Lactobacillus inoculants , for 3 months. Untreated and L. plantarum-treated silages were sampled at several times during ensiling, while L. buchneri-treated silage was sampled only at 3 months. Bacterial composition was studied using next generation sequencing approach. Dominant bacteria, before ensiling, were Pantoea (34.7%), Weissella (28.4%) and Pseudomonas (10.4%), Exiguobacterium (7.8%), and Paenibacillus (3.4%). Exogenous inoculants significantly affected bacterial composition and dynamics during ensiling. At 3 months of ensiling, Lactobacillus dominated the silage bacterial population and reached an abundance of 59.5, 92.5, and 98.2% in untreated, L. plantarum- and L. buchneri-treated silages, respectively. The bacterial diversity of the mature silage was lower in both treated silages compared to untreated silage. Functional profiling of the bacterial communities associated with the wheat ensiling demonstrated that the abundant pathways of membrane transporters, carbohydrate and amino acids metabolisms followed different pattern of relative abundance in untreated and L. plantarum-treated silages. Only three pathways, namely base-excision repair, pyruvate metabolism and transcription machinery, were significantly different between untreated and L. buchneri-treated silages upon maturation. Lactic acid content was higher in L. plantarum-treated silage compared to untreated and L. buchneri-treated silage. Still, the pH of both treated silages was lower in the two Lactobacillus-treated silages compared to untreated silage. Aerobic stability test demonstrated that L. plantarum-, but not L. buchneri-supplement, facilitated silage deterioration. The lower aerobic stability of the L. plantarum-treated silage may be attributed to lower content of acetic acid and other volatile fatty acids which inhibit aerobic yeasts and molds. Indeed, high yeast count was recorded, following exposure to air, only in L. plantarum-treated silage, supporting this notion. Analysis of bacterial community of crop silage can be used for optimization of the ensiling process and the selection of appropriate inoculants for improving aerobic stability.Knowledge regarding bacterial dynamics during crop ensiling is important for understanding of the fermentation process and may facilitate the production of nutritious and stable silage. The objective of this study was to analyze the bacterial dynamics associated with whole crop wheat silage with and without inoculants. Whole crop wheat was ensiled in laboratory silos, with and without  Lactobacillus buchneri  convert water-soluble carbohydrates into organic acids, mainly lactic acid, resulting in forage acidification. Accumulation of LAB during the fermentation results in rapid pH decline that enables forage preservation for long time. Basically, ensiling fermentation process comprises four main stages: At initial stage, air is still present between the crop particles and the pH value is above 6.0. At this stage a variety of microorganisms are active. Secondly at the stage of fermentation, air is depleted through the activity of aerobic microorganisms and LAB become the dominant flora. At this stage the pH decreases to a value depending on forage composition and ensiling conditions. After successful fermentation, silage remains stable, as long as no air penetrates into it. At this stage sometimes, lactic acid is converted gradually to acetic acid by hetero-lactic bacteria, such as buchneri . The lasbuchneri . Anaerobbuchneri . LAB speiococcus .Ensiling has become a common global practice for feed preservation, especially in wet climates where the conservation of dried forage is problematic . The majL. plantarum and L. buchneri, two common silage additives, and the bacterial population of wheat silage, is also missing.Ensiling is a natural preservation process, which involves complex microbial interactions. A significant effort is required for detailed characterization of the ecology of the microorganisms involved in the ensiling process and associated spoilage . Most ofL. plantarum MTD1  (Lp-Treated). The inoculant was applied at a final concentration of ca. 106 CFU/g crop by suspending an adequate amount of the bacteria (based on its concentration in the inoculant product) in 25 ml distilled water and spraying the suspension on 10 kg chopped wheat spread on 2 × 1 m plastic sheeting, followed by thorough mixing. Untreated freshly chopped wheat crops were sampled in triplicates, before ensiling (time 0). Triplicate jars of the untreated and Lp-treated silages were sampled, at 6 h after ensiling, and on days 1, 2, 7, 15, 30, and 90. A similar triplicates of ensiling jars was inoculated with L. buchneri  (Lb-treated), at a final concentration of ca. 106 CFU/g. Lb-treated- jars were sampled only after 90 days of ensiling in order to compare the effects of L. plantarum and L. buchneri inoculants on the quality and bacterial community of mature silage. Following sampling on day 90, the silages were subjected to an aerobic stability test for five additional days, as described before . Silage treatments included natural microbiome without additives (Untreated) and addition of d before . Visual Dry matter (DM) was determined in triplicate by oven drying for 48 h at 60°C. A mixture 40 g of silages in 360 ml of distilled water was stomached for 3 min , the filtered aqueous extract was used for estimation of lactic acid content, ethanol and acetic acid. Lactic acid content was determined by the spectrophotometric method of g for 30 min at 4°C. The supernatant was discarded and pellet was used for microbial DNA isolation using PowerSoil DNA Isolation Kit , following manufacturer’s protocol. The quality and quantity of the DNA were assessed at A260/280 nm by NanoDrop 2000 spectrophotometer . The purified DNA was stored at −20°C until used.Untreated and inoculant-treated samples, collected at the different time points, were stored at −20°C before further analysis. Isolation of bacterial DNA and purification were performed as described previously . Silage Bacterial identification was performed using amplicon sequencing of 16S rRNA gene. PCR amplification was carried out to amplify the V4–V5 conserved regions of bacterial 16S rRNA gene sequences using primers 515F and 926R . LibrarySequence processing was performed using Mothur software, version 1.39.1 . SequencStatistical analysis of silage parameters, relative abundances and diversity indices was performed by Analysis of Variance (ANOVA) and pairs Tukey HSD test using JMP Pro 13.0.0 software . Two dimensional non-metric multidimensional scaling (NMDS) plots were generated based on the weighted unifrac distance matrix calculated between different samples using the OTU table with OTU clustering at the species level . Differe1 to generate the predicted KEGGs (Kyoto encyclopedia of genes and genomes) pathways Orthology (KO) classification  was used to predict the functional profiling based on the 16S rRNA gene sequences of the bacterial communities associated with the wheat ensiling process . For thification . The finfication  to test Lactobacillus counts were significantly higher in Lb-treated silages and molds were not detected in any of the treatments. Yeast counts were significantly higher in untreated than the Lp-treated silages, and were below the detection level in Lb-treated silages  in case of untreated silages (P < 0.05) than both untreated and Lp-treated silages  at 6 h of ensiling . The ric silages . In Lb-t silages .P < 0.05) than that of Lp-treated silage during the entire ensiling from day 2 to day 90  at time 0 and lowest at day 1 (1 ± 0.02). The diversity then increased to (1.6 ± 0.04) at day 2, decreased up to day 15 and sharply increased up to day 30 of ensiling. The diversity of Lp-treated silage was lower than the untreated silage at 6 h, and continued to decrease up to day 7 (0.2 ± 0.04). The diversity then increased up to day 15 (0.7 ± 0.08) when it decreased until day 30 (0.4 ± 0.04). A gradual increase in the diversity (from 0.4 ± 0.04 to 0.9 ± 0.02) was observed until day 90. The diversity of untreated silage is significantly higher  during various ensiling times . A remarProteobacteria were the dominant population in pre-ensiled wheat (time 0), with an abundance of 53.6%, and their abundance decreased during ensiling down to 0.12, 2, and 0.7% in untreated, Lp- and Lb-treated silages, respectively. The Firmicutes dominated the mature silage population with an abundance of 99.8, 97.9, and 99.2% in untreated and Lp- and Lb-treated silages, respectively , Weissella (28.4%), Pseudomonas (10.4%), Exiguobacterium (7.4%), and Paenibacillus (3.4%). After 6 h of ensiling, the abundance of Pantoea increased to 46.3%, but then decreased continuously during silage maturation and at the terminal stage of 90 days they represented only 0.02% of the overall population. The relative abundance of Weissella kept on fluctuating during the course of ensiling. Pseudomonas decreased continuously during ensiling and was not found in mature silage. Enterococcus increased significantly at day 7 where it represented 33.9% of relative abundance and then decreased to reach 1.8% in mature silage. Lactobacillus was initially presented at very low abundance (0.53%), which decreased after 6 h of ensiling to 0.09%, but then increased continuously up to 59.5% relative abundance in mature silage  after 6 h of ensiling, then it decreased during ensiling up to 0.16% at the terminal stage. Weissella abundance was increased at 6 h (30.5%) and on day 1 (44.7%), then declined and in the mature silage it was represented by 4.4% of the bacterial populations. Similar to untreated samples, Pseudomonas decreased during ensiling and was not found at 90 days of ensiling. The relative abundance of Enterococcus was maximum at day 1 (2.3%), and then decreased to reach 0.28% at 90 days. Lactobacillus species were in low abundance (4.8%), at 6 h of incubation, and rapidly increased to 34.8% at day 1, and at day 2, they already encompassed more than 95% of the population. Their relative abundance was maximum at day 7 (96.8%), and dropped to 92.4% at 90 days  , Pediococcus (5.8%), Clostridium_sensu_stricto_12 (1.6%), Enterococcus (1.1%). In contrast, the abundance of Lactobacillus in treated silages was much higher. In Lp-treated silage, Lactobacillus species represented 92.4% of the entire bacterial population followed by Weissella (4.4%) and Enterococcus (0.28%). In Lb-treated silages, Lactobacillus were the most dominant genus covering 98.2% of the population followed by Clostridium_sensu_stricto_12 (0.7%) and Enterococcus (0.1%) (There was a significant difference in the relative abundance of  < 0.05) . Althougs (0.1%) . Only 21s (0.1%) .Lactobacillus remained the most dominant population in all silages (Lactobacillus significantly increased from 59.5 to 85% (P < 0.05), while in Lb-treated silage, its abundance remained constant (98%) after air exposure. A significant change was observed in Lp-treated silages, where Lactobacillus dropped from 92.4% in mature silage to 49.8% after air-exposure, whereas the genus Clostridium_sensu_stricto_1 increased from 0.03 to 45.3%. Other prevalent genera that were detected, in air-exposed untreated silage were Weissella (12.7%), Enterococcus (0.5%) and 12 other genera with relative abundance below 0.05%. In total, 16 and 17 genera were identified in untreated and Lp-treated silages, respectively, while 30 genera were detected in Lb-treated silages . The most abundant predicted pathways, in pre-ensiled wheat, were  transporters (7.7%), ABC transporters (4.3%), DNA repair and recombination proteins (2.5%), two-component systems (2.4%), purine metabolism (2.1%), secretion systems (2.1%), transcription factors (2%), other ion-coupled transporters (1.7%), bacterial motility proteins (1.6%) and ribosome (1.6%). Variations in the abundance of the pathways were observed during ensiling and after aerobic exposure in both untreated and treated silages , and werThe abundant pathways of membrane transporters  and metabolisms (carbohydrate and amino acids) followed different pattern of relative abundance in untreated and Lp-treated silages –8. CompaWheat is a major crop used for silage in Israel and China . While rL. plantarum inoculant overcame this effect and resulted in significantly faster and improved ensiling. The considerable fermentative activity of the inoculated LAB was also observed in wheat ensiling with high (884 g/kg) DM content , whereas Proteobacteria occupied only 16.2% of the population  was also observed in three out of four good-quality wheat silages taken from commercial bunker silos, whereas less than 10% of Lactobacillus was present in the fourth sample  found in Lp-treated silage as compared to the untreated silages (54 genera) after 3 months of ensiling . Future Lactobacillus population under these conditions. Notably, the most abundant predicted pathway in both silages was transporters, the 2nd most abundant pathway/function was “General function prediction only, Unclassified,” which mainly consists of “poorly characterized” pathways. This finding underlines the importance of yet unknown pathways/functions in the ensiling process.In order to predict the potential metabolic pathways involved in ensiling of wheat crop, metagenomics functional prediction analysis was performed. The relative abundance of pathways was evaluated using KEGG classification (levels 1–3). Overall, the relative abundance of pathways (at level 3) varied in untreated compared to Lp-treated silages. Low variation of metabolic pathways is observed on days 30 and 90 in the untreated silage, as well as during all time points in the Lp-treated silage. These findings are likely related to the high dominance of distinct 2 level. During these stages, metabolic byproducts released by some of the populations may be taken by others, using transporters, in order to obtain essential nutrients for energy and growth. As the pH level decreases up to d15, the silage population becomes more homogeneous and dominated by LAB. Consequently, less transporters are required. In contrast, in the case of Lp-treated silage, the pH level decreases sharply, as early as d2, and Lactobacillus dominated the population, so less transport function was required.The putative abundance of transporters was much higher in untreated silage until d15, compared to Lp-treated silage. To the best of our knowledge, such high abundance of transporters during ensiling was not reported before. These findings may infer a commensal relationship among the microbial populations in the untreated silage. Indeed, the initial ensiling stages are characterized by the presence of heterogeneous aerobic and facultative anaerobic populations, which consume the oxygen trapped in the chopped forage and increase COLactobacillus population. In contrast, carbohydrate metabolisms increased in untreated silages until d15 to a much higher levels, and then reduced at d30 and remained constant until d90, at a level comparable to that of Lactobacillus treated silages. This is possibly due to the high initial activity of the heterogeneous microbial population at the early ensiling stages. Once oxygen was consumed, and pH level reduced, LAB became the dominant population and anaerobic fermentation took place at d30 through d90.A similar phenomenon was observed regarding carbohydrate metabolism. In the Lp-treated silage, a constant level of carbohydrate metabolisms observed throughout the ensiling stages, which apparently reflects the metabolism of the dominant de novo. In contrast, as LAB do not synthesize all their essential amino acid, they rely on proteolytic systems to provide essential amino acids for their growth  and molds , which degrade silages subsequently to severe extent  silage (Aerobic exposure of mature wheat silage for 5 days (aerobic stability test), resulted in a rapid deterioration of Lp-treated silage compared to untreated and Lb-treated silage. A possible explanation for the lower aerobic stability of Lp- versus Lb-treated silage is that nd molds . Indeed,) silage  and othe) silage .L. buchneri is a heterofermentative species, which produce a mix of products, and is able to ferment lactic acid to acetic acid , unlike the laboratory silos’ model, and were dominated by Bacillaceae, Thermoactinomycetaeae grp I and Enterobacteriaceae , a significantly higher content of lactic acid was evident in Lp-treated compared to untreated silage. This may set the stage for the emergence of ubstrate . Indeed,ioration . While, eriaceae . These dPredicted pathways that might be associated with aerobic stability were analyzed by PICRUSt. Pathway analysis using KEGG classification at level 2, did not yield significant differences between untreated and Lp-treated silage, consequently, KEGG pathway analysis at level 3 was employed. A total of seven pathways were significantly different between untreated and Lp-treated silages after aerobic exposure. In untreated silages, pathways for ethylbenzene degradation, D-Alanine metabolisms, terpenoid backbone synthesis and ion channel systems were more abundant, while in Lp-treated silages two component systems, novobiocin biosynthesis were higher. It is possible that production of antibiotics restricted the multiplication of some bacterial communities, which are competing for substrates with other bacterial and fungal communities. Since, the PICRUSt data are based on bacterial 16S sequences, further metagenomics studies should examine if antibiotics biosynthesis pathways are indeed expressed during ensiling in order to support this notion. Future studies based on metagenomics, covering both bacterial and fungal communities will be required for a more comprehensive understanding of the various metabolic networks and interactions present in the silage microbiome.L. plantarum, L. casei, and L. buchneri were added at a ratio of 2:1:1 .Finally, it should be noted the data obtained from functional metagenome prediction must be analyzed cautiously, since the prediction relies on genomic data and 16SrRNA amplification, rather than on direct mRNA measurements . TherefoPantoea, Weissella, Pseudomonas, Exiguobacterium, and Paenibacillus. During wheat ensiling with and without LAB-inoculant, the bacterial community shifted dramatically and the terminal silage was dominated mainly by Lactobacillus. There was a substantial difference in the abundance of Lactobacillus, between untreated and LAB-supplemented silages. Exogenous L. plantarum significantly affect the bacterial composition and dynamics during ensiling and resulted in a higher dominance of LAB in the mature silage and a higher content of lactic acid. Notably, addition of L. plantarum, but not L. buchneri reduced aerobic stability of the wheat silage. The lower aerobic stability of the L. plantarum-treated silage may be attributed to the different fermentation pathways in the two LAB inoculants. PICRUSt analysis provided knowledge regarding the role of transporters and carbohydrate metabolism in the initial stages of ensiling until stabilization of the bacterial populations. Since, ensiling is an uncontrolled fermentation process, depending on multiple parameters, the ability to monitor the bacterial dynamics, through the process, holds promise for a better understanding and improvement of the silage quality.The bacterial community of fresh whole crop wheat was found to be dominated by SSS conceived and coordinated the study and edited the manuscript. JK, ZW, and SSS designed the experiments. JK, YC, RP, and ZW ensiled the wheat crop and collected the samples. JK carried out the molecular biology and bioinformatics analyses. YC and YK carried out the microbiological and biochemical analyses. JK and ZW drafted the manuscript with input from all co-authors. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human T cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a subset of infected subjects. Two viral proteins, Tax-1 and HTLV-1 basic leucine zipper factor (HBZ), play important roles in the pathogenesis of both diseases. We recently demonstrated that HBZ, previously considered a nuclear protein, is exclusively localized in the cytoplasm of peripheral blood mononuclear cells (PBMCs) of HAM/TSP patients. Here, the analysis of a larger panel of HAM/TSP cases confirmed that HBZ is a cytoplasmic protein, while Tax-1 preferentially localized in the cytoplasm with fewer speckle-like dots in the nucleus. More importantly, here we report for the first time that HBZ, when expressed in asymptomatic carriers (AC), is also confined in the cytoplasm. Similarly, Tax-1 was preferentially expressed in the cytoplasm in a significant proportion of AC. Interestingly, in both HAM/TSP and AC patients, the expression of HBZ and Tax-1 was rarely found in the same cell. We observed only few cases coexpressing the two oncoprotein in a very limited number of cells. In representative AC and HAM/TSP patients, cells expressing cytoplasmic HBZ were almost exclusively found in the CD4+ T cell compartment and very rarely in CD8+ T cells. Interestingly, at least in the cases analyzed, the expression of thymocite-expressed molecule involved in selection (THEMIS) is dispensable for the cytoplasmic localization of HBZ in both AC and HAM/TSP. The study of an HTLV-1-immortalized cell line established from an HAM/TSP patient confirmed HBZ as a resident cytoplasmic protein not shuttling between the cytoplasm and nucleus. These results extend our previous observation on the dichotomy of HBZ localization between HAM/TSP and ATL, pointing to the exclusive either cytoplasmic or nuclear localization in the two diseased states, respectively. Moreover, they show a rather selective expression in distinct cells of either HBZ or Tax-1. The unprecedented observation that HBZ is expressed only in the cytoplasm in AC strongly suggests a progressive modification of HBZ localization during the disease states associated to HTLV-1 infection. Future studies will clarify whether the distinct HBZ intracellular localization is a marker or a causative event of disease evolution. HTLV-1 encodes two regulatory genes, tax and rex, and three accessory genes  in the plus strand of the provirus (HTLV-1 bZIP factor (HBZ), is transcribed from the minus strand of the proviral genome  is the first discovered human oncogenic retrovirus  and is tprovirus . Anotherl genome . It has l genome . Tax-1 pl genome . Howeverl genome . Moreovel genome . Therefoin vitro . There ain vitro . The SP1in vitro . The seqin vitro , the splin vitro . All thein vitro . HBZ disin vitro  and two in vitro , which lpatients . It was patients . The amopatients . Subsequpatients ,b, in HApatients . More repatients , dependipatients . Interespatients . Consistpatients . The disWe obtained PBMCs from HTLV-1 asymptomatic donors and HAM/TSP patients as part of the NIH natural history protocol # 98-N-0047. All individuals gave written informed consent. The patients’ data were analyzed anonymously.HTLV-1-immortalized CB T cells  primers and probes and Bio-Rad 2× Supermix and then emulsified with droplet generator oil using a QX-100 droplet generator according to the manufacturer’s instructions (Bio-Rad). The following primers and probe were used to amplify and detect a 154-base-pair region of HTLV-1 tax: ddPCR HTLV-1 tax F: 5′-CTTATTTGGACATTTACCGATG-3′; ddPCR HTLV-1 tax R: 5′-TGAGGCCGTGTGAGAGTAGA-3′; ddPCR HTLV-1 tax probe: 6FAM-TGATTTCCGGGCCCTGC-MGBNFQ. The droplets were then transferred to a 96-well reaction plate (Eppendorf) and heat-sealed with pierceable sealing foil sheets (Thermo Fisher Scientific). The duplex PCR amplification was performed in this sealed 96-well plate using a GeneAmp 9700 thermocycler (Applied Biosystems). Following PCR amplification, the 96-well plate was transferred to a QX100 droplet reader (Bio-Rad). For PVL calculation, QuantaSoft software version 1.3.2.0 (Bio-Rad) was used to quantify the copies per microliter of each queried target per well. All samples were tested in duplicate, unless otherwise specified, and PVL is reported as the average of the two measurements. The PVL was calculated using the following formula: PVL = [quantity of HTLV-1 tax/(quantity of RPP30/2)] × 100%.HTLV-1 PVL was measured using ddPCR (Bio-Rad) as previously described . DNA was2. Cells were then washed and processed for confocal microscopy analysis by using the following antibodies: anti-HBZ 4D4-F3 mAb, anti-Tax-1 mAb (clone 168 A51-2 from the NIH AIDS Research and Reference Reagent Program), and anti-RelA rabbit polyclonal antibody , followed by the secondary antibodies as specified in the figure legends.CB-CD4/HTLV1 cells cultured on glass coverslips precoated with poly-L-lysine were incubated with 20 nM leptomycin B  or the vehicle methanol for 3 h at 37°C, 5% CO2), three times, fixed in methanol 7 min at −20°C, and blocked with 1% bovine serum albumin (BSA) in 1× PHEM for 1 h at room temperature (RT). Cells were then stained overnight with anti-HBZ 4D4-F3 mAb, anti-Tax-1 mAb (clone 168 A51-2 from the NIH AIDS Research and Reference Reagent Program), anti-vimentin rabbit polyclonal antibody , rabbit anti-CD4 mAb , and rabbit anti-THEMIS mAb , diluted in PHEM buffer containing 0.5% BSA. The slides were then washed five times with cold 1× PHEM and incubated in the dark for 2 h at RT with the following secondary antibodies from Life Technology : goat anti-mouse IgG1 coupled to Alexa Fluor 546 to detect HBZ, goat anti-mouse IgG2a conjugated to Alexa Fluor 488 to detect Tax-1, and goat anti-rabbit IgG conjugated to Alexa Fluor 488 or to Alexa Fluor 546 to detect vimentin, CD4, or THEMIS. For co-staining with directly labeled antibodies, after extensive washing with 1× PHEM, anti-CD8 rabbit mAb directly conjugated to Alexa Fluor 647  and mouse anti-human CD25 mAb directly conjugated to Alexa Fluor 488  were added after the indirect immunofluorescence for 2 h at RT. Similarly, after indirect immunofluorescence, the nuclei were stained by incubating the cells with DRAQ5 Fluorescent Probe  for 30 min at RT. CIB-CD4/HTLV1 cells were cultured on glass coverslips precoated with poly-L-lysine for 5 h and processed for confocal microscopy analysis as described above.Peripheral blood mononuclear cells from AC, HAM/TSP patients, or normal control individuals, after rapid thawing, were washed with warm RPMI medium and immediately processed for immunofluorescence and flow cytometry analysis or for confocal microscopy, as described . For floAfter washing, the slides were mounted on coverslips with the Fluor Save reagent  and examined by a confocal laser scanning microscope . Images were acquired and analyzed by LAS AF Lite Image  and/or Fiji (ImageJ) software.6 CD-CD4/HTLV-1 cells, using an NE-PER Nuclear Cytoplasmic Extraction Reagent kit (Thermo Fisher Scientific) according to the manufacturer’s instruction. Briefly, cells were washed twice with cold phosphate buffer saline (PBS) and centrifuged at 500g for 3 min. The cell pellet was suspended in 200 μl of cytoplasmic extraction reagent I (CERI), supplemented with 0.1% protease inhibitor cocktail (Sigma), by vortexing. The suspension was incubated on ice for 10 min followed by the addition of 11 μl of a second cytoplasmic extraction reagent II (CERII), vortexed for 5 s, incubated on ice for 1 min, and centrifuged for 5 min at 16,000g. The supernatant fraction (cytoplasmic extract) was transferred to a pre-chilled tube. The insoluble pellet fraction, which contains crude nuclei, was resuspended in 100 μl of nuclear extraction reagent (NER), supplemented with 0.1% protease inhibitor cocktail (Sigma), by vortexing for 15 s, incubated on ice for 40 min, and then centrifuged for 10 min at 16,000g. Both the nuclear and cytoplasmic protein extracts were used for the subsequent immunoprecipitation experiments.Endogenous HBZ protein was precipitated from both the nuclear and cytoplasmic protein fractions prepared from 8 × 10After preclearing with protein A/G-Sepharose beads, the nuclear and cytoplasmic extracts were incubated with anti-HBZ 4D4-F3 mAb for 1 h on ice and then immunoprecipitated with protein A/G-Sepharose beads overnight at 4°C, under rotation. The precipitated proteins were resolved on 9% sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting with the anti-HBZ 4D4-F3 mAb, followed by Mouse True Blot ULTRA-anti mouse Ig HRP (e Bioscience). This specific secondary antibody was used to avoid the detection of both heavy and light chains in the precipitated proteins. Ten percent of the nuclear and cytoplasmic protein extracts were analyzed by Western blotting with anti Nup98  and anti-β-tubulin (Sigma) mAbs to assess the purity of nuclear and cytoplasmic protein extracts, respectively.By analyzing the subcellular distribution of endogenous HBZ in PBMCs of four HAM/TSP patients, we previously demonstrated for the first time that HBZ is a cytoplasmic protein expressed in a discrete percentage, up to 11%, of the cells. Unlike HAM/TSP, we were unable to detect HBZ-positive cells in PBMCs of four AC ,b. To beInterestingly, expression of both HBZ and Tax-1 was found in PBMCs of only four ACs, but in two ACs coexpression of HBZ and Tax-1 within the same cell was detected in a small proportion of cells  and only 2 and 0% of HBZ and Tax-1 positive cells, respectively. Of interest is the observation that this AC with an uncommonly high PVL was the spouse of a HAM/TSP patient and is clinically being followed for any signs and symptoms of neurological disease.In line with our previous findings ,b, additAlthough CD4+/CD25+ Tregs can be infected by HTLV-1 and HAM/TSP patients have been shown to have a high number of CD4+/CD25+ Tregs with impaired function and higher HBZ mRNA levels , the resFurther studies by confocal and biochemical analysis conducted on CB-CD4/HTLV-1, a T cell line established from an HAM/TSP patient , confirmIn conclusion, we believe that the data presented in this research add new relevant information for a better definition of the pathological states associated with HTLV-1 infection, particularly in relation to the distinct subcellular expression of HBZ in the different pathological contests and related pathologies. Whether HBZ cytoplasmic and nuclear localization in the natural history of HTLV-1 infection represents a marker of infection or is part of the mechanism governing the evolution toward HAM/TSP or ATL is the challenge for future investigation.GF, MB, and RA conceived the work. GF, MB, and AT performed the experiments. All authors wrote, revised, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: We found previously that ursolic acid (UA) administration could alleviate cigarette smoke-induced emphysema in rats partly through the unfolded protein response (UPR) PERK-CHOP and Nrf2 pathways, thus alleviating endoplasmic reticulum stress (ERS)-associated oxidative stress and cell apoptosis. We hypothesized that other UPR pathways may play similar roles in cigarette smoke extract (CSE)-induced emphysema. So, we sought to investigate the dynamic changes and effects of UPR and the downstream apoptotic pathways. Further, we investigated whether UA could alleviate CSE-induced emphysema and airway remodelling in rats, whether and when it exerts its effects through UPR pathways as well as Smads pathways.Methods: One hundred eight Sprague Dawley (SD) rats were randomly divided into three groups: Sham group, CSE group, and UA group, and each group was further divided into three subgroups, administered CSE (vehicle) for 2, 3, or 4 weeks; each subgroup had 12 rats. We examined pathological changes, analyzed the three UPR signaling pathways and subsequent ERS, intrinsic and extrinsic apoptotic pathway indicators, as well as activation of Smad2,3 molecules in rat lungs.Results: Exposure to CSE for 3 or 4 weeks could apparently induce emphysema and airway remodeling in rats, including gross and microscopic changes, alteration of mean alveolar number (MAN), mean linear intercept (MLI), and mean airway thickness in lung tissue sections. UA intervention could significantly alleviate CSE-induced emphysema and airway remodeling in rats. UA exerted its effects through ameliorating apoptosis by down regulating UPR signalling pathways and subsequent apoptosis pathways, as well as, downregulating p-Smad2 and p-Smad3 molecules.Conclusions: UA attenuated CSE-induced emphysema and airway remodeling, exerting its effects partly through regulation of three UPR pathways, amelioration downstream apoptotic pathways, and alleviating activation of Smad2 and Smad3. Chronic obstructive pulmonary disease (COPD) is a severe respiratory disease characterized by chronic respiratory syndrome and airway limitation, the pulmonary functional decline is progressive and ultimately results in respiratory failure. The two main pathologic manifestations of COPD—emphysema and airway-vessel remodeling—determine the phenotype of the disease .Nearly 90% of COPD cases are caused by cigarette smoke . CigaretIn recent years, unfolded protein response (UPR) mediated ERS associated cell apoptosis is attracting more and more researchers’ attention on its effect in development of COPD . Except Our previous work confirmed that PERK pathway mediating ERS associated cell apoptosis participated in CS induced emphysema in rats . IRE1 paIn addition, CS-induced oxidant stress and cell apoptosis could promote TGF-β1 secretion, one of the main inducers of cell proliferation and airway-vessel remodeling in COPD . Among iUrsolic acid (UA) is a pentacyclic triterpenoid compound widely distributed in natural plants, such as apple peels, herbal medicines, and many other edible plants . UA has Antibodies against eIF-2a and CHOP were obtained from Santa Cruz Biotechnology . Antibodies against p-eIF2α, Bcl-2, Bax, Caspase12, and p-IRE1 were purchased from Abcam Technology . Antibodies against PERK, p-PERK, Cleaved-Caspase3, Caspase8, and Caspase9 were purchased from Cell Signaling Technology . Antibodies against ATF6 were purchased from Wanlei Biology Technology . Cigarettes were acquired from Marlboro, USA. UA was bought from Wanxianghengyuan Biotechnology , and dissolved in phosphate-buffered saline (PBS) with 1% Tween80 before use. The Bicinchoninic acid (BCA) protein assay kit was acquired from Pierce , and the super enhanced chemiluminescence kit was purchased from Applygen Technologies Inc. .Briefly, rats were randomized into one of five treatment groups (N = 10 each): Sham, CS, UA10, UA20, and UA40. UA rats were administrated 10, 20, or 40 mg/kg body weight UA via gavage 30 min before the first CS exposure every day. Sham and CS rats were given vehicle instead. CS and UA rats were exposed to smoke of 16 filters removed 1R3F cigarettes for 30 min a time, two times a day, 6 days a week, for 3 months. Rats in the Sham group were exposed to normal air.CSE was prepared as previously reported . BrieflyOne hundred eight 6-week-old male SD rats were maintained with a standard laboratory diet and free access to water. After a 2-week adaptation period, the rats were randomly assigned into three intervention groups (n = 36 each): control group (Sham), CSE group (CSE), and UA 20 mg/kg group (UA). Rats in each group were further divided into three subgroups: 2-week group (2W), 3-week group (3W), and 4-week group (4W) (n = 12 each). Rats in the CSE and UA groups were intraperitoneally injected with 1 ml CSE-PBS solution on days 1, 8, 15, and 21. Rats in the Sham group were intraperitoneally injected with 1 ml PBS. Rats in the UA group were administered 20 mg/kg UA intragastrically daily in a volume of 3 ml. Rats in the Sham and CSE groups were administrated an equal volume of vehicle. The body weights of rats were measured.2O, and fixed in 10% formalin for 48 h. The paraffin-embedded sections of the left lung were used for histopathological examination. The right lung was frozen in liquid nitrogen for 5 min before storing in −80°C freezer for further Western blot analysis.The rats’ chests were opened and blood was drawn from the inferior vena cava. The left lung was inflated with 0.5% low-melting agarose at a constant pressure of 25 cm HTo assess the morphological changes that characterize emphysema and airway remodeling in the experimental rat lungs, the mean linear intercept (MLI), mean alveolar number (MAN), and bronchial wall thickness were measured in lung tissue sections by hematoxylin and eosin (HE) staining. Lung tissue sections were examined by light microscopy at 50× and 100× magnification. Inter-alveolar wall distance was measured by MLI, defined as the total length of the test line divided by the number of intersected alveolar walls . AlveolaSelected samples were embedded in paraffin, sectioned (4-μm thickness), paraffinized, and rehydrated. Then antigen retrieval was performed using a microwave in 10 mM citrate buffer (pH 6). Endogenous peroxidase was blocked with 3% peroxide for 5 min and then incubated with 10% unimmunized serum. Sections were incubated with primary 8-OHdG (1:500), Cleaved-Caspase3 (1:500), p-Smad2 (1:500), TGF-β1 (1:500), and α-SMA  antibodies overnight at 4°C, then washed and incubated with the secondary antibody  for 1 h at room temperature. Staining was developed by incubating with Diaminobenzidine (DAB) and DAB enhancer. Protein expression levels were measured using Image Pro Plus software. One field in a different quadrant of each section was assessed.g for 30 min at 4°C to remove cellular debris. The protein concentration was determined using BCA protein assay kit. Equal amounts of proteins (20–60 μg) were separated on 8–10% SDS-PAGE gels and transferred to PVDF membranes . The membranes were blocked in 5% non-fat milk at room temperature for 2 h, and then incubated with diluted primary antibodies overnight at 4°C. Blots were stripped and re-probed with anti-β-actin antibody to demonstrate equal loading. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibody, the chemiluminescent signal was detected using the Super Enhanced Chemiluminescence Kit. Band density was quantified using Quantity One software .Whole cell lysates were prepared from the rat lung tissues. Briefly, lung tissues were resuspended in an ice-cold lysis buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS, 1 mM protease inhibitor cocktail and protein phosphatase inhibitor  and then homogenized for 15 s at 4°C four to five times. Cell lysates were centrifuged at 12,000 × Data were represented as the Mean ± SD. SPSS13.0 software and Graph Pad Prism 5.0 were used for statistical analysis. One-way ANOVA was used to compare differences among groups. P-value of <0.05 was considered to indicate statistical significance.We used Western blot method to assess the activation of IRE1 and ATF6 pathways in CS induced emphysema rat lung. Our results demonstrated that CS exposure for 3 months up-regulated protein expression of phosphorylated IRE1(p-IRE1), as well as XBP1 molecule that functions downstream. UA administration alleviated the activation of IRE1 pathway. However, the ATF6 pathway seem to not be activated. Figure 1Emaciation is an important complication and an indicator for severity and poor prognosis of COPD. Rats that received peritoneal injection of CSE for 3 or 4 weeks weighed less than those in the corresponding Sham groups. UA alleviated the degree of weight loss-induced in rats exposed to CSE for 3 or 4 weeks Figure 2Tissue morphological analyses were performed to determine the protective effect of UA on CSE-induced emphysema in rats. We found that peritoneal injection of CSE for 3 weeks induced emphysema. Alveolar structure appeared disorganized, alveolar walls were thinned or ruptured, fused into bullae, and pulmonary fibrosis was observed. More severe emphysema was observed in rats administered CSE for 4 weeks. UA improved CSE-induced emphysema of rats , UA reduced expression of activated ATF6 in rat lungs in the CSE2W group. Injection of CSE for 2, 3, and 4 weeks all could increase expression of Cleaved-Caspase12. UA alleviated the activation of Caspase12 in rat lungs.The intrinsic and extrinsic apoptotic pathways indicators were activated by CSE injection, but timing and duration differed between groups, while UA administration alleviated the upregulation Figure 5Expression of Bcl-2 was down-regulated in CSE2W and CSE3W groups, and UA treatment ameliorated CSE-induced Bcl-2 down-regulation.Bax expression was up-regulated in all CSE groups, and UA treatment down-regulated CSE-induced Bax expression.Caspase9 was activated by CSE in CSE2W and CSE3W groups, Cleaved-Caspase9 was more strongly induced in CSE3W than CSE2W animals. UA could attenuate the up-regulation of Cleaved-Caspase9.Caspase8 was activated by CSE in CSE3W and CSE4W groups, UA attenuate the up-regulation of Cleaved-Caspase8.Caspase3 was activated by CSE in CSE2W, CSE3W and CSE4W groups, Cleaved-Caspase3 was more strongly induced in CSE4W than CSE2W animals. UA attenuate the up-regulation of Cleaved-Caspase 3 expression.Peritoneal injections of CSE for 3 or 4 weeks induced apparent airway remodeling-associated changes. Airway walls were thickened, inflammatory cells infiltrated into the airway wall, and collagen deposition was increased around the airway. UA administration alleviated CSE induced changes above  is an organ responsible for protein synthesis and maturation. The accumulation of unfolded proteins in the ER lumen activate a series of UPR signaling cascades in response to cellular stress such as oxidant stress, hypoxia, and high-temperature exposure . These rpoptosis . This prpoptosis . Of the poptosis . HoweverTGF-β1 is a pro-fibrotic factor that regulates expression of extracellular matrix proteins such as procollagen I and III, fibronectin, versican, and tenascin . VariatiIn the current study, we found that injections of CSE for 3 or 4 weeks could induce significant emphysema, and pathologic changes were more severe in CSE4W group. In that case, we call pathological changes in CSE2W, CSE3W, CSE4W rat pre-emphysema, middle-emphysema, severe-emphysema, respectively. ERS specific apoptotic indicator Cleaved-Capase12 was activated in all CSE groups, and thus ERS-associated apoptosis may exert its action in all stages of CSE-induced emphysema. Intrinsic apoptotic index Cleaved-Caspase9 was upregulated in CSE2W and CSE3W groups, while, extrinsic apoptotic index Cleaved-Caspase8 was upregulated in CSE3W and CSE4W groups. Communal downstream indicators of apoptosis Cleaved-Caspase3 expression was upregulated in CSE2W, CSE3W, and CSE4W group. CSE-induced expression of three transmembrane molecules  associated with ERS. However, the phase and duration of these actions differed. PERK-CHOP pathway molecules were up-regulated in CSE2W, CSE3W, and CSE4W groups, which may indicate that the PERK pathway exerts its effect in all stages of CSE-induced emphysema through ERS-associated apoptosis, and the out-of-sync of the peak may be related to the activation order of the pathway and the presence of feedback effect . IRE1 wavia RPAP2 to abort failed ER-stress adaptation and trigger apoptosis, which may explain our result that IRE1 pathway was only activated in early and middle stage of emphysema, and may participant in intrinsic apoptotic pathways, and at the severe or called end stage of emphysema, receptor mediate extrinsic apoptosis pathway activation may be due to the PERK and RPAP2 pathways.Recently, researchers reported PERK shuts IRE1 down in the terminal stage of UPR, and that it exerts this effect through a phosphatase known as RPAP2 (RNA polymerase II-associated protein 2). RPAP2 reverses IRE1 phosphorylation, oligomerization, and RNase activation. This inhibits IRE1-mediated adaptive events, including activation of the cytoprotective transcription factor XBP1s, and ER-associated degradation of unfolded proteins. Furthermore, RIDD termination by RPAP2 unleashes DR5-mediated caspase activation and drives extrinsic cell death . In shorUA intervention could alleviate the upregution of these three ERS transmembrane molecules and inhibit ERS-associated apoptosis in rat lungs. UA intervention alleviated CSE-induced activation of intrinsic and extrinsic apoptosis pathways also, through all these apoptotic pathways. UA alleviated CSE-induced lung cell apoptosis, and subsequently alleviated CSE-induced emphysema of rats.CSE intervention also could upregulate p-Smad2 and p-Smad3 expression in the lungs of rats, implicating these proteins participated in CSE induced airway remodeling in the rat lungs. UA alleviated airway-vessel remodeling, partly through downregulating p-Smad2 and p-Smad3 expressions.in vitro after CSE exposure.However, the failure to upregulation of ATF6 pathway remain to be investigated. Soon afterwards, we will discuss the interaction and the regulatory mechanisms of the three UPR pathways UA could alleviate CSE-induced emphysema and airway remodeling, a process involving all three UPR pathways. The process also involved suppression of ERS-associated apoptosis, attenuation of intrinsic and extrinsic apoptosis pathways, and down-regulation of p-Smad2/3.This study was approved by the ethics committee of the first affiliated hospital of Chinese Medical University.QW participated in the design of the study and modification of the manuscript. JK conceived of the study and modified the manuscript. LL carried out the studies, performed the statistical analysis, and drafted the manuscript. GH and DH participated in its design and coordination, and performed the statistical analysis. All authors read and approved the final manuscript.This work is supported by grant from the National Key Technology Research and Development Program of the 13th National 5-year Development Plan (2016YFC1304103) and Liaoning Provincial Innovation Team Project (LT2013015).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.


The same genes and signaling pathways control the formation of limbs in vertebrates, arthropods and cuttlefish. Related research article Tarazona OA, Lopez DH, Slota LA, Cohn MJ. 2019. Evolution of limb development in cephalopod mollusks. eLife8:e43828. doi: 10.7554/eLife.43828Drosophila melanogaster has identified a number of genes and signaling pathways that guide the development of the legs along the three axes of the body: anterior-posterior (head-to-tail), dorsal-ventral (top-to-bottom), and proximal-distal, which runs from the body to the tips of the limbs (reviewed in A human leg appears to have little in common with an insect leg, apart from the fact that both are used for walking, so most zoologists think that the limbs of vertebrates and arthropods evolved independently and are not, therefore, homologous structures. Ask a developmental geneticist, however, and the answer will not be so clear. Research in the fly In flies, two proteins, Extradenticle and Homothorax, are co-expressed in the part of the leg closest to the body and they establish an initial proximal-distal axis in the developing limb. The distal part is further refined by ‘leg-gap genes’, which are triggered by molecular signals active in this area, such as the Wnt, Hedgehog and Bmp signaling pathways. Two of these pathways, Wnt and Hedgehog, are also involved in setting up the anterior-posterior axis of the leg; another two, Wnt and Bmp, collaborate to form the dorsal-ventral orientation.Subsequent research revealed that an almost identical set of genes and signaling pathways control the development of limbs in chicken and mouse (reviewed in The truth may actually lie somewhere in between. According to the ‘co-option hypothesis’, a developmental program evolved in the common ancestor of the bilaterians – a group that includes most animals except for primitive forms like sponges – to shape an appendage that later disappeared during evolution. However, the program itself survived in arthropods, vertebrates and possibly other bilaterians, where it would have been independently repurposed to build limbs . As suchSepia officinalis and Sepia bandensis (In 2005, Pueyo and Couso proposed a way to test the co-option hypothesis: "if conservation of similar features are found in the tentacles of a cephalopod [...], then conservation of an ancestral appendage developmental program cannot be ignored" . Now, inandensis .Extradenticle and Homothorax, and a distal part that expresses homologs of the leg-gap genes as well as components of the Wnt, Hedgehog, and Bmp signaling pathways. In addition, the expression patterns of the genes closely resemble those in arthropod and vertebrate limbs.Just like arthropod and vertebrate limbs, cuttlefish appendages have a proximal part that co-expresses These results alone are intriguing, but Tarazona et al. took the analysis one step further, examining how Bmp and Hedgehog signaling helped to form cephalopod limbs. Tiny beads soaked with a chemical that inhibits Bmp signaling were implanted on the dorsal side of the arms and tentacles, which led to suckers abnormally appearing in this area. These results show that, as in arthropods and vertebrates, Bmp signaling is required for proper dorsal-ventral development in cephalopods.The team also tested the role of Hedgehog signaling. Tissue from donor embryos that expressed Hedgehog was transplanted into developing cuttlefish, which generated a second anterior-posterior axis in the arms and tentacles. On the other hand, repressing the pathway with the drug cyclopamine dramatically reduced the axis in the limbs. These stunning results leave no doubt that the segmental legs of arthropods, the limbs of vertebrates, and the arms or tentacles of cephalopods use very similar developmental genetic mechanisms. While this does not indicate that these limbs are homologous, the findings strongly support the co-option hypothesis.To rephrase the conclusions by Tarazona et al., the ancestral appendage program is probably not a ‘Sleeping Beauty’ which lies dormant in the genome of limbless creatures, waiting to be repurposed once in a while. Instead, it was always patterning some kind of appendage, implying that limbless animals evolved from animals with limbs. Extinct members of the major branches of bilaterians all appear to have some sort of frontal extensions . Did the


Various stimuli induce phosphorylation of ERK1/2in vivo and in vitro. Phosphorylated ERK1/2 moves to the nucleus, activates many transcription factors, regulates gene expression, and controls various physiological processes, finally inducing repair processes or cell death. With the aging of the population around the world, the occurrence of ischemia-reperfusion injury (IRI), especially in the brain, heart, kidney, and other important organs, is becoming increasingly serious. Abnormal activation of the ERK1/2 signaling pathway is closely related to the development and the metabolic mechanisms of IRI. However, the effects of this signaling pathway and the underlying mechanism differ between various models of IRI. This review summarizes the ERK1/2 signaling pathway and the molecular mechanism underlying its role in models of IRI in the brain, heart, liver, kidneys, and other organs. This information will help to deepen the understanding of ERK1/2 signals and deepen the exploration of IRI treatment based on the ERK1/2 study.Extracellular signal-regulated kinase 1/2 (ERK ERK1/2 plays a key role in signal transduction from surface receptors to the nucleus. Activated ERK1/2 phosphorylates substrates in the cytoplasm or nucleus, and thereby induces expression or activation of specific proteins, leading to regulation of cell proliferation, differentiation, apoptosis, and other processes.Extracellular signal-regulated kinase 1/2   . It is treat IRI . HoweverERK gene is located on chromosome 1p34~35, has a total length of 3,118 bp, and harbors an open reading frame encoding 978 amino acids. In the early 1990s, 1/2 because it is activated by various extracellular signals and has a relative molecular weight of 44/42 kDa. The ERK family is divided into five subgroups called ERK1–ERK5. Among these, ERK1 and ERK2 are the most widely studied and are generally referred to as ERK1/2 because they exhibit 90% homology (The homology .1/2 is an important subfamily of the MAPK family and is part of a three-stage enzymatic cascade reaction (1/2 kinase (MEK) as a MAPKK, and ERK1/2 as a MAPK. ERK1/2 is normally located in the cytoplasm. Intracellular and extracellular stimuli induce serine/threonine phosphorylation in the VIII region of ERK1/2via the classical Ras-Raf-MEK-ERK1/2 pathway, which leads to activation of ERK1/2. This well-studied pathway is involved in various physiological and pathological processes, such as cell growth, development, proliferation, and differentiation.ERKreaction , which c1/2, which phosphorylates cytoplasmic target proteins or regulates the activities of other protein kinases. More importantly, activated ERK1/2 enters the nucleus and phosphorylates various transcription factors, such as Elk-l, c-Myc, STATs, Jun, Fos, ATF2, and Max. These factors regulate transcription of their target genes and thereby change the expression or activities of specific proteins, ultimately leading to the regulation of cellular metabolism and function  is the most effective method to treat ischemic injury. However, reperfusion can aggravate reversible ischemic injury and induce irreversible damage. This effect, called IRI, is involved in the pathogenesis of organ transplantation and various diseases and affects the prognosis of patients with ischemic diseases. Elucidation of the mechanism underlying IRI is important to ameliorate or prevent this condition and to treat ischemic diseases.The mechanism underlying IRI is very complicated and involves primary injury in the early stage of ischemia and secondary injury after reperfusion. Tissue damage due to IRI not only depends on the degree of blood flow reduction, but is also related to intracellular calcium overload , oxidati1/2 signaling pathway and IRI in different tissues and organs is discussed in this review.The relationship between the ERK1/2 is widely found in the nervous system. Phosphorylation of ERK1/2 is indispensable for its functions, such as inducing proliferation and differentiation, and inhibiting programmed cell death upon brain injury  inhibits the MAPK-ERK1/2-CREB signaling pathway and thereby reduces Mfn2 expression, represses mitophagy, and activates mitochondrial apoptosis in reperfused brains. Upregulation of NR4A1 is a key step in the initiation of CIRI  signaling via VEGF receptor 2 (Flk-1) and activates ERK1/2 through an oxidative stress-dependent mechanism. Phosphorylated ERK1/2 activates Elk-1 and other transcription factors, such as NF-κB, and promotes cell death  injury in the heart  is a type of myocardial injury caused by restoration of coronary blood flow after ischemia. This eventually leads to death of myocardial cells that initially survived after ischemia and seriously threatens human health . Frequenhe heart . Activatytokines . Oxidatiytokines . The MAPnst MIRI .1/2 signaling pathway. Carvacrol protects against MIRI by activating various signaling pathways such as MAPK/ERK1/2 and thereby reducing apoptosis  regulates expression of FUNDC1 through the MAPK/ERK1/2-CREB pathway, which reduces mitochondrial apoptosis of cardiomyocytes and improves MIRI  is proinflammatory, contributes to MIRI, and silencing of FPR1 gene have a negative effect on ERK1/2 signaling pathway and reduce inflammation, apoptosis, and ventricular remodeling  is a common clinical problem observed after liver transplantation, hepatectomy, severe trauma, and hemorrhagic shock, and is caused by sudden restoration of blood supply following hypoxia . Reperfu1/2 signaling pathway and HIRI has been a focus of research in recent years. Various signaling proteins, including members of the MAPK family, are activated upon HIRI   is characterized by restriction of renal blood flow and subsequent restoration of normal blood flow and oxygen supply. This aggravates renal damage by increasing inflammation es (ROS) , and by es (ROS) . Acute ies (ROS) . RIRI caes (ROS) , and hases (ROS) . This coes (ROS) . ERK1/2 l injury , suggesting RIRI .1/2 signaling pathway was reported to elicit beneficial effects on RIRI in most studies. Dexamethasone induces acetylation of the p65 subunit of NF-κB by inhibiting MAPK/ERK1/2 activation and subsequent translocation of HMGB1, leading to attenuation of inflammation in RIRI  participates revealed that a complex signaling network, which includes the PI3K/Akt, VEGF, and MAPK/ERK1/2 signaling pathways, mediates survival and proliferation of renal cells . Inhibit in RIRI . Stannio pathway . C-reacty injury . Rapid ay injury . Pretreaynthesis . In cont by RIRI .Further investigation of these pathways is required and may help to develop a new strategy to treat or prevent RIRI.1/2 is activated during human lung transplantation (Lung transplantation provides hope for many patients with end-stage lung disease. Acute lung injury (ALI) induced by lung IRI (LIRI) is an important problem following lung transplantation . During antation  and promantation .1/2 signaling pathway is involved in development of LIRI; therefore, inhibition of this pathway may alleviate LIRI. Hypoxia and inflammation induce VEGF production  is a well-known risk factor for clinical morbidity and causes delayed dysfunction due to induction of ischemia and subsequent activation of the inflammatory pathway . Severe 1/2 signaling pathway plays an important role in improvement of IIRI and is beneficial for repair. IIRI results in tissue damage, loss of visceral barrier function, and distant complications, including multiple organ dysfunction syndrome. Reperfusion induces activation of PI3K/Akt and MEK/ERK1/2, which are involved in recovery, leading to restoration of tissue and barrier function. Endogenous HB-EGF is induced after IIRI, which may activate PI3K/Akt and MEK/ERK1/2via ErbB1 to improve IIRI and facilitate recovery of intestinal barrier function  occurs during surgical repair of thoracic and celiac aneurysms resulting from temporary aortic occlusion. SIRI can lead to spinal cord dysfunction and paraplegia, which is the most serious complication of thoracic and abdominal aortic surgery, with a reported incidence of higher than 10% . SIRI ha1/2 signaling pathway protects against SIRI. Both standard and modified distal posterior adaptation elicits neuroprotective effects against spinal cord ischemia by increasing expression of p-Akt and p-ERK1/2 (1/2 expression, and activating the Akt and ERK1/2 signaling pathways (Most studies reported that activation of the ERKp-ERK1/2 . Xenon-dpathways .1/2 signaling pathway may help to identify a new therapeutic approach for SIRI.SIRI causes great suffering to patients and imposes a great burden on their families and society. Therefore, the mechanism underlying SIRI must be urgently studied, and methods to prevent and treat this condition must be developed. Further investigation of the ERK1/2 pathway is one of the most important signal transduction pathways and regulates many biological responses. It even plays a key role in behavioral responses and cognitive processes, such as learning and memory. ERK1/2 is abnormally expressed upon IRI in vivo and in vitro. Even transient local ischemia can change ERK1/2 expression and induce corresponding effects.The ERK1/2 signaling pathway protects against or aggravates IRI. This pathway elicits different effects, including inhibition of apoptosis and induction of necrosis, depending on the period of ischemic injury and tissue region. The mechanism underlying these effects must be investigated further.The period, degree, and location of injury may determine whether the ERK1/2 signaling pathway can induce many pathological changes, such as tumorigenesis. Consequently, many MEK and ERK1/2 inhibitors, such as the non-steroidal anti-inflammatory drug NS398, have been developed. This drug blocks the Ras/c-Raf interaction and inhibits activation of the ERK1/2 signaling pathway (1/2 signaling pathway. Investigation of the regulatory mechanism of this pathway will help to identify proteins that directly regulate ERK1/2 and to determine their involvement in ERK1/2-related diseases. This approach will facilitate the development of drugs with improved targeting and fewer side effects.Abnormalities in the ERK pathway , thereby1/2 signaling pathway in IRI and the underlying mechanisms. The effect of the ERK1/2 signaling pathway on IRI remains controversial. Further comprehensive and in-depth studies are required to determine whether the ERK1/2 signaling pathway protects against or aggravates IRI. Elucidation of the underlying mechanisms will help to establish a theoretical basis for the clinical treatment of IRI.This review summarizes the role of the ERKTK wrote the manuscript and generated the figures. ML helped to design the figures. BJ and BB proposed suggestions for revisions. BC revised the manuscript. CW designed the content and provided intellectual insight.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.


Diverse signaling cues and attendant proteins work together during organogenesis, including craniofacial development. Lip and palate formation starts as early as the fourth week of gestation in humans or embryonic day 9.5 in mice. Disruptions in these early events may cause serious consequences, such as orofacial clefts, mainly cleft lip and/or cleft palate. Morphogenetic Wnt signaling, along with other signaling pathways and transcription regulation mechanisms, plays crucial roles during embryonic development, yet the signaling mechanisms and interactions in lip and palate formation and fusion remain poorly understood. Various Wnt signaling and related genes have been associated with orofacial clefts. This Review discusses the role of Wnt signaling and its crosstalk with cell adhesion molecules, transcription factors, epigenetic regulators and other morphogenetic signaling pathways, including the Bmp, Fgf, Tgfβ, Shh and retinoic acid pathways, in orofacial clefts in humans and animal models, which may provide a better understanding of these disorders and could be applied towards prevention and treatments. Summary: Investigation of the crosstalk between the Wnt and other signaling pathways will improve understanding of orofacial cleft development and provide opportunities for treatment and prevention. C6 motif: the six-amino-acid C-terminal domain of Axin proteins. It is implicated in JNK activation, but has no effect on Wnt signaling.Epithelial-mesenchymal transition (EMT): the induction of adhesive epithelial cells to become migratory and proliferative cells during developmental processes, including in palatogenesis.Goltz-Gorlin syndrome: a rare genetic disorder, also known as focal dermal hypoplasia (FDH), characterized by distinctive skin abnormalities, including CLP in some cases, and other defects that affect eyes, teeth, and the skeletal, urinary, gastrointestinal, cardiovascular and central nervous systems. Mutations in PORCN, an upstream regulator of Wnt signaling, are associated with FDH.Maxillary prominences: a pair of developmental structures at the lateral edges of the oral cavity that give rise to the upper jaw elements, including the maxillary bone.Medial edge epithelium (MEE): the distalmost edge of the palatal epithelium that surrounds the proliferating mesenchyme. On each palatal shelf, this layer will meet and fuse during secondary palatogenesis.Midline epithelial seam (MES): the layer of epithelial cells that separates the two lateral pools of the mesenchyme after palatal shelf fusion. MES cells undergo apoptosis to allow the formation of a continuous mesenchyme layer across the secondary palate.Nasal prominences: two pairs of medial and lateral extensions derived from the unpaired frontonasal prominence during early craniofacial development, which fuse on either side with the maxillary prominences to form the primary palate and nostrils, and separate the nasal cavity from the oral cavity.Neurulation: a stage in vertebrate embryogenesis in which the neural plate folds to form the neural tube.Palatal shelf: a pair of palatal structures elongated from the maxillary prominences between the nasal prominence and the first branchial arch/mandibular process, which eventually fuse to separate the oral and nasal cavities.Primary palate: a rostralmost palatal structure formed by the fusion of the nasal and maxillary prominences to separate the nasal pits from the oral cavity.Regulator of G protein signaling (RGS) domain: a motif required for the protein's activity in accelerating the GTPase activity of G-proteins. The RGS domain in Axin proteins is required for binding APC in Wnt signaling.Robinow syndrome: congenital syndrome characterized by craniofacial, skeletal and urogenital defects, which frequently includes orofacial clefts and has been associated with mutations in noncanonical Wnt signaling genes, including WNT5A and ROR2.Rugae: the series of ridges produced by folding of the anterior wall of the palate behind the incisive papillae.Secondary palate: a roof structure of the oral cavity that arises from the fusion of the left and right palatal shelves posterior to the primary palate.Sporadic or familial CLP: occurrence of CLP within families or close relatives is referred to as familial CLP, whereas appearance of the phenotype without apparent genetic predisposition is termed sporadic CLP.Submucous cleft palate: a form of cleft palate in which the two palatal shelves incompletely fuse such that the oral and nasal cavities are separated from each other by soft tissue, but a continuous layer of bone has not developed across the midline.Syndromic or non-syndromic CLP: CLP patients carrying additional dysmorphic or clinical features are syndromic; non-syndromic CLP is not associated with other phenotypes.Tetra-amelia syndrome: a congenital syndrome characterized by limb malformation, often coupled with craniofacial and urogenital defects, associated with WNT3 mutations.Unilateral and bilateral CLP: a cleft can occur either at one  or both sides  of the face.Van der Woude syndrome: a congenital syndrome characterized by craniofacial, limb, and cardiac defects, associated with mutations in the transcription factors downstream of canonical Wnt signaling.Orofacial clefts, mainly cleft lip and/or cleft palate, are among the commonest structural birth defects . The occMurine and human facial formation follow a similar developmental trajectory, and facial structures arise from several primordial tissues as described below . Facial In mice, the palatal shelves first emerge from the maxillary prominences at E11.5 and continue to proliferate, elongating ventrally between E12 and E14 . The elo2+ pathway (Wingless-type MMTV integration site (Wnt) signaling is required for body axis patterning, cell fate specification, cell proliferation and cell migration during embryonic development . Wnt sig pathway . This ReWnts are secreted lipid-modified signaling proteins that are evolutionally conserved and play vital roles in development, homeostasis and disease. Nineteen Wnt ligand proteins encoded by respective genes in mammalian genomes act through a variety of receptors and co-receptors, including ten seven-transmembrane frizzled (Fzd) receptors, two single-transmembrane co-receptors Lrp5/6, and the receptor tyrosine kinase-like receptors Ror1/2 and Ryk.2+ pathway, which is less understood.In the Wnt-producing cells, the nascent Wnt proteins are palmitoylated by porcupine O-acyltransferase (Porcn), followed by their secretion in the extracellular matrix via Wnt ligand secretion mediator Wls; . Wnt sigMsx1/Msx2 in orofacial primordia , the casein kinase CK1, the protein phosphatase 2A (PP2A) and the E3-ubiquitin ligase β-TrCP. Upon the binding of a Wnt ligand to a Fzd receptor, the Fzd recruits a Dvl cytoplasmic phosphoprotein and a Lrp co-receptor recruits an Axin, which inhibits the destruction complex. This stabilizes cytoplasmic β-catenin, resulting in its accumulation and translocation into the nucleus. There it binds to Tcf/Lef1 transcription factors to regulate the transcriptional activation of critical Wnt target genes in various cells/tissues, such as the orofacial cleft-associated genes rimordia .2+ to trigger a number of downstream effects, such as cell migration and proliferation.Non-canonical PCP pathway: The binding of a Wnt ligand to Ror or Ryk receptors promotes the interaction of a Dvl with disheveled-associated activator of morphogenesis 1 (Daam1), which activates several downstream GTPases, including the Rac proteins and ras homolog family member A (RhoA). This results in the restructuring of actin to change cell shape, polarity and movement. Dvl can also activate phospholipase C to generate inositol triphosphate, which activates the release of CaGPR177) through the Golgi apparatus to the cell surface for secretion , an important gene in the production of collagen. Mutations in either gene are associated with NSCLP  phenotype  (DVL1 (RYK), have also been linked to NSCLP in Vietnamese and Japanese patients   and the 2)  were linked with abnormal craniofacial features, but not with orofacial clefts (AXIN2 and glycogen synthase kinase 3 beta (GSK3B) in NSCLP families across multiple populations identified intronic SNPs that contribute to orofacial clefts In vitro Gsk3b, which encodes a β-catenin-degrading enzyme in the canonical Wnt signaling pathway, results in mice displaying full cleft palate  domain (ΔC6)  domain  or the C) domain  that encin (ΔC6) . Intriguding CLP   expression during palatogenesis Wnt5a mutAlthough mouse models have vastly contributed to our understanding of Wnt signaling in palatogenesis, other models, such as the zebrafish, provide unique insight into craniofacial formation and the basic requirements for palate formation . CanonicOrofacial clefts have also been observed in chick embryos , where WXenopus embryos for transplanting tissue and local chemical perturbation have provided a suitable clefting model , during palatogenesis remain to be elucidated CDH1 haveal bones   and interferon regulatory factor 6 (IRF6), detected in families with Van der Woude syndrome , tend tofication . Exome s studied  , and a separate interaction between IRF6 and WNT2, in association with NSCLP  and in the axon guidance signaling molecule netrin 1 (NTN1)  .Wnt signaling does not act in isolation during lip/palate development. Several other signaling pathways are involved in orofacial development, including the fibroblast growth factor (Fgf), bone morphogenic protein (Bmp), transforming growth factor beta (Tgfβ), sonic hedgehog (Shh) and retinoic acid (RA) signaling pathways ;. This sFgf8 expression in early facial patterning (Pax9) expression during palatogenesis (Fgf9 and Fgf18) and their receptors (Fgfr1 and Fgfr2) has also been associated with a cleft palate phenotype (in utero in Pax9-null mouse embryos (Spry2) is also a candidate Wnt/β-catenin target gene Fgf8 exprogenesis . Knockouhenotype   embryos   embryos . Knockou embryos . Tbx22 i embryos . Sproutyget gene , and Sprget gene  e palate . Moreovee palate .Msx1 and Msx2 expression in the orofacial primordia, but Lrp6 ablation does not affect Bmp4 during primary lip/palate formation and fusion, suggesting that the Bmp and Wnt pathways may converge by way of common activation of Msx1/Msx2 Msx1 and sx1/Msx2 . This imsx1/Msx2 .Many Bmp receptors are expressed in differing patterns along the anterior-posterior (A-P) axis of the palatal shelves during palatogenesis. Submucous cleft palate  results Epithelial Wnt/β-catenin signaling also regulates Tgfβ signaling. Wnt-mediated Tgfβ3 activation is required for MEE cell apoptosis during palatal shelf closure  Fig. 2)Fig. 2), Ift88) or kinesin family member 3A (Kif3a) in mice causes CLP Fig. 3). uses CLP  uses CLP , whereasuses CLP . Loss ofuses CLP . In addil clefts . Both Ifl clefts  VAX1 is a candidate human NSCLP gene  transcription factors are important for neural patterning, and they mediate signaling between Shh and Wnt . Sfrp anCLP gene , and VaxCLP gene . PalatalCLP gene . Wnt/β-cCLP gene .Wnt5a/Ror2 may act upstream of the non-canonical Wnt signaling molecule Prickle1  Fig. 1)Fig. 1), Shh has been reported to activate Fox genes during lip and palate development . Foxf2 aRA plays an important role in normal palatogenesis , and excCDH1 during EMT in cancer cells, in turn activating the Wnt signaling pathway  transcript regulation, have garnered increasing attention in recent years. These epigenetic mechanisms play a role in regulating many Wnt pathway components . Studies pathway . The miR mutants . In zebr mutants . FurtherFZD5  into ignaling . In addiignaling  and coulignaling . Wnt5a aignaling , may alsin vitro fertilization with selected gametes that do not possess the risk-imparting allele.Given the heterogeneous causes of orofacial clefts and the variability in the genotypes of affected individuals, it is unlikely that we will see a ‘one-size’ approach to non-surgical orofacial cleft treatment any time soon. However, successful studies using mutant mouse models are promising for the prospect of pathway-specific treatments to allow for prenatal intervention when a fetal genotype renders human embryos at risk of orofacial cleft development. Implementation of promising therapies to human patients would impinge on the timing of application and on the accurate detection of improper palatogenesis. Even if parents carrying alleles linked to orofacial clefts were to commit to prenatal genotyping, many palatal development processes occur early in gestation. Even with improved protocols for correcting the levels of a target signaling factor in patients, implementing postconception measures at such an early stage of pregnancy, before many mothers know that they are pregnant, is challenging. Further challenges arise from the high variability in the manifestation and penetrance of orofacial cleft phenotypes. The complexity of the molecular processes that govern orofacial development makes it difficult to predict the therapeutic requirement. Moreover, a particular intervention may not be applicable to more than a minor subset of cases, even in individuals with defects in a particular gene or pathway. A deeper understanding of the pathways that govern palatogenesis may allow Interactions between Wnt/β-catenin signaling and other morphogenetic signaling pathways are widely employed in many different developmental programs. The nature of embryonic development creates significant potential for off-target effects and disruption of other essential developmental processes in both mother and child if molecular treatments to correct palatogenesis errors are applied systemically. Before treatments targeting signaling pathways could be considered for clinical trials with human patients, such risks would need to be thoroughly explored and addressed, and likely require new delivery techniques more advanced than those currently available. Additionally, because NSCLP is not life threatening, many parents may be unwilling to attempt untested and potentially dangerous approaches, despite the burden and difficulty of current treatments. However, in the future, a more complete understanding of morphogenetic pathway crosstalk and the systemic impact of perturbations to them may eventually allow the progression of molecular clinical approaches to a point at which they are considered safe. A robust understanding of how signaling pathways function in all systems and processes during development will be able to not only inform studies related to orofacial clefts, but also contribute to the development of treatments for other syndromes and disorders with Wnt pathway etiologies. Despite the many barriers that still remain, knowledge of developmental mechanisms is helping to, and will continue to, facilitate the refinement of techniques for the application of that knowledge to develop the means to safely and effectively treat congenital disorders like orofacial clefts.


They can be made fluorescent with perfect photostability by creating nitrogen-vacancy (NV) color centers in the diamond lattice. The resulting fluorescent nanodiamonds (FND) have been used since the late 2000s as fluorescent probes for short- or long-term analysis. FND can be used both at the subcellular scale and the single cell scale. Their limited sub-diffraction size allows them to track intracellular processes with high spatio-temporal resolution and high contrast from the surrounding environment. FND can also track the fate of therapeutic compounds or whole cells in the organs of an organism. This review presents examples of FND applications (1) for intra and intercellular molecular processes sensing, also introducing the different potential biosensing applications based on the optically detectable electron spin resonance of NV ODMR can only be observed in NV− defects. Furthermore, the NV defects within the ND lattice confer a very high resolution for imaging single molecules. NDs also benefit from having a large variety of surface functionalizations [Nanodiamonds (NDs) are diamond phase carbon nanomaterials that were initially used for their strong abrasive properties and as lubricant additives  for induizations . Pristinizations ,11,12. CEpifluorescence confocal imaging is the simplest method used to observe fluorescent nanodiamonds due to an excitation at a wavelength of 532 nm and an emission between 600 and 800 nm for the NV defects. When in vivo or ex vivo actions are performed, time-delayed detection is then preferred. The radiative lifetime of NV centers in nanodiamonds of approximately 25 ns  is one oThis short review focuses on some biomedical applications of fluorescent nanodiamonds, taking advantage of their unique photophysical properties to use them for (1) intracellular and molecular processes sensing and (2) for long-term tracking in organisms for therapeutic or diagnostic purposes.From their first application in cellular studies, NDs have been observed by various microscopy techniques to be internalized efficiently in a large variety of cellular types, from human cancer cell lines  to mouseThe first task involved identifying NDs’ internalization pathways. By using drugs that selectively inhibit the different uptake pathways, several studies highlighted that clathrin-mediated endocytosis was the dominant internalization process of HPHT nanodiamonds smaller than 100 nm ,18. The 2) and transferrin grafted FNDs (FND-Tf) showed that the latter, which triggers receptor-mediated endocytosis, has a higher uptake efficiency compared to the former, which probably acts via electrostatic interactions between the NDs and the cell membrane [In addition, a comparison of the internalization process between positively charged amine functionalized FND  is an ideal candidate molecule for such targeting as it has receptors on a large variety of cancer cells. Taking advantage of FND fluorescence, Li et al. demonstrated that the grafting of Tf at the surface of FND improved their internalization in HeLa cells . They alIn addition to the observation of their internalization in cells, the perfectly stable fluorescence conferred by the NV centers to NDs is particularly well suited for tracking intracellular processes such as endosomal transports. This process is impeded in neurodegenerative diseases, such as Parkinson and Alzheimer diseases . The latIntercellular communication is essential in various biological processes, such as immunity, tissue regeneration, and development. This communication is completed through different gateways, including chemical synapses, gap junctions, and tunneling nanotubes (TNTs) . The latAcid-cleaned FNDs have been reported to be spontaneously internalized, but in some cases, only the labeling of the cell membrane is desired without the internalization of the nanodiamonds. Such labeling is made with PEG-modified FNDs that are covalently coated with neoglycoproteins or with streptavidin. One application of this function is immunostaining to label the membrane of specific cells, including hepatocytes, which can be further sorted by flow cytometry . 2-PEG-COOH, on which streptavidin and then BSA and biotin-TGF were attached. This complex efficiently labeled TGF-beta receptors, and the authors were able to track their three-dimensional (3D) motion with a high localization accuracy (10 nm in x and y and 19 nm in z). The trajectory analysis highlighted three different behavioral states of TGF-beta receptors: immobile, intermediate, and fast diffusion [Such functionalization and labeling was also used to study some transmembrane signaling functions and membrane endogenous proteins dynamics in live cultured-cells. Sotoma et al.  targetediffusion . They sh−, whereas the hydrogen-terminated surface only emits neutral NV0 [− to NV0 PL intensity has been used in a purely optical method to detect the release in the cytoplasm of negatively charged nucleic acid transported by cationic polymer-coated FNDs [The NV center charge state in a nanodiamond depends on the Fermi level of the host matrix, which can be adjusted by varying the surface functionalization. The oxygen-terminated FND favors emission of negatively charged NVtral NV0 . Each chted FNDs .– electron spin resonance is shifted by the Zeeman effect, providing an optical means for magnetometry [− center electron spin resonance frequency also experiences a shift upon a thermal variation that allows the measurement of temperature changes with a noise as small as 9–10 mK·Hz−1/2 [NV centers in diamonds are remarkable not only for their perfectly stable fluorescence but also for the optically detectable electron spin resonance of the negatively charged form. In the presence of an external magnetic field, the NVetometry , or for etometry . NV-magnetometry . Althougetometry . The NV−K·Hz−1/2 ,36, in aK·Hz−1/2 . The locK·Hz−1/2 , making K·Hz−1/2 ,39.– electron spin can be coherently manipulated with light and microwave at the ODMR microwave frequency, which provide additional sensing approaches. In particular, modification of the spin longitudinal relaxation time T1 can be measured by purely optical manipulations [T1 reflects the magnetic noise surrounding the electron: an increase in noise results in a decrease in T1. Taking advantage of these properties, Rendler et al. [− center in diamond spin decoherence can also be used to improve the sensitivity of electromagnetic field detection, which has been proposed for the real-time monitoring of membrane ion channel activity [T2 of NV− spins in 45-nm FNDs was monitored with spin echoes sequences during the addition of Mn2+ ions in solution. The authors measured a decrease in T2 induced by only about 2500 Mn2+ ions surrounding the FND. This type of approach was further improved, leading to the detection of only 12 ferritin molecules at the FND surface [T1 relaxometry and T2 measurements. The challenge of translating these first experiments into real biosensing applications relies on making them compatible with the complex ionic environment of the biological medium.Furthermore, the state of the NVulations ,41,42. Tr et al.  performeactivity . McGuinnactivity  complete surface ,47 usingNanodiamonds have been studied since the late 2000s as biomolecule delivery agents due to their high biocompatibility  and vers188Re-DND [18F-DND [125I-DND [99Tc-DND [When using ND for drug delivery in model living organisms like mice   that pr99Tc-DND  by using99Tc-DND  using anCaenorhabditis elegans worm during 12 h due to their coupling to FND. Time-delayed fluorescence imaging was used to achieve a higher signal over background imaging of FND in C. elegans compared with confocal fluorescence  was completed in small pigs . This sthe liver . FurtherFNDs were also used to label and then track quiescent cancer stem cells (CSCs) . CSCs arAfter assistance in diagnosis, support in the follow-up of the proliferation of the cancer is necessary. To this aim, Hui et al.  imaged iTogether, these studies are improving accurate tumor cell and metastasis detection. We can envision the development of diagnostic devices based on tracking circulating tumor cells (CTC) in patients’ blood after labeling of the tumor with fluorescent nanodiamonds.− centers that allow the selective imaging of NV-containing nanodiamonds in an autofluorescent background [Nanodiamonds containing NV centers appear to be valuable bioimaging and biosensing probes. They allow the observation, characterization, and measurement of subcellular processes, from cellular uptake to intracellular physico-chemical parameters. NDs are also valuable for biomolecular dynamics sensing. Fluorescent nanodiamonds have also shown superior properties for the long-term tracking of stem cells or cancerous cells, offering possible applications in disease status monitoring. Despite the promising biomedical results presented in this review, a bottleneck still exists for fully in vivo live observation and imaging of fluorescent nanodiamonds. In vivo observations are complicated by the high autofluorescent background from tissues. This could be circumvented by taking advantage of the specific ODMR response of NVckground ,78. A sickground  was succ


Thymic stromal lymphopoietin (TSLP) is a pleiotropic cytokine originally isolated from a murine thymic stromal cell line. TSLP exerts its biological effects by binding to a high-affinity heteromeric complex composed of thymic stromal lymphopoietin receptor chain and IL-7Rα. TSLP is primarily expressed by activated lung and intestinal epithelial cells, keratinocytes, and fibroblasts. However, dendritic cells (DCs), mast cells, and presumably other immune cells can also produce TSLP. Different groups of investigators have demonstrated the existence of two variants for TSLP in human tissues: the main isoform expressed in steady state is the short form (sf TSLP), which plays a homeostatic role, whereas the long form (lfTSLP) is upregulated in inflammatory conditions. In addition, there is evidence that in pathological conditions, TSLP can be cleaved by several endogenous proteases. Several cellular targets for TSLP have been identified, including immune  and non-immune cells (platelets and sensory neurons). TSLP has been originally implicated in a variety of allergic diseases . Emerging evidence indicates that TSLP is also involved in chronic inflammatory  and autoimmune  disorders and several cancers. These emerging observations greatly widen the role of TSLP in different human diseases. Most of these studies have not used tools to analyze the expression of the two TSLP isoforms. The broad pathophysiologic profile of TSLP has motivated therapeutic targeting of this cytokine. Tezepelumab is a first-in-class human monoclonal antibody  that bin The  methods , while tosome 18 . Human aosome 18 , 8. The osome 18 . TSLP isosome 18 –15 and losome 18 –21, skinosome 18 –25, and osome 18 . Howeverosome 18 , human Dosome 18  and mastosome 18 , 29, humosome 18 , macrophosome 18 , synoviaosome 18  and cancosome 18 , murine osome 18 , and canosome 18 .+ DCs and plasmacytoid DCs to induce functional Th2 and regulatory T (Treg) cells . In fact, TSLP activates TSLPRg) cells , 36 and g) cells . Interesg) cells –40 and cg) cells , 34, 41.g) cells –45.in vitro human blood DC maturation and chemokine (CCL17) production induced by TSLP   , 16, 20, aureus)  and fung aureus) , mechani aureus) , allerge aureus) , cigaret aureus) , 52, and aureus)  can indu+ T cells. Culture of TSLP-activated DCs together with naive syngeneic CD4+ T cells leads to T cell proliferation, which suggests a role for TSLP in CD4+ T cell homeostasis. However, when TSLP-stimulated DCs prime CD4+ T cells in an antigen-specific manner, the resulting T cells show characteristic features of T helper type 2 (Th2)-differentiated cells   . These dd IL-13) .+ Th2 cells potentially via DCs and/or basophils  production by human cord blood-derived cultured mast cells and by human peripheral blood-derived mast cells when combined with IL-33  .Harada and collaborators first identified two variants for TSLP in human bronchial epithelial cells . The autXie et al. studied the differential expression and modulation of the two isoforms in primary human keratinocytes . They coin vitro and in vivo , whereas lf TSLP increases the release of IFN-γ. Importantly, they reported that lf TSLP activates a canonical TSLPR on human immune cells, whereas sf TSLP induces or inhibits signaling through an unknown receptor  upregulate the long isoform and downregulate the short isoform, whereas the opposite is true after challenge with a commensal E. coli strain. The latter observation suggests that the dysbiosis observed in barrier surfaces could impact the expression of TSLP. More recently, Dong et al. reported that inflammatory stimuli upregulate lf TSLP mRNA but not sf TSLP in human bronchial epithelial cells , EoE, allergic rhinitis, etc. Because of the pathophysiological relevance of long TSLP expression in all these diseases, the cytokine has been repeatedly suggested as a valid target for therapy with antibodies that would target TSLP and/or prevent its binding to TSLPR –45. SincGenetic analysis has shown an association of polymorphisms in TSLP with several allergic diseases, including asthma and airway hyperresponsiveness, IgE concentrations, and eosinophilia , 108. AdTSLP have been detected in genome wide studies for asthma, rhinitis, and atopy  reduces airway inflammation and remodeling by inhibiting Stat3 signaling through TSLP downregulation . Interes2 mAb , and epidermal barrier function of AD remain to be elucidated  is a heterogeneous enteropathy caused in genetically susceptible individuals by the ingestion of gluten . TSLPR ain vitro and development of autoimmune hemolytic anemia in vivo  of TSLP in humans , 101. sfAn additional level of complexity derives from the observation that, in pathological conditions, TSLP can be cleaved by several endogenous proteases , 63, 106The role of TSLP in different types of cancer is controversial , 174. ThGiven the role of TSLP in experimental and clinical asthma , 180 andThymic stromal lymphopoietin was initially considered a key cytokine that initiates and promotes type 2 immunity . IncreasGiven the complexity of the interactions of different isoforms and cleavage products of TSLP with a plethora of immune cells in various organs and in different diseases, several fundamental questions remain to be elucidated. A better understanding of the impact of TSLP isoforms and cleavage products on immune cells will be critical for the development of safe and effective TSLP targeted therapies in inflammatory disorders and cancer.All authors contributed equally to reviewing the current literature and writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.