior, suboptimal fine motor ability, and impaired social and verbal development [Increased offspring neurocognitive functioning has been demonstrated in many double-blind, placebo-controlled randomized clinical trials conducted in mothers receiving either supplementation with DHA or consuming DHA-rich foods ,149,162.elopment .n-6 and n-3 PUFAs consumption only at lactation stage, while at post-weaning period the reversal was only partial [n-3 PUFA deficiency was shown to impair the mesocortical and mesolimbic dopaminergic systems [n-3 supplementation was not able to completely reverse such alterations associated with fronto-limbic dysfunction in PFC [n-3 supplementation efficacy. In good agreement, restoration of n-3 DHA levels through supplementation varies in the PFC compared to the striatum and cerebellum [n-3 supplementation [Interestingly, it has been shown that perturbations in dopaminergic system, a neurotransmitter system strictly involved in SSD, can be completely reversed by adequate levels of  partial . Thus, t partial ,165. In  systems ,166,167 n in PFC ,169. Agarebellum . The speentation ,152,171.n-3 PUFAs was associated with an increased risk of ASD development [n-6 PUFAs, usually associated with increased inflammatory response. Thus, as the same Authors stated, possible confounding factors may have affected the results and more accurate studies need to be performed. Indeed, in rodents, it has been reported that lifelong nutritional exposure to a diet rich in n-6 PUFAs associated with prenatal stress contributes to the production of autistic-like behavior [n-3 PUFA deficiency has been implicated in synapse plasticity [n-6/n-3 PUFA ratio was found leading to profound changes in myelination and thus in connectivity [n-3 PUFA intake, especially in perinatal period, is linked to decreased BDNF content [Autistic Spectrum Disorders are characterized by defects in communication and social behavior and are associated with repetitive behaviors . The etielopment . In the behavior . Many libehavior  and n-3 asticity . In addiasticity ,188,189.ectivity ,191. ASDectivity  and PUFAectivity ,194, furectivity ,196,197 ectivity ,199,200.ectivity . Deficie content , thus mon-3 PUFAs or low dietary intake after birth may cause ADHD [n-3 and n-6 PUFA supplementation. However, the Authors underlined the urgency of future research prompted to correct the current weaknesses, such as small sample sizes, variability of selection criteria, variability of the type and dosage of supplementation, short follow-up times and other methodological weaknesses [n-3 PUFA oxidation used as of non-invasive quantification of their metabolism [Attention Deficit Disorder is mainly occurring in children and is characterized by inattentive, impulsive and hyperactive behavior. The link between ADHD disorders and PUFA consumption originates from the study of Stevens et al. (1995) that found significant reduction in plasma DHA and EPA levels in affected patients . Furtheruse ADHD ,205. Unfuse ADHD  or benefuse ADHD . In 2012aknesses . In partaknesses  while PUaknesses ,210,211.tabolism .n-3 PUFAs received for three weeks in utero and for two weeks post-natally caused profound changes in the dopaminergic system. In particular, it was shown a substantial enrichment in D2 receptors in discrete regions of the mesolimbic and mesocortical pathways as well as in a large number of brain areas of n-3 PUFA-deficient pups. The Authors hypothesized that such high expression of D1 and D2 receptors may be attributed to a behavioral hypersensitivity caused by the possible impairment of DA production during brain development [From a neurochemical point of view, deficits in cortical dopamine neurotransmission have been reported in patients with ADHD. Alteration in dopamine neurotransmission has been associated with lower brain DHA concentrations . It has elopment .n-3 PUFA deficient diets identified many effects on dopaminergic systems, including lower levels of dopamine [n-3 PUFA deficient diet soon after conception [Studies in animals fed with dopamine ,214,215,dopamine , and incdopamine  in the Pnception .n-3 PUFA supplementation showed anxiolytic effects [n-3 PUFAs during pregnancy may lead to beneficial effects in children also by reducing anxiety symptoms in childbearing women.On the other hand, other indirect evidence for PUFA’s beneficial effect during gestation for ADHD comes from the study of Monk et al.  indicati effects . Therefon-3 PUFA deficiency have been linked to the development of neuropsychiatric conditions that become overt in early childhood, such as ASD; later childhood, such as ADHD; adolescence, such as SSD; or in adolescence and adulthood, such as depression and anxiety disorders. The influence of PUFAs in these conditions could be explained based on their significant effects on brain development and maturation.In the present review, we have summarized the main literature data regarding the potential role that maternal PUFA consumption may have on the risk for developing psychiatric complications in later adulthood. In this regard, maternal malnutrition and particularly N-3 supplementation during gestation and lactation periods has been shown beneficial in some studies, although many efforts need to be done in this regard. Better structured protocols and the choice of proper timeframe of interventions would surely lead to more consistent results.In conclusion, the available results, although not completely conclusive, suggest that changes in lifestyle, particularly during pregnancy, could result helpful in preventing or reducing the risk of developing neuropsychiatric disorders later in life.All these neuropsychiatric diseases share some common features, such as alteration in vulnerable brain region, especially in brain areas crucially involved in the etiopathogenesis of these pathologies, such as PFC or subcortical circuitries. Given the crucial role played of DHA in this area, especially during brain development and maturation, special attention should be paid in the choice of a correct diet. Many recommendations have been released by national and international authorities ,220,221


Stenotrophomonas maltophilia is a common nosocomial pathogen that causes high morbidity and mortality. Because of its inherent extended antibiotic resistance, therapeutic options for S. maltophilia are limited, and sulfamethoxazole/trimethoprim (SXT) is the only first-line antimicrobial recommended. However, with the spread of dihydropteroate synthase (sul1 and sul2) genes, global emergence of SXT resistance has been reported. There is an urgent need to develop a rapid and sensitive but cost-efficient method to monitor the dissemination of sul genes. In this study, we developed loop-mediated isothermal amplification (LAMP) assays for sul1 and sul2 using real-time turbidity and hydroxy naphthol blue coloration methods. The assays could quickly detect sul genes with high sensitivity and specificity. The LAMP detection limit was 0.74 pg/reaction of extracted genomic DNA for sul1 and 2.6 pg/reaction for sul2, which were both 10-fold more sensitive than the corresponding traditional PCR assays. Additionally, the LAMP assays could positively amplify DNA from sul1-producing strains, but not from the negative controls. We then used the LAMP assays to investigate the dissemination of sul genes among S. maltophilia isolates from patients in three hospitals in Beijing, China. Among 450 non-duplicated samples collected during 2012–2014, 56 (12.4%) strains were SXT-resistant. All these SXT-resistant strains were positive for sul genes, with 35 (62.5%) carrying sul1, 17 (30.4%) carrying sul2, and 4 (7.1%) carrying both sul1 and sul2, which indicated that sul genes were the predominant resistance mechanism. Of 394 SXT-susceptible strains, 16 were also sul-positive. To provide epidemiological data for the appropriate choice of antimicrobials for treatment of sul-positive S. maltophilia, we further tested the susceptibility to 18 antimicrobials. Among these, sul-positive strains showed the highest susceptibility to tetracycline derivatives, especially minocycline . Ticarcillin-clavulanate and new fluoroquinolones (moxifloxacin and levofloxacin) also showed some in vitro activity. Apart from these three kinds of antimicrobials, other agents showed poor activity against sul-positive strains. Stenotrophomonas maltophilia is the third most common non-fermentative gram-negative bacillus isolated from hospitals and has emerged as an important opportunistic pathogen  is the only first-line antimicrobial recommended for treatment because of its good antibacterial activity in vitro and low incidence of resistance , the sul genes have generally been recognized as the predominant resistance mechanism  elements. The global emergence of SXT resistance mediated by the dissemination of sul genes has been reported  is a novel DNA amplification method. It was first developed in the year 2000 and can efficiently and specifically amplify DNA under isothermic conditions within 60 min using Bst DNA polymerase  and further confirmed by species-specific PCR  and sul2  positive control DNAs were used in the establishment of the LAMP assays and were confirmed to carry sul genes by PCR and sequencing of the PCR products. S. maltophilia ATCC 13637, K279a, and P129 were used as negative controls for estimating the specificity of the LAMP assays. Whole genome sequencing of these strains has been performed and they were confirmed to carry genes encoding multidrug-efflux pumps, β-lactamases, and aminoglycoside-modifying enzymes, but they did not carry sul genes  according to the manufacturer’s recommended protocol. The extracted DNA was used as a template in the LAMP and PCR reactions.Whole genomic DNA (including plasmid DNA) was extracted using the protocol described by a previous study . The bacsul1 detection was performed using the PCR primers shown in Table 1. The target products were 250 bp for sul1 and 276 bp for sul2. The PCR cycling parameters were: initial PCR activation, 95°C for 5 min; amplification, 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s; final extension step, 72°C for 10 min. A 25-μl reaction volume was used, and the products were separated by 1% (w/v) agarose  gel electrophoresis and stained with ethidium bromide. Images were documented with a Gel DocTM EQ imaging system .To compare the sensitivity of LAMP assays and traditional PCR, sul1-specific and sul2-specific PCR and LAMP primers, respectively. The primers were designed and analyzed by PrimerExplorer software1. The sequences and locations of primers for sul1 and sul2 are both shown in Table 1.The sequences CP011493 (80815–81654) and CP009257 (2540172–2540987) obtained from the NCBI GenBank database were used for the design of Bst DNA polymerase, and 2 μl template using the Loopamp DNA Amplification Kit  for real-time turbidimetry or with the further addition of 1 μl HNB solution  for visual detection. Primers were used at the concentrations of 1.6 μM for the forward internal primer and backward internal primer, 0.8 μM for the loop forward primer and loop backward primer, and 0.2 μM for the forward outer primer and backward outer primer. The reactions were performed in the reaction tubes  for 60 min at 65°C.The LAMP reactions were performed in a final volume of 25 μl containing 12.5 μl reaction mixture, 1 μl Two different methods, namely sample turbidity and HNB coloration, were used to detect the LAMP products. Real-time changes in turbidity were monitored by spectrophotometric analysis by recording the optical density (650 nm) every 6 s with a Loopamp Realtime Turbidimeter . For direct visual detection, 1 μl of HNB detection reagent was added to the reaction. The amount of DNA template used in the LAMP reaction mixture was equal to that used in PCR reactions. Each experiment was performed at least three times.sul-positive strains were determined for each isolate by the Mueller–Hinton agar  macrodilution method. Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, and Escherichia coli ATCC 25922 were used as quality control strains to ensure the accuracy and reliability of the assays. Interpretive breakpoints for susceptibility are available only for SXT, ceftazidime, minocycline, levofloxacin, chloramphenicol, and ticarcillin/clavulanate .Eighteen clinically used antibacterial agents were tested in our study, as follows: SXT, ceftazidime, minocycline, levofloxacin, chloramphenicol, ticarcillin/clavulanate, tigecycline, doxycycline, moxifloxacin, cefepime, cefoperazone/sulbactam, meropenem, amikacin, colistin, fosfomycin, aztreonam, azithromycin, and rifampicin. Tigecycline was obtained from Wyeth Pharmaceutical ; moxifloxacin was obtained from Bayer Healthcare ; Colistin was purchased from Sigma-Aldrich ; other antimicrobial agents were purchased from the National Institute for the Control of Pharmaceutical and Biological Products . The antimicrobial powders were used to prepare stock solutions, as described by the Clinical and Laboratory Standards Institute . MICs ofvulanate . Other asul1-specific and sul2-specific LAMP primers were designed and analyzed using PrimerExplorer software (Table 1). To compare the detection limit of the LAMP method with that of the conventional PCR method, genomic DNA was extracted from S. maltophilia X2 and serially diluted 10 times from 37.0 ng/μL to 3.7 fg/μL. As shown in Figure 1, the detection limits of the real-time turbidity and HNB methods were 0.74 pg/reaction of extracted whole genomic DNA, 10-fold more sensitive than that of the PCR assay (7.4 pg/reaction) . Similarly, the detection limit of LAMP specific to sul2 was 2.6 pg/reaction, which is 10 times more sensitive than that obtained for the conventional PCR method .The sul1 was evaluated using X2, P121, and K106 carrying sul1 as positive controls, S. maltophilia ATCC 13637, K279a, and P129 as negative controls, and distilled water as blank control. As shown in Figure 3, both methods positively amplified DNA from sul1-producing strains, but not from the negative and blank controls. The results showed that the assay was highly specific for detecting sul1. The same results were observed for the developed sul2 LAMP assay .The specificity of the LAMP assay for detecting S. maltophilia strains were collected in our study, and 56 (12.4%) strains were resistant to SXT. As shown in Table 2, all these 56 SXT-resistant strains were tested positive for sul genes, among which 35 (62.5) strains carried sul1, 17 (30.4%) strains carried sul2, and 4 (7.1%) strains carried both sul1 and sul2. Among 394 SXT-susceptible strains, 16 (4.1%) carried sul genes.During 2012–2014, 450 non-duplicated S. maltophilia were 95.4% for minocycline, 68.1% for doxycycline and tigecycline, 50.0% and 59.7% for levofloxacin and moxifloxacin, respectively, and 69.4% for ticarcillin-clavulanate. The susceptibility rates for 12 other antimicrobial agents were less than 50%, while for the agents meropenem, amikacin, colistin, ceftazidime, chloramphenicol, aztreonam, sulfamethoxazole/trimethoprim, azithromycin, and rifampicin, S. maltophilia had resistance rates exceeding 70% (Table 3).Among 18 clinically used antimicrobials, the susceptibility rates of Stenotrophomonas maltophilia is an opportunistic pathogen, mainly causing pneumonia and bloodstream infections in immunocompromised patients, with high morbidity and mortality  of these strains tested positive for sul genes. These sul-positive strains showed poor susceptibility to most clinically used antimicrobials apart from tetracycline derivatives, ticarcillin-clavulanate, moxifloxacin, and levofloxacin. The developed LAMP assays provide a useful tool for the surveillance of sul genes among clinical S. maltophilia strains, since these assays are more sensitive, faster, and more cost-efficient than the conventional PCR method.We have developed rapid, simple, and cost-efficient LAMP assays for the detection of the SXT resistance genes YoL and YuL conceived and designed experiments. JZ, YX, WL, WN, CW, and RW executed experiments. JZ, YX, and WN performed and wrote the manuscript. CW and RW helped to edit the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.