OsMKK3, and investigated its function. It was observed that mechanical wounding, infestation of brown planthopper (BPH) Nilaparvata lugens, and treatment with methyl jasmonate (MeJA) or salicylic acid (SA) could induce the expression of OsMKK3. The over-expression of OsMKK3 (oe-MKK3) increased levels of jasmonic acid (JA), jasmonoyl-L-isoleucine (JA-Ile), and abscisic acid (ABA), and decreased SA levels in rice after BPH attack. Additionally, the preference for feeding and oviposition, the hatching rate of BPH eggs, and BPH nymph survival rate were significantly compromised due to over-expression of OsMKK3. Besides, oe-MKK3 also augmented chlorophyll content but impaired plant growth. We confirm that MKK3 plays a pivotal role in the signaling pathway. It is proposed that OsMKK3 mediated positive regulation of rice resistance to BPH by means of herbivory-induced phytohormone dynamics.Plants undergo several but very precise molecular, physiological, and biochemical modulations in response to biotic stresses. Mitogen-activated protein kinase (MAPK) cascades orchestrate multiple cellular processes including plant growth and development as well as plant responses against abiotic and biotic stresses. However, the role of MAPK kinases (MAPKKs/MKKs/MEKs) in the regulation of plant resistance to herbivores has not been extensively investigated. Here, we cloned a rice MKK gene,  In natural ecosystems, plants frequently experience herbivore attacks. Plants possess complicated defense systems to respond against insect attack and protect themselves. These defense strategies include physical barriers, metabolic adjustments, signaling cascades, and expression of different herbivory-associated genes ,2,3,4,5.Nicotiana attenuata, oral secretions (OS) of Manduca sexta rapidly induced expression of NaSIPK and NaWIPK and elicited JA, jasmonoyl-L-isoleucine (JA-Ile), SA, and ET biosynthesis [OsRab7 enhanced the rice resistance to drought and heat stress [NaMPK4 enhanced the plant resistance to M. sexta through a JA-independent defense pathway in N. attenuate [Nicotiana tabacum ectopically expressing AhMPK3 increased plant resistance to Spodoptera litura [Nezara viridula in Glycine max induced the expression of different protein kinases such as GmMPKK1, GmMPK3, and GmMPK6 [Cicer arietinum infested by Helicoverpa armigera, e.g., CaMAPK2, CaMAPK3, CaMAPK5, CaMAPK7, and CaMAPK8 [Oryza sativa, OsMPK3 and OsMPK4 were induced by Chilo suppressalis and positively regulated rice resistance by modulating JA signaling [OsMPK20-5, which negatively regulated the rice resistance to BPH by modulating ethylene and nitric oxide accumulation [MAPKs comprise 11 (I–XI) domains that have also been recorded in all serine/threonine protein kinases . Phosphoynthesis . A transt stress . Similarttenuate . Transgea litura . Damage d GmMPK6 . Similar CaMAPK8 . In Oryzignaling ,29. Browmulation . Though OsMKK3 is an ortholog of AtMKK3 [Arabidopsis thaliana, MKK3 is involved in defense against pathogens, drought tolerance, JA signal transduction, and ABA and auxin responses [Xanthomonas oryzae-infested rice plants, OsMKK3 phosphorylated OsMPK7 and activated OsWRKY30. Furthermore, overexpression of OsMKK3 enhanced disease resistance and up-regulated genes related to pathogenesis, maintenance of the cell wall, and metabolism [f AtMKK3 . In Arabesponses ,34,35,36tabolism . AlthougO. sativa) crop is severely damaged by various insect pests. Among them, brown planthoppers  destroy rice crops by phloem sucking and cause heavy losses in growth and productivity [O. sativa induced the biosynthesis of JA, JA-Ile, SA, ABA, ET, and H2O2, which regulate the plant defense response to herbivores [OsMKK3, for its probable role in rice against BPH attack. It was confirmed that OsMKK3 was induced by mechanical damage and BPH infestation. Moreover, overexpression of OsMKK3 enhanced rice resistance to BPH through increased JA, JA-Ile, and ABA levels as well as decreased SA levels. Taken together, our data suggest that OsMKK3 plays a positive role of rice immune responses elicited by BPH.Rice  and 4 hpt, respectively  containing a single insertion through southern blot (OsMKK3 in transgenic lines which over-expressed OsMKK3 (oe-MKK3), exhibiting 12.9- and 16.6-fold higher expression levels of OsMKK3 in the OE3-26 and OE3-47 lines, respectively, in comparison with wild-type plants (WILD) without wounding treatment, and 17.6-fold and 16.6-fold higher expression levels of OsMKK3 in the OE3-26 and OE3-47 lines respectively comparing to WILD at 8 h post wounding  and wild-type plants (WILD). Under BPH infestation, the levels of these two signals were much higher in oe-MKK3 lines than WILD. At 8 hours post BPH infestation (hpi), the JA levels exhibited remarkable differences between oe-MKK3 and WILD; meanwhile, the significant difference for JA-Ile lasted from 8 hpi to 48 hpi. The levels of these two signals showed the most remarkable difference at 8 hpi; to be specific, OE3-26 and OE3-47 exhibited 2.6- and 1.9-fold higher levels of JA as well as 3.3- and 2.6-fold higher levels of JA-Ile, respectively, in comparison with WILD at 8 hpi  to regulate this signaling. Numerous studies have reported that this accumulation of JA and ABA is supported by the fact that JA and ABA interact synergistically, whereas they have antagonistic interactions with SA in plant defense [Arabidopsis, AtMPK3 and AtMPK4 negatively regulated flg22-induced SA accumulation [N. tabacum, NtMPK3 and NtMPK6 suppressed wound-induced SA accumulation [OsMPK6 increased X. oryzae-induced SA accumulation and overexpression of OsMPK4 elevated C. suppressalis-induced SA levels in rice [OsMKK3 plants to the negative regulation of SA via its downstream MAPKs or the antagonistic interaction between JA and SA. Additionally, our results indicated that SA elicited the expression of OsMKK3 which was a negative regulator of SA. Similarly, AtMPK3 could be induced by SA but negatively regulated SA accumulation in Arabidopsis [OsMKK3 might have a negative feedback to SA via its downstream MAPKs. JA and ABA signaling pathways positively mediate the rice resistance to BPH whereas SA signaling pathway is opposite [OsMKK3 enhanced the rice resistance to BPH may result from the increased biosynthesis of JA, JA-Ile as well as ABA and decreased SA. However, some studies report that SA signaling pathway is a positive regulator, while the JA signaling pathway is a negative regulator in rice resistance to BPH [MKK3 plays a crucial role in the defense of plants to biotic stress. Overexpression of OsMKK3 enhanced rice resistance to X. oryzae through the OsMKK3–OsMPK7–OsWRKY30 pathway, which induced up-regulated genes related with pathogenesis, maintenance of the cell wall, and metabolism [OsMPK3 and OsMPK4 positively regulated rice resistance to C. suppressalis by JA and trypsin proteinase inhibitors, while OsMPK20-5 negatively regulated rice resistance to BPH by ethylene and NO [NaWIPK and NaSIPK act as positive regulators of M. sexta-induced expression levels of NaWRKY6 and CDPKs as well as accumulation of phytohormones  and defense metabolites  [OsMKK3 may activate downstream MAPKs and WRKYs, causing accumulation of phytohormones and defense metabolites to enhance rice resistance to BPH. However, the mechanistic details about how OsMKK3 influenced signaling pathways in resisting BPH are largely unknown and should be studied further. In the light of our results, it is worth discussing the interesting facts revealed by other authors. In line with previous studies, we confirmed that MKK3 plays a pivotal role in signaling pathways. For instance, in mulation . Likewisesponses . A simillmonella . These f cascade ,54,55. Ibidopsis , OsMKK3  in rice . This po defense ,56. Numemulation , and in mulation . On the  in rice ,59. Therbidopsis ,60. We sopposite ,62,63,64opposite ,46. There to BPH ,47,65,66tabolism . Moreovee and NO ,29,30. Nlatiles) . Taken tOsMKK3, we noticed its involvement in plant growth and chlorophyll content in this study. Overexpression of OsMKK3 impaired plant growth compared with wild-type plants, i.e., shortened plant height and root length, and increased chlorophyll content  were pre-germinated and grown in an incubator at 28 ± 2 °C with a 14-h light period. Seedlings (10 days) were shifted to hydroponic boxes (20 L) containing nutrient media and placed in growth room . After 25–30 days, seedlings of good health and with consistent growth were transplanted in 300-mL hydroponic jars with nutrient solution. Four days later, plants were used for experiments.Seeds of wild-type rice  and two overexpressed A BPH colony was collected from rice fields in Zhejiang province and reared on BPH-susceptible rice variety TN1 under favorable conditions .OsMKK3. The forward and reverse primers MKK3-F (5′-AGGTCCGCCCTATCCAGC-3′) and MKK3-R (5′-TAACTGCAAACAAAGCAATCCCTAG-3′) were designed according to the OsMKK3 (Os06g27890) sequence. In pMD19-T vector , PCR products were cloned and sequenced.PCR was used to amplify the cDNA of OsMKK3 full-length cDNA was cloned with pCAMBIA-1301 vector. Rice variety Xiushui 11 was transformed with this vector by using Agrobacterium. Using GUS staining and southern blot, homozygous T2 transgenic plants were screened and confirmed [To prepare an overexpression construct, onfirmed . T2 homoFor mechanical injury, 2-cm-long basal portions of leaf sheaths were separately wounded for 200 pricks with a needle. Non-wounded plants were considered control for comparisons (Con). For recording responses to BPH attack, each plant covered with a glass cylinder was individually attacked with 15 gravid female BPHs. Non-infested plants were considered as the control for comparisons (Con). For the treatment of methyl jasmonate (MeJA) and SA, each plant absorbed the nutrient solution which was mixed with the ethyl alcohol (70%), dissolving MeJA (100 μM) and SA (100 μM), respectively. Plants supplied with ethyl alcohol (70%) only were used as controls (BUF). The experiment was performed with six replications.OsMKK3 (Os06g27890) together with an internal standard OsACTIN (Os03g50885) for normalizing concentrations of all the sample cDNA. Details on OsMKK3 and OsACTIN primers and probes are provided in The SV Total RNA Isolation System was used to isolate plant total RNA, with subsequent reverse transcription to cDNA by CFX96 Real-Time system . We performed qRT-PCR for The plant height and root length were measured with the help of a ruler. Plant height and root length of transgenic plants  and wild-type plants (WILD) were measured at 0, 15 and 30 days (d) after transfer to hydroponic boxes. For the determination of chlorophyll content, 30-days-old three-identical-position leaves on each plant were selected. Chlorophyll was measured by the chlorophyll meter SPAD-502 Plus. Thirty replications of each rice line at each growth stage were performed for this experiment. OsMKK3 on BPH preference for feeding and oviposition, individual plants  were confined in a glass jar. Gravid females BPH (15) were released in every jar and number of females BPH was recorded on every plant after 1, 2, 4, 8, 12, 24, 48, and 72 h. After 72 h of female BPH infestation, every plant was dissected and observed under microscope for recording the number of eggs. Data were collected in 10 replicates.To observe the impact of overexpressing Survival rates and developmental duration were recorded to assess impact of transgenic plants on BPH nymphs. Fifteen BPH nymphs (newly hatched) were released on each plant  confined with a glass cylinder. Until their final emergence as adults, all the surviving nymphs were counted and recorded each day. Post-emergence, the BPH nymph survival rate and developmental duration were calculated for each plant. The experiment of each rice line was replicated 10 times.To compare the difference of the hatching rate of BPH eggs between transgenic plants and wild-type plants, 10 gravid female BPHs were allowed to lay eggs on each plant for 24 h. The numbers of BPH nymphs (newly-hatched) on transgenic plants  and wild-type plants (WILD) were noted until the total absence of nymphs. Plant was dissected under microscope and the number of unhatched eggs was recorded to evaluate the hatching rate. The experiments for each rice line were replicated 15 times.Every plant was infested by 15 gravid BPH females confined in a covered glass jar. We harvested the sheaths of non-infested plants as 0 hpi. The sheaths were collected at 3, 8, 12, 24, and 48 hpi. After grinding the samples in liquid nitrogen, we extracted the JA, JA-Ile, and ABA as well as SA of samples by ethyl acetate, which contained their labelled internal standards, through vortex, centrifugation, and concentration. Then we analyzed their contents by an HPLC-MS-MS according to Lu et al. . Six repEvery plant with a closed glass jar received 15 gravid BPH females. The production of ethylene was measured at 24, 48, and 72 hpi by GC-MS based on the method of Lu et al. . Ten rep2O2 of samples by cold water through vortex and centrifugation. Then, we determined H2O2 concentrations of all the samples by the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit . A standard curve  was used to normalize the H2O2 concentration of every sample. Six replications of each rice line at each time point were performed for this experiment.Every plant was infested by 15 gravid BPH females covered with a glass cylinder. We harvested the sheaths of non-infested and infested plants at 0 hpi. At 8, 12, and 24 hpi, we ground the sheaths in liquid nitrogen and extracted the HOsMKK3 expression level, plant height, root length, chlorophyll, and BPH hatching rate as well as the level of phytohormones and H2O2 between oe-MKK3 and wild-type plants by one-way ANOVA with PASW statistics software. Meanwhile, we also analyzed the differences in means of OsMKK3 expression levels between controls and treated plants by Student’s t-test with PASW statistics software.We analyzed the differences with respect to OsMKK3 is a positive regulator of BPH-induced rice defense through the JA, ABA, and SA signaling pathways, whereas it is a negative regulator of rice growth. This provides new evidence for the crucial role of MAPKs in plant defense against herbivores as well as a theoretical basis for controlling pests in the fields.We conclude that


Mitochondrial sirtuins (Sirts) control important cellular processes related to stress. Despite their regulatory importance, however, the dynamics and subcellular distributions of Sirts remain debatable. Here, we investigate the subcellular localization of sirtuin 4 (Sirt4), a sirtuin variant with a mitochondrial targeting sequence (MTS), by expressing Sirt4 fused to the superfolder green fluorescent protein (Sirt4-sfGFP) in HeLa and pancreatic β-cells. Super resolution fluorescence microscopy revealed the trapping of Sirt4-sfGFP to the outer mitochondrial membrane (OMM), possibly due to slow mitochondrial import kinetics. In many cells, Sirt4-sfGFP was also present within the cytosol and nucleus. Moreover, the expression of Sirt4-sfGFP induced mitochondrial swelling in HeLa cells. In order to bypass these effects, we applied the self-complementing split fluorescent protein (FP) technology and developed mito-STAR , a tripartite probe for the visualization of Sirt4 distribution between mitochondria and the nucleus in single cells. The application of mito-STAR proved the importation of Sirt4 into the mitochondrial matrix and demonstrated its localization in the nucleus under mitochondrial stress conditions. Moreover, our findings highlight that the self-complementation of split FP is a powerful technique to study protein import efficiency in distinct cellular organelles. The sirtuin (Sirt) isoforms Sirt3, Sirt4, and Sirt5 harbor an N-terminal mitochondrial targeting sequence (MTS), responsible for the localization of these enzymes in the lumen of mitochondria . SeveralMitochondria comprise about 1500 proteins, of which the vast majority are nuclear encoded and hence translated by cytosolic ribosomes . DistincReliable methods that enable the investigation of the subcellular protein transport, distribution, and translocation of Sirts in intact living cells are essential to further reveal their involvement in distinct cellular stress responses. Most studies that have aimed to reveal the subcellular distribution of Sirts have applied methods such as immunohistochemistry to label (over)expressed Sirts fused with respective tags ,55,56,5711 fragment is a short peptide, consisting of only 17 amino acids (AAs) [1–10 constructs targeted to both the lumen of mitochondria and the nucleus, respectively. We named the novel tripartite probe mito-STAR, which means, in long form, mitochondrial sirtuin 4 tripartite abundance reporter. Our data demonstrate that the tripartite probe is suitable for the visualization of imported Sirt4 in both the mitochondrial matrix and nucleus upon the induction of mtUPR.Here, we fused Sirt4 with a very fast-maturing FP variant, the superfolder GFP (sfGFP) , which eds (AAs) , which wEscherichia coli (E. coli) cells were obtained from New England Biolabs . Agar−Agar Kobe I, CaCl2, D-Glucose, HEPES, KCl, MgCl2, NaCl, NaOH, Trypton/Pepton, and yeast extract were purchased from Carl Roth . Agarose was purchased from VWR International . Oligomycin A and antimycin were purchased from Sigma Aldrich . The cells were imaged in a 2 Ca-buffer (2CaB) containing, in mM, 2 CaCl2, 140 NaCl, 5 KCl, 1 MgCl2, 1 HEPES, and 10 d-Glucose, where the pH was adjusted to 7.4 for all .Cell culture materials were purchased from Greiner Bio-One . For plasmid expression, chemically competent 10-beta 1–10, Sirtuin 4-sfCherry211, and MTS-sfCherry21–10-SC-Sirt4-sfCherry211-SC-sfCherry21–10-NLS. MTS refers to the mitochondrial targeting sequence, using an N-terminal tandem repeat of the COXVIII; 2A peptide refers to the self-cleavage peptide; NLS refers to the indication of a 2A self-cleavage peptide; NLS refers to the nuclear localizing sequence as a repetition of three times [Cloning was performed according to standard cloning protocols provided by the manufacturer, and the final plasmid was verified by sequencing . Primers for cloning were purchased from ThermoFisher Scientific . PCR reactions were performed using Herculase II fusion DNA polymerase . Mt-sfGFP was achieved using an N-terminal tandem repeat of the COXVIII targeting sequence . SfGFP-Nee times .−1 penicillin, and 100 mg·mL−1 streptomycin INS-1 832/13 (INS-1). The cells were cultivated in Gibco RPMI 1640 media (ThermoFisher). The cells were transfected at a confluency of 60–70%, in 30 mm imaging dishes  and on 1.5 H high-precision glass cover slips , using the transfection reagent PolyJet  24 h prior to measurement. PolyJet was incubated for 24 h and the cells were transfected with 1 µg of DNA. All cells were maintained in a humidified incubator . All experiments were performed 24 h after transfection.HeLa cells were grown in DMEM (Sigma Aldrich), containing 10% FCS, 100 U·mL2, 135 NaCl, 5 KCl, 1 MgCl2, 1 HEPES, 2.6 NaHCO3, 0.44 KH2PO4, 0.34 Na2HPO4, 10 d-glucose (Roth), 0.1% vitamins, 0.2% essential amino acids, and 1% penicillin/streptomycin (Gibco), all at pH 7.4. The cells were then incubated in a loading buffer containing either 0.2 µM MitoTrackerTM Green FM (MTG) or 0.2 µM MitoTrackerTM Red FM (MTR) for 20 min. Afterwards, the cells were washed three times with a loading buffer and imaged in 2CaB. MTR was imaged with an excitation of 561 nm, and emission was captured at 644 nm. MTG was excited at 488 nm and the emission was captured at 516 nm. Tetramethylrhodamine methyl ester  loading was done likewise for 40 min with a TMRM concentration of 50 nM. TMRM was excited at 550 nm and emissions were caught at 600 nm. TMRM remained on the cells during measurement.For colocalization analysis, the cells were first washed once with a loading-buffer containing, in mM, 2 CaCl1–10, Sirt4-sfCherry211, mito-STAR, or mCherry-TOM22, and stained with MTG, MTR, or TMRM, respectively, and imaged using SIM microscopy or ACLSM, respectively. For colocalization studies, the ImageJ plugin ‘coloc2’ was used to measure the Pearson correlation coefficient.The cells were transfected with Sirt4-sfGFP, mt-sfGFP, seCFP-OMP25, MTS-sfCherry2For depolarization, the mitochondria cells were treated with 10 µM of oligomycin A and 5 µM of antimycin A. Incubation of the cells with oligomycin A and antimycin A was done 8 h after transfection and lasted overnight prior to measurements.3D analysis and models of all probes were predicted with the online tool Phyre2  , based on a Zeiss Observer Z.1 inverted microscope, equipped with a Yokogawa CSU-X1 Nipkow spinning disk system, a piezoelectric z-axis motorized stage , and a CoolSNAP HQ2 CCD Camera . The probes were excited at 488 nm and 561 nm, respectively and emission was captured at 516 nm and 644 nm, respectively, using a 100× objective. Data acquisition and control were done using the VisiView Premier Acquisition software .The SIM setup consisted of a 405-, 488-, 515-, 532- and a 561-nm excitation laser. A CFI SR Apochromat TIRF × 100-oil (NA 1.49) objective was mounted on a Nikon Structured Illumination Microscopy (N-SIM) System, with standard wide-field and SIM filter sets, and was equipped with two Andor iXon3 EMCCD cameras mounted to a two-camera imaging adapter , in order to achieve super-resolution. For calibration and reconstruction of the SIM images, ImageJ , Bethesda, MA, USA) and Metamorph (Molecular Devices) were applied. Laser adjustment was checked by projecting the laser beam through the objective at the top cover of the bright field arm of the microscope prior to each measurement. This process was automatically run by the NIS-Elements Advanced Research (AR) . The cells were transfected using PolyJet with the respective constructs and stained, if necessary, with MTG and/or MTR, respectively .p), minor (x), and major (y) axes of the mitochondria, the ImageJ particle analyzer was used. Aspect ratio (AR) was defined as For the evaluation of mitochondrial area (a), perimeter (For the determination of the form factor (FF), the following formula was applied: Image analysis was performed with the freeware program ImageJ.t-test or a one-way ANOVA with a Bonferroni post-hoc test. The number of independent experiments is indicated in the figure legends. The total number of measured cells is also indicated in the figure legends.The obtained data were analyzed using GraphPad Prism 5 Software  and Excel . For image analysis, MetaMorph (Molecular Devices) and the freeware program ImageJ  were applied. To calculate the ratio values of the nuclear fluorescence to the cytosolic fluorescence or the mitochondrial fluorescence, respectively, first, the ACLSM images were background subtracted on MetaMorph using a background region of interest (ROI). Each further step was performed with MetaMorph. For the determination of the cytosolic and the nuclear fluorescence, region measurement was performed by drawing a region randomly in the cytosol or by bordering the complete nucleus. If complete nucleus bordering was not possible, for instance, because of mitochondrial structures, a region measurement was done by randomly picking a position in the nucleus. For the evaluation of the mitochondrial fluorescence, an auto threshold for light objects in MetaMorph was used to set a mask. The threshold was manually set to encompass mitochondrial structures. Hence, the average mitochondrial fluorescence within the mask was determined. We applied GraphPad Prism 5 for the statistical analysis, using either an unpaired double-sided Student’s Nucleus/FMitochondria ≥ 0.33, denoted by gray areas, which were defined as areas of significant extra-mitochondrial localization; First, we investigated the subcellular localization of Sirt4-sfGFP  in livinTM Red FM, which integrates into the inner mitochondrial membrane (IMM) [n  =  30/59 cells/mitochondria), indicating their colocalization to the OMM. Moreover, Sirt4-sfGFP neither merged with the red fluorescence of TMRM of the IMM  , resulte the IMM c,d, nor  the IMM e,f. Thes the IMM .Calculation of the form factor and aspect ratio, as the ratio between the major (height) and minor (width) axes of the ellipse equivalent to the mitochondrion, revealed that both the form factor and the minor of mitochondria in cells expressing Sirt4-sfGFP were significantly higher compared to cells expressing mt-sfGFP . This an11, in which a 17-AA short β-barrel strand of sfCherry2 was fused to the C-terminus of Sirt4 , using an approved nuclear localizing sequence (NLS) in a three-time repetition [1–10, Sirt4-sfCherry211, and SfCherry21-10-NLS, we exploited the P2A peptide, which has a strong degree of self-cleavage activity . In 70% of INS-1 cells, we observed an exclusive mitochondria-located fluorescence signal, indicating an efficient import of Sirt4-sfCherry211 into mitochondria  under conditions of mitochondrial stress, this dramatically increased the Sirt4 level in the nucleus. These results again support the idea that Sirt4 contributes to mitochondria-to-nucleus retrograde signaling. Moreover, our data emphasize that this technique is useful to study the involvement of other proteins, such as Sirt3 and Sirt5, in the retrograde signaling pathway with high resolution. We also highlight that this approach is applicable to screen compounds that target mitochondrial protein import, such as stendomycin [Using high and super-resolution fluorescence microscopy, we have proven that mitochondria located Sirt4-sfGFP integrates into the OMM. This finding is unexpected, in light of the many other studies that have demonstrated the localization and function of overexpressed Sirt4 within the mitochondrial matrix ,60,87. Indomycin .In summary, our work unveils that sfGFP fused to mitochondria targeted proteins, in combination with (super) high-resolution microscopy, could be exploited to identify mitochondrial stress responders exhibiting slow and poor mitochondrial importation activity. Moreover, we introduced a novel expandable tripartite probe, which bypasses the rather peculiar effects of sfGFP fusion, enabling the quantification of the distribution of a protein of interest between two distinct compartments by fluorescence microscopy.


Glucocorticoids are steroid hormones that regulate inflammation, growth, metabolism, and apoptosis via their cognate receptor, the glucocorticoid receptor (GR). GR, acting mainly as a transcription factor, activates or represses the expression of a large number of target genes, among them, many genes of anti-inflammatory and pro-inflammatory molecules, respectively. Transrepression activity of glucocorticoids also accounts for their anti-inflammatory activity, rendering them the most widely prescribed drug in medicine. However, chronic and high-dose use of glucocorticoids is accompanied with many undesirable side effects, attributed predominantly to GR transactivation activity. Thus, there is a high need for selective GR agonist, capable of dissociating transrepression from transactivation activity. Protopanaxadiol and protopanaxatriol are triterpenoids that share structural and functional similarities with glucocorticoids. The molecular mechanism of their actions is unclear. In this study applying induced-fit docking analysis, luciferase assay, immunofluorescence, and Western blot analysis, we showed that protopanaxadiol and more effectively protopanaxatriol are capable of binding to GR to activate its nuclear translocation, and to suppress the nuclear factor-kappa beta activity in GR-positive HeLa and HEK293 cells, but not in GR-low level COS-7 cells. Interestingly, no transactivation activity was observed, whereas suppression of the dexamethasone-induced transactivation of GR and induction of apoptosis in HeLa and HepG2 cells were observed. Thus, our results indicate that protopanaxadiol and protopanaxatriol could be considered as potent and selective GR agonist. Glucocorticoids (GCs) are steroid hormones which exert their actions via binding to their cognate receptors, the glucocorticoid receptors (GRs). Glucocorticoid receptors belong to the large superfamily of nuclear receptors that act mainly as transcription factors . GCs conDue to the crucial role of GCs in the regulation of human physiology and especially due to their strong anti-inflammatory, immunosuppressive and tissue-specific apoptotic activities, GCs are widely used for the treatment of acute and chronic inflammatory diseases, autoimmune diseases ,12, as wOn the other hand, the supra-physiological doses and chronic use of exogenous GCs, for medical purposes, often gives rise to many undesirable side effects, including diabetes induction, muscle wasting, and fat redistribution, effects associated with the GCs-induced gluconeogenesis and activation of catabolic actions such as lipolysis and protein degradation ,18,19. TThus, there is a high need for potent selective steroidal or non-steroidal glucocorticoid receptor agonist (SEGRAs) or modulators (SEGRMs) that possess the desirable anti-inflammatory, immunosuppressive or anti-cancer activities of the classic GCs, but exert no or reduced side effects ,22,23,24S) and 20(R)-type structures, depending on the position of the chiral carbon substitution [S)-type protopanaxadiol) and the Re, Rf, Rg1, Rg2, Rh1 (the 20(S)-type protopanaxadiol [Triterpenoids, that share structural similarities with GCs proved to be potent and selective glucocorticoid receptor modulators. Experimental observations showed that triterpenoids, such as avidin D , echynoctitution  . NevIn this study the efficacy of the PPD and PPT compound to exert dissociative glucocorticoid like activity was examined in human HeLa, HEK293 and HepG2 cells, via assessment on their effect on GR potential binding, nuclear translocation, regulation of GR transactivation and transrepression activities and induction of apoptosis.Structural similarities of PPD and PPT with the natural cortisol and the synthetic corticosteroid, dexamethasone (DEX) , prompte2 and the C-12 hydroxyl O atom. This frees the C-12 hydroxyl H atom to form an intra-molecular hydrogen bond with the C-20 hydroxyl but it also closes to hydrogen bonding distance with the Asn564 (H3) sidechain carbonyl (an interaction present in the GR-DEX complex through its C-11 hydroxyl). For PPT, this hydrogen bond interaction with the Asn564 amide C=O is present through its C-20 hydroxyl group. The C-20 hydroxyl group of PPD, however, is not involved in any hydrogen bond interactions although it is close to, but not within hydrogen bonding distance of the previously mentioned flipped sidechain C=O of Gln642 (H7). Lastly, the C-6 hydroxyl of PPT, absent for PPD, forms weak hydrogen bond contact with the Met601 (H5) sidechain S atom. The tail group –CH2CH2CHC(CH3)2 of both PPD and PPT ligands is orientated between H7 and H11, and with no bad contacts.The predicted binding interactions of PPD and PPT are shown in Overall, therefore, although the hydrogen bond networks formed by PPD and PPT hydroxyls have differences, interactions with key residues for translation are observed similar to DEX, retaining helices such as H3 in it canonical position and exploiting other favorable hydrogen bond interactions with key residues in H5 (Arg611) and H7 (Gln642) . A thorocrit = 2.7; DFbetween groups = 3; DFwithin groups = 106; DFtotal = 109, p < 0.001). 10 μM of PPD and PPT caused approximately 50 and 60 %, respectively, nuclear localization of GR, compared to the 85 and 40 % nuclear GR localization of the DEX-induced and vehicle-treated HeLa cells, respectively .Structural similarities of PPD and PPT with corticosteroids as well as results from the induced-fit docking analysis , showingectively . In ordeectively . Quantifectively . Statistcrit = 4.0; DFwithin groups = 75; DFtotal = 80, n > 10, p < 0.001, analysis of the results by two way ANOVA followed by Tukeys’ post-hoc test). Interestingly, PPT and PPD showed a suppressive effect against the DEX-induced GR transactivation. Incubation of the cells with DEX when combined with PPD and PPT showed approximately 20% inhibition of the DEX-induced GR transactivation. . In addition, evaluation of the effect of the PPD and PPT compound on the GR target, gluconeogenic phosphoenolopyruvate carboxykinase (PEPCK) enzyme [PPD- and PPT- induced GR nuclear translocation prompted us to evaluate the effect of both compounds on GR transcriptional activation. Luciferase reporter gene assays, using a MMTV-GRE (Mouse Mammary Tumor Virus-Glucocorticoid Response Elements) promoter-driven luciferase construct , reveale) enzyme  showed dcrit = 4.5; (B) F = 78.1; Fcrit = 3.9; (C) F=276.4; Fcrit = 4.5, p < 0.001), whereas DEX caused statistically significant suppression of the TNF α - induced transcriptional activity of NF-κΒ, by 40–55 %. A similar effect was also observed by PPD and PPT ((A) F = 35.2; Fcrit = 3.2; DFwithin groups = 16, DFtotal =23; (B) F = 10.9; Fcrit = 2.7; DFwithin groups = 92; DFtotal = 99; (C) F = 4.4, Fcrit = 3.2; DFwithin groups = 16; DFtotal = 23). PPD suppressed the TNF α-induced NF-κΒ activity by 28–45%, whereas PPT exerted 38–65 % inhibition. Our results indicate that increase in PPD and PPT concentration from 1 μΜ to 10 μM did not substantially strengthen their inhibitory activity. Evaluation of the anti-inflammatory effect of 10 μM of PPD and PPT, via transcriptional inactivation of the TNF α-activated NF-κΒ, was not observed in GR low level COS-7 cells ; F = 0.8, Fcrit = 3,3 (Treatment); F = 2.0, Fcrit = 3.2, (TNF/Treatment); DFwithin groups = 16, DFtotal = 23); “Treatment” corresponds to treatment either with vehicle, DEX, PPD or PPT), providing circumstantial evidence for the involvement of GR in the PPD- and PPT-induced NF-κΒ transcriptional inactivation. Cytotoxicity analysis in HeLa cells, applying the sulphorodamine B assay [Anti-inflammatory activity of PPD and PPT was assessed via evaluation of their actions on NF-κB activity. Thus, effect of PPD and PPT on the NF-κΒ activity was assessed in GR-positive HeLa A,B and H-7 cells D P2/Akt) pathway, signal transducer and activator of transcription 5/peroxisome proliferator-activated receptor gamma coactivator 1-alpha (STAT5/PPARgamma) pathway, phosphoinositide-3 kinase/protein kinase B (PI3K/Akt) pathway, AMP-activated protein kinase/c-Jun terminal NH2-terminal kinase (AMPK-JNK) pathway, NF-κB pathway, endoplasmic reticulum stress, inflammatory, antioxidant and apoptotic pathways, glucose import and glucose metabolism pathways . NeverthDue to the structural similarities of ginsenosides with steroid hormones they could be considered as potential modulators of steroid hormone receptors. Accumulating evidence indicate that ginsenosides are involved in the regulation of nuclear receptors . Thus, iOur data from induced fit docking analysis showed that the aglycone form of the PPD- and PPT- type compound is capable of binding to GR ligand binding domain. In fact, comparable binding strengths to DEX are predicted as well as similar interactions with GR LBD helices/residues, previously cited as important for nuclear translocation ,36. As w−7 M, has also been reported in various types of cells, including HeLa cells [PPD- and PPT- induced nuclear translocation was not accompanied by GR transactivation, as indicated by the results from the GRE-driven luciferase reporter gene expression assay, showing no PPD- and PPT- induced GRE driven luciferase gene expression. Nevertheless, both compounds were capable of suppressing the DEX-induced GR transactivation. Moreover, PEPCK protein level were considerably decreased in HeLa cells, cultured in hormone-free medium, and treated with PPT or PPD in combination or not with DEX. In addition, moderate reduction in PEPCK protein levels by PPT, but not by PPD, was observed in HepG2 cells. Since liver is the main gluconeogenic organ, the discrepancy of the results in the two cell lines may be attributed to the more pronounced gluconeogenetic properties, of the hepatocarcinoma HepG2 compared to that of the HeLa cells, relied on the differential expression of GR and its co-activators or co-repressors, as well as, to the differential recruitment of these factors in the GR transcription complex, triggered by binding of PPD or PPT to GR . Our obsLa cells ,56,57,58La cells , howeverThe antidiabetic-hypoglycemic and anti-inflammatory activity of PPD- and PPT- type ginsenosides has been known for a very long time. The molecular mechanisms underlying these actions are unclear and under investigation. Thus, multiglycosylated PPD- and PPT-type ginsenosides, such as Rb1 and Rg1, respectively, have been shown to exert antihyperglycemic activity via induction of increase in insulin sensitivity and insulin uptake ,61, suppEven though PPD and PPT did not induce GR transactivation, both compounds were capable of suppressing the TNF-α -induced NF-κB activity in HeLa- as well as in HEK293- GR positive cells. 1 μM of the compounds were approximately as effective as 10 μM to suppress the TNF-α -induced NF-κB activity in HeLa cells, in agreement with results from fluorescence microscopy analysis, showing that concentrations higher than 1 μΜ of the compounds are effective enough to trigger the GR nuclear translocation. Similar effects have also been observed by the synthetic glucocorticoid, DEX. Thus, the ability of DEX to fully activate GR nuclear translocation and transcriptional activation, in a broad range of concentrations, varying from nM to μΜ, has been reported ,65. FurtThe suppressive effect of PPD and PPT on NF-κΒ activity in combination with their inability to induce GR transactivation renders them potential promising lead molecules for the development of novel potent drugs with anti-inflammatory activity but with no or reduced side effects. Moreover, the possible additive effect of the compound when administered in combination with other GR modulators, that also exert GR transrepression activity, may be of significant pharmacological importance. Thus, co-administration of the PPD and PPT compounds with RU486, the well-known GR antagonist which also exert immunosuppressive actions, through suppression of NF-κB, AP1 activity and cytokines production ,67,68, cIn line with our observations, multiglycosylated PPD- and PPT-type ginsenosides such as Rb1 and Rg1 have been shown to exert anti-inflammatory activity, through (a) reduction in pro-inflammatory cytokines and mediators, including TNF-α, interleukins IL-6, IL-1β, (b) regulation of ΝF-κB and JNK signaling pathway, and (c) increase in nitric oxide synthesis and release ,70,71. GGlucocorticoids are also well known for their tissue and cell type-specific pro-apoptotic actions  renderinIn this study we showed that PPD and PPT compound exert apoptotic activities, which are probably mitochondrial-mediated as indicated by the activation of caspase 9 and 3 upon PPT and PPD incubation of HeLa and HepG2 cells. In addition, co-incubation of HepG2 cells with DEX and the PPD or PPT compound caused increase in procaspase 9 indicating, cell type-specific interference of PPD and PPT compounds with GR signaling. The observed induction of apoptosis is indicated to be at early stage, since reduction in procaspase 9 protein level is higher than that of the pro-caspase 3, in the case of HepG2 cells, and also, no statistical significance inhibition of HeLa cells growth by the PPD and PPT compound was observed, at this time point. The exact mechanism of induction of apoptosis remains to be elucidated. However, it is possibly related to the steroid-like activities of the compounds.To conclude, in this study, we provide evidence that glucocorticoid receptor may constitute, among others, a potential molecular target for PPD and PPT compounds, to drive their pharmaceutical actions. Thus, we showed that PPD and PPT compound can effectively bind to GR and activate its nuclear translocation. Activated GR exert transrepression activity, suppressing NF-κB activation, whereas no transactivation of GR was observed. Moreover, reduction in PEPCK expression by PPD and PPT was observed, possibly due to the induced reduction in GR protein levels. To our knowledge this is the first study to describe potent anti-hyperglycemic, anti-inflammatory actions and apoptotic activities of the aglycone form of the PPD- and PPT-type ginsenoside, revealing also potential dissociative activity of PPD and PPT compound on GR transcriptional activation. Although further analysis is required to delineate the exact mechanisms of actions and interfering of PPD and PPT with GR signaling, our study contribute to the establishment of new lead molecules for the development of selective glucocorticoid receptor agonists with potential for therapeutic use as anti-inflammatory and anticancer drug, with reduced side effects.Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and lipofectamin 2000 were obtained from Invitrogen . Molecular weight protein markers were from Fermentas  complete protease inhibitors cocktail were purchased from Roche  and TNFα was purchased from Immuno Tools  All other chemicals including dexamethasone (DEX) were purchased from Sigma-Aldrich . PPD and PPT were purchased from Extrasynthese, Genay Cedex, France.The GR-H300 affinity purified polyclonal GR antibody, which recognizes an epitope corresponding to amino acids 121-420 of human GR, and was commercially provided by Santa Cruz Biotechnology, was used. Monoclonal antibodies against β-actin  and caspase 9 , and rabbit polyclonal antibodies against phosphoenolpyruvate carboxykinase (PEPCK) , caspase 3 , Bcl-2  were also applied.2 humidity. 48–72 hrs before treatment, cells were cultured in phenol red free-DMEM medium supplemented with 10% charcoal-dextran- stripped FBS , 2 mM glutamine, and 100 Units/mL penicillin/streptomycin.The human hepatocarcinoma HepG2 and HEK293 cells were obtained from the American type culture collection (ATTC). HeLa and COS-7 cells were a kind gift from Dr. M Alexis, from the National Hellenic Research Foundation (NHRF) Greece. All cell lines were maintained in DMEM, supplemented with 10% FBS, 2 mM glutamine, and 100 Units/mL penicillin/streptomycin. Cells were cultivated at 37 °C in 5% COCells grown on coverslips, in DMEM hormone-free medium  for 48 hrs. Cells were then transiently transfected with a pEGFPC2GR construct . Brieflywww.berthhold.com). Relative luciferase activity was expressed as normalized luciferase activity against β-galactosidase activity (RLU).NF-κB and GR transcriptional activity was measured applying luciferase reporter gene assay. Briefly, HeLa cells grown on 24-well plates were co-transfected, using Lipofectamin 2000, with a NF-κB-Luciferase (NF-κB-Luc) (for assessment of NF-κB activity), or an MMTV-GRE (Glucocorticoid response elements of the murine mammary tumour virus DNA) promoter-driven luciferase construct (GR-Luc reporter gene construct) (for assessment of GR activity) and a β-galactosidase reporter construct ,37. The Cells grown on 6 well plates, for 48 hrs in hormone depleted medium, were incubated for additional 48 hrs with 10 μΜ of PPD, 10 μΜ of PPT and/or 1 μΜ DEX. Cells were washed in PBSX1, lysed in buffer A  supplemented with cocktail preotease inhibitors . After Bradford protein determination , cell ext-test or by One-Way analysis of variance (ANOVA)   or Two-W (ANOVA)  followedhttps://pubchem.ncbi.nlm.nih.gov/). The GR protein was prepared for docking using the solved crystallographic complex (PDB code: 3BQD) with deacylcortivazol, exploiting its more open binding pocket for the study of bulkier ligands [O-glucoside derivative [The ligands  were prepared for docking calculations using LigPrep v3.6  with the ligands ,80, and rivative .a values at pH 7. Subsequent optimization of hydroxyl groups, histidine sidechain C/N atom “flips” and protonation states, and side-chain O/N atom flips of Asn and Gln was based on optimizing hydrogen bonding patterns. Finally, an “Impref” minimization was performed using the OPLS3 force field [The initial setup of the GR structure for calculations was performed using Schrodinger’s “Protein Preparation Wizard” . Water mce field  to removIFD calculations  consiste


The results showed that TP53, PIK3CA, APC, and EGFR were the most frequently mutated genes. Stage IV patients had significantly higher ctDNA concentration than Stage I patients, and increased ctDNA concentration correlated with increased tumor size. Additionally, ctDNA detection was found to be a greater predictor of disease when compared with five known commonly used tumor biomarkers. The present study supports the use of ctDNA as a liquid biopsy to gain clinical tumor information that may facilitate early diagnosis and treatment and improve CRC patient prognosis.Characterization, diagnosis, and treatment of colorectal cancers (CRC) is difficult due to limited biopsy information, impracticality of repeated biopsies, and cancer biomarker fallibility. Circulating tumor DNA (ctDNA) has recently been investigated as a non-invasive way to gain representative gene mutations in tumors, in addition to monitoring disease progression and response to treatment. We analyzed ctDNA mutations and concentrations in 47 early- and late-stage CRC patients using a targetted sequencing approach using a panel that covers 50 cancer-related genes. ctDNA mutations in 37 genes were identified in 93.6% of the patients ( Colorectal cancer (CRC), with over 1.3 million new cases worldwide annually, can be difficult to detect at early stages due to non-specific symptoms which may lead to delayed diagnosis . CharactKRAS mutations during anti-EGFR antibody treatment [Recently, circulating tumor DNA (ctDNA) is being investigated as a non-invasive, easily obtainable sample to gain information, such as genetic mutations and tumor burden, in various types of tumors ,8. Some reatment ,10. Othereatment . Despitereatment ,13. Whilreatment ,15.The goal of the present study was to use a highly sensitive targetted sequencing approach to identify ctDNA mutations in a panel of 50 cancer-related genes in 47 early- and late-stage CRC patients. Additionally, we compared ctDNA concentrations with other known cancer biomarkers mutations to analyze if ctDNA is a better predictor for disease, and ctDNA concentrations were compared amongst patients of different cancer stages to investigate, if these factors were correlated.This prospective study with 47 CRC patients was approved by the Department of General Surgery, Beijing Friendship Hospital. All the patients provided appropriate written informed consent for the use of blood under the approval of the Ethics Committee of Department of General Surgery, Beijing Friendship Hospital. All samples and medical data used in the present study were irreversibly anonymized. Blood samples were collected from each patient during surgical tumor resection and traditional serum tumor markers  were measured at the same time.g. Supernatants were further centrifuged for 10 min at 6000 g and plasma was collected and stored at −80°C until further use. At least 1 ml plasma was used to extract ctDNA with the QIAamp Circulating Nucleic Acid kit  following the manufacturer’s instructions. DNA was quantitated with the Qubit 2.0 Fluorometer and the Qubit dsDNA HS Assay kit  using the recommended protocol. ctDNA was sequenced using the SV-CA50-ctDNA panel of 50 genes frequently mutated in CRC to identify candidate mutations for ctDNA analysis. Patients with solid tumors are enrolled in the group’s San Valley database. The SV-CA50-ctDNA panel allows simultaneous detection of hundreds of variants across 50 genes relevant to solid tumors and includes solid tumor genes targetted by on-market oncology drugs and a published evidence, which includes content aligned to approved therapies and indications [EDTA tubes containing blood samples were centrifuged for 10 min at 1600 Ion proton library preparation and DNA sequencing was performed as described in our previous studies . An adapThe Ion OneTouch system and the Ion PI Template OT2 200 Kit v3 (Life Technologies) were used for sample emulsion PCR and emulsion breaking, per the manufacturer’s instructions. After recovering ion sphere particles (ISPs), Dynabeads MyOne Streptavidin C1 beads on the Ion One Touch ES (enrichment system) (Life Technologies) were used to enrich template-positive ISPs, which was confirmed using the Qubit 2.0 Fluorometer. Finally, the Ion PI Sequencing 200 Kit v3 (Life Technologies) was used for sequencing barcoded samples on Ion PI v2 Chips for 100 cycles on the ion proton, as per the recommended protocol.ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, and VHL. This 50-gene panel and 2856-associated mutational hotspot loci was selected based on the Chinese cancer sequence database  that contains sequencing data from more than 30000 cancer patients and 13 different cancer types.The SV-CA50-ctDNA panel  was used to detect somatic mutations in the following 50 cancer-related genes: P -value <0.01. Next, mutations were examined visually using integrative genomics viewer (IGV) software (http://www.broadinstitute.org/igv) or SAMtools software (http://samtools.sourceforge.net) and possible DNA strand-specific errors were removed. Finally, variants were set within the 2856-defined mutational hotspot loci.The ion proton platform-specific pipeline software Torrent Suite was used to process initial sequencing data to generate sequence reads, trim adapter sequences, and filter and remove poor signal-profile reads. Additionally, the Torrent Suite Software with a variant ‘caller v4.0’ plug-in was used to generate initial variant calling, followed by three filtering steps to eliminate faulty base calling and generate final variant calling. First, the following definitions were made: average total coverage depth >10000; each variant coverage >10; variant frequency of each sample >0.1%; and In addition to mutation analysis, plasma samples were analyzed for the tumor biomarkers CEA, AFP, CA19-9, CA125, and CA72-4 using an electrochemiluminescence immunoassay , following the manufacturer’s instructions. The following cut-off values were used to identify samples with elevated biomarker expression: CEA >5.0 ng/ml, AFP >5.4 ng/ml, CA19-9 >37.0 U/ml, CA125 >35.0 U/ml, and CA72–4 >5.3 U/ml. All the measurements were performed in duplicate.P-values using GraphPad QuickCalcs Online Calculator for Scientists . All P-values are two-tailed and statistical significance was defined as P<0.05.Fisher’s exact test was used to calculate TP53, EGFR, APC, PIK3CA, and KRAS genes , and in other genes but at lower frequencies. The number of mutations in these genes per patient, along with patient characteristics, is illustrated in Forty-seven CRC patients were enrolled in the study, the clinical features of them were represented in Amongst all patient samples examined in the present study, we found that the average number of mutated genes per patient was 5.0 , and the average number of total mutations per patient was 9.9 . Three patients  had no mutations in the 50 genes screened and seven patients had only one gene with mutation(s). Remaining 37 patients had two or more genes with mutations  and the average number of mutations per patient in this group was 12.3 . The number of mutated genes and total number of mutations varied slightly between patients of different stages: Stage I had an average of 3.0 mutated genes and 5.0 total mutations per patient; Stage II averaged 4.1 mutated genes and 6.2 mutations per patient; Stage III averaged 6.7 mutated genes and 15.1 mutations per patient; and Stage IV patients had an average of 4.1 mutated genes and 8.0 mutations per patient.TP53. The other commonly mutated genes, PIK3CA, APC, EGFR, and KRAS, were found in 20/47 (42.6%), 16/47 (34.0%), 16/47 (34.0%), and 13/47 (27.7%) of patients, respectively. Approximately 70% of the patients (33/47) had multiple mutations within a single gene, most commonly in TP53 and EGFR. Interestingly, patients with the highest ctDNA concentrations (over 1.0 ng/μl) had fewer mutated genes and nearly half as many mutations per patient when compared with patients with lower ctDNA concentrations .The most common gene with mutations found in 38/47 (80.9%) of the patients was P=0.0149) . Results showed that the concentration of ctDNA increased with cancer stage and a significant difference was observed in the ctDNA concentration from Stage I patients compared with Stage IV patients (=0.0149) A. FigureIn addition to measuring the ctDNA concentration for each patient, we also measured the concentration of several known tumor biomarkers such as CEA, AFP, CA19-9, CA125, and CA72-4. Number of patients with one or more elevated biomarkers increased with cancer stage: 20.0 in Stage I, 68.8 in Stage II, 84.2 in Stage III, and 100.0% in Stage IV patients. TP53 was mutated in 43/47 (80.9%) of patients, and mutations were also frequently detected in EGFR, APC, PIK3CA, and KRAS. One potential advantage of the present study over others is that compared with PCR-based assays to analyze one or two genes, the SV-CA50-ctDNA panel contain the important mutations about CRC treatment sensitively; hence removing the bias present in PCR-based analyses of a few genes.The present study analyzed plasma ctDNA in 47 early- and late-stage CRC patients using targetted sequencing to identify mutations in a defined panel of 50 known cancer-related genes and oncogenes. We found that 93.6% of patients, including all Stage I patients had detectable levels of ctDNA and mutations were identified in 39/50 (78%) genes in the cancer panel. Most patients (80.9%) had multiple mutations within a single gene and/or across multiple genes. Our mutation analysis revealed that KRAS, NRAS, BRAF, and PIK3CA, may lead to drug resistance due to constitutive EGFR activation [PIK3CA mutations identified in ctDNA were found to contribute to acquired cetuximab resistance in patients with metastatic CRC. Recent studies have monitored acquired KRAS mutations in ctDNA during anti-EGFR antibody treatment of advanced CRC patients. These mutations, which render certain drugs ineffective, were detectable in ctDNA prior to radiographic detection of disease progression and decreased when treatment was discontinued [KRAS mutations in CRC patients [PIK3CA, EGFR, KRAS, NRAS, and BRAF. Because of their implications in treatment, a targetted approach that can identify ctDNA mutations in several key genes simultaneously would aid in treatment and potentially improve patient outcome.Many of the mutated genes in our study are significant in a clinical setting and would potentially influence drug treatments. Alterations in genes involved in the pathways downstream of EGFR activation, such as tivation . Xu et ativation  reportedontinued ,21. A repatients . We founConsistent with the previous studies showing that ctDNA levels correspond to tumor burden ,24, we fWe also compared ctDNA concentration with the expression of five clinically relevant biomarkers, including CEA, CA19-9, CA125, CA72-4, and AFP. These biomarkers have been found to be expressed at higher levels in CRC patients prior to surgery and upon disease recurrence, and are associated with more advanced disease . AFP hasIn conclusion, the present study supports the use of targetted sequencing to identify mutations in ctDNA from early- and late-stage CRC patients. Our study is clinically significant as cancer patients may benefit from ctDNA analysis as it is a relatively easy and a non-invasive way to supplement traditional diagnostics with real-time tumor information and exhibit a much greater sensitivity than other tumor biomarkers. Further research with larger groups of patients is required before standardized detection and sequencing methods are established.


Cellular components are attractive targets for antiretroviral therapy because they do not mutate as readily as do viral proteins do –3. The i+ T cells positively correlate with RNA viral loads [in vitro where CCR5 levels inversely correlate with rates of HIV-1 entry inhibition [in vivo [The CCR5 viral coreceptor, one of a family of chemokine receptors belonging to the G protein-coupled receptor family , is expral loads  and progal loads  in untrehibition ,27, espehibition , 22. Thehibition , have gihibition , 30, 31,hibition , CCR5 anhibition , 34, and[in vivo ,29,34,35[in vivo .in vitro synergy with other CCR5 blockers, including CCR5 monoclonal antibodies (mAbs) [in vitro.Several small-molecule CCR5 inhibitors have been developed in the last decade ,38. At ps (mAbs) –35,43,44Two other CCR5 inhibitors reached clinical trial phases, but both were discontinued for the different reasons. Aplaviroc (APL) administration gave significant reduction of plasma HIV-1 RNA copies during the first ten days of treatment , but devCenicriviroc (CVC), an experimental drug candidate for blocking CCR5 receptors, is in the phase III clinical trials . Like MVin vitro [Resistance to MVC has been reported previously –54, and in vitro , but is in vitro .ex vivo by several gene editing technologies, including CRISPR and zinc finger nuclease (ZFN) proteins. Genome editing of the HIV co-receptor CCR5 by CRISPR-Cas9 protects CD4+ T cells from HIV-1 infection [+ T-cells are safe to give to humans and how the procedure would affect HIV-1 status . Another clinical trial on CCR5-modified CD4+ T cells for HIV infection is about to start in mid-December 2018.Lastly, other alternative ongoing efforts on blocking CCR5 function have focused on deleting the CCR5 gene nfection . A complThe success of current cART therapies is limited by the emergence of drug-resistance, potential drug toxicity, the need for sustained adherence and costs. Advances in cART have generally resulted in reduced viral spread, but not in full viral clearance. There are numerous ongoing efforts to explore the most effective ways to intensify standard cART activity –61 and tCCR5 blockers have great therapeutic potential for prevention and treatment of HIV-1 infection and perhaps (and importantly) a reduction of establishment, size, and/or persistence of reservoirs of latent HIV-1. Due to the potential beneficial effects of CCR5 inhibitors, their inclusion in clinical regimens may offer new possibilities for treating HIV-1 infection and associated disease.


The importance of local DNA structures in the regulation of basic cellular processes is an emerging field of research. Amongst local non-B DNA structures, G-quadruplexes are perhaps the most well-characterized to date, and their presence has been demonstrated in many genomes, including that of humans. G-quadruplexes are selectively bound by many regulatory proteins. In this paper, we have analyzed the amino acid composition of all seventy-seven described G-quadruplex binding proteins of Homo sapiens. Our comparison with amino acid frequencies in all human proteins and specific protein subsets  revealed unique features of quadruplex binding proteins, with prominent enrichment for glycine (G) and arginine (R). Cluster analysis with bootstrap resampling shows similarities and differences in amino acid composition of particular quadruplex binding proteins. Interestingly, we found that all characterized G-quadruplex binding proteins share a 20 amino acid long motif/domain (RGRGR GRGGG SGGSG GRGRG) which is similar to the previously described RG-rich domain (RRGDG RRRGG GGRGQ GGRGR GGGFKG) of the FRM1 G-quadruplex binding protein. Based on this protein fingerprint, we have predicted a new set of potential G-quadruplex binding proteins sharing this interesting domain rich in glycine and arginine residues. The discovery of the B-DNA structure in 1953 ,3,4,5. HA tetrameric arrangement of guanines was first described using crystallographic methods in 1962 , and manProtein-DNA interactions are essential for all organisms. Besides proteins that bind to a particular DNA sequence, there is a group of proteins that bind specifically to various local DNA structures, e.g., to single stranded DNA , crucifoAlthough information about G-quadruplex binding proteins has increased rapidly, there are no statistical studies focused on their amino acid composition. Many recent studies used amino acid composition to predict intricate protein functions ,39,40,41http://bsbe.iiti.ac.in/bsbe/ipdb/index.php) ..http://pTo find out if there are significant correlations between particular amino acids within the set of 77 quadruplex binding proteins, we used an amino acid composition matrix of the above 77 quadruplex binding proteins in R package “corrplot” . The comA tree diagram was constructed using the R package “pvclust” . Bootstrhttp://nslab.mbu.iisc.ernet.in/clap/run.html); default parameters were used. CLAP is an alignment free approach that computes local similarities among selected sequences, and allows comparisons of proteins with multiple domains. Such clusters show high functional and domain architectural similarities [All 77 quadruplex binding proteins sequences were uploaded to the CLAP web server , and the FIMO tool [p-value threshold was 0.01. All 2106 hits are listed in All 77 quadruplex binding protein sequences were uploaded to the MEME web server  allows discovery of novel, gapped motifs in protein sequences [To investigate whether the 77 quadruplex binding proteins share a common sequence motif, we performed a GLAM2 search. The GLAM2 web tool  [We used the STRING web server (db.org/) ,68 with p-value threshold was 0.1. The best 100 results were filtered using Excel.The FIMO tool was used for the prediction of new quadruplex binding proteins . AnalysiThe G4IPDB (a database for G-quadruplex structure forming nucleic acid interacting proteins) has been recently established . This daBased on relative enrichment or a depletion of greater than 10% in comparison to the expected values in the human proteome, the most distinctive enrichments were found for glycine (G), arginine (R), lysine (K) and aspartate (D), while the most notable depletions were detected for tryptophan (W), leucine (L), isoleucine (I), histidine (H), cysteine (C) and threonine (T) . Among tp-values are listed in To obtain statistical information about amino acid composition differences, we used the Composition Profiler program. We compared the amino acid compositions of all quadruplex binding proteins with three specific protein groups—first: A random subset of the human proteome (5000 proteins); second: A well-defined group of nucleic acid binding proteins including isoforms , third: Golgi apparatus proteins including isoforms (2565 sequences), all obtained from the UNIPROT Reviewed Database. The relative enrichments or depletions of quadruplex binding protein amino acid compositions in comparison with these protein groups are shown in Differences in amino acid composition compared to a random subset of the human proteome were confirmed A. The la2-mer amino acid composition of quadruplex binding proteins showed that the most abundant is GG, followed by SS, EE, and AA, which are also very frequent in DNA-binding proteins . The maip-value > 0.05) are crossed out.The correlation diagram D demonstA—FUS, ROA1, ROA2, ROA3, HNRPF, TADBP, YBOX1, SFPQ, HNRPK, HNRPR, ILF3, HNRPL; B—EGR1, MYF5, TOPRS, NEIL2, TNR4, MYOD1, p53, MYF6, MYOG, IGF2, TERT, NEIL1, ACD, RECQ4, TINF2; C—NUCL, NPM, TOP1, EBNA1BP2, POTE1, CAMP, NDKB, DHX36, SF3B3, ILF2, NOA1, DHX30, DHX15, DPOE1, ERCC2, EFHD2, ERCC3, TERF1, SAFB1, SAFB2, DNM3B, FMR1, U2AF2, TERF2, DSRAD, SPAST, DNM3A, TE2IP, DNMT1, NEIL3, ADA10, IF16, PARP1, DDX21, POLH, BRCA1, NF2L2, ATRX, BLM, FANCJ, RFA2, RFA1, ELAV1) of proteins supported by approximately unbiased values . Most proteins in group A recognize RNA-quadruplexes, whereas proteins in group B are DNA-quadruplex binding proteins, suggesting that amino acid residue composition is important for distinguishing between DNA and RNA quadruplex binding proteins, and implying that different molecular mechanisms of recognition may be involved in various groups of quadruplex binding proteins. Group C contains proteins that bind only DNA or only RNA quadruplexes, or bind to both DNA and RNA quadruplexes. Three proteins, CNBP, VEGFA, and MAZ, were not located in these clusters, and will be discussed below.To compare the relationship of quadruplex binding proteins according to their amino acid composition, we used statistical clustering  using GLAM2 software ,65. Our Interestingly, this motif is formed almost exclusively by R and G amino acid residues with a few alternations of S. Using the UGENE software, we display an overlay of this sequence for individual proteins .q ≤ 0.05 significance, 8 proteins with q ≤ 0.1 significance and 14 proteins with q > 0.1 significance  to a motif derived from the FMR1 protein. The direct interaction of FMR1 protein with quadruplex DNA has recently been shown . The cryificance . The aliWe also found that many quadruplex binding proteins contain two or more NIQI motifs, often repeated two or more times in the same region of the protein . DHX36 aq ≤ 0.1 significance. Besides already known quadruplex binding proteins, we found proteins with highly significant NIQI motifs, from which we selected 100 proteins with the best match to the NIQI motif, suggesting that these proteins could be novel quadruplex binding proteins  and depletions . We discovered a new 20 amino acid motif (termed NIQI) common to human quadruplex binding proteins, comprised mainly of glycine and arginine residues. Based on this model, we identified additional human proteins that contain highly homologous sequences, and which are therefore potential quadruplex binding proteins. We expect that our findings will contribute to the identification and characterization of G-quadruplex binding proteins, and to the development of tools for the optimization of proteins, peptides, or other small molecules that bind to these structures for potential clinical application.


P < 0.001). Among the cytokines, only IL-6 was significantly elevated from POD 0 to POD 1. In the regression model, the change in IL-6 levels between POD 0 and POD 1  and high malnutrition risk  were significantly associated with changes in fatigue scores. Conclusions: The perioperative changes in plasma IL-6 levels are positively associated with changes in the fatigue scores of GA patients undergoing gastric surgery. Targeting the IL-6 signaling cascade or new fatigue-targeting medications may attenuate perioperative fatigue, and further clinical studies should be designed to validate this hypothesis.Background: Gastric adenocarcinoma (GA), one of the most common gastrointestinal cancers worldwide, is often accompanied by cancer cachexia in the advanced stage owing to malnutrition and cancer-related symptoms. Although resection is the most effective curative procedure for GA patients, it may cause perioperative fatigue, worsening the extent of cancer cachexia. Although the relationship between cytokines and cancer fatigue has been evaluated, it is unclear which cytokines are associated with fatigue in GA patients. Therefore, this study aimed to investigate whether the changes in cytokine levels were associated with the perioperative changes in fatigue amongst GA patients. Methods: We included GA patients undergoing gastric surgery in a single academic medical center between June 2017 and December 2018. Fatigue-related questionnaires, serum cytokine levels -1, IL-2, IL-5, IL-6, IL-12 p70, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor), and biochemistry profiles  were assessed at three time points (preoperative day 0 (POD 0), post-operative day 1 (POD 1), and postoperative day 7 (POD 7)). We used the Brief Fatigue Inventory-Taiwan Form to assess the extent of fatigue. The change in fatigue scores among the three time points, as an independent variable, was adjusted for clinicopathologic characteristics, malnutrition risk, and cancer stages. Results: A total of 34 patients were included for analysis, including 12 female and 22 male patients. The mean age was 68.9 years. The mean score for fatigue on POD 0, POD 1, and POD 7 was 1.7, 6.2, and 3.6, respectively, with significant differences among the three time points ( Worldwide, gastric adenocarcinoma (GA) is among the most common causes of cancer mortality—GA ranks fifth in terms of the incidence  and third in terms of mortality  . TherefoFatigue is one of the most widely reported complaints during perioperative periods and is described as involving tiredness, lethargy, sluggishness, changed emotional state, and a desire to sleep more . PostopeThe pathogenesis of cancer-related fatigue is multifactorial, and many mechanisms may contribute to its development, including anemia, anti-cancer treatment, hormones, cancer progression, or cytokines ,13,14,15Fatigue was associated with cytokine dysregulation  in patients with breast or prostate cancer ,21,22,23Adult GA patients (aged ≥ 20 years) undergoing gastric operations were included in this study between June 2017 and December 2018 at the National Taiwan University Hospital. The exclusion criteria were previous abdominal surgery, inability to communicate verbally and completely fill out the questionnaires, or severe renal or hepatic impairment. This study was reviewed and approved by the Research Ethics Committee of the National Taiwan University Hospital (201705112RINB) and followed all ethical recommendations. All participating patients were informed about the study processes and signed the informed consent form. We used the Brief Fatigue Inventory-Taiwan Form (BFI-T), a scale with 11 points (0 to 10), to assess fatigue . For eacg at 20 °C. Subsequent collection of the serum fraction was performed by using standard serum separator tubes, which were then stored at −80 °C until further use for analysis. Serum levels of IFN-gamma, IL-1, IL-2, IL-5, IL-6, IL-12 p70, TNF-alpha, and GM-CSF were determined by using ProcartaPlex multiplex immunoassays panels, affymetrix , and Luminex 100/200. Furthermore, serum levels of IL-10 cytokine were analyzed via the ELISA kit  according to the manufacturer’s instructions. The cytokines evaluated included serum interferon-gamma (IFN-gamma), interleukin (IL)-1, IL-2, IL-5, IL-6, IL-12 p70, tumor necrosis factor-alpha , and granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, biochemistry profiles included the serum white blood cell (WBC) counts, C-reactive protein (CRP), albumin, and prealbumin. These aforementioned tests were performed on POD 0, POD 1, and POD 7. We obtained early morning, fasted venous blood samples from each patient under aseptic techniques. Serum was isolated via centrifugation at 1000× Clinicopathologic and medical data were obtained by reviewing the medical charts of patients. We collected the following data on the comorbidity of patients before gastrectomy by using the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM codes): anemia (ICD-9-CM: 285.x), myocardial infarction (410.x and 412.x), mild liver disease (571.2 and 571.4–571.6), hyperlipidemia (272.0–272.2), diabetes mellitus , chronic obstructive pulmonary disease , renal failure (584.x–586.x), and hypertension (401.x–405.x). Next, the Charlson comorbidity index (CCI) scores were calculated to determine the baseline comorbidity of each patient . Further2 test or Fisher’s exact test if the numbers were <5. Continuous data were compared using the nonparametric Mann–Whitney U test, and some continuous variables were dichotomized according to predetermined thresholds, which were analyzed as categorical variables. The comparison among the three time-points was performed using the ANOVA test. In addition, we performed linear regression analysis to assess the correlation between changes in fatigue scores and the changes in the cytokine levels after adjusting for clinicopathologic characteristics, CCI scores, and cancer staging. The independent variable was the change in fatigue scores among time points, and the dependent variables were identified according to the bivariate correlations. All statistics were 2-tailed, and differences were considered statistically significant at P < 0.05.In this study, all statistical analyses were performed using STATA version 15 . Data are presented as mean (standard deviation), number (percentage), or coefficients and 95% confidence interval (CI). We compared categorical variables using the χIn this study, we enrolled 34 GA in the analysis , includiP < 0.001; P < 0.001). The mean score of fatigue on POD 0, POD 1, and POD 7 was 1.7, 6.2, and 3.6, respectively. The fatigue score was significantly higher on POD 1 and POD 7 than on POD 0 (both P < 0.001), while there were no significant differences in the levels between POD 0 and POD 7  and between the changes in biochemistry profiles and fatigue scores (P = 0.037) and high malnutrition risk  were positively significantly associated with increased scores for perioperative fatigue. Among the cytokines, only IL-6 was included as a dependent variable to predict the association between the change in fatigue scores from POD 0 to POD 1. The scatter plot graph illustrated the association between perioperative changes in IL-6 and fatigue scores (e scores . It showe scores . The anaThe results of our study showed that gastric surgery contributed to perioperative fatigue in GA patients. During the perioperative period, the highest score of fatigue occurred on POD 1, but patients partially recovered on POD 7 compared to the preoperative baseline. Considering the perioperative change in cytokine levels, IL-6 significantly increased on POD 1 compared to POD 0, which was associated with the severity of fatigue on adjusted analysis.IL-6, a pleiotropic cytokine involved in both wound healing and regeneration in mitotic tissues , is a prIn addition to the production of IL-6 by muscles or osteoblasts, several types of cancer and associated stromal cells are involved in the production of this cytokine , which cAs IL-6 has emerged as a cytokine causing fatigue in GA patients, therapeutic strategies focusing on IL-6 and associated signaling could be used to attenuate fatigue . In animIn our study, malnutrition was the other risk associated with perioperative fatigue, and one recent study focusing on colectomy patients had similar finding . To our Another explanation for the relationship between elevated IL-6 and fatigue may be related to the anxiety in cancer patients. Anxiety is another common mental illness in cancer patients, who often worry about the cancer stage or oncological outcomes. Studies showed that the patient with chronic anxiety were associated with elevated serum IL-6 values, which caused further neurobehavioral complications . As a reThe findings of our study should be evaluated in the context of some limitations. First, the study is limited to an Asian population, and further studies are warranted to validate these findings in Western populations. Second, the impact of the changes of IL-6 serum values on fatigue in the late postoperative period should also be investigated in the future. More than 30% of GA patients had elevated baseline levels of plasma IL-6 and the levels of plasma IL-6 were associated with the worsening of perioperative fatigue in GA patients. Nevertheless, further clinical studies are needed to validate whether targeting the IL-6 signaling cascade or new fatigue-targeting medications may attenuate fatigue in GA patients undergoing surgical procedures.


The proportion of nanoparticles varied by process from 40 to 95%. Twenty employees, working in nanocomposite materials research were examined pre-shift and post-shift using spirometry and fractional exhaled nitric oxide (FeNO) in parallel with 21 controls. Pro-inflammatory leukotrienes (LT) type B4, C4, D4, and E4; tumor necrosis factor (TNF); interleukins; and anti-inflammatory lipoxins (LXA4 and LXB4) were analyzed in their exhaled breath condensate (EBC). Chronic bronchitis was present in 20% of researchers, but not in controls. A significant decrease in forced expiratory volume in 1 s (FEV1) and FEV1/forced vital capacity (FVC) was found in researchers post-shift (p ˂ 0.05). Post-shift EBC samples were higher for TNF (p ˂ 0.001), LTB4 (p ˂ 0.001), and LTE4 (p ˂ 0.01) compared with controls. Nanocomposites production was associated with LTB4 (p ˂ 0.001), LTE4 (p ˂ 0.05), and TNF (p ˂ 0.001), in addition to pre-shift LTD4 and LXB4 (both p ˂ 0.05). Spirometry documented minor, but significant, post-shift lung impairment. TNF and LTB4 were the most robust markers of biological effects. Proper ventilation and respiratory protection are required during nanocomposites processing.Thousands of researchers and workers worldwide are employed in nanocomposites manufacturing, yet little is known about their respiratory health. Aerosol exposures were characterized using real time and integrated instruments. Aerosol mass concentration ranged from 0.120 mg/m Thousands of workers are engaged in the research, development, and commercial scale production of nano-enabled composites. Yet, limited data is available on exposures and more so on health effects, in occupational settings that produce and use nanomaterials ,2,3 despMolecular epidemiological studies to date on workers handling engineered nanomaterials, suggest respiratory health impairment. Elevated pro-inflammatory markers, including-inflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin (IL) 6 and IL 8 in the biological fluids (blood) of workers ,10, highIn the context of non-invasive sampling of the deep airways ,17, EBC Several lung injury biomarkers have been measured successfully in EBC using highly sensitive liquid chromatography mass spectrometric (LC/MS) techniques. LTB4 is a potent inducer of chronic inflammation because of the activation of leukocytes. Cysteinyl LTs  contract airway smooth muscles and increase vascular permeability . Proinfl2 [2O3) [2 or Fe-oxides to nanocomposite researchers and we have shown that nanocomposites workers have high markers of oxidative stress in EBC [In our previous studies, we report on elevated inflammatory and oxidative stress markers in EBC of workers exposed to aerosols containing about 80–85% nanoparticles of TiO2 ,13 or Fe2 [2O3) . We haves in EBC . In thiss in EBC  in EBC. We have used sensitive techniques such as liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) or matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF).The primary aim of this work was to evaluate, for the first time, markers of inflammation in the EBC of workers exposed to nanoparticles during various tasks involved in the synthesis and post-processing of nanocomposite materials.A detailed description of workplace processes was provided in a preceding paper . The focResearchers would normally work in two workshops: welding and smelting of mixtures containing nano-additives in workshop 1, and machining of the finished nanocomposite in workshop 2. All tasks occur simultaneously in both workshops and lasted on average 2.5 h. For simplicity, we refer to these examinations as pre-shift and post-shift, even though the remainder of their total 8 h shift was spent in their offices. Total duration of nanoparticles production in both workshops lasted about 4 h, each worker was exposed for about 2.5 h.On the day of medical examination, in workshop 1, 11 researchers performed welding on metal surfaces on mild steel . In the same workshop 1, an alloy , mixed with modifying salts , was smelted in the smelting oven at 760 ⁰C.2 fillers, (1.0% w/w nanoSiO2) was performed by the remaining nine researchers. Neither ventilation nor respiratory protection of any kind was used in both shops.In workshop 2, machining of surfaces of nanocomposite blocks of geopolymers and epoxide resins with SiOReal-time nanoaerosol monitoring was conducted with several aerosol spectrometers, including a scanning mobility particle sizer , and an aerodynamic particle sizer , covering the size range of aerosol particles from 6 nm up to 20 µm. Additionally, an ultrafine condensation particle counter , was used to measure the total particle number concentration (3 nm–1 µm), as well as three optical particle sizers  were used to measure number size distribution in the range of 300 nm–10 µm.The measurement of aerosols started 15 min before the beginning of the working tasks. In workshop 1, it lasted 95 min during welding and 100 min during smelting, in workshop 2, it lasted 120 min during machining. Total number of samples measured using SMPS/APS was 109.Area and time integrated sampling was conducted using a Berner low pressure impactor  to sample aerosol particles onto 10 stages corresponding to their aerodynamic diameter covering the 25 nm–13.6 µm size range .Elemental analysis of size-resolved aerosol samples from the BLPI was performed using a scanning electron microscope equipped with energy-dispersive X-ray spectroscopy .Pre-shift and post-shift EBC samples were collected in 20 nanocomposites researchers . They were exposed for an average of 2.5 h in the workshops, the remainder of their total 8-h shift was spent in their offices. For simplicity, we refer to these examinations as pre-shift and post-shift, even though they were in fact post-exposure task measurements.Similarly, 21 controls  working as office employees in the same town were examined.First, they answered questions from a standardized questionnaire concerning their personal and occupational history. Chronic bronchitis was defined clinically as chronic, productive cough for three months in each of two successive years, and dyspnea was defined according to The New York Heart Association (NYHA).All participants then underwent a physical examination, followed by collection of their EBC. Lastly, fractional exhaled nitric oxide (FeNO) and spirometry were also conducted. The researchers were examined twice, at first before exposure and then after the 2.5-h exposure. The controls were examined only once during the same time frame as the researchers.The study was approved by the Ethical Committee of the Charles University according to the Helsinki criteria. All participants were informed of the study aim and signed an informed consent form before the study began.FeNO was measured by a portable Hypair FeNO analyzer . According to ATS/ERS recommendations, a FeNO result greater than 50 ppb was considered elevated, whereas values 25–50 ppb were considered borderline .Spirometry was performed by a SpiroPro, Jaeger, Germany. The measurement included forced vital capacity (FVC), inspiratory vital capacity (VCIN), peak expiratory flow (PEF), and forced expiratory volume in 1 s (FEV1). These parameters were considered lower than normal if they were less than 80% of the predicted values , and if the FEV1/FVC ratio was less than 0.75.EBC samples were collected using Ecoscreen Turbo . All subjects breathed tidally for about 15 min through a mouthpiece connected to a condenser (−20 °C) while wearing a nose-clip. A minimum exhaled air volume of 120 L was maintained through the EcoVent device . All samples were immediately spiked with deuterium labelled standards, immediately frozen and stored at −80 °C for subsequent processing.Analyses of low-molecular biomarkers LTs and LXs were performed using LC-ESI-MS/MS, consisting of a quaternary pump, Accela 600, and Accela autosampler coupled with a triple quadrupole mass spectrometer TSQ Vantage, equipped with heated electrospray ionization (HESI) . Then, solid-phase extraction (SPE) for rapid and effective isolation of biomarkers from the EBC, and MS/MS detection were used, as previously described .ILs and TNF were analyzed by a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) using an Autoflex mass spectrometer  with a MALDI sample target  ,29. To ep < 0.05. All analyses were conducted using SPSS version 22.0 .Basic descriptive statistics  were computed, which were subsequently tested for normality using the Kolmogorov–Smirnov test. To compare frequency counts of demographic categorical variables  in groups of workers versus controls, the Fischer exact test was used. Differences in interval variables  were tested using the Mann–Whitney U test . The paired sample t-test (or the Wilcoxon signed-rank test) was used to compare workers’ pre-shift and post-shift values of the markers of inflammation. The bivariate relationship between variables under study was assessed using the Spearman correlation coefficient. Multiple regression analysis was used to predict markers in EBC by a set of predictors. Two sets of regression models were specified: (a) for controls and pre-shift data , and (b) for controls and post-shift data . Statistical significance was set at 3 during smelting, 0.804 mg/m3 during machining, and 1.840 mg/m3 during welding. Total median particle number concentration was 1.3 × 105 particles/cm3 (#/cm3) and the interquartile range (IQR) was from 1.2 × 105 to 1.5 × 105 #/cm3 during welding, 4.8 × 104 #/cm3 (IQR 3.1 × 104 to 9.0 × 104 #/cm3) during smelting, and 5.4 × 105 #/cm3 (IQR 3.1 × 105 to 6.8 × 105 #/cm3) during machining. The highest proportion of particles smaller than 100 nm in diameter was found during smelting, at 95%, followed by machining (61%), and welding (40%). Chemical analysis of airborne aerosols for all three operations revealed the following elements presented in their descending order of abundance: Fe, Mn, Si, Na, S, Cl, Al, Ca, K, Mg, and Ti. Chemical analysis of the nano-sized fraction showed prevailing Fe, Mn, Si, Al, S, Cl, and K [Exposure data have been provided in detail in a preceding manuscript  and havel, and K .The group characteristics did not differ significantly, including smoking  and daily alcohol consumption (90% in workers and 85% in controls). No difference was seen between the characteristics of the subgroups of researchers working in workshop 1 and workshop 2.Dyspnea, associated with minor limitations in ordinary physical activity, and chronic bronchitis were found only in exposed subjects . Cough wLung function parameters were within the normal range in both researchers and controls. However, the post-shift FEV1, %FEV1, and FEV1/FVC ratio declined significantly in researchers relative to the pre-shift measurements; the changes were modest B. DuratiMedian and mean FeNO levels in researchers did not differ significantly from the controls  and postSeveral pre-shift and post-shift pro-inflammatory markers were elevated relative to controls as shown in For some pro-inflammatory markers, most notably LTE4, IL 9, and IL 10, the post-shift values were higher, whereas for other markers, either there were no differences or no clear trends.There was no significant difference in pH of EBC samples of both groups. Amylase concentrations were less than 0.01% of those in saliva in all samples.Duration of employment (years) as a nanocomposite researcher correlated positively with pre-shift TNF and negatively with post-shift anti-inflammatory LXB4. Allergic rhinitis, cough, and chronic bronchitis in the exposed subjects correlated with lower pH in both pre- and post-shift EBC samples, as presented in The levels of several markers in the pre-shift and the post-shift samples correlated, for example, with pH, LTs, FeNO, and IL 4.In addition, some correlations between different pre-shift and post-shift markers were found. They are shown in Confirmed significant association between nanocomposites handling and pre-shift and post-shift LTB4, LTE4, and TNF, as shown in Markers of oxidative stress were presented in an accompanying manuscript . Here, wA large body of in vivo and in vitro nanotoxicology studies have shown that nanoparticles induce intracellular reactive oxygen species, and pro-inflammatory mediators ,31. MuchThis study deals with long-term employed subjects in nanocomposites research, where the proportion of nanoparticles in the aerosol during handling of nanocomposites ranged from 40% to 95%. The chemical analysis of the nano-sized fraction in these processes identified predominantly Fe, Mn, Al, S, and Si . High co2 exposed workers, relative to controls [In our earlier studies, LTs were elevated in TiOcontrols . LTB4 oncontrols .2 production workers and 2.5 h in nanocomposite workers.In this study of nanocomposite manufacturing workers, we did find elevated LTB4 and LTE4, but not LTC4 and post-shift LTD4. These differences may reflect different exposure profiles, including exposure intensities, size distributions, chemical composition, as well as frequency and duration of tasks. For example, exposure duration was on average 4 h in TiO2 , but 40% higher than LTB4 in the EBC of office workers of the same factory who spent about 15 min daily in the workshops (28 pg/mL) [LTB4 levels in nanocomposite research workers in this study were ~30% lower than in workers exposed to nanoTiO8 pg/mL) .p = 0.039).Both pre-shift and post-shift TNF overexpression were positively associated with exposure, as well as with several other oxidative stress and inflammatory markers in other human studies ,33,34. TBoth pro-inflammatory cytokines/chemokines (IL 5 and IL 9), and anti-inflammatory IL 4, IL 10, IL 13, LXA4, and LXB4 were examined.The anti-inflammatory eicosanoids mediators that are derived from the arachidonic acid are involved in the resolution of acute inflammatory responses and initiate tissue repair in response to injury, infection, or allergy ,36,37. LWe have confirmed that these mediators end up in the EBC and we could see several negative correlations of the anti-inflammatory LXB4 with the pro-inflammatory markers, especially TNF and with several markers of oxidative stress .Pro-inflammatory IL 9 is an important player in the pathogenesis of bronchial hyperresponsiveness that affects various cell types involved in immunity and inflammation . Again, 2 studies [FeNO, which decreased during the shift, does not seem useful, accordingly to both our nanoTiO studies  and the  studies . This ag studies . Another studies .Our findings are consistent with limited epidemiological studies in nanomanufacturing workers, in which reduction of lung function parameters and an increase of TNF or oxidative stress markers were described ,43,44. I2, as well as their respective office workers attending the workshops only for a brief period of time [In addition, markers of oxidation of lipids, nucleic acids, and proteins used in our studies were so sensitive that they could distinguish between industrial production workers exposed to TiO of time ,46,47.It is important to mention that the main source of airborne nanoparticles in the workshops originated from manufacturing and post-processing of nanocomposites, including smelting, welding, possibly handling of nano- powders (which was not monitored here), and mechanical processing such as trimming and polishing. As such, these nanoparticles are of mixed origin  and mixed composition. It is not possible, and perhaps not advisable, to try and parse out the relative contribution of such nanoparticles on the health outcomes. On the contrary, such research settings should adopt better exposure controls and increase researchers’ awareness about such exposures and their possible health effects. In occupational cohorts exposed to nanoparticles and other particles in general, respiratory disorders typically develop under chronic exposures over many years to decades . PredictThe main limitation of this study is the relatively low number of exposed subjects. Unfortunately, the research and development nature of the nanocomposite sector  is characterized by small and unstable occupational cohorts of workers. As a result, the number of similar studies is also remarkably limited.Another limitation is that we could not measure personal exposures and link exposures with airway dosimetry. A one-day exposure characterization is not representative of long-term exposures to these workers. Therefore, we plan to continue to monitor them over time and generate personal exposure data and better exposure summaries.2 and nano Fe-oxides, and even in this group of nanocomposites research workers. Better exposure controls and increased awareness are needed in this as well as similar nanomanufacturing sectors.This study provides evidence that occupational exposure to inhaled nanomaterials generated during various tasks associated with nanocomposite synthesis, production, and post-processing (in both workshops) causes respiratory impairments and subclinical spirometry changes. Several markers of inflammations were elevated both pre- and post-shift, with an underlying chronic neutrophilic, not eosinophilic, inflammation, according to FeNO results and clinical findings. The length of the employment in the nanocomposites production correlated with TNF and a decline in the FEV1/FVC ratio, which supports the chronic effect of exposure to nanomaterials. These findings and those from our earlier studies provide evidence in favor of the usefulness of monitoring inflammatory markers, such as TNF, in EBC. Overexpression of TNF in EBC complements our earlier findings of DNA, protein, and lipid damage in the EBC of workers with nanoTiO