This work reports the interlayer difference of exciton and phonon performance between the top and bottom layer of a bilayer-stacked two-dimensional materials structure (BSS). Through photoluminescence (PL) and Raman spectroscopy, we find that, compared to that of the bottom layer, the top layer of BSS demonstrates PL redshift, Raman  Owing tD materia2 . Additio2 ,5. Furth2 , nonline2 ,7, Raman2 ,9, and t2 ,13,14,152 . Van Hov2  and angl2  are anot2  and mirr2 , imply tHowever, all these findings about BSS focus only on the interlayer-coupling-induced effects, while ignoring the difference between the top and bottom layer of BSS. The BSS sample fabricated by transfer method can be divided into three different regions: stacked region where top and bottom layer overlap each other, bottom region (bottom layer excluding stacked region), and top region (top layer excluding stacked region). Although bottom and top regions are both supposed to be in direct contact with the substrate, there exists great difference between the top-substrate and the bottom-substrate coupling. Substrate coupling can affect 2D materials in many aspects. For example, on the one hand, substrate contact can employ strain on 2D materials, leading to phonon variations measured by Raman spectroscopy ,22,23. O2 monolayer with a two-dimensional structure demonstrates intriguing effects in various aspects, including optical [2 is expected to have strong photoluminescence (PL) emission, which has been confirmed both experimentally and theoretically. Excited by 532 nm laser at ambient conditions, monolayer MoS2 is reported to have two prominent PL peaks at 625 nm (B peak) and 670 nm (A peak) [2 monolayer [−1 , respectively.As one sort of transitional metal dichalcogenide (TMD) materials, MoS optical ,33,34, e optical ,36,37, a optical ,39,40. E optical , monolay(A peak) ,43,44. T(A peak) . Also, tonolayer , located2 (BSM) samples fabricated by transfer, exciton and phonon performance in the top and bottom regions are remarkably different. Despite the fact that both top and bottom layers of BSM are transferred, compared to the bottom region, the top region demonstrates PL intensity reduction and peak redshift, implying less p-doping to top region from substrate. Meanwhile, redshift of in-plane Raman mode 2 samples are fabricated. It is found that, compared to the supported region, the suspended region of monolayer MoS2 demonstrates redshift in PL and Raman peaks, which are consistent with those in the top region of BSM, thus providing evidence for coupling difference between top-substrate and bottom-substrate.Through photoluminescence and Raman spectroscopy, we found that, in bilayer-stacked MoS2 bilayer-stacked structure on SiO2 substrate, we are sure this work can be informative to film–substrate interaction study on other 2D materials and substrates.Since the interlayer difference in BSS can complicate the experimental results, and is also affected by interlayer coupling, our findings are of great significance to distinguish between contributions from interlayer coupling and film–substrate interaction, which is of great significance to researches on interlayer-coupling-induced effects like optoelectric effects in VdW hetero-structure and twisted angle dependence in tBLS. Furthermore, our findings are universal and, apart from MoS2/(001)Si substrate (SiO2 layer is 300 nm thick), with a size of approximately 50 μm by chemical vapor deposition (CVD). Then, we transferred two sheets of monolayer MoS2 to one substrate, by which method we can obtain tens of BSM samples with various angles in a single step. It should be noted that, in this work, both top and bottom layer of the BSM undergo transfer process to avoid preparation method induced difference. During the transfer process, any solvent that may cause doping in MoS2 was avoided. Universally used transfer methods like, PMMA-way (poly-methyl-methacrylate) [2 flow at 300 °C for 2 h) to remove residues and enhance interlayer coupling. For this study, a convenient fabrication process is employed to obtain BSM samples. First, all single-layer flakes are deposited on a SiOcrylate)  and PVA-crylate)  introduccrylate)  is chosecrylate)  and anne2 samples, they are fabricated by PLLA transfer onto SiO2/(001)Si substrate (SiO2 layer is 300 nm thick) with 300 nm-depth holes. These holes are of a radius 5 μm each, fabricated by reactive ion etching (RIE) method in SF6/CHF3 mixed gas flow (30 sccm).As for free-standing monolayer MoS−1 and 1 nm.In this work, all bright field optical micrographs are taken by Nikon LV150 microscope, using 50× objective lens . Dark-field optical micrographs are taken by ZEISS Axio Scope A1 microscope, using a 50× objective lens . Atomic force microscopy (AFM) images are taken by NT-MDT Prima AFM system, using semi-contact scanning mode . PL and Raman spectroscopy are measured by WITec Alpha300R confocal Raman system, using a 50× objective lens . A 532 nm laser is used as the excitation source. For Raman measurements, the laser power is 1 mW, while for PL measurements laser power is 0.5 mW, sufficiently low to avoid heating effects. Optical gratings used for Raman and PL measurements are 1800 L/mm and 600 L/mm, respectively, providing respective spectral resolution smaller than 1 cmOne as-fabricated BSM sample is shown in As is plotted in 1g Raman shift difference among the top region and bottom region is below 19 cm−1, which is the signature of monolayer MoS2, indicating these samples are stacked by two individual monolayer MoS2. For each sample, compared to the bottom region, the top region exhibits PL intensity reduction and redshift  area of this sample totally excluded the film–substrate interaction. In Interlayer difference and spatial inhomogeneity of PL and Raman spectroscopy in BSS possibly come from substrate-coupling difference between the layers, and among the top layer, respectively. To confirm this, we fabricated a freestanding sample in a by tran2 thickness), not sufficiently macroscopic to be detected by bright-field/dark-field optical microscope and atomic force microscopy, they can remarkably reduce film–substrate coupling. A schematic illustration is shown in 2 is resulted from exciton (radiative wavelength: ~660 nm) and trion recombination (radiative wavelength: ~680 nm) [2 is an n-type semiconductor, the silicon oxide depletes equilibrium electrons in regions of strong coupling with substrate [ex) while suppressing the trion formation rate (Γf) at the same time, as is shown in For one thing, PL emission of MoS~680 nm) . Accordi~680 nm) ,25,26, tFor another, the in-plane Raman mode l strain ,22. At tl strain ,55. As aThe discussions above provide a reliable explanation for our findings. Admittedly, interference effects can induce intensity change of PL and Raman. However, on the one hand, the minor height fluctuations in BSS film cannot result in remarkable interference variations. On the other hand, interference-induced intensity change would be broad band, which is not consistent with our experimental results. Therefore, we conclude that substrate-coupling-induced strain and doping to BSS play the dominant part in interlayer difference and spatial inhomogeneity of phonon and exciton performance.2 fabricated by the transfer method. PL and Raman spectroscopy of freestanding monolayer MoS2 are also measured for comparison. Interlayer difference and spatial inhomogeneity of exciton and phonon performance are experimentally observed in as-fabricated BSS samples, which we attribute to film–substrate coupling-induced strain and doping. Additionally, our findings prove that, even surface fluctuations less than one-atom-layer thickness can be easily identified by Raman and PL spectroscopy. This work will be of great use to inform future researches on BSS including tBLS, VdW homostructure and heterostructure, and improve our understanding of substrate effects on optical and transport properties of 2D materials.In summary, we conducted a systematic investigation on PL and Raman spectroscopy of bilayer-stacked MoS


Oxidative stress plays a crucial role in the development of diseases induced by welding fumes. To our knowledge, little information is available on the relationship between multiple heavy metal exposure and oxidative stress in welders. We assessed the relationship between multiple heavy metal exposure and oxidative damage by analyzing 174 nonsmoking male welders in a shipyard. Urinary metals were used as the internal dose of exposure to metals in welding fumes, and urinary 8-hydroxy-2’-deoxyguanosine (8-OHdG) was used as an oxidative DNA damage marker. The relationship between workers’ metal levels and 8-OHdG was estimated using a multiple linear regression model. The geometric mean levels of urinary chromium (Cr), nickel (Ni), cadmium (Cd), and lead (Pb) were considerably higher in welders than in controls. Urinary Cr and Ni were determined as effective predictors of urinary 8-OHdG levels after adjusting for covariates. Oxidative DNA damage was associated with both Cr and Ni of welding fume exposure in shipyard welders . In this study, we investigated the significantly positive relationship between urinary metals  and 8-OHdG in nonsmoking shipyard welders. Moreover, the use of particulate respirators did not reduce metal exposure and oxidative damage. Therefore, we infer that hazard identification for welders should be conducted. Welding fumes were classified as a possible human carcinogen group 2B) by the International Agency for Research on Cancer (IARC) in 1990 B by the ,4. In adWelding fumes consist of many toxic materials, including several metals such as chromium (Cr), nickel (Ni), cadmium (Cd), and lead (Pb) , which p2−), singlet oxygen (1O2), and H2O2 [The most common DNA lesion caused by the reaction of hydroxyl radicals with guanosine at the C-8 position in DNA is 8-Hydroxy-2’-deoxyguanosine (8-OHdG) . DNA damand H2O2 ,17. The and H2O2 . Such daand H2O2 ,20.For the last several decades, epidemiologists have been warning of the hazards of welding fumes; however, until now, welding fume-induced health problems remain an occupational hazard that must be solved. The adverse effects of heavy metals from welding fume exposure are observed in both humans and animals ,22,23; hWe conducted a cross-sectional study to assess the relationship between metal exposure and oxidative damage. The study participants were recruited from a shipyard located in Taiwan during their annual physical examination. We collected their spot urine for urinary metal, 8-OHdG, and creatinine analyses. Sociodemographic information, such as sex, age, education, and lifestyle , was obtained using a self-reported questionnaire. The history of diagnosed illness, information on chemical exposure, and working experience with a specific welding fume exposure, including the frequency of the particulate respirator use, were also obtained from the questionnaire. Participants with highly diluted (urinary creatinine < 30 mg/dL) or highly concentrated (urinary creatinine > 300 mg/dL) urine samples were also excluded from our statistical analysis according to the WHO guidelines for the biological monitoring of chemical exposure in the workplace ,25. MorePersonal air samples from the workplace were collected using active samplers with cellulose-ester filters (diameter: 37 mm and pore size: 0.8 μm). Metals were sampled at a flow rate of 2000 ± 30 mL/min. Personal samplers were placed nearly at a height of the breathing level of workers in welding areas for consecutive 8-hour workdays. The concentrations of Cr, Ni, Cd, and Pb were measured through inductively coupled plasma mass spectrometry . The detection limits for Cr, Ni, Cd, and Pb were 5.2 ng/L, 6.3 ng/L, 1.2 ng/L, and 8.0 ng/L that were obtained using seven repeated analyses of deionized water.Participants were asked to wash their hands with soap prior to urine collection; spot urine samples were collected after the shift ended on a weekend. Urine samples were stored at −20 °C until analysis. Urinary metals, namely Cr, Ni, Cd, and Pb, were analyzed through ICP-MS . Urinary 8-OHdG levels were measured through high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) as described in . A detect-test or regression analysis was done. Student’s t and Chi-Square statistics were used to compare the personal covariates, which were the urinary 8-OHdG and metals of welders and office workers. The association among urinary metals, personal covariates, work experience, and oxidative damage biomarkers was assessed using multiple linear regression; moreover, to minimize the effect of residual confounding of sociodemographic factors, such as sex, age, and race/ethnicity, on urinary biomarkers, urinary creatinine was adjusted as an independent variable in the linear regression analysis [Urinary 8-OHdG, metals, and creatinine levels were first natural log-transformed to normalize their distributions before the Student’s analysis . The levp > 0.05). The proportion of education above college was higher in office workers (84.91%) than in welders (26.45%) (p < 0.001), and approximately 44% of welders regularly wore particulate respirators during their work shift.The characteristics of the study participants are presented in 3; 6.47 and 5.29 μg/m3; 0.40 and 2.95 μg/m3; and 5.39 and 4.24 μg/m3, respectively.The metal concentrations in ambient air were obtained using personal air sampling as listed in t-test, a considerably higher amount of urinary biomarkers was observed in welders than in office workers.The relationship between the levels of heavy metals in air and urine samples was determined using the Pearson correlation analysis . Individ−3 level for occupational exposure in shipbuilding welders, rather than Ni [Urinary Cr, Ni, and Cd levels were positively correlated with the 8-OHdG concentration. The geometric mean 8-OHdG concentration (5.02 μg/g creatinine) in welders in this study was higher than that in gas metal arc welders working with stainless steel, flux-cored arc welders working with mild steel, tungsten inert gas welders, and welders wearing a powered air-purifying respirator in an earlier study (median = 3.79–4.41 μg/g creatinine) , as well than Ni .2.5 during the welding process was related to increased urinary 8-OHdG levels [Welding fumes contained various contaminants generated from heated metal work pieces and flux materials. Some of the metals and fluxes were vaporized and then condensed as nanometer-sized particles ,35,36,37G levels .Oxidative stress occurs through the imbalance of free radicals and antioxidants due to either the depletion of antioxidants or the accumulation of free radicals. Consequently, free radicals attack and oxidize cell components, such as lipids, proteins, and nucleic acids, resulting in cell dysfunction ,39,40. MThe half-life of urinary Cr excretion was 10.75 years in workers who performed the plasma cutting of stainless steel . After cThe results of the WELDOX study group indicated increased urinary Cr and Ni concentrations in welders working in confined spaces and decreased urinary Cr and Ni concentrations in welders working in sufficient ventilation and using respiratory mask . AdditioWe investigated the relationship between multiple metal exposure and urinary 8-OHdG; however, some limitations were observed. First, the study failed to measure some potentially useful elements, such as volatile organic carbon and other metals including iron and zinc . MoreoveIn this study, the use of particulate respirators was not an effective predictor for both urinary metals and 8-OHdG in welders. The levels of urinary Cr, Ni, and 8-OHdG were significantly higher in welders than in office workers. The levels of Cr and Ni in urine samples were positively associated with the 8-OHdG levels of shipyard welders, indicating that preventive measures must be developed immediately, including the use of effective gloves and respirators and enforcement of ventilation to reduce welding fume exposure and promote the health of shipyard welders.


Recent studies have shown that the actin cytoskeleton is not only an upstream regulator of Ca2+ signaling, but also a downstream regulator. Ca2+ has been shown to regulates actin dynamics and rearrangements via different mechanisms in plants, and on this basis, the upstream signaling encoded within the Ca2+ transient can be decoded. Moreover, actin dynamics have also been proposed to act as an upstream of Ca2+, adjust Ca2+ oscillations, and establish cytosolic Ca2+ ([Ca2+]cyt) gradients in plant cells. In the current review, we focus on the advances in uncovering the relationship between the actin cytoskeleton and calcium in plant cells and summarize our current understanding of this relationship.In plant cells, calcium (Ca Moreover, even the endoplasmic reticulum (ER) is expected to contain large amounts of Ca2+ [2+ stockpiles can be used for elevating the [Ca2+]cyt level under stress conditions or growth signaling [2+ is toxic to plants if high levels of the ion remain in the cytosol for a long period [2+ [2+]cyt homeostasis plays a vital role in a myriad of physiological functions, and changes in [Ca2+]cyt result from the influx and efflux of external and internal Ca2+ stores cyt levels, and that this increase in [Ca2+]cyt was abolished by the calcium channel blocker La3+ or Gd3+ cyt concentration in response to gravity are also widely accepted in plant biology [2+]cyt through mechanosensitive (MS) channels induced by changes in the gravity vector was attenuated by actin-specific reagents, implying that actin dynamics adjust calcium homeostasis by regulating the activity of MS Ca2+-permeable channels [2+-permeable channels and are involved in many developmental and stress-related processes [2+-permeable channels by influencing the annexin-actin interaction [2+]cyt changes may also derive from the release of the ion from the mitochondria and vacuole [Arabidopsis root hairs indicated that disruption of actin dynamics by latrunculin B (Lat B) and jasplakinolide (Jas) decreased the Ca2+ concentration in the mitochondria and induced an instantaneous elevation of [Ca2+]cyt, followed by a continuous decrease cyt in response to salt stress, and plants lacking the subunit proteins in the Arp2/3 complex showed enhanced increases in [Ca2+]cyt in response to salt stress, decreased mitochondria movement, and hypersensitivity to salt. Furthermore, ARP2/3 complex promotes actin assembly around mitochondria and drives mitochondrial movement cyt promotes actin filament disassembly, but low [Ca2+]cyt promotes the assembly of a tip-localized actin spot. Moreover, abolishing the Ca2+ gradient leads to dramatic actin accumulation at the protonema tip. Together, these data indicate that the tip calcium gradient regulates actin accumulation to promote tip growth [2+.Calcium signaling also regulates the dynamics of the actin cytoskeleton ,19,49,50len tube ,54,55,56p growth . Calcium2+), pH, phosphatidylinositol  bisphosphate (PIP2), and phosphatidic acid (PA) [2+ is the most important second messenger for the regulation of plant development and stress signaling [Actin dynamics are regulated spatially and temporally by different classes of ABPs, such as G-actin sequestration proteins, nucleating proteins, severing, and depolymerizing proteins, bundling and crosslinking proteins and end-capping proteins . The actcid (PA) . Among tignaling .2+-dependent ABPs regulate actin dynamics in the tip growth of pollen tubes, including profilin, LIM domain-containing proteins (LILIM1 and PLIM2c), MICROTUBULE-ASSOCIATED PROTEIN18 (MAP18), MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25), Rho-like GTPase of plants (ROP)-interactive and CRIB motif-containing protein1 (RIC1), and Villins (VLNs) [2+-dependent manner [2+-dependent/regulated ABPs are discussed below and summarized in From pollen hydration and germination to pollen tube tip growth, the process is accompanied by actin rearrangement regulated via numerous ABPs and calcium oscillations ,15,58,59s (VLNs) ,65,66,67t manner ,69,70. T2+-dependent ABPs, the major group is the villin/gelsolin/fragmin superfamily, which is a class of multifunctional ABPs that remodel actin dynamics by nucleating, severing, depolymerizing, and bundling actin [2+-binding site, based on protein crystallography [Lilium) pollen, which can bind and bundle actin filaments in a Ca2+-dependent manner [VLN1-VLN5) in the Arabidopsis genome, and all of them bundle actin filaments in a Ca2+-insensitive manner [2+-sensitive manner [2+ binding sites in the villin/gelsolin/fragmin superfamily, and as regards to gelsilin, Ca2+ binds type-1 sites at the interface of gelsolin is important for its interaction with actin, while Ca2+ binds type-2 sites buried within gelsolin activate this protein [Arabidopsis have just one type-1 site in the G1 domain. In contrast to VLN1, which has only two type-2 sites in the G2/4 domains, VLN2-5 has one type-2 site in the G1 domain and another two to three type-2 sites in the G2/4/6 domains [2+-sensitive [Arabdopsis root hairs, VLN4 caps and severs actin filaments in the presence of 5 μM Ca2+ but fails to do so in the presence of 0.5 μM Ca2+; these Ca2+ concentrations resemble those at the tip region and the shank region, respectively. Therefore, it may be that VLN4 needs to stabilize the actin cytoskeleton via its bundling activity in the shank region, while it promotes actin turnover by severing and capping actin filaments at the tip region [2+-dependent actin filament severing, actin filament bundling, and actin filament capping activities. OsVLN2 promotes recycling of PIN2 and polar auxin transport by regulating actin dynamics to modulate morphogenesis of plant architecture [Among these Cang actin ,87. Theslography ,88,89,90t manner ,73,74. Te manner ,84,85,91e manner ,84,85,91 protein . Khuranaensitive ,84,85,91p region . To gainitecture .Papaver rhoeas (poppy) pollen, Huang et al. isolated and characterized an 80 kDa gelsolin-like protein, PrABP80, which possesses six tandem G domains and exhibits Ca2+-dependent actin-filament-severing and barbed-end-capping activities. This calcium-mediated actin filament depolymerization is vital for the self-incompatibility response in P. rhoeas pollen [Mimosa pudica, which possesses three G domains, severs actin filaments, and enhances actin polymerization in a Ca2+-dependent manner [Lilium davidii pollen, the Ren group also identified a fragmin-like protein, LdABP41, which possesses three G domains, and the smallest member of this superfamily, ABP29, which possesses only two G domains; they found that these two proteins nucleate and sever actin filaments in a Ca2+-sensitive manner [2+-dependent manner was identified in apples . Moreover, MdMVG can physically interact with S-RNase, and this interaction inhibits the actin filament-severing activity of MdMVG in vitro. Therefore, S-RNase interacts with MdMVG to inhibit its actin filament-severing activity, while Ca2+ binds to MdMVG to enhance its severing activity and then regulates self-pollen tube growth during the early stage of self-pollination induction [In t manner . From Lie manner ,75. Recenduction .Arabidopsis, cotton (Gossypium hirsutum), lily (Lilium longiflorum), sunflower (Helianthus annuus), and tobacco (Nicotiana tabacum) [Arabidopsis can bind to actin filaments and bundle them in response to Ca2+, and their activity is downregulated by high concentrations of Ca2+ [LIM is a family of proteins containing two LIM domains that essentially consist of two zinc fingers linked together by a short, two-amino-acid spacer and function as a module for protein–protein interaction . Plant Ltabacum) ,99,100,  of Ca2+ ,64. One Arabidopsis PM-associated cation-binding proteins (PCaPs), namely, PCaP1 and PCaP2, which can bind to the PM and to calcium, have been proven to be microtubule (MT)-associated proteins, named MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) and MICROTUBULE-ASSOCIATED PROTEIN18 (MAP18), respectively [2+-dependent actin-filament-severing activity, and this activity is essential for determining the direction of pollen tube growth [2+-dependent actin filament-severing activity [2+-dependent manner, and the distribution of RIC1 at the apical PM exhibits oscillation in concert with pollen tube growth. Moreover, high concentrations of Ca2+ enhance actin-filament-severing activity of RIC1 to regulate the abundance and oscillatory amplitude of fine actin filaments in pollen tubes [2+ might coordinate diverse actin filament severing proteins to regulate the proper organization, abundance, and dynamics of actin filaments for pollen tube tip growth [Two ectively ,103,104.ectively ,65. Zhu e growth . The secactivity ,102. Addactivity ,107,108,activity . Zhou eten tubes . Consideen tubes , these sp growth .2+ could inhibit the travel of 170 kDa myosin along the actin filaments responsible for cytoplasmic streaming. Moreover, the light chain of myosin-CaM might be involved in Ca2+ regulation [2+-dependent manner both in vitro and in vivo [2+-dependent manner, such as p34 and p35 from tomato, annexin from Mimosa, and AnxGb6 from cotton [Profilin as a key player in the early step of actin assembly is a G-actin binding protein with a molecular mass of 12–15 kDa capable of maintaining a pool of monomeric actin in cells ,112,113.gulation . Annexin in vivo ,119,120.m cotton ,77,79.Collectively, these studies demonstrate that calcium can regulate actin dynamics by directly binding to ABPs to regulate their activity.2+ has been demonstrated to activate the CDPK family; the activated CDPKs subsequently transfer Ca2+ signals to downstream phosphorylation substrates to decode information related to Ca2+ oscillations and spikes [2+ via activation/deactivation of CDPKs.Posttranslational modifications are very important for the activity of some proteins ,122. Amod spikes ,125,126.d spikes ,128, sugArabidopsis, maize, tobacco, and moss [ADF/cofilin is a family of ABPs that can bind both G-actin and actin filaments and then depolymerize and sever actin filaments to promote rapid actin turnover ,131,132.and moss ,135,136.and moss ,19. Howeand moss ,19. Uno and moss . Recentland moss . While Aand moss ,133, sug2+ sensor in plants, has been suggested to be involved in regulating the dynamics of the actin cytoskeleton [2+ signals, interacts with and activates CIPK24/SOS2 to participate in salt-stress sensing and tolerance [sos3 are abnormal in response to salt stress, and external calcium or a low concentration of latrunculin A (Lat A) can partially rescue this phenomenon [Additionally, the CBL family, which serves as another Caskeleton . CBLs peskeleton ,142,143.olerance . Ye et aenomenon . CollectIn plants, ROP GTPase, also called RAC-GTPase, plays a fundamental role in several important cellular processes, such as tip growth of pollen tubes and root hairs, regulation of the actin cytoskeleton, and hormone and stress response ,146,147.2+ gradient by interacting with reactive oxygen species (ROS), phosphoinositides, and pH gradient signaling in the tip regions of pollen tubes [2+ gradients through their downstream plant-specific family of ROP effectors, RICs [2+ gradients: RIC3 directly stimulates Ca2+ influx in the tube apex, while RIC4-mediated actin assembly might inhibit the accumulation of Ca2+ at the tip [2+ gradient and the assembly of tip actin filaments, respectively. Then, elevated Ca2+ might induce actin disassembly through some ABPs, such as profilin or members of the villin/gelsolin/fragmin family [2+ gradient and tip growth [2+ gradients mainly via its downstream effectors, the RIC family  with a nonphosphorylatable Ala substitution at the Ser-6 position shows high activity to counteract the inhibiting effect of NtRac1 overexpression on pollen tube tip growth, while the phosphomimic form of NtADF1(S6D) shows a reduced ability to counteract this inhibiting effect . Moreove2+ ,19. In a2+ . Overall2+-permeable channels [Overall, recent studies have shown that calcium can adjust the actin cytoskeleton by directly binding to ABPs and regulating their activity or by indirectly regulating their activity via calcium-stimulated protein kinases, such as CDPKs ,18. On tchannels . Althoug


Vibrio mediterranei is a Gram-negative bacterium of the family Vibrionaceae. Vibrio mediterranei strain 117-T6 was pathogenic to Pyropia yezoensis, a red seaweed cultivated in China, by causing death to its conchocelis. Vibrio mediterranei is a Gram-negative bacterium of the family Vibrionaceae. Vibrio mediterranei strain 117-T6 was pathogenic to Pyropia yezoensis, a red seaweed cultivated in China, by causing death to its conchocelis. Here, we report the complete genome sequence of Vibrio mediterranei 117-T6. Vibrio is a major group of the family Vibrionaceae, and its members are widespread in marine environments, or associate with hosts in those environments, and can be pathogenic or nonpathogenic . Pyropia species are popular edible red algae with great commercial importance and have a long history of cultivation in northeast Asia. In this paper, the complete genome sequence of V. mediterranei strain 117-T6 was determined to facilitate further research into its virulence factors.The genus thogenic , 2. Sinc in 1986 , the bac in 1986 –6. V. V. mediterranei 117-T6 was cultured in tryptic soy broth (TSB) medium containing 1% NaCl at 28°C for 12 h with shaking at 120 rpm. The genomic DNA (gDNA) was extracted using the Ezup column bacteria gDNA purification kit  according to the manufacturer’s protocols. The gene library was constructed using a SMRTbell template prep kit 1.0 and was quality controlled using Qubit 3.0  and a 2100 bioanalyzer . After library preparation, the genome was sequenced by single-molecule real-time sequencing (SMRT) using the PacBio sequel system   . The genN50, 9,729 bp; 233-fold coverage). The reads were assembled, and we found that the complete genome sequence of V. mediterranei 117-T6 consists of two circular chromosomes  and one plasmid . The total length of V. mediterranei 117-T6 was 5,976,027 bp with a GC content of 44.04%, including 5,539 coding DNA sequences (CDS), 31 rRNAs, 115 tRNAs, and 264 noncoding RNAs (ncRNAs). The number of tandem repeat finder (TRF) repeats of chromosome1, chromosome2, and the plasmid were 48, 20, and 9, respectively, and the number of simple sequence repeats (SSRs) were 9, 3, and 2, respectively. The plasmid contained 2 clustered regularly interspaced short palindromic repeat (CRISPR) sequence cassettes.A total of 178,807 filtered subreads with a total length of 1,332,991,254 bp were generated after sequencing . The raw data have been submitted to the Sequence Read Archive (SRA) under run number SRR8294759.The complete genome sequence for


Chemokines are small secreted proteins that orchestrate migration and positioning of immune cells within the tissues. Chemokines are essential for the function of the immune system. Accumulating evidence suggest that chemokines play important roles in tumor microenvironment. In this review we discuss an association of chemokine expression and activity within the tumor microenvironment with cancer outcome. We summarize regulation of immune cell recruitment into the tumor by chemokine-chemokine receptor interactions and describe evidence implicating chemokines in promotion of the “inflamed” immune-cell enriched tumor microenvironment. We review both tumor-promoting function of chemokines, such as regulation of tumor metastasis, and beneficial chemokine roles, including stimulation of anti-tumor immunity and response to immunotherapy. Finally, we discuss the therapeutic strategies target tumor-promoting chemokines or induce/deliver beneficial chemokines within the tumor focusing on pre-clinical studies and clinical trials going forward. The goal of this review is to provide insight into comprehensive role of chemokines and their receptors in tumor pathobiology and treatment. Migration of the immune cells to specific organs is controlled in part by small proteins called chemokines  , 2. ChemSince chemokines and their receptors are highly promiscuous, with most chemokines binding multiple receptors, and receptors binding multiple chemokine ligands. One must consider this complexity in reference to functional significance of each chemokine or receptor in reference to cancers .FM, TFH, Th22, Tregs, ILCs, NKT), NK cells, B cells and immature DCs  and B cells to the TME through interaction with CCR7 (Of key importance in immune surveillance is the recruitment of dendritic cells (DCs), CD4+Th1 cells, and CD8+ T effector cells to the tumor microenvironment. Chemokine receptors CCR4, CCR5, CXCR3, CXCR4, CCR6, and CCR7 play a pivotal role in the regulation of T cell homing to inflammatory sites . T cellsture DCs –16 are rith CCR7 –20. Dendith CCR7 –23. Whenith CCR7 . As suchith CCR7 .In contrast, tumor-promoting leukocytes are comprised of macrophages expressing arginase, IL4, IL10, and IL13, as well as myeloid-derived suppressor cells (MDSCs), T regulatory cells (Tregs) and specific B cell subsets. Ligands for chemokine receptors CCR1, CCR2, CCR3, and CCR5, CCR8, CXCR1, CXCR2, and CXCR4 recruit macrophages to the TME –39. NeutFor naïve T cells to become activated, antigen presenting DCs migrate from the developing tumor to the lympth node where they present antigen to the T cells via the T cell receptor (TCR) and stimulate a process that leads to T cell activation. CD4 cells can be activated by antigen presenting cells (APCs) and mature into helper cells [T helper type I cells (Th1) or T helper type II cells (Th2)]. Th1 cells produce cytokines including interferon-γ (IFNγ), tumor necrosis factor-alpha (TNFα), while Th2 cells secrete IL-4, IL-5, IL-10, and IL-13. The cytokines produced by the DCs influence the differentiation of naïve helper T cells into either Th1 or Th2 cells. For example, if DCs secrete IL-12, the naïve helper T cells differentiate into Th1 cells. Th1 cells express CD40L on their plasma membrane and this ligand binds to CD40 expressed by the DC or other APC. Engagement of CD40 on the DCs or other APC primes them to a higher activation level resulting in elevated expression of class I MHC, B7 and co-stimulating molecules such as 4-1BBL. When CD8+ T cells come into contact with one of these highly activated DCs, its TCRs recognizes the peptides presented by the MHC Class I molecule on the DC/APC. This, in turn, leads to the activation of CD8 T cell upon binding of its TCR to the MHC presented peptide. DiffereH1 cells , cancer-associated fibroblasts, myeloid cells, and neutrophils, providing a very rich “soil” to facilitate the recruitment of immune cells into the tumor microenvironment (TME). For example, tumor cells, macrophages, and neutrophils produce CXCL1, CXCL2, CXCL5, and CXCL8 and these chemokines recruit MDSCs, both the PMN-MDSCs and the Monocytic-MDSCs , 69. Thend CCL28 . Mature nd CCL28    , 73 or Clas.org) , 75, chelas.org) . Furtherlas.org) . Moreovelas.org)  and poorlas.org) . Interes relapse . Of note relapse .Thus, primary tumor data indicate that chemokines play an important role in tumor progression, which, in part, may relate to the direct effect of chemokines on cancer cell growth and metastasis . HoweverTumor cells express a wide range of chemokine receptors, and there are extensive reports that tumor cells utilize both autocrine and paracrine pathways to respond to chemokines with altered migration, proliferation, and gene expression. Importantly, chemokine receptors have been reported to play a crucial role in maintenance of cancer stem cells. For example, a CXCR1 blockade has been shown to selectively target breast cancer stem cells  and its Similarly, CCL2 expression by cancer-associated fibroblasts has been shown to support the growth of breast cancer stem cells , while C2O2 and tumor-secreted CCL2 is a critical mediator of optimal anti-metastatic entrainment of G-CSF-stimulated neutrophils. Tumor entrained neutrophils inhibit seeding in the pre-metastatic lung  and CXCR3 ligands (CXCL9 and CXCL10) has been associated with limited infiltration of antigen-specific T cells . The criCertain C-C chemokines can also contribute to T cell recruitment into the tumor. Clinical data indicate that CCR5 ligands, CCL4, and CCL5, can promote anti-melanoma immune response. This observation is based on our analysis of the TCGA set of 287 melanoma samples which identified a robust association of the CD8+ T cell marker CD8A with the expression of chemokine CCL5 . One of Besides CXCR3 and CCR5 ligands, additional chemokines are now emerging as key players in the regulation of anti-tumor immunity. For example, CXCL16 has been implicated in driving immune response against liver cancer by recruiting anti-tumor NKT cells. Sinusoidal endothelial cells were the major source of CXCL16 which was induced by gut microbiome-mediated primary-to-secondary bile acid conversion . Cremonein vivo analysis of anti-tumoral T cell chemotaxis using competitive homing assay showed that key tumor-derived chemotactic factors are CXCR3 ligands, while CCL5, which was also produced by melanoma tumors, is dispensable for direct homing of T cells into the tumor   . On the ome.org) . IFNγ reome.org) –128. Thiome.org) . This saome.org) . This CRome.org) .In addition to driving adaptive immune resistance, IFNγ also promotes chemokine expression which, in turn, can recruit additional immune cells into the tumor . Based oAccumulating evidence suggests that CXCR2 and CXCR4 are promising therapeutic targets in multiple malignancies. There are now over 2,400 publications describing a role for CXCR4 in cancer and over 300 publications describing a role for CXCR2 in cancer progression. These receptors are expressed on tumor cells, endothelial cells, leukocytes, including MDSCs. These studies provide significant evidence that CXCR2 and CXCR4 promote tumor growth through a variety of mechanisms , 132. FoCurrently, clinical trials are ongoing with both CXCR2 and CXCR4 antagonist –153.Chemokines control infiltration of diverse immune cells into the tumors. The immune cell infiltrate, in turn, is essential for mounting an effective anti-tumor immune response with immunotherapy. Thus, therapies that induce chemokine secretion in tumors and restore immune cell entry into non-inflamed tumors are likely to facilitate immunotherapy response. One of the previously explored approaches to induce infiltration of T cell into the tumors was to inject them directly with interferons. In a mouse model interferon injection into melanoma tumor-induced chemokine production and improved response to anti-PD-L1 therapy . One draWe have discovered that senescent-inducing drugs increase chemokine secretion by melanoma cells . SenesceAnother approach to re-activate chemokine expression in immunologically cold tumors is by targeting the epigenetic blocks that impede chemokine expression in tumor cells. For instance, treatment of ovarian cancer cells with epigenetic modifiers reversed the EZH2 and DNMT1 suppression of expression of the CXCR3 ligands, CXCL9, and CXCL10, resulting in T cell influx into the tumor and improved response to T cell transfer and anti-PD-L1 blockade therapy . InteresAlso, viral delivery of chemokines can be used to increase T cell homing into the tumor and promote immunotherapy response. For instance, intra-tumoral injection of vaccinia virus delivering CXCL11 promoted response to adoptive T cell therapy and vaccines . In addiIn the last 30 years, we have made extensive progress in identifying chemokines and chemokine receptors, characterizing their roles in the development of the immune system, in angiogenesis, wound healing, inflammation, tumorigenesis, and host defense. Extensive effort was put into developing antagonists of chemokine receptors and some of these were investigated in various clinical trials. CCR5 antagonists, like maraviroc, have been developed and used in AIDs patients with some success . CXCR2 aAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Sclerotinia sclerotiorum. Here, we identified a viral RNA sequence contig, representing a novel virus, Sclerotinia sclerotiorum deltaflexivirus 2 (SsDFV2), from an RNA_Seq database. We found that SsDFV2 was harbored in the hypovirulent strain, 228, which grew slowly on potato dextrose agar, produced a few sclerotia, and could not induce typical lesions on detached rapeseed (Brassica napus) leaves. Strain 228 was also infected by Botrytis porri RNA Virus 1 (BpRV1), a virus originally isolated from Botrytis porri. The genome of SsDFV2 comprised 6711 nucleotides, excluding the poly (A) tail, and contained a single large predicted open reading frame encoding a putative viral RNA replicase. Phylogenetic analysis demonstrated that SsDFV2 is closely related to viruses in the family Deltaflexiviridae; however, it also differs significantly from members of this family, suggesting that it may represent a new species. Further we determined that SsDFV2 could be efficiently transmitted to host vegetative incompatible individuals by dual culture. To our best knowledge, this is the first report that a (+) ssRNA mycovirus can overcome the transmission limitations of the vegetative incompatibility system, a phenomenon that may facilitate the potential use of mycoviruses for the control of crop fungal diseases.Various mycoviruses have been isolated from  Sclerotinia sclerotiorum (Lib.) de Bary, an ascomycetous fungus, is a devastating necrotrophic plant pathogen with wide host range that encompasses economically important crops, including vegetable crops, and is responsible for heavy global agricultural losses each year [S. sclerotiorum depends mainly on chemical fungicides, since no resistant cultivars are available. Therefore, it is important to explore novel, environmentally friendly alternatives to chemical fungicides or to reduce amounts of chemical fungicides used.ach year . The conCryphonectria parasitica, to control chestnut blight in European countries has become a textbook example of the biological control of plant fungal diseases [C. parasitica that can function as super donors for hypovirus transmission have been constructed. These engineered strains overcome the limitations of hypovirus transmission caused by host vegetative incompatibility and have clear potential for use in control of chestnut blight in forests [Mycoviruses replicate in fungal cells and are widespread in nature. Among plant fungal pathogens, some mycoviruses can confer hypovirulence to their hosts and, therefore, have potential as biological agents for disease control ,3,4. Mycdiseases ,8,9; how forests ,11, and  forests , suggestS. sclerotiorum have been identified, including viruses from the families, Hypoviridae, Alphaflexiviridae, Endornaviridae, Genomoviridae, Mymonaviridae, Narnaviridae, Reoviridae, Megabirnaviridae, Narnaviridae, and the new families such as Deltaflexiviridae and the proposed Fusariviridae [S. sclerotiorum strains isolated from Australia [S. sclerotiorum are varied and widespread, and may provide new insights into virus taxonomy and ecology [Numerous mycoviruses from iviridae ,19,20,21ustralia . Togethe ecology ,23,24.S. sclerotiorum from diseased rapeseed (Brassica napus) plants in China, and performed RNA-sequencing analysis of pooled samples. We found that one contig (contig 1178), with an incomplete ORF encoding a putative RNA-dependent RNA Polymerase (RdRp), had similarities with a previously reported Soybean leaf-associated mycoflexivirus 1 (SlaMFV1) in the RNA-sequencing database [S. sclerotiorum [S. sclerotiorum that is phylogenetically related to flexivirus. Further RT-PCR analysis indicated that strain 228 carries this novel flexivirus. In this study, we identified and characterized this novel virus and investigated its properties, including its impact on virulence and transmission among strains of S. sclerotiorum.To identify novel viruses with potential to control fungal diseases, we initially collected a total of 173 abnormal strains of database . Furtherrotiorum . The preS. sclerotiorum strain 228 was isolated from a sclerotium collected from diseased rapeseed in a field in Hubei province, PR China. Strains N36, Ep-1PNA367, and 1980, were fused as reference control strains in this study. Virus-infected strain 228 and virus-free strains N36, Ep-1PNA367, and 1980 were grown on laboratory-produced potato dextrose agar (PDA) at 20–22 °C, and stored on PDA slants at 4–6 °C. Sclerotia were dried at room temperature and maintained at −20 °C for long term conservation. In this study, strains Ep-1PNA367 and 1980 incorporating a hygromycin-resistance gene (Ep-1PNA367hph and 1980hph) were used to determine virus transmission between vegetative incompatible strains. Virus was introduced into strains Ep-1PNA367 and 1980 by dual culture; the resulting virus-containing strains were named Ep-1PNA367VI and 1980VI.The biological properties of strain 228, including colony morphology, growth rate, and virulence were compared to these of strain N36, as described previously by Xie et al., 2009 . The colDouble-stranded RNA (dsRNA) was extracted and purified from strain 228 cultured on cellophane membranes laid on PDA plates (9 cm diameter). Fresh mycelia were harvested by roiling the colony on cellophane membrane using a sterile blade 2–3 days post-inoculation and ground into a fine powder using liquid nitrogen and a mortar and pestle. dsRNA extraction in the presence of cellulose  was performed by chromatography as previously described in detail by Xie et al., 2015 . ExtractS. sclerotiorum was constructed and viral genome contigs identified by comparison with the NCBI viral reference amino acid sequence database using USEARCH [An RNA_Seq database of 173 strains of  USEARCH . Among vTo obtain a complete cDNA sequence of the purified dsRNA segment, the PC3-T7 loop adapter  was ligahttp://www.genome.jp/tools/motif/). Multiple sequence alignments were performed using CLUSTALW  [https://www.ncbi.nlm.nih.gov/tools/ vecscreen/). All phylogenetics trees were constructed using the neighbor-joining method using MEGA version 7.0 [A motif search was conducted using Transfac  . Vector sion 7.0 . PhylogeS. sclerotiorum, fresh mycelial agar discs from strains 228 and Ep-1PNA367hph, or 228 and 1980hph, were dual-cultured on PDA plates. After 7 days of dual culture, mycelial agar discs from the growth side of strain Ep-1PNA367hph were transferred to fresh hygromycin-containing PDA plates, and then to fresh PDA plates overlaid with cellophane for further incubation for 3 days before viral detection [hph were dual cultured on PDA for 7 days, and then 10 mycelial agar discs from the growth side of strain Ep-1PNA367hph were transferred to a fresh hygromycin-containing PDA plate  with a normal growth rate and virulence. Although strain 228 could form sclerotia given time, only a few sclerotia were produced from colonies at later stages a. StrainSclerotinia sclerotiorum deltaflexivirus 2 (SsDFV2).An RNA sample was extracted from strain 228 and subjected to RT-PCR amplification using primer pairs designed based on the sequence of contig 1178. RNA samples from strains N36 and Ep-1PNA367 were used as controls. As expected, a DNA fragment of approximately 750 bp was amplified from strain 228 cDNA, while no DNA fragment of the expected size was amplified from strain N36 and Ep-1PNA367 . SequencBotrytis porri RNA virus 1 (BpRV1). As BpRV1 has two dsRNA fragments, the previously reported sequence of BpRV1 dsRNA2 in the NCBI database was used to design another primer pair, which was used to amplify an RT-PCR product of approximately 500 bp, confirming its presence in S. sclerotiorum strain 228 , revealed that the ORF-encoded protein contained three putative conserved domains: viral methyltransferase (Mtr), Viral (Superfamily 1) RNA helicase (Hel), and RNA dependent RNA polymerase (RdRp)  . Thus, wDeltaflexiviridae. We aligned the conserved domains  of the putative RNA replicase of SsDFV2 with those of homologous domains from selected other viruses, using pairwise comparison to determine the percentage of amino acid sequence identity , 38% , and 35%  sequence identity to the viral methyltransferase domains of Fusarium graminearum deltaflexivirus 1 (FgDFV1), SlaMFV1, and SsDFV1, respectively , 42%  and 43%  identity to those of FgDFV1, SlaMFV1, and SsDFV1, respectively  domain homologous region within the putative RNA replicase of SsDFV2 showed significant sequence identity (approximately 13%–46%) with the Hel encoded by viruses from the order Tymovirales, with 46% , 50%  and 50%  identity with those of FgDFV1, SlaMFV1, and SsDFV1, respectively.The RdRp domain of SsDFV2 contains six typical viral RdRp motifs c and exhThe SsDFV2 replicase consists of 2114 amino acids, and is larger than SsDFV1 (2078 amino acids); there is an amino acid identity of 446/1185 (38%) and similarity of 625/1185 (52%) between these two viruses (NCBI Blastp). Compared with the majority of closely related viruses, including FgDFV1 and SlaMFV1, the amino acid identities were 441/1159 (38%) and 405/1134 (36%), with similarities of 602/1159 (51%) and 588/1134 (51%), respectively (NCBI Blastp). These data suggest that SsDFV2 differs significantly from previously reported viruses.Deltaflexiviridae.Phylogenetic trees based on multiple alignments of the entire replicase, methyl transferase, helicase, or RdRp domains of SsDFV2 with those of other selected viruses from the order Tymovirales were constructed . This phhph, on PDA plates. After the two colonies intermingled, ten mycelial agar discs were taken from the Ep-PNA367hph colony and transferred to hygromycin-containing PDA plates for further growth. Next, the newly developed Ep-PNA367hph colonies were screened for virus by RT-PCR amplification. SsDFV2 was successfully detected in all newly developed colonies, while BpRV1 was not be detected in any of the same colonies , the SsDFV2-infected strain, Ep-1PNA367VI, was dual cultured with strain 1980g colony , confirmBotrytis sp. Although strain 228 appeared hypovirulent, whether SsDFV2 alone could confer hypovirulence to its host was not known. Therefore, we separated the two viruses by dual culturing strain 228 with strain Ep-1PNA367hph, and then transmitted SsDFV2 from the recipient Ep-1PNA367hph strain to strain Ep-1PNA367. The SsDFV2-infected strain, Ep-1PNA367VI, showed a slower growth rate (2.4 cm/day) than the virus-free strain, Ep-1PNA367 (2.81 cm/day), along with delayed sclerotial development. Strain Ep-1PNA367VI also exhibited slightly lower pathogenicity than its virus-free counterpart, evidenced by the smaller lesions (4.5 cm) it produced on fresh rapeseed leaves, whereas the virus-free strain generated larger lesions (5.7 cm)  . Thus, SS. sclerotiorum using RNA_Seq analysis followed by RACE cloning. According to phylogenetic analysis, SsDFV2 is a member of the order Tymovirales. Recently, the order Tymovirales was divided into five families, Alphaflexiviridae, Betaflexiviridae, Deltaflexiviridae, Gammaflexiviridae, and Tymoviridae ; based on genomic organization, phylogenetic relationships, and virion morphology [Deltaflexiviridae.In the present study, we isolated and characterized a novel mycovirus, SsDFV2, from strain 228 of rphology . PhylogeDeltaflexiviridae. The SsDFV2 has only one gene, which encodes an RNA replicase, while SsDFV1 and SlaMFV1 each have four genes encoding a replicase and three small proteins, and FgDFV1 has five genes [Alphaflexiviridae, Deltaflexiviridae, and Gammaflexiviridae, are reported to infect fungi, Botrytis virus X, in the family Alphaflexiviridae, has flexuous rod-shaped particles and five genes [Gammaflexiviridae, also has flexuous rod-shaped particles, while its genome consists of two genes [Sclerotinia sclerotiorum debilitation-associated RNA virus (SsDRV), in the family Alphaflexiviridae, does not have any coat protein, and its genome comprises a single gene [Fusariviridae, the numbers of genes differ among the members, and all reported fusariviruses lack genes encoding coat proteins [Hypoviridae.The genome organization of SsDFV2 is significantly different from other reported viruses in the family ve genes ,23,24. Sve genes , and Botwo genes . Sclerotgle gene . Recentlgle gene ,36, suggproteins ,38,39,40Rosellinia necatrix is reported to be infected with multiple viruses [Ophiostoma novo-ulmi [S. sclerotiorum, including the hypovirulent strains 382, 16235, SZ-150, and Ep-1PN, and there is also one example of a virulent strain, Sunf-M, reported to be co-infected with two or three mycoviruses [S. sclerotiorum. A possible reason for the phenomenon of multi-infection of this species by mycoviruses is that S. sclerotiorum can form sclerotia, a fungal dormancy structure that can survive for long periods of time in disadvantageous conditions and directly germinate to infectious hyphae for plant invasion. A high frequency of multi-infection was also found in the sclerotial-forming fungi, Rhizoctonia solani and Botrytis cinerea [The co-infection of a single fungal strain by multiple mycoviruses is a common phenomenon in nature . Strain  viruses  and 12 dovo-ulmi ,44, whilovo-ulmi . There aoviruses ,46. In t cinerea ,49,50,51 cinerea ,53,54,55 cinerea ,57, whicGenomoviridae, which can directly infect host hyphae and has an insect transmission vector [The transmission of mycoviruses in host populations will be key for the use hypovirulence-associated mycovirus to facilitate control of crop fungal diseases. Mycoviruses may transmit vertically, via fungal spores, or horizontally, via hyphal anastomosis, with very rare exceptions, including the fungal DNA virus SsHADV-1, in the family n vector , and a pn vector . There an vector ; howevern vector . In this


Epithelial–mesenchymal transition (EMT) is a reversible cellular process, characterized by changes in gene expression and activation of proteins, favoring the trans-differentiation of the epithelial phenotype to a mesenchymal phenotype. This process increases cell migration and invasion of tumor cells, progression of the cell cycle, and resistance to apoptosis and chemotherapy, all of which support tumor progression. One of the signaling pathways involved in tumor progression is the MAPK pathway. Within this family, the ERK subfamily of proteins is known for its contributions to EMT. The ERK subfamily is divided into typical (ERK 1/2/5), and atypical (ERK 3/4/7/8) members. These kinases are overexpressed and hyperactive in various types of cancer. They regulate diverse cellular processes such as proliferation, migration, metastasis, resistance to chemotherapy, and EMT. In this context, in vitro and in vivo assays, as well as studies in human patients, have shown that ERK favors the expression, function, and subcellular relocalization of various proteins that regulate EMT, thus promoting tumor progression. In this review, we discuss the mechanistic roles of the ERK subfamily members in EMT and tumor progression in diverse biological systems. Epithelial–mesenchymal transition (EMT) is a process of trans-differentiation by which epithelial cells change to a mesenchymal phenotype . At the The extracellular-signal regulated kinase (ERK) participates in many physiological and pathological processes such as proliferation, cell migration, invasion, and metastasis ,5. Two dEMT is a process of trans-differentiation by which epithelial cells change to a mesenchymal phenotype . In highIn addition to its participation in normal physiological processes, EMT can be triggered by many oncogenic signaling pathways, hypoxia, and reactive oxygen species-mediated injury and signals of tumor microenvironment . This reDuring EMT, several signaling pathways are activated. Among these are the TGF-β, Wnt, Hedgehog (Hh), β-catenin, Notch, Nanog, and STAT3, as well as miRNAs and cytokines . During The ERK pathway comprises several proteins that belongs to the family of mitogen-activated protein kinases (MAPK). These regulate diverse physiological and cellular processes, such as gene expression, cell division, survival, apoptosis, differentiation, and motility ,16. In m16p11.2 and 22q11.21; they have 10 and 9 exons, and molecular weights of 44 and 42 kDa, respectively .,95.94,95Due to the variety of cellular processes and responses that the ERK pathway can trigger, it must be tightly regulated. The ERK1/2 regulatory systems involve cytoplasmic anchors, adapter proteins and phosphatases . The cytThe kinase suppressor of RAS (KSR) is another important adapter protein for ERK1/2-dependent signal transduction. KSR is a scaffolder that increases the signaling efficiency of the pathway by anchoring kinases such as B-RAF, RAF-1, MEK1/2, and ERK1/2, to the cell membrane ,106. TheDual-specificity MAPK phosphatases (MKPs/DUSPs) also regulate the ERK signaling pathway. MKP3/DUSP6 can dephosphorylate ERK1/2 in both Tyr/Thr residues to inactivate it . The nuc2O2, suggesting that ERK5 is a redox-sensitive kinase [ERK5 is activated in response to serum growth factors, such as EGF, NGF, and hyperosmolarity ,114,115,e kinase . ERK5 ace kinase ,118. It e kinase  transmite kinase . These ie kinase ,119,120,e kinase . Upon activation, ERK5 translocates to the nucleus and phosphorylates diverse substrates including some TFs, including the myocyte enhancer factor 2 (MEF2), Sap1a (ETS domain transcription factors), c-Myc, serum and glucocorticoid-activated kinase (SGK), connexin 43, and BAD ,114. AllThe mechanisms of activation and biological effects of the atypical members of ERK3/4 and ERK7/8 are poorly understood ,22. The 2O2 stress [ERK7/8 phosphorylation occurs upon stimuli like serum, estrogens , glucoco2 stress ,133. The2 stress . Evidenc2 stress ,125. On 2 stress . This ev2 stress . c-Fos and c-Myc. A common substrate for ERK7 and ERK7/8 is the myelin basic protein (MBP) [The regulation of ERK8 activity in mammalian cells is determined by the relative rates of ERK8 autophosphorylation at Ser126 and its dephosphorylation at Thr175 catalyzed by PP2A or PTP1B . Some ofin (MBP) . ERK8 isin (MBP) . Despitein (MBP) ,46,131. In the last few decades, several mutations have been identified in molecules involved in MAPKs signaling pathway. Overall, these mutations lead to the hyperactivation of ERK1/2, and are associated with tumor progression. Ras gene occur in approximately 15%–30% of cancers [H-Ras), 13 (K-Ras), and 61 (N-Ras) are oncogenic as well [The mutations in the small GTPase RAS were the first oncogenic mutations described in a murine model of leukemia more than 30 years ago ,135. Mut cancers ,137. Acc cancers . The mos cancers . RAS mut cancers ,139,140. as well ,142. Als as well .B-Raf gene, approximately 30 missense mutations in the kinase domain are oncogenic [It is estimated that in the Ser/Thr kinase ncogenic . Particuncogenic . Mutant ncogenic . Interesncogenic . On the ncogenic . A common mutation in B-RAF is the substitution Val600Glu, which occurs in at least 7% of tumors . This muA study of patients with multiple myeloma showed that 24% of the individuals have mutations in N-RAS, 25% in K-RAS, and 9% in B-RAF . In mostAdditional mutations in ERK1/2 have been reported in several types of cancer and cell lines. For instance, the in vitro models A375, 293T, SKMEL-19, and WM266.4 cell lines showed that the mutants ERK1 Tyr53His/ERK2 Tyr36His/Asn, ERK1 Gly54Ala/ERK2 Gly37Ser, ERK1 Pro75Lys/ERK2 Pro58Lys, ERK1 Tyr81Cys/ ERK2 Tyr64Asn, and ERK1 Cys82Tyr/ERK2 Cys65Tyr had resistance to ERK inhibitors, favoring tumor progression . On the Overall, the mutations described for the different proteins of the MAPK pathway ultimately lead to the phosphorylation of ERK. However, several studies in patients have elucidated the relationship between the phosphorylation of ERK and tumor progression. These findings are described in the following sections.During EMT, various proteins associated with diverse signaling pathways are post-translationally modified. The most relevant are MAPKs, tyrosine kinase, the cadherin–catenin complex, and cyclin-dependent kinase. These are the major players of the cell cycle, thus deregulation of their phosphorylation–dephosphorylation state can contribute to cancer progression . FurtherTo study the frequency of ERK activation in primary liver cancers, p-ERK was analyzed using a tissue microarray (TMA) containing 78 hepatocellular carcinoma specimens by immunochemical analysis. Levels of p-ERK in different tumor tissues were classified as high expression in 11 cases , low expression in 11 cases , or negative in 56 cases  and p-ERK was detected in both the cytoplasm and nucleus . PreviouIn this section, we highlight the experimental evidence obtained from different cancer cell lines, animal models, and biopsies of patients, which have established the relationship between ERK and EMT in different types of cancer .Zeb1 through Fra1 [A549 lung cancer cells and MLCC mesenchymal lung cancer cells stimulated with TGFβ showed increased levels of p-ERK1/2. This correlated with enhanced expression of β3 integrin, vimentin, fibronectin, Slug, MMP-9, and decreased levels of E-cadherin . In the ugh Fra1 . Overexpugh Fra1 . In the ugh Fra1 . In the ugh Fra1 . In lungugh Fra1 .Erk5 knockdown in A549 cells resulted in a decreased expression of Snail and ZEB1, both transcriptional repressors of CDH1 [In studies using lung tissue from BALB/c mice exposed to tobacco, a decrease of E-cadherin and ZO-1 and an increase of N-cadherin and vimentin were found to induce EMT. Tobacco also increases the activity of ERK1/2 while decreasing the activity of ERK5 . Althoug of CDH1 . An addi of CDH1 . TGF-β is well-known as a classical inducer of EMT in breast cancer. Studies using the highly invasive triple-negative cell line MDA-MB-231 showed that inhibition of TGF-β blocks the ERK/NF-kB/Snail signaling cascade and contributes to the recovery of the epithelial phenotype while decreasing cell migration . In the CDH1, a decrease of the mesenchymal marker SERPINE1 (the plasminogen activator inhibitor), and an associated decrease in cell migration and invasion [Studies of other members of the ERK family have shown that in MDA-MB-231 and MCF7 cell lines, the overexpression of ERK3 induces changes in the actin cytoskeleton, decreasing the stress fibers in the periphery and promoting cell movement . Interesinvasion . Furtherinvasion . Regarding colorectal cancer, HGF induces EMT and an increase in p-ERK and AKT in the CT26 murine colorectal cancer cell line, that in turn regulates the expression of Slug . The colA study performed on 160 biopsies of patients with colon cancer showed that 54% of the cases had increased levels of p-ERK and were strongly detected in cells located at the infiltrative tumor edge. Of the cases, 75% of the samples also had high expression of FRA1, which was also stronger in the infiltrative tumor cells and colocalized with p-ERK . Also, tTGF-β was shown to induce EMT in the cervical cancer cell lines SiHa and C33A. In these cases, both the decrease of E-cadherin and increase of vimentin and fibronectin were regulated by CK17 (cytokeratin 17); in these models, CK17 is regulated by ERK1/2 . On the ERK1/2 signaling is also involved in prostate cancer via different activation pathways. For instance, in vitro studies using the DU145 prostate cancer cells showed that the knockdown of ZEB1 decreases the phosphorylation of ERK1/2, without affecting the expression of total ERK1/2 . This inIn hepatocellular carcinoma (HCC) cells cultured in medium conditioned with cells isolated from hepatocellular carcinoma patients , an incrIn human osteosarcoma U2OS cells, TGF-β1 induces an increase in AKT and ERK1/2 phosphorylation. Both phosphorylation pathways decrease E-cadherin levels and increase the levels of vimentin, Snail, and Slug . AdditioIn melanoma cell lines generated from invasive and pre-metastatic tumors, the EMT-associated TF Twist is commonly overexpressed. Interestingly, Twist overexpression is enhanced in cell lines with B-RAF mutations . LikewisT24 bladder cancer cells treated with benzidine, a cancer inducer, present a significant increase in p-ERK1/2 and p-ERK5, while in EJ bladder cancer cells, only an increase in p-ERK5 was observed . In addiThe knockdown of caveolin 1 (Cav-1) in the mouse peritoneal mesothelial cell line MC induces a decrease in E-cadherin and ZO-1 and an increase in the levels of α-SMA, vimentin, Snail, and also in the activity of ERK1/2. In this model, ERK inhibition with CI-1040 reduces Snail expression, allowing the expression of E-cadherin, ZO-1, and Cav1. The morphology to an epithelial phenotype is also recovered . In gastric cancer, miR-592 induces EMT and promotes its progression via PI3K/AKT and MAPK/ERK signaling pathways by directly binding to 3′UTR of Spry2 and triggering its RNA degradation, thus modifying E-cadherin, vimentin, and N-cadherin expression, which are hallmarks of EMT . The EMT in glioma tissues is correlated with high expression of ring finger protein 138 (RNF138), which regulates caspase 3, E-cadherin, p-ERK1/2, vimentin, MPP-2, HIF-1α, and VEGF . In thisTo date, designing therapeutic targets for the RAS-RAF-MEK-ERK1/2 signaling cascade have been focused on RAF and MEK; some of these inhibitors are approved and largely used in clinical therapies ,28,29. H50 of 0.51 μM, and 0.33 μM, respectively). This small molecule also inhibits the activity of the MAPK p38α, but at a higher IC50 (10 μM). At the cellular level, FR180204 decreased the phosphorylation of ERK1/2 target proteins like the myelin basic protein [The small molecule FR180204 is amongst the first inhibitors shown to impair the kinase activity of ERK1/2 in vitro . Structu protein . 50 of 1 nM against ERK2 and 4 nM for ERK4. It also inhibits the activating phosphorylation of ERK by MEK (100 nM) and inhibits phosphorylation of the ERK downstream substrate RSK. SCH772984 is less effective against MEK1 (IC50 >10 µM) and at 1 µM, this small molecule partially inhibits CLK2, FLT4, VEGFR3, GSG2, MAP4K4, MINK1, PKCµ, and TTK. Further, prolonged inhibition of ERK with this compound inhibits C-RAF phosphorylation dependent on ERK at Ser289, Ser296, and Ser301 [ClinicalTrials.gov Identifier: NCT01358331). This study showed that MK-8353 was well tolerated by patients; however, it presented limited anti-tumor activity that did not correlate to the pharmacodynamic parameters observed in patients with B-RAF Val600 mutated melanoma [SCH772984 is based in a 3(S)-thiomethyl pyrrolidine structure and is a potent and highly selective inhibitor of ERK1/2. SCH772984 has an ICd Ser301 . The strd Ser301 . Since td Ser301 . Howeverd Ser301 , which id Ser301 . Mechanid Ser301 . Thus, bd Ser301 ,216. SCHmelanoma . A highly selective ATP-competitive inhibitor of ERK1/2 is VTX-11e. Cell-based assays showed that VTX-11e impairs the phosphorylation of the kinase’s substrate, p90RSK . Novel d50 less than 0.3 nM against ERK1/2 in vitro and in A375 cells the IC50 was 12 nM for p-ERK and 60 nM for p-90RSK, suggesting a dual mechanism of action similar to that observed for VTX-11e [50 of 6.9 nM against ERK2 activity and an IC50 of 53 nM in an A375 cell proliferation assay [Tetrahydro-pyrazolopyridine-based ERK2 inhibitors continue to be developed. This chemical structure is known to improve potency and lipophilic efficiency of the drugs, which is achieved by a lipophilic aryl-Y interaction with the enzyme. Upon cleavage of the lipophilic moiety, the ligand-bound binding cleft undergoes reconfiguration to establish a polar contact between the released N-H and a neighboring Asp of the kinase, thus impairing its function ,220. Fur VTX-11e . This noon assay . WhetherERK inhibitors that impair the kinase’s catalytic activity induce the accumulation of phosphorylated ERK, which releases it from cytoplasmic MEK1/2 to be translocated to the nucleus ,158,216.i values of 0.31 and 0.14 µM, respectively [Covalent ERK1/2 inhibitors have been designed to target signaling pathways in a sustained fashion. These molecules interact with their targets by initially establishing a reversible electrophilic interaction facing toward a nucleophilic residue to form a covalent bond with the drug, which may or may not be reversible ,225. Somectively ,230. TheAllosteric ERK1/2 inhibitors impede the interaction of the kinase with the protein’s interactors upstream and downstream of the signaling cascade, in a highly specific manner ,231. BioMutations on ERK2 helped to determine potential residues which may be relevant for the establishment of the kinase’s interactions and may be interesting targets for allosteric inhibitors . Among tCumulative research described here speaks to the wide range of potential mechanisms to impair ERK activity, the ultimate and fundamental effector of the RAS/RAF/MEK/ERK cascade. Due to its unique structural properties, mechanisms of action, feedback regulation, and numerous substrates, ERK1/2 is an ideal candidate for developing single or combined strategies to prevent tumor progression. However, the same appealing properties of ERK as a target make the development of drugs against this kinase challenging, as the potential for off-target effects from these novel compounds is also elevated. Thus, future strategies for development of drugs against ERK should utilize comprehensive, interdisciplinary methods to prevent deleterious reactions. However, taking into account the promising outcomes of ERK inhibitors in preclinical research, ERK1/2 is an optimal target to overcome acquired drug resistance in the RAS-RAF-MEK-ERK pathway.Kinases from the ERK family are able to promote EMT and enhance the cellular migration and invasion properties of tumor cells, as well as cell-cycle progression and resistance to apoptosis and drugs. ERK proteins are overexpressed and hyperactive in diverse cancer forms. Upon activation of the ERK pathway, several proteins that regulate EMT are activated, promoting tumor growth and metastasis. With ERK being the ultimate effector of the MAPK cascade, it is an ideal target to design novel therapies for patients that have developed drug resistance for other members of this signaling pathway.


Metabolomics is the identification and quantification of all or specified metabolites in a living system under a specific condition or disease. Metabolomics in cirrhosis can be used in diagnosing complications, determining prognosis and assessment of response to therapy. In this review, we summarized representative applications of metabolomics in cirrhosis and significant metabolites associated with cirrhosis and its complications. Metabolites are endogenous or exogenous small low-molecular weight downstream intermediate or end-products of genes and proteins in a living organism. The composition of all metabolites generated by a system in a living organism  constitute a metabolome ,2. MetabIdentification of metabolites in biological samples requires two major analytical consecutive steps including separation of metabolites using gas chromatography, high-performance liquid chromatography or capillary electrophoresis followed by identification and quantification of metabolites using mass spectrometry (MS) or nuclear magnetic resonance (NMR) spectroscopy ,2,3,4. BDetection and quantitation of the analytes using MS is dependent on separation of analytes prior to MS analysis ,2,3,4. TAfter chromatographic separation, the MS step starts with ionization of the analytes ,2,3,4,5.m/z) obtained on MS [For data analysis, the platform displaying the best characteristics is chosen. Raw data consist of integrated peak areas quantified by the area under the receiver operating characteristic curve, where the units are ion counts ,2,3,4. Med on MS ,2,3,4. Ped on MS ,2,3,4. Med on MS ,2,3,4.t-tests, analysis of variance (ANOVA), volcano plots (plots log of fold change in metabolite(s) value(s) versus negative log of p value) examine a single variable of interest (metabolite) [R2X, R2Y, and Q2Y that range between 0 and 1 [R2X and R2Y are percentages of the variance in the independent and dependent variables, respectively explained by the model [Q2Y is the estimated predictive accuracy of the model [R2X, R2Y, and Q2Y close to 1 [After the metabolomic data are pre-treated , univariate and multivariate analysis can be performed by different techniques. The primary goal of metabolomics data analysis is metabolite biomarker discovery for disease diagnosis and treatment. Univariate analysis techniques such as abolite) . In contabolite) . PCA is abolite) . PLS andabolite) ,6. Metri 0 and 1 ,6. R2X ahe model ,6. Q2Y ihe model ,6. High ose to 1 ,6.In addition to univariate and multivariate analysis techniques, pathway and network analysis is performed to identify alterations in metabolic pathways and gain a more profound insight in complex disease processes ,8.Several metabolomic studies were conducted in patients with cirrhosis to identify metabolomic signatures related to the cause and complications of cirrhosis including decompensation, acute-on-chronic liver failure, hepatorenal dysfunction, hepatic encephalopathy, hepatocellular carcinoma, prognosis and response to treatment . Herein,1H NMR-spectroscopy and performed PCA and OPLS-DA for data analysis. Their results showed that there was a distinct metabolomic signature that differentiated patients with cirrhosis from healthy controls  [Studies showed that a set of metabolites is capable of differentiating patients with cirrhosis from healthy controls. Qi et al.  conducte1H NMR-spectroscopy differentiated patients with cirrhosis with hepatitis B  and alcoholic cirrhosis  from healthy controls.In another study conducted among 41 patients with hepatitis B and alcoholic cirrhosis and 20 healthy controls, Qi et al.  reported1H NMR-spectroscopy reported significantly decreased acetoacetate, choline, isoleucine, LDL, leucine, unsaturated lipid, valine, and VLDL, and increased acetate, α-ketoglutarate, glycerol, glutamine, 1-methylhistidine, N-acetylglycoproteins, phenylalanine, pyruvate, taurine, and tyrosine, in 36 patients with cirrhosis compared to 63 healthy controls . Similarly, Gao et al.  using 1HUsing quantitative hepatic phosphorus-31 magnetic resonance spectroscopy, Corbin et al.  reportedTaken altogether, although small sample size was the major limitation of these studies, their findings suggest presence of altered lipid metabolism, tricarboxylic acid cycle, and energy metabolism in patients with cirrhosis compared to healthy individuals ,10,11,12R2Y = 0.784, Q2Y = 0.598, validation with an accuracy of 85%) [Qi et al.  showed t of 85%) .Using quantitative hepatic phosphorus-31 magnetic resonance spectrometry, Corbin et al.  reportedCollectively, results of these studies suggest alterations in the tricarboxylic acid cycle, dysfunction or deficiency of liver-specific phenylalanine hydroxylase that converts phenylalanine into tyrosine, altered amino acid metabolism and lipid synthesis in decompensated cirrhosis ,12,14.Rachakonda et al.  conducteAscha et al.  conducte1H NMR-spectroscopy, Qi et al. [R2Y = 0.873, Q2Y = 0.826). Increased isobutyrate and creatine and decreased glutamate, glutamine and acetoacetate levels were observed in patients with hepatitis B compared with those with alcoholic cirrhosis [Using i et al.  differenirrhosis .1H NMR spectroscopy showed that an OPLS model was capable of differentiating patients with acute on chronic liver failure from those with chronic liver failure  [A study conducted among 30 acute on chronic liver failure and 93 chronic liver failure (compensated or decompensated cirrhosis) patients using  = 0.73) . This mo = 0.73) . Authors = 0.73) .S-adenosylhomocysteine (SAH) and myo-inositol were the 10 most significantly increased ones when patients with high liver and kidney liver disease severity were compared with those with low liver and kidney disease severity [A unique metabolomic signature consisting of 34 metabolites that significantly increased in patients with high liver and kidney disease severity has recently been described in patients with cirrhosis . Among tseverity . This stseverity .1H NMR-spectroscopy analysis conducted by Jimenez et al. [n = 39) compared with patients with cirrhosis without minimal hepatic encephalopathy (n = 62) . A study conducted among 24 outpatients with cirrhosis and minimal hepatic encephalopathy by Saito et al. [l-carnitine .An z et al.  showed io et al.  identifiA study conducted by Weiss et al.  among 14Bajaj et al.  conducteIn another study, Bajaj et al.  conducteResults of a prospective study of 24 patients with hepatopulmonary syndrome and 27 controls showed increased plasma levels of several primary and secondary bile acids, bilirubin, biliverdin, endothelin, fatty acids, nitric oxide synthase signaling regulators, sphingosine metabolites, urobilinogen and decreased plasma levels of monoglycerol .1H NMR-spectroscopy, showed that there were increased hepatic alanine, choline, glutamate, glutamine, glycine, lactate, leucine, and phosphorylethanolamine and decreased hepatic glucose, glycogen, and triglyceride levels in hepatic tissues involved with HCC compared to tissues free of HCC. Similarly, they showed increased hepatic alanine, choline, glutamate, glutamine, glycine, lactate, leucine, and phosphorylethanolamine and decreased glucose, glycerophophocholine, glycogen, phosphocholine, and triglycerides in high-grade HCC compared to low-grade HCC [n-acetylglycoproteins, phenylalanine, pyruvate, and tyrosine in 39 patients with hepatocellular carcinoma compared to 63 healthy controls . Collectively, these findings suggest an increase in glycolysis, alterations in tricarboxylic acid cycle, and impaired lipid synthesis [Several studies reported diagnostic and prognostic metabolites associated with HCC. Yang et al. , using 1rade HCC . Gao et rade HCC , using Nynthesis ,27.Patterson et al. , using uLu et al. , using lGrammatikos et al.  reportedp = 0.00025; for validation cohort: Area under the receiver operating characteristics curve = 0.803) [Soga et al.  performe= 0.803) . Authors= 0.803) .S-adenosylhomocysteine, glucuronate, trans-aconitate, 3-ureidopropionate, 3-(4-hydroxyphenyl)lactate, 3-methoxytyramine sulfate, arabitol/xylitol, N-formylmethionine, phenyllactate and 7-methylguanine significantly predicted mortality in patients with cirrhosis [Metabolomic analysis has also been used to predict mortality in patients with cirrhosis. In a study that included 103 patients with cirrhosis, the multivariate Cox regression analysis controlled for measured glomerular filtration rate and demographics showed that metabolites including irrhosis .1H NMR spectroscopy and reversed-phase ultra-performance liquid chromatography coupled to time-of-flight MS in 80 patients with decompensated cirrhosis and validated them in an independent cohort of 101 patients with decompensated cirrhosis, 20 patients with stable cirrhosis and 47 healthy subjects. They reported that lysophophatidylcholines and phosphatidylcholines that play a key role in regulation of cell senescence, hepatic repair, immunomodulation and lipolysis were significantly decreased in non-survivors compared to survivors and were inversely correlated with cell death marker levels including M30 and M65 [McPhail et al.  identifi and M65 . A prosp and M65 . Similar and M65 . Collect and M65 ,36,37.In a study conducted among 48 patients with cirrhosis, tyrosine itself and in combination with MELD score predicted 3-month and 6-month survival without liver transplantation with a greater accuracy compared with the MELD score alone .Application of metabolomics in cirrhosis is broad and rapidly evolving. In this review, we presented several metabolomic studies conducted in patients with cirrhosis. Although these studies have identified multiple promising metabolites and/or metabolomic signatures to diagnose, differentiate and determine prognosis; only few of them interpreted data by performing pathway and network analyses to provide mechanistic and therapeutic insights in disease phenotypes. We recommend that following important steps should be considered when conducting metabolomics studies in patients with cirrhosis: (1) Clinical phenotypes related to etiology and complications of cirrhosis should be as distinctive as possible; (2) pathway and network analysis should be performed to identify the most altered pathways to discriminate among clinical phenotypes rather than simply providing a list of metabolites; (3) multi-omics, which is an integrative data analysis of metabolomics with proteomics, genomics, epigenomics, transcriptomics and microbiomics should be taken into consideration as it has recently gained ground to provide better mechanistic understanding of complex disease phenotypes in cirrhosis and its complications ,39,40,41


Luteolin improved chronic low-grade inflammation by modulating the TLR signaling pathway, resulting in reduced pro-inflammatory cytokines and macrophage accumulation. A positive relationship was detected between gene expressions of Tlr5, Map2k7, Mapk12, Mapk13, and Mapk9 and lipogenesis in epididymal white adipose tissue (eWAT) of luteolin-treated mice, which was linked to attenuation of hepatic lipotoxicity by increasing free fatty acid (FFA) flux to the WAT. Luteolin prevented fibrosis by decreasing extracellular matrix accumulation and cathepsin gene expressions, while enhancing the hepatic antioxidant system. Emr1 and Ccl7, important markers inducing low-grade inflammation, were affected by advanced age and greater body weight, which were normalized by luteolin treatment. Luteolin improved insulin resistance by normalizing pancreatic islet dysfunction and differentially modulating the plasma glucagon-like peptide-1 and gastric inhibitory polypeptide levels. Our results suggest that luteolin ameliorates diet-induced obesity and its comorbidities. Overall, this study provides novel insights into the effect of luteolin on the links among adiposopathy, insulin resistance, hepatic steatosis, and fibrosis.This study was to investigate the protective role of luteolin on inflammation-mediated metabolic diseases, focusing on the role of luteolin in the modulation of the Toll-like receptor (TLR) signaling pathway. C57BL/6J mice were fed a normal, high-fat, or high-fat + 0.005% ( Obesity is defined as excessive fat accumulation and can lead to metabolic complications, including dyslipidemia, adipokines dysregulation, insulin resistance (IR), inflammation, fibrosis, and steatohepatitis. White adipose tissue (WAT) is responsible for the storage of energy in the form of triacylglycerol, and thus protects other organs and tissues from ectopic fat accumulation resulting in lipotoxicity. WAT is also a critical endocrine organ capable of controlling the energy homeostasis and metabolism of several tissues through the secretion of several adipokines/cytokines such as leptin, adiponectin, and TNF-α that are known to mediate lipid metabolism, inflammation, and IR .Obesity promotes not only adipocyte hypertrophy but also WAT macrophage accumulation, which can contribute to inflammation; this then triggers the production and deposition of extracellular matrix (ECM), including collagen, to induce adipose tissue fibrosis, possibly as a result of the pro-inflammatory adipokines/cytokines released by adipocytes and infiltrated macrophages . In obesLuteolin  is a flavone found in leaves, vegetables, fruits, and natural herbal drugs such as parsley, thyme, peppermint, and celery. Some of the proven biological effects of LU are a variety of anti-inflammatory actions. However, little is known about the detailed mechanisms linked to the anti-metabolic syndrome action of luteolin based on the integration of transcriptional profile and phenotypic biomarkers. Accordingly, the aim of this study was to investigate the protective role of oral luteolin treatment on inflammation-mediated metabolic diseases in diet-induced obese C57BL/6J mice, focusing on its role in modulating the Toll-like receptor (TLR)-signaling-pathway-triggered up-regulation of pro-inflammatory cytokines. C57BL/6J is the most widely used inbred strain and is susceptible to diet-induced obesity, type 2 diabetes, and atherosclerosis. These results should provide novel insights into the effect of luteolin on the links among adiposopathy, IR, hepatic steatosis, and fibrosis.n = 10), high-fat diet , and HFD with 0.005% (w/w) luteolin   for 16 weeks. The HFD is the AIN-76 semipurified diet containing 40 kcal% fat by calories: 15% (w/w) corn oil and 85% (w/w) lard of total fat. All experimental diets were prepared every week and stored in a dark room at −4 °C. Body weight and blood glucose levels were measured every 1 and 2 weeks, respectively. At the end of the experimental period, all mice were anesthetized with ether after a 12 h fast. Blood was taken from the inferior vena cava for the determination of glucose, plasma lipid, and hormone concentrations. The liver and adipose tissues were removed, rinsed with physiological saline, weighed, immediately frozen in liquid nitrogen, and stored at −70 °C until analysis. The study was performed under protocols approved by the Ethics Committee at Kyungpook National University .Four-week-old male C57BL/6J mice were purchased from the Jackson Laboratory . The 36 mice were individually housed at room temperature (24 °C) and under a 12 h light/dark cycle, and were fed the AIN-76 semipurified diet for a one-week acclimation period after arrival. After acclimation, the mice were randomly divided into three groups consisting of normal diet . For the IPGTT analysis performed in the 15th week after the start of the diet experiments, mice fasted for 12 h were injected intraperitoneally with glucose (0.5 g/kg body weight), and the blood glucose level was determined in the tail vein blood at 0, 30, 60, and 120 min later. Plasma biomarkers were measured using fluorescent magnetic bead-based assays : insulin, glucagon, leptin, resistin, plasminogen activator inhibitor-1 (PAI-1), glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) (Bio-Plex Pro™ mouse diabetes assays), IL-6, TNF-α, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein 1 beta (MIP-1β) (Bio-Plex Pro™ mouse cytokine assays), and adiponectin (Bio-Plex Pro™ mouse diabetes adiponectin assay). The plasma adipsin , soluble CD antigen 163 , glutamic oxaloacetic transaminase (GOT) , and glutamic pyruvic transaminase (GPT)  levels were analyzed using a commercially available kit.The plasma and hepatic FFA levels were both measured using an enzymatic assay kit , except that the hepatic FFA level was determined after extraction according to the methods of Folch .v/v) paraformaldehyde/PBS and embedded in paraffin for immunohistochemistry staining for CD68 and MCP-1 and Masson’s trichrome staining. The pancreas tissue sample was fixed in 4% (v/v) paraformaldehyde/PBS and embedded in paraffin for immunohistochemistry staining of insulin and glucagon. Stained slices were viewed under a microscope set at 200× magnification.The liver, epididymal adipose, and pancreas tissues were eliminated from each mouse. The liver and epididymal adipose tissue samples were subsequently fixed in 10% , and three pooled RNA sample sets were then constructed for the ND, HFD, and LU groups as previously described . RNA wasCt−ΔΔ method [Total RNA (1 μg) was used to synthesize cDNA via the QuantiTect Reverse Transcription kit , and mRNA expression was quantified by RT-qPCR using the SYBR green PCR kit  and the CFX96TM real-time system . Gene-specific mouse primers were used as indicated in t method .Total RNA was amplified and purified using the Ambion Illumina RNA amplification kit . Biotinylated cRNA (750 ng per sample) was hybridized to Illumina MouseWG-6 v2 Expression BeadChips  according to the manufacturer’s instructions. Hybridized arrays were washed and stained with Amersham Fluorolink streptavidin-Cy3  following the standard protocol provided in the bead array manual. Hybridization quality and overall chip performance were determined by visual inspection of both the internal quality controls and the raw scanned data in the Illumina BeadStudio software. Probe signal intensities were quantile-normalized and log-transformed. Microarray analysis was performed in the ArrayAssists software  and with the R programing language.www.genome.jp/kegg) was consulted for the analysis of gene functions involved in inflammation. This microarray data was deposited in the Gene Expression Omnibus (GEO) database (GEO accession numbers: GSE111412).The statistical differential gene expression analysis between groups was performed using the nonparametric Rank Prod approach. Oligonucleotides showing changes between groups with a false discovery rate lower than 0.05 were considered significant. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways mapper  and were analyzed using SPSS . Statistical differences between groups  were determined using Student’s Luteolin treatment significantly reduced the body weight gain and weights of WAT, including depots of epididymal, perirenal, retroperitoneal, mesenteric, subcutaneous, and interscapular WAT, compared with the HFD group. Luteolin also slightly decreased the liver weight, but there was no significant difference between the HFD and LU groups . Plasma Cd68, Ccl2, Emr1, Csf14, and Saa3) in adipocytes was down-regulated by luteolin treatment , interleukins , and cytokines  was significantly decreased, while the expression levels of anti-inflammatory genes such as Cxcl9 and Il15ra were elevated in comparison to levels detected in the HFD group (Irf5 and Irf8) was also significantly altered by luteolin treatment. Specifically, Tlr1, Tlr2, Tlr4, Tlr6, Tlr7, Tlr8, Tlr13, Irf5, and Irf8 were significantly down-regulated, while Tlr5 was markedly up-regulated by luteolin , Mapk12 and 13(p38), Mapk9, Mapk8ip1 (Jnk), and Cxcl9 (Mig) expression was consistently down-regulated by the HFD at most time points except at the early stage (between weeks 2 and 4 of DIO development)  . These rCcl7, Emr1, Evi2a, Gusb, H2-DMb2, Lcp1, LOC100041137, Ly9, Ptpre, Rgs1, sc1000528.1_16, Sirpa, Sfrp5, and Acacb) were associated with both advanced age and diet-induced obesity. In line with our previous study, among these genes, Ccl7, Emr1, Evi2a, Gusb, H2-DMb2, Lcp1, LOC100041137, Ly9, Rgs1, and Sirpa expression levels were positively correlated with body weight, while the Acacb expression level was negatively correlated with body weight in the present study  in adipocytes; this, in turn, induced a significant decrease in hepatic and adipocyte macrophage infiltration and accumulation as evidenced by immunohistochemical staining of CD68 and MCP-1.Excessive adipose tissue is linked with increased angiogenesis, macrophage infiltration, production of ECM components, and the production and release of several inflammatory mediators, which cause a state of chronic low-grade inflammation and promote obesity-linked metabolic disorders such as IR ,20. In tTlr4, Cd14, and Irf5, all of which are used as markers of visceral adipose tissue inflammation and linked with obesity and/or IR [Emr1 and Ccl7 are associated with advanced age as well as obesity-related inflammation. Adipocyte Emr1 and Ccl7 mRNA expression levels increased with advancing age in the normal diet group and were further up-regulated when given the HFD; however, luteolin decreased the Emr1 and Ccl7 mRNA expression levels in proportion to body weight compared with the levels in the HFD group. Thus, the Emr1 and Ccl7 genes could be targets for ameliorating the deleterious effects of the obesity-induced inflammation response since these genes are not only very sensitive to body weight but also important factors inducing chronic low-grade inflammation. It is plausible that luteolin prevented adipose tissue expansion, the concomitant activation of TLR-mediated inflammatory signaling cascades, and induction of CD antigens. Luteolin consequently decreased the expression levels of pro-inflammatory cytokines, chemokines, and interleukins, thereby ameliorating the chronic low-grade inflammation in DIO mice. Interestingly, luteolin treatment led to an increase in the expression of Tlr5 (TLR5), Map2k7 (MKK7), Mapk12 and Mapk13 (p38), and Mapk9 (JNK) genes, although it decreased the overall expression of genes involved in the TLR signaling pathway, suggested as a major pathway in response to luteolin treatment; these gene expression patterns were reversed by the HFD. Previous studies have indicated a positive role for p38, MKK, and JNK in inducing adipogenesis [−/− mice exhibit hyperphagia and develop hallmark features of metabolic syndrome, including dyslipidemia and IR. Therefore, our present observation suggests that p38, MKK, and JNK could be associated with lipogenesis rather than inflammation in the TLR signaling pathway, and that Tlr5 may be a key transcription factor for luteolin-mediated lipotoxicity protection.Oligonucleotide microarray profiling also revealed that luteolin treatment down-regulated the expression of adipocyte genes involved in inflammation compared with the effects of the HFD. Luteolin markedly decreased the mRNA expression levels of pro-inflammatory chemokines, interleukins, and cytokines along with their upstream signaling pathway genes such as TLRs, CDs, and IRFs, including nd/or IR ,25. Furtogenesis ,27,28. Oogenesis . Moreoveogenesis  reported2O2 and TBARS levels, which reflects improved hepatic lipotoxicity along with reduced plasma GOT and GPT levels. The inflammatory response is complex and often involves reactive oxygen species (ROS) such as H2O2 [The anti-inflammatory effects coupled with the decrease in adiposity conferred by luteolin were strongly linked to the improvement of fibrosis in the adipose tissue as evidenced by the results of Masson’s trichrome staining. Fibrosis, attributed to the excessive deposition of ECM proteins, including collagen, is a ubiquitous tissue response to chronic inflammation ,31. Furt as H2O2 . ROS-ind as H2O2 . Thus, aTlr5 expression. Moreover, luteolin significantly increased the plasma GLP-1 level but decreased the plasma GIP level compared with the HFD, leading to amelioration of IR. The two incretin hormones GLP-1 and GIP have dissimilar metabolic effects, with GLP-1 being more favorable for glucose and lipid homeostasis, as GLP-1 inhibits glucagon, increases insulin sensitivity and action, and has an anti-inflammatory effect in the liver against steatosis [The improved chronic low-grade inflammation by luteolin through modulation of the TLR signaling pathway was likely associated with a normalization of pancreatic beta-cell mass and insulin levels, leading to the prevention of IR. IR is always associated with inflammation with regard to adiposopathy. Adipokine/cytokine dysregulation caused by adiposopathy impairs insulin secretion and action in pancreatic beta-cells and induces global IR . Moreoveteatosis . In contteatosis ,39.Tlr5 is a possible key transcription factor for preventing lipotoxicity, although this observation requires additional experiments for verification. In summary, the data obtained from our animal study indicate that luteolin can suppress diet-induced obesity and modulate obesity-associated metabolic disorders. Luteolin decreased adipokine/cytokine dysregulation, macrophage infiltration, and ECM accumulation in the adipose tissue through modulating the TLR signaling pathway, and reduced excessive ROS production via an increase in hepatic antioxidant capacity. Luteolin improved and normalized pancreatic islet dysfunction by differentially regulating plasma GLP-1 and GIP, which contributes to improvement of lipotoxicity, fibrosis, and IR. The noticeable improvement in lipotoxicity by luteolin is linked with increased adipocyte lipogenesis mediated via p38, MKK, and JNK;